- Email: [email protected]
Activation-induced T cell apoptosis by monocytes from stem cell products
International Immunopharmacology 1 Ž2001. 1307–1319 www.elsevier.comrlocaterintimp
Activation-induced T cell apoptosis by monocytes from stem cell products q Kazuhiko Ino a , Ana G. Ageitos b, Rakesh K. Singh c , James E. Talmadge c,) a
Department of Obstetrics and Gynecology, Nagoya UniÕersity School of Medicine, 65 Surumai-cho, Showa-ku, Nagoya 466-8550 Japan b Oncology Department, Fundacion Jimenez Diaz, SerÕicio de Oncologia, AÕada Reyes Catolicos, 2, Madrid 28015 Spain c Department of Pathology and Microbiology, UniÕersity of Nebraska Medical Center, 987660 Nebraska Medical Center, Omaha, NE 68198-5600, USA Received 31 October 2000; received in revised form 15 February 2001; accepted 28 February 2001
Abstract We recently found that mobilized peripheral blood stem cell ŽPSC. products Žfrom both cancer patients and normal donors. contain high levels of CD14q monocytes, which can inhibit the proliferation of allogeneic and autologous T cells. We found in our studies that using CD14q monocytes from mobilized PSC products Žfrom normal and cancer patient donors., normal apheresis products or normal peripheral blood ŽPB. can affect lymphocyte function and apoptosis-dependent T cell activation. However, it appears that the apoptosis is dependent on the frequency of monocytes, which is increased by both mobilization and apheresis. Both phytohemagglutinin ŽPHA.- and interleukin ŽIL.-2-induced proliferation of steady-state peripheral blood mononuclear cells ŽPBMC. were markedly inhibited by co-culture with irradiated CD14q monocytes, although inhibition was significantly greater with PHA than with IL-2 stimulation. IL-2 Žpredominately CD56q NK cells. or anti-CD3 monoclonal antibody ŽmAb. and IL-2-expanded lymphocytes Žactivated T cells. were inhibited by PSC monocytes to a significantly greater level as compared to steady-state lymphocytes. Indeed, no inhibition of T cell proliferation was observed when lymphocytes were co-cultured in the absence of mitogenic or IL-2 stimulation. In contrast, an increased proliferation was observed in co-cultures of CD14q monocytes and steady-state or activated lymphocytes without mitogenic stimulation. Cell cycle analysis by flow cytometry revealed a significant increase in hypodiploid DNA, in a time-dependent manner, following co-culture of monocytes and PBMC in PHA, suggesting that T cell apoptosis occurred during PHA-induced activation. These results demonstrate that PSC-derived monocytes inhibit T cell proliferation by inducing the apoptosis of activated T cells and NK cells, but not steady-state cells. This suggests a potential role for monocytes in the induction of peripheral tolerance following stem cell transplantation. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Monocytes; Peripheral tolerance; T cells; IL-2; Mitogenesis
q )
This research was supported in part by grant aRO1-CA61593 from the National Institutes of Health. Corresponding author. Tel.: q1-402-559-5639; fax: q1-402-559-4990. E-mail address: [email protected] ŽJ.E. Talmadge..
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 0 6 2 - 5
1308
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1. Introduction High-dose therapy ŽHDT. supported by stem cell transplantation is increasingly used for the treatment of advanced cancer patients w1,2x. Immune reconstitution and hematopoietic recovery are more rapid following growth factor-mobilized peripheral blood stem cell transplantation ŽPSCT. than following transplant with bone marrow autografts w3–5x. Nonetheless, immune dysfunction is observed following PSCT, despite the restoration of normal lymphocyte numbers, and may be associated, at least in part, with the immunologic characteristics of the infused peripheral stem cell ŽPSC. products w4–16x. We have previously shown that this is due to the frequency of monocytes within mobilized stem cell products w17x and the expression of Fas-L w18,19x. We reported recently that high levels of cellulardependent T cell inhibiting activity are present in both granulocyte macrophage colony-stimulating factor ŽGM-CSF.-mobilized PSC products and peripheral blood ŽPB. following autologous PSCT w5,6,17,18,20–23x. We also have found that this activity is associated with CD14q monocytes, which can suppress T cell function in vitro via cell-to-cell contact w17x. Since immune function is determined by a balance of positive and negative immunoregulatory cytokines, the CD14q cells observed at a high frequency Ž30–40%. in PSC products have the potential to inhibit immune recovery following HDT and PSCT w6x. Furthermore, monocyte-dependent T cell inhibitory ŽMDTI. activity has the potential to reduce therapeutic responses to adjuvant immunotherapy following autologous PSCT. Conversely, MDTI activity may prevent an increase in acute graft-vs.-host disease ŽGVHD. in allogeneic PSCT patients, despite the infusion of 100-fold more T cells than is infused in bone marrow transplant ŽBMT. patients, as observed by recent clinical studies w19,24,25x. Here we report the MDTI activity by CD14q monocytes isolated from mobilized PSC products for lymphocytes with or without mitogenic or interleukin ŽIL.-2 stimulation. Our results indicate that monocytes inhibit the proliferation of activated, but not steady-state, lymphocytes. Further, we provide preliminary data suggesting apoptosis as one mechanism of MDTI, and that MDTI is observed in mobi-
lized PSC products from both normal and cancer patients.
2. Materials and methods 2.1. Cell preparation PSC product from either the second or third apheresis was collected with a Cobe spectra ŽCobe BCT, Lakewood, CO. from 10 cancer patients Žsix with non-Hodgkin’s lymphoma and four with breast cancer. following administration of GM-CSF ŽImmunex, Seattle, WA. at a dose of 250 m grm2 to mobilize progenitor cells. Written informed consent for PSC collection and PSCT was obtained from each patient. All samples were obtained according to protocols approved by the Institutional Review Board of the University of Nebraska Medical Center ŽUNMC.. PSC products Ž2 ml. were diluted in Hanks Balanced Salt Solution ŽHBSS, Gibco, Grand Island, NY. with 10 mM HEPES ŽResearch Organics, Cleveland, OH. and peripheral blood mononuclear cells ŽPBMC. separated by centrifugation on a Ficoll-Hypaque ŽOrganon Teknika, Durham, NC. gradient. After washing, cells were resuspended in complete medium consisting of RPMI 1640 medium ŽGibco. supplemented with 10% fetal bovine serum ŽFBS, Gibco., 40 m grml gentamicin ŽGibco., 2 mM L-glutamine, and 10 mM HEPES. PBMC from the PB of healthy donors were also obtained following Ficoll-Hypaque gradient. 2.2. Isolation of CD14 q monocytic cells from PSC products [26–28] CD14q monocytic cells were isolated from PSC products using Percoll density gradients centrifugation. In brief, Ficoll-purified PSC were suspended in iso-osmotic complete medium adjusted to 285 mosM. The stock solution of Percoll ŽSigma, St. Louis, MO. was prepared by mixing 9.25 parts of concentrated Percoll and 0.75 parts of 10 = concentrated phosphate buffered saline ŽPBS.. The osmolarity of this solution was adjusted to 285 mosM. Iso-osmotic complete medium and Percoll solution were mixed to prepare seven different concentrations of Percoll
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Žvrv.: 38.6, 47.5, 52.1, 56.5, 61.1, 65.6, 70.1%. Gradients consisted of seven fractions layered in a discontinuous manner using 1.5 ml of each with fraction 7 Ž70.1%. at the bottom of a 15 ml conical tube. Thereafter, 3 ml of the PSC suspension Žup to 1 = 10 8 cells. were carefully placed on the top of the gradients, and centrifuged at 550 = g for 30 min at room temperature. Cells at each interface were collected and washed twice in HBSS and resuspended in complete medium. Following separation, most monocytes were contained in the low density fraction from the band at the 38.6–47.5% interface, whereas the majority of lymphocytes were in the higher density fractions from the bands at 47.5– 52.1% and 52.1–56.5%. To obtain further purified CD14q monocytic cells, the contaminated CD4q and CD8q T cells in the low density fraction were depleted by immunomagnetic bead cell isolation ŽMiniMACS, Miltenyi Biotec, Auburn, CA. using magnetic microbead-conjugated anti-CD4 and CD8 monoclonal antibodies ŽmAbs.. The monocyte purity was assessed by fluorescent cytometry using antiCD14 mAb Ždescribed below. and morphology using Wright-Giemsa stained smears. The CD4q and CD8q T cell-depleted low density PSC were 85% to 95% Ž89% on average. purified CD14q monocytic cells. Cell viability was determined by Trypan blue dye exclusion Žviability always exceeded 95%.. 2.3. Treatment of responder cells with anti-CD3 mAb and r or IL-2 To generate activated effector cells such as lymphokine-activated killer ŽLAK., natural killer ŽNK. cells and activated T cells, PBMC were treated as described in previous reports w29,30x. In brief, Ficoll-purified PBMC from normal donors were added at a concentration of 2 = 10 6 cellsrwell in complete medium including 100 Urml of recombinant human IL-2 ŽChiron, Emeryville, CA. in a 24-well culture plate. Cells were cultured for 72 h at 378C in a humidified 5% CO 2 atmosphere. The cells were then harvested by pipetting vigorously, washing once with HBSS, and resuspending in complete medium. In some experiments, cells were pretreated with anti-CD3 cross-linking mAb ŽOKT3, Ortho Biotech, Raritan, NJ. before the start of culture with
1309
IL-2 to upregulate T cell receptors on CD3q T cells. PBMC were incubated with 100 ngrml of anti-CD3 mAb for 30 min in a 15 ml conical tube in a 378C water bath with gentle agitation. These anti-CD3 mAb coated cells were then cultured with 100 Urml IL-2 for 72 h. Both activated effector cell populations were used as responder cells for a subsequent MDTI assay. 2.4. Immunofluorescent staining and flow cytometric analysis Three-color fluorescent cytometry analysis was used for cell surface immunophenotyping of responder lymphocytes. Cells were counted and adjusted to 1 = 10 6rml in PBS containing 2% FBS. Aliquots of 1 = 10 5 cells were stained at 48C for 30 min with mAbs specific for the following cell surface markers: Fluorescein isothiocyanate ŽFITC.-labeled anti-CD3 ŽBecton Dickinson, San Jose, CA., phycoerythrin ŽPE.-labeled anti-CD4, CD56, CD25 ŽBecton Dickinson., and biotin-conjugated anti-CD8 ŽBecton Dickinson. with CD19 ŽCoulter, Hialeah, FL. added with streptavidian allophycocyanin ŽAPC. as the third fluorochrome. To assess the purity of CD14q monocytes isolated from PSC products, we also used biotin-conjugated anti-CD14 mAb ŽCoulter.. Background staining using isotype controls was done to determine thresholds for positive cells. All three-color data were acquired on a FACStar Plus ŽBecton Dickinson. and analyzed using the Cellquest software ŽBecton Dickinson.. 2.5. MDTI actiÕity assay The methodology for the co-culture assay to measure MDTI activity has been previously described w5,6,17x. Briefly, freshly purified normal PBMC or IL-2-cultured cells Žwith or without treatment with anti-CD3. as responder cells Ž1 = 10 5 . were co-cultured with varying numbers of irradiated Ž500 cGy. inhibitor cells Žisolated CD14q cells. at inhibitor to responder ŽI:R. ratios of 4:1, 2:1, 1:1, and 0.5:1. Cells were cultured in complete medium with or without an optimal concentration of phytohemagglutinin ŽPHA. Ž0.1 m grml. or IL-2 Ž100 Urml. in 96-well flat bottom microplates for 72 h at 378C in a
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1310
humidified 5% CO 2 atmosphere. In some wells, responder cells alone were cultured with PHA or IL-2 as a control. The proliferative response of responder cells was determined by 3 H-thymidine incorporation over the final 18 h of culture. All experiments were performed in triplicate. The percent MDTI activity was calculated using the following formula Žcpm s counts per minute.:
The mean and standard error of the mean ŽS.E.M.. are shown for all data. The unpaired Student’s t-test was used to compare groups. A p value of less than 0.05 was considered significant.
% inhibition
3. Results
s 1 y Ž mean cpm in tested wells with co ycultured cells . r Ž mean cpm in control wells without co ycultured cells . = 100. 2.6. Measurement of apoptosis by flow cytometry Ficoll-purified PBL Ž6 = 10 5rwell. were co-cultured with irradiated CD14q inhibitor cells Ž1.2 = 10 6rwell. in 24-well culture plates in the presence of 0.1 m grml PHA. In some wells, only responder PBL were cultured with the same concentration of PHA as a control. After culture for 2, 4, 12, 24, and 48 h, non-adherent cells Žlymphocytes. were collected by repeated pipetting and washing in PBS twice. To measure the percentage of apoptotic cells, cell cycle analysis was carried out as reported by Telford et al. w31x. In brief, cells Ž2 = 10 6 . were resuspended in 1 ml of ice-cold 70% ethanol with vigorous mixing. The cells were incubated at 48C for 1 h. The ethanol-fixed cells were then washed in ice-cold PBS and resuspended in 1 ml of a staining reagent consisting of 0.1% Triton X-100, 0.1 mM EDTA, 50 m grml RNase A Ž50 Urmg. and 50 m grml propidium iodide ŽPI, Sigma.. Samples were stored in the dark at 48C overnight. Samples were acquired on a FACStar Plus ŽBecton Dickinson. using the 488 nm argon laser and analyzed by Cellquest and Modifit software ŽBecton Dickinson.. Only lymphocytes were gated and evaluated for apoptosis. Contaminating CD14q monocytic cells were removed by gating based on differences in forward and side scatter intensity. Apoptotic cells were determined by the hypodiploid areas Žbelow the G0rG1 region. in the PI-staining profiles.
2.7. Statistical analysis
3.1. MDTI actiÕity against PHA or IL-2-induced lymphocyte proliferation We evaluated the MDTI activity of CD14q cells purified from PSC products for lymphoproliferative responses to either PHA or IL-2 using fresh Žsteadystate. or activated lymphocyte responders. The PHA-induced mitogenesis of normal allogeneic PBMC was significantly Ž p F 0.05. inhibited compared to control Žwithout monocyte co-culture. in a dose-dependent manner when co-cultured with irradiated CD14q inhibitor cells for 72 h at inhibitor to
Fig. 1. Monocyte-dependent T cell inhibitory ŽMDTI. activity of peripheral stem cell ŽPSC.-derived CD14q cells against phytohemagglutinin ŽPHA. or interleukin ŽIL.-2-induced proliferation of fresh peripheral blood mononuclear cells ŽPBMC.. Normal peripheral blood mononuclear cells ŽPBMC. were co-cultured with irradiated monocytes for 72 h at the indicated inhibitor:response ŽI:R. ratios with phytohemagglutinin ŽPHA. or IL-2, and percent monocyte-dependent T cell inhibitory ŽMDTI. activity was calculated. Points, mean"standard error of the mean ŽS.E.M... ) Significant difference compared to the IL-2 stimulation group Ž pF 0.05.. aSignificant difference compared to control Žwithout co-culture. Ž pF 0.05..
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1311
Table 1 Effect of activation on PHA-induced proliferation of PBMC in the presence or absence of CD14q monocytes Responder peripheral blood mononuclear cells ŽPBMC. were untreated or pretreated with interleukin ŽIL.-2 alone or anti-CD3 and IL-2 for 72 h. The responder cells were then co-cultured with irradiated CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 72 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. Mitogenic proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M.. for the 3 H-thymidine incorporation expressed as counts per minute Žcpm.. Responder cells PBMC Ž n s 6.
IL-2-pretreated Ž n s 3. Anti-CD3q IL-2-pretreated Ž n s 3.
cpm PI b % Inhibition cpm PI b % Inhibition cpm PI b % Inhibition
Control Žwithout co-culture.
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1
I:R s 1:1
I:R s 0.5:1
89,839 " 2713 – – 91,771 " 6006 – – 87,085 " 1004 – –
3653 " 1247 a 0.04 " 0.01c 96 " 1%c 13,188 " 2534 a 0.15 " 0.05 85 " 5% 7147 " 3325a 0.08 " 0.04 92 " 4%
55,489 " 6915a 0.62 " 0.08 d 38 " 8%d 88,098 " 16,138 0.96 " 0.15 4 " 15% 85,964 " 1008 0.98 " 0.01 2 " 1%
77,953 " 4295a 0.88 " 0.06 d 12 " 6%d 113,613 " 10,904 1.26 " 0.14 y26 " 14% 105,371 " 11,241 1.21 " 0.13 y21 " 13%
31,264 " 6333 a 0.35 " 0.07 d 65 " 7%d 58,804 " 11,070 a 0.67 " 0.15 33 " 15% 48,970 " 2003 a 0.56 " 0.03 44 " 3%
a
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s cpm with co-cultured inhibitor cellsxrwcpm without co-cultured inhibitor cellsx. c Significant difference in PI and % monocyte-dependent T cell inhibitory ŽMDTI. activity compared to IL-2-pretreated group Ž p - 0.05.. d Significant difference in PI and % MDTI activity compared to IL-2-pretreated and anti-CD3 and IL-2-pretreated groups Ž p - 0.05.. b
response ratios ŽI:R. of 4:1 to 0.5:1 ŽFig. 1 and Table 1.. The proliferation of PBL to IL-2 stimulation was also significantly Ž p F 0.05. inhibited by co-culture
at I:R of 4:1 and 2:1 ŽTable 2., although inhibition was significantly greater with PHA stimulation than with IL-2 stimulation ŽFig. 1..
Table 2 Effect of activation on IL-2-induced proliferation of PBMC in the presence or absence of CD14q monocytes Responder peripheral blood mononuclear cells ŽPBMC. were untreated or pretreated with interleukin ŽIL.-2 alone or anti-CD3 and IL-2 for 72 h. The responder cells were then co-cultured with irradiated CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 72 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. IL-2-induced proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M... Responder cells PBLs Ž n s 6.
IL-2-pretreated Ž n s 3. Anti-CD3q IL-2-pretreated Ž n s 3. a b
Control Žwithout co-culture. cpm PI b % Inhibition cpm PI b % Inhibition cpm PI b % Inhibition
18,003 " 1868 – – 47,156 "20,896 – – 87,085 " 1004 – –
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1 a
3042 " 659 0.16 " 0.03 84 " 3% 8734 " 2774a 0.30 " 0.17 70 " 17% 11,900 " 7528 a 0.15 " 0.07 85 " 7%
a
10,407 " 1288 0.57 " 0.03 43 " 3% 18,208 " 2417 0.58 " 0.25 42 " 25% 45,591 " 22,528 0.70 " 0.27 30 " 27%
I:R s 1:1
I:R s 0.5:1
16,985 " 2688 0.94 " 0.10 6 " 10% 36,638 " 4935 1.00 " 0.27 0 " 27% 62,088 " 25,160 0.99 " 0.36 1 " 36%
20,309 " 2505 1.13 " 0.08 y13 " 8% 50,773 " 8685 1.34 " 0.30 y34 " 30% 74,476 " 25,400 1.19 " 0.34 y19 " 34%
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s wcounts per minute Žcpm. with co-cultured monocytesxrwcpm without co-cultured monocytesx.
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1312
The PSC-derived inhibitory cells also inhibited the PHA- or IL-2-induced proliferation of activated responder cells. Following preculture of PBMC for 72 h with IL-2 alone, CD56q NK cells were significantly increased to 69.62 " 12.3% on average Ž n s 4. as compared to unstimulated PBMC Ž7.0 " 1.9%., while CD3q CD25q activated T cells were significantly increased to 61.1 " 0.52% on average Ž n s 3. following anti-CD3 and IL-2 treatment as compared to unstimulated PBMC Ž2.11 " 0.75%.. The proliferation of the IL-2-activated NK cell predominant population was significantly Ž p F 0.05. inhibited when co-cultured with monocytes at I:R of 4:1 and 2:1 with PHA stimulation and at 4:1 with IL-2 stimulation. Similarly, the proliferative response to PHA and IL-2 of anti-CD3- and IL-2-activated T cells was significantly Ž p F 0.05. inhibited at the same I:R. However, both types of activated cells were significantly less sensitive to inhibition with PHA stimulation as compared to steady-state PBMC ŽTable 1., while there was no difference in sensitivity to inhibition among the three responders with IL-2 stimulation ŽTable 2.. In contrast to the significantly inhibited PHA or IL-2 mitogenic response, no MDTI activity was observed to any responder cells Žwhether previously
activated or not. when cells were co-cultured with monocytes and without mitogenic or IL-2 stimulation ŽTable 3.. These results indicate that PSC-derived cells can exert their MDTI activity against T cells and NK cells undergoing activation, but have less activity against activated cells and no activity against steady-state cells in the absence of mitogenic stimulation. 3.2. MDTI actiÕity from PBMC and PSC products from normal donors MDTI activity could be associated not only with the frequency of monocytes, but also the mobilizing cytokine, the presence of neoplastic disease, and the number of prior chemotherapy cycles w22x. In previous studies, we reported that there is a direct association between MDTI activity and the frequency of monocytes contained within the product w17x and the number of cycles of prior chemotherapy w22x. In the studies shown in Fig. 2, we compared at an I:R of 1:4 and 1:2 the MDTI activity of G-CSF-mobilized PSC products from metastatic breast cancer patients to G-CSF-mobilized PSC products and non-mobilized, apheresis products obtained from volunteer normal donors and normal PBMC products. The
Table 3 Effect of activation on steady state proliferation of PBMC in the presence or absence of CD14q monocytes Responder peripheral blood mononuclear cells ŽPBMC. were untreated or pretreated with interleukin ŽIL.-2 alone or anti-CD3 and IL-2 for 72 h. The responder cells were co-cultured with irradiated CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 72 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. Proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M... Responder cells PBLs Ž n s 6.
IL-2-pretreated Ž n s 3. anti-CD3q IL-2-pretreated Ž n s 3. a
Control Žwithout co-culture. cpm PI b % stimulation cpm PI b % stimulation cpm PI b % stimulation
559 " 16 – – 14,199 " 8188 – – 11,694 " 5020 – –
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1 508 " 85 0.98 " 0.10 9.1 " 1.5 c 12,718 " 6288 0.92 " 0.17 10.4 " 5.1 10,656 " 4469 0.90 " 0.05 8.9 " 3.7
1944 " 517 a 2.12 " 0.67 247.8 " 66 21,384 " 7110 a 3.50 " 0.97 50.6 " 16.8 28,425 " 13,240 a 2.25 " 0.21 143 " 66.6
I:R s 1:1
I:R s 0.5:1
2220 " 484 a 2.64 " 0.96 297.1 " 64.8 30,090 " 14,998 a 3.96 " 0.89 111.9 " 55.8 36,782 " 18,914 a 2.60 " 0.64 214 " 54
2425 " 406 a 3.17 " 0.82 333.8 " 55.9 38,133 " 19,385a 4.30 " 0.70 168.6 " 85.7 43,995 " 24,864 a 3.09 " 0.82 276.2 " 156.1
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s wcounts per minute Žcpm. with co-cultured monocytesxrwcpm without co-cultured monocytesx. c Percent stimulations wŽcpm with co-culture monocytes. y Žcpm without co-culture monocytes.xrcpm without co-culture monocyte. b
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1313
Fig. 2. Monocyte-dependent T cell inhibitor ŽMDTI. activity of various peripheral blood mononuclear cells ŽPBMC. source. This includes peripheral stem cell ŽPSC. obtained following granulocyte-colony stimulating factor ŽG-CSF. mobilization of autologous cancer patients Žauto Mob PSC., G-CSF mobilized normal PSC Žnorm Mob PSC. and normal non-mobilized apheresis cells Žnorm non-Mob PSC.. In these studies, normal PBMC were co-cultured with irradiated sources of cellular inhibitors for 72 h at the indicated inhibitor:responder ŽI:R. ratio with phytohemagglutinin ŽPHA. and percent MDTI activity calculated and expressed as the mean " standard error of the mean ŽS.E.M... In these studies, there was an N of 19 for auto Mob PSC, 18 for norm Mob PSC, 9 for normal non-MOB PSC and 13 for norm PBL. The insert graphic shows the percent of monocytes contained within the various products" S.E.M. ) Significantly different from normal peripheral blood leukocytes Žnorm PBL.. aSignificantly different from the auto Mob PSC, p F 0.05 by Student’s t-test.
normal PBMC cells and normal, non-mobilized PSC cells had minimal MDTI activity. However, normal, mobilized PSC products, used to transplant allodonors, and mobilized PSC cells from autologous donors demonstrated significant levels of MDTI activity. As shown in the insert to Fig. 2, there was a direct relationship between the frequency of monocytes within these products and the extent of MDTI activity, verifying these results and previous reports on the frequency of monocytes and MDTI activity w17x. Further, as shown in Table 1, pure populations of monocytes from G-CSF-mobilized products are capable of high levels of MDTI activity, suggesting the need for a high I:R ratio. In contrast to mobilized products from normal donors and cancer patients, normal PMNC have minimal MDTI activity. Nonmobilized PSC products have a slight, but significant, increase in monocyte frequency, but not a significant increase in MDTI levels. These results also suggest a relationship between MDTI activity
and percent monocytes. Despite minimal MDTI activity by normal PBMC if the monocytes are enriched by Percoll isolation w17x, they also demonstrate MDTI activity ŽTable 4.. In these studies we examined the MDTI of enriched monocytes on the PHA response of autologous and allogeneic lymphocytes. As can be seen in Table 4, there was a significant inhibition of both allogeneic and autologous lymphocyte response to PHA mitogenesis ranging from 49% to 56%. Although not as extensive as that observed with purified monocytes from mobilized apheresis products from cancer patients, it does suggest that normal monocytes are capable of MDTI activity in response to PHA mitogenesis. 3.3. Induction of T cell apoptosis by monocytes To determine the possible mechanism of MDTI activity, a cell cycle study was undertaken to deter-
1314
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Table 4 Effect of PHA induced proliferation of autologous and allogeneic PBMC in the presence or absence of isolated, normal CD14q monocytes Responder, autologous or allogenic normal peripheral blood mononuclear cells ŽPBMC. were co-cultured in the presence or absence of irradiated, normal CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 96 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. Mitogenic proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M.. for the 3 H-thymidine incorporation assay, and compared with controls. Responder cells Autologous lymphocytes Ž n s 6. Allogeneic lymphocytes Ž n s 9. a b
cpm PI b % stimulation cpm PI b % stimulation
Control Žwithout monocyte co-culture.
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1
I:R s 1:1
109,983 " 21,173 – – 118,026 " 23,043 – –
44,384 a " 11,944 0.44 " 0.11 56.4 " 11.2 62,985a " 16,618 0.51 " 0.11 49.2 " 11.1
105,752 " 17,545 1.00 " 0.05 0.1 " 5.4 105,206 " 18,850 0.92 " 0.04 8.1 " 4.4
88,703 a " 14,985 0.83 " 0.07 16.6 " 6.9 93,304 a " 20,274 0.78 " 0.09 21.6 " 9.1
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s cpm with co-cultured inhibitor cellsrcpm without co-cultured inhibitor cells.
mine the induction of apoptosis by flow cytometry using PI-stained cells. After PBMC were co-cultured with irradiated CD14q cells in the presence of PHA,
only non-adherent responder cells were collected and evaluated. As shown in Fig. 3, the fragmented DNA Žhypodiploid areas. of responder lymphocytes were
Fig. 3. DNA histograms by flow cytometry using propidium iodide ŽPI.-stained lymphocytes. Peripheral blood mononuclear cells ŽPBMC. were co-cultured with CD14q monocytes with phytohemagglutinin ŽPHA. stimulation at an inhibition:responder ŽI:R. ratio of 2:1 for 0 Žcontrol. ŽA., 4 ŽB., 24 ŽC., or 48 h ŽD.. Then only lymphocytes were gated and evaluated for DNA histograms by flow cytometric analysis as described in Materials and methods. Percent apoptotic cells were determined as the cells below the G0rG1 peak ŽM1 region.. In this representative sample, % apoptotic cells in A, B, C, and D were 0.4%, 2.1%, 21.0%, and 32.2%, respectively.
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Fig. 4. T cell apoptosis induced by peripheral blood stem cell ŽPSC.-derived monocytes. Peripheral blood mononuclear cells ŽPBMC. were cultured with or without CD14q monocytes with phytohemagglutinin ŽPHA. stimulation at a inhibition:response ŽI:R. ratio of 2:1 for the indicated time and non-adherent cells were collected and stained with propidium iodide ŽPI.. Then only lymphocytes were gated and evaluated for DNA histograms by flow cytometric analysis. Percent apoptotic cells were determined as the cells below the G0 rG1 peak. Points, mean"standard error of the mean ŽS.E.M.. Ž ns 4.. ) Significant difference compared to control Žwithout co-culture. Ž pF 0.01..
clearly observed after co-culture for 24 and 48 h. Since we previously found that irradiated CD14q cells alone Ž89% purity., at least within the irradiation dose used in this study, showed no apoptosis at any culture time Ž2–48 h. Ždata not shown., this result demonstrates the apoptosis of responder T cells during PHA stimulation. The frequency of apoptotic cells was increased by co-culture in a time-dependent manner and significantly increased Ž p F 0.01. as compared to control Žwithout co-culture. at 24- and 48-h incubation ŽFig. 4..
4. Discussion Cells with T cell inhibitory activity have been found in sites of intense hematopoiesis w32–39x and can be increased by tumor secretion or exogenous administration of hematopoietic growth factors such as GM-CSF or IL-3 w37–39x. However, the cellular lineage and mechanism of the MDTI action remains controversial w32–39x and few studies focus
1315
on human cells. Schmidt-Wolf et al. found one cellular subset in human bone marrow that had a CD4y CD8y ab TCRq phenotype w33x while Pak et al. reported that CD34q progenitor cells inhibited immune function in patients with a GM-CSF-secreting cancer w37x. In contrast, several studies demonstrated the immune inhibitor is from the macrophagermonocyte lineage w35,39–41x. In our earlier studies w5,6x. we found, for the first time, that growth factor-mobilized human PSC products from cancer patients contained high levels of MDTI activity, which is associated with GM-CSF mobilization andror leukapheresis. This profound inhibition of T cell function is observed not only in the PSC product itself but also in the PB of patients post-transplantation w5,18x. More recently, we isolated and characterized the origin of this inhibitor cell activity in PSC products and found that the MDTI activity is associated with a CD14q monocytic cell w17,18,21–23x. Since about 40% of GM-CSF-mobilized and apheresed PSC and more than 10% of PB leukocytes up to day 30 post-PSCT are CD14q monocytes w5,6x, this high frequency of inhibitor cells could have a significant effect on immune recovery following PSCT. Increased levels of T cell apoptosis are observed following allogeneic PSCT and may occur by similar mechanisms w9,10,42x. Further, the MDTI activity may reduce the efficiency of adjuvant immunotherapy, including IL-2 administration posttransplant or adoptive immunotherapy using ex vivo expanded and activated effector cells. Thus, in this paper we examine the MDTI activity of PSC-derived CD14q cells for proliferative response of steady-state PBL as well as activated T cells, NK and LAK cells. The data show that PSC-derived cells responsible for MDTI activity can significantly inhibit the proliferation of responder lymphocytes, although previously activated T cells and NKrLAK cells are less sensitive to MDTI activity as compared to the strong inhibition observed in PBMC undergoing PHA blastogenesis. Further, no MDTI activity was found for any responder cells when cells were co-cultured without mitogenic or IL-2 stimulation. Indeed, we observed significantly increased 3 H-thymidine uptake in the PBMC rather than inhibition when co-cultured without stimulation at I:R of 2:1 to 0.5:1, although counts per minute values were very low Žless than 3000.. These results suggest that the cells
1316
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
responsible for MDTI activity can exert their inhibitory activity against activated lymphocytes, but not against steady-state, unstimulated lymphocytes. Our data also suggest that monocytes exert their greatest effect on the initial step of T cell activation from resting cells to activated cells with primary stimulation, while exerting less effect on previously activated cells. Proliferation of T and NK cells is dependent on the interaction of IL-2 with its receptors w29,30,43,44x. Monocytes can negatively control T cell activation and proliferation w39,45–49x by various mechanisms, including prostaglandins w39,46x, nitric oxide w46–49x and tumor necrosis factor ŽTNF. w49x. However, in earlier studies, we found that inhibition by PSC-derived monocytes requires direct cell-to-cell contact and is not abrogated by the anti-TNF neutralizing antibody w17x. More recently, macrophage- or antigen-presenting cell ŽAPC.-induced T cell apoptosis has been proposed w50–52x. Munn et al. w52x suggested that macrophages exposed to macrophage-CSF ŽM-CSF. can induce the selective deletion of mature T cells at a point before their clonal expansion. In their study, a form of activation-induced T cell death Žapoptosis. was proposed. They found that T cell proliferation was inhibited by M-CSF-derived macrophages in a contact-dependent manner. The macrophage-induced apoptosis was characterized as affecting resting T cells before blastogenesis and cell division and preventing functional activation and clonal expansion. We demonstrate herein that GM-CSF-mobilizedPSC-derived monocytes inhibited T cell proliferation upon stimulation. Interestingly, the results from our cell cycle studies clearly show that PSC-derived MDTI activity-induced T cell apoptosis in the coculture during PHA stimulation, which is consistent with the activation-induced apoptosis reported by Munn et al. w52x. Recent studies suggest that activation-induced apoptosis is mainly involved in FasrFas ligand ŽFasL. interactions w51,53–55x. Suss ¨ et al. report that some APCrdendritic cells that also express high levels of FasL can kill T cells via FasrFasL-induced apoptosis w51x. Flow cytometry studies from our laboratory revealed that Fas was expressed on more than 70% of CD3q T cells after 24–48 h co-culture with PHA stimulation, while FasL was expressed on less than 10% of CD3q T cells and on 10–20% of CD14q monocytes Žunpub-
lished data.. Furthermore, we have demonstrated that MDTI activity was significantly reduced by the addition of anti-FasL neutralizing antibody at concentrations of 1–10 m grml w19x. We, therefore, believe that T cell apoptosis by PSC-derived cells occurs, at least in part, by FasrFasL interactions between T cells and monocytes, or T cells and T cells, resulting in a profound inhibition of T cell function and peripheral tolerance post-transplant w18x. Further studies are being undertaken to clarify the detailed mechanism for T cell apoptosis induced by PSC-derived monocytes. Clinically, PSC-derived MDTI activity may have a significant regulatory role in the immune function following SCT. While the highest MDTI activity is seen in mobilized apheresis products from tumorbearing patients, it is also found in mobilized apheresis products from normal donors used in allotransplantation. In contrast, apheresis products from normal, non-mobilized apheresis products and normal PBMC do not have significant MDTI activity. It should be noted that when monocytes from normal PBMC or normal apheresis products are enriched for andror isolated, MDTI activity is observed, suggesting that the lack of activity in normal products is due to a low percentage of monocytes. Based on these results, and our prior studies, there is a clear relationship between the frequency of monocytes in a product and MDTI activity w17x. Furthermore, there is a direct link between the number of cycles of prior chemotherapy and MDTI activity w22x. There is also an association between MDTI activity and the activating effects of G-CSF-mobilization on monocytes and the increased frequency of monocytes available for enrichment by apheresis. Therefore, we suggest that MDTI activity is associated with mobilization, the frequency of monocytes, FasL expression, and the extent of prior chemotherapy that the donor has received. Thus, it appears that one possible therapeutic strategy to induce immune tolerance following autologous PSCT is to increase the frequency of CD14q FasLq cells from apheresed PSC products by T cell depletion with retention of monocytes before infusion to patients. Additionally, the existence of MDTI in PSC products may be associated with a decrease in acute GVHD after allogeneic PSCT, as reported by recent clinical studies w9,10,24,25,42x.
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Acknowledgements We would like to thank the individuals in the Cell Processing Laboratory at UNMC for collecting PSC samples. Our thanks also to Tina Winekauf for secretarial assistance in the preparation of this manuscript and Julie Sweeney for technical assistance in the flow cytometric study.
w11x
w12x
References w1x Kessinger A, Bierman PJ, Vose JM, Armitage JO. High-dose cyclophosphamide, carmustine, and etoposide followed by autologous peripheral stem cell transplantation for patients with relapsed Hodgkin’s disease wpublished erratum appears in Blood 1991 Dec 15; 78 Ž12. 3330x. Blood 1991;77:2322–5. w2x Vose JM, Anderson JR, Kessinger A, Bierman PJ, Coccia P, Reed EC, et al. High-dose chemotherapy and autologous hematopoietic stem-cell transplantation for aggressive nonHodgkin’s lymphoma. J Clin Oncol 1993;11:1846–51. w3x Chao NJ, Schriber JR, Grimes K, Long GD, Negrin RS, Raimondi CM, et al. Granulocyte colony-stimulating factor AmobilizedB peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy. Blood 1993;81:2031–5. w4x Roberts MM, To LB, Gillis D, Mundy J, Rawling C, Ng K, et al. Immune reconstitution following peripheral blood stem cell transplantation, autologous bone marrow transplantation and allogeneic bone marrow transplantation. Bone Marrow Transplant 1993;12:469–75. w5x Talmadge JE, Reed EC, Kessinger A, Kuszynski CA, Perry GA, Gordy CL, et al. Immunologic attributes of cytokine mobilized peripheral blood stem cells and recovery following transplantation. Bone Marrow Transplant 1996;17:101–9. w6x Mills KC, Gross TG, Varney ML, Heimann DG, Reed EC, Kessinger A, et al. Immunologic phenotype and function in human bone marrow, blood stem cells and umbilical cord blood. Bone Marrow Transplant 1996;18:53–61. w7x Peters BG, Adkins DR, Harrison BR, Velasquez WS, Dunphy FR, Petruska PJ, et al. Antifungal effects of yeast-derived rhu-GM-CSF in patients receiving high-dose chemotherapy given with or without autologous stem cell transplantation: a retrospective analysis. Bone Marrow Transplant 1996;18:93–102. w8x Avigan D, Wu Z, Joyce R, Elias A, Richardson P, McDermott D, et al. Immune reconstitution following high-dose chemotherapy with stem cell rescue in patients with advanced breast cancer. Bone Marrow Transplant 2000;26: 169–76. w9x Lin MT, Tseng LH, Frangoul H, Gooley T, Pei J, Barsoukov A, et al. Increased apoptosis of peripheral blood T cells following allogeneic hematopoietic cell transplantation. Blood 2000;95:3832–9. w10x Brugnoni D, Airo P, Pennacchio M, Carella G, Malagoli A,
w13x
w14x
w15x
w16x
w17x
w18x
w19x
w20x
w21x
w22x
1317
Ugazio AG, et al. Immune reconstitution after bone marrow transplantation for combined immunodeficiencies: downmodulation of Bcl-2 and high expression of CD95rFas account for increased susceptibility to spontaneous and activation-induced lymphocyte cell death. Bone Marrow Transplant 1999;23:451–7. Mackall CL, Stein D, Fleisher TA, Brown MR, Hakim FT, Bare CV, et al. Prolonged CD4 depletion after sequential autologous peripheral blood progenitor cell infusions in children and young adults. Blood 2000;96:754–62. Verfuerth S, Peggs K, Vyas P, Barnett L, O’Reilly RJ, Mackinnon S. Longitudinal monitoring of immune reconstitution by CDR3 size spectratyping after T-cell-depleted allogeneic bone marrow transplant and the effect of donor lymphocyte infusions on T-cell repertoire. Blood 2000;95: 3990–5. Talmadge JE, Reed E, Ino K, Kessinger A, Kuszynski C, Heimann D, et al. Rapid immunologic reconstitution following transplantation with mobilized peripheral blood stem cells as compared to bone marrow. Bone Marrow Transplant 1997;19:161–72. Kiesel S, Pezzutto A, Korbling M, Haas R, Schulz R, Hunstein W, et al. Autologous peripheral blood stem cell transplantation: analysis of autografted cells and lymphocyte recovery. Transplant Proc 1989;21:3084–8. Scambia G, Panici PB, Pierelli L, Baiocchi G, Rumi C, Menichella G, et al. Immunological reconstitution after high dose chemotherapy and autologous blood stem cell transplantation for advanced ovarian cancer. Eur J Cancer 1993;29A: 1518–22. Olsen GA, Gockerman JP, Bast Jr. RC, Borowitz M, Peters WP. Altered immunologic reconstitution after standard-dose chemotherapy or high-dose chemotherapy with autologous bone marrow support. Transplantation 1988;46:57–60. Ino K, Singh RK, Talmadge JE. Monocytes from mobilized stem cells inhibit T cell function. J Leukocyte Biol 1997;61: 583–91. Singh RK, Varney ML, Buyukberber S, Ino K, Ageitos AG, Reed E, et al. Fas–FasL-mediated CD4q T-cell apoptosis following stem cell transplantation. Cancer Res 1999;59: 3107–11. Talmadge JE, Singh RK, Kazuhiko I, Ageitos A, Suleyman B. Potential for cytokine and product manipulation to improve the results of autologous transplantation for rheumatoid arthritis. J Rheumatol 2000, in press. Ageitos AG, Ino K, Ozerol I, Tarantolo S, Heimann DG, Talmadge JE. Restoration of T and NK cell function in GM-CSF mobilized stem cell products from breast cancer patients by monocyte depletion. Bone Marrow Transplant 1997;20:117–23. Ageitos AG, Singh RK, Ino K, Ozerol I, Tarantolo S, Reed EK, et al. IL-2 expansion of T and NK cells from growth factor-mobilized peripheral blood stem cell products: monocyte inhibition. J Immunother 1998;21:409–17. Ageitos AG, Varney ML, Bierman PJ, Vose JM, Warkentin PI, Talmadge JE. Comparison of monocyte-dependent T cell inhibitory activity in GM-CSF vs. G-CSF mobilized PSC products. Bone Marrow Transplant 1999;23:63–9.
1318
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
w23x Ozerol I, Ageitos A, Heimann DG, Talmadge JE. Impaired T and NK cell response of bone marrow and peripheral blood stem cell products to interleukin ŽIL.-2. Int J Immunopharmacol 1999;21:509–21. w24x Bensinger WI, Weaver CH, Appelbaum FR, Rowley S, Demirer T, Sanders J, et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. wsee commentsx Blood 1995;85:1655–8. w25x Korbling M, Przepiorka D, Huh YO, Engel H, van Besien K, Giralt S, et al. Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 1995;85:1659–65. w26x Osborne MP, Rosen PP. Detection and management of bone marrow micrometastases in breast cancer. Oncology 1994;8: 25–31. w27x Segre M, Tomei E, Segre D. Cyclophosphamide-induced suppressor cells in mice: suppression of the antibody response in vitro and characterization of the effector cells. Cell Immunol 1985;91:443. w28x Hock H, Dorsch M, Diamantstein T, Blankenstein T. Interleukin 7 induces CD4q T cell-dependent tumor rejection. J Exp Med 1991;174:1291–8. w29x Talmadge JE, Wiltrout RH, Counts DF, Herberman RB, McDonald T, Ortaldo TR. Proliferation of human peripheral blood lymphocytes induced by recombinant human interleukin-2: contribution of large granular lymphocytes and T lymphocytes. Cell Immunol 1986;102:261–72. w30x Yannelli JR, Crumpacker DB, Good RW, Friddell CD, Poston R, Horton S, et al. Use of anti-CD3 monoclonal antibody in the generation of effector cells from human solid tumors for use in cancer biotherapy. J Immunol Methods 1990;130: 91–100. w31x Telford WG, King LE, Fraker PJ. Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Proliferation 1991;24:447–59. w32x Maier T, Holda JH, Claman HN. Natural suppressor ŽNS. cells, members of the LGL regulatory family. Immunol Today 1986;7:312–8. w33x Schmidt-Wolf IG, Dejbakhsh-Jones S, Ginzton N, Greenberg P, Strober S. T-cell subsets and suppressor cells in human bone marrow. Blood 1992;80:3242–50. w34x Palathumpat V, Dejbakhsh-Jones S, Holm B, Wang H, Liang O, Strober S. Studies of CD4-CD8-alpharbeta bone marrow T-cells with suppressor activity. J Immunol 1992;148:373– 80. w35x Noga SJ, Wagner JE, Horwitz LR, Donnenberg AD, Santos GW, Hess AD. Characterization of natural suppressor cell populations in adult rat bone marrow. J Leukocyte Biol 1988;43:279–87. w36x Sykes M, Eisenthal A, Sachs DH. Mechanism of protection from graft-vs.-host disease in murine mixed allogeneic chimeras: I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component. J Immunol 1988;140:2903–11. w37x Pak AS, Wright MA, Matthews JP, Collins SL, Petruzzelli
w38x
w39x
w40x
w41x
w42x
w43x
w44x
w45x
w46x
w47x
w48x
w49x
w50x
GJ, Young MRI. Mechanisms of immune suppression in patients with head and neck cancer: Presence of immune suppressive CD34q cells in cancers that secrete granulocytermacrophage colony-stimulating factor. Clin Cancer Res 1995;1:95–103. Young MRI, Young ME, Wright MA. Stimulation of immune-suppressive bone marrow cells by colony-stimulating factors. Exp Hematol 1990;18:806–11. Oghiso Y, Yamada Y, Ando K, Ishihara H, Shibata Y. Differential induction of prostaglandin E2-dependent and-independent immune suppressor cells by tumor-derived GMCSF and M-CSF. J Leukocyte Biol 1993;53:86. Wanebo HJ, Riley T, Katz D, Pace RC, Johns ME, Cantrell RW. Indomethacin sensitive suppressor-cell activity in head and neck cancer patients. The role of the adherent mononuclear cell. Cancer 1988;61:462–74. Farinas MC, Rodriguez-Valverde V, Zarrabeitia MT, ParraBlanco JA, Sanz-Ortiz J. Contribution of monocytes to the decreased lymphoproliferative response to phytohemagglutinin in patients with lung cancer. Cancer 1991;68:1279–84. Hebib NC, Deas O, Rouleau M, Durrbach A, Charpentier B, Beaujean F, et al. Peripheral blood T cells generated after allogeneic bone marrow transplantation: lower levels of bcl-2 protein and enhanced sensitivity to spontaneous and CD95mediated apoptosis in vitro. Abrogation of the apoptotic phenotype coincides with the recovery of normal naiverprimed T-cell profiles. Blood 1999;94:1803–13. Caligiuri MA, Zmuidzinas A, Manley TJ, Levin H, Smith KA, Ritz J. Functional consequences of IL-2-receptor expression on resting human lymphocytes: identificaiton of a novel NK-cell sub-set with high-affinity receptors. J Exp Med 1990;171:1509–26. Minami Y, Kono T, Miyazaki T, Taniguchi T. The IL-2 receptor complex: its structure, function, and target genes. Annu Rev Immunol 1993;11:245–67. Krakauer T. A macrophage-derived factor that inhibits the production and action of interleukin-2. J Leukocyte Biol 1985;38:429–39. Schleifer KW, Mansfield JM. Suppressor macrophages in African trypanosomiasis inhibit T cell proliferative responses by nitric oxide and prostaglandins. J Immunol 1993;151: 5492–503. Mills CD. Molecular basis of AsuppressorB macrophages. Arginine metabolism via the nitric oxide synthetase pathway. J Immunol 1991;146:2719–23. Albina JE, Abate JA, Henry Jr. WL. Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway. J Immunol 1991;147:144–8. Tomioka H, Sato K, Maw WW, Saito H. The role of tumor necrosis factor, interferon-gamma, transforming growth factor-beta, and nitric oxide in the expression of immunosuppressive functions of splenic macrophages induced by Mycobacterium aÕium complex infection. J Leukocyte Biol 1995;58:704–12. Wu MX, Daley JF, Rasmussen RA, Schlossman SF. Mono-
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319 cytes are required to prime peripheral blood T cells to undergo apoptosis. Proc Natl Acad Sci U S A 1995;92:1525– 9. w51x Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via FasrFas-ligand-induced apoptosis. J Exp Med 1996;183:1789–96. w52x Munn DH, Pressey J, Beall AC, Hudes R, Alderson MR. Selective activation-induced apoptosis of peripheral T cells imposed by macrophages: a potential mechanism of antigenspecific peripheral lymphocyte deletion. J Immunol 1996; 156:523–32.
1319
w53x Scott DW, Grdina T, Shi Y. T cells commit suicide, but B cells are murdered! J Immunol 1996;156:2352–6. w54x Ju ST, Panka DJ, Cui H, Ettinger R, el Khatib M, Sherr DH, et al. Fas ŽCD95.rFasL interactions required for programmed cell death after T-cell activation. Nature 1995;373: 444–8. w55x Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley KA, et al. Fas ligand mediates activation-induced cell death in human T lymphocytes. J Exp Med 1995;181: 71–7.
Activation-induced T cell apoptosis by monocytes from stem cell products q Kazuhiko Ino a , Ana G. Ageitos b, Rakesh K. Singh c , James E. Talmadge c,) a
Department of Obstetrics and Gynecology, Nagoya UniÕersity School of Medicine, 65 Surumai-cho, Showa-ku, Nagoya 466-8550 Japan b Oncology Department, Fundacion Jimenez Diaz, SerÕicio de Oncologia, AÕada Reyes Catolicos, 2, Madrid 28015 Spain c Department of Pathology and Microbiology, UniÕersity of Nebraska Medical Center, 987660 Nebraska Medical Center, Omaha, NE 68198-5600, USA Received 31 October 2000; received in revised form 15 February 2001; accepted 28 February 2001
Abstract We recently found that mobilized peripheral blood stem cell ŽPSC. products Žfrom both cancer patients and normal donors. contain high levels of CD14q monocytes, which can inhibit the proliferation of allogeneic and autologous T cells. We found in our studies that using CD14q monocytes from mobilized PSC products Žfrom normal and cancer patient donors., normal apheresis products or normal peripheral blood ŽPB. can affect lymphocyte function and apoptosis-dependent T cell activation. However, it appears that the apoptosis is dependent on the frequency of monocytes, which is increased by both mobilization and apheresis. Both phytohemagglutinin ŽPHA.- and interleukin ŽIL.-2-induced proliferation of steady-state peripheral blood mononuclear cells ŽPBMC. were markedly inhibited by co-culture with irradiated CD14q monocytes, although inhibition was significantly greater with PHA than with IL-2 stimulation. IL-2 Žpredominately CD56q NK cells. or anti-CD3 monoclonal antibody ŽmAb. and IL-2-expanded lymphocytes Žactivated T cells. were inhibited by PSC monocytes to a significantly greater level as compared to steady-state lymphocytes. Indeed, no inhibition of T cell proliferation was observed when lymphocytes were co-cultured in the absence of mitogenic or IL-2 stimulation. In contrast, an increased proliferation was observed in co-cultures of CD14q monocytes and steady-state or activated lymphocytes without mitogenic stimulation. Cell cycle analysis by flow cytometry revealed a significant increase in hypodiploid DNA, in a time-dependent manner, following co-culture of monocytes and PBMC in PHA, suggesting that T cell apoptosis occurred during PHA-induced activation. These results demonstrate that PSC-derived monocytes inhibit T cell proliferation by inducing the apoptosis of activated T cells and NK cells, but not steady-state cells. This suggests a potential role for monocytes in the induction of peripheral tolerance following stem cell transplantation. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Monocytes; Peripheral tolerance; T cells; IL-2; Mitogenesis
q )
This research was supported in part by grant aRO1-CA61593 from the National Institutes of Health. Corresponding author. Tel.: q1-402-559-5639; fax: q1-402-559-4990. E-mail address: [email protected] ŽJ.E. Talmadge..
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 0 6 2 - 5
1308
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1. Introduction High-dose therapy ŽHDT. supported by stem cell transplantation is increasingly used for the treatment of advanced cancer patients w1,2x. Immune reconstitution and hematopoietic recovery are more rapid following growth factor-mobilized peripheral blood stem cell transplantation ŽPSCT. than following transplant with bone marrow autografts w3–5x. Nonetheless, immune dysfunction is observed following PSCT, despite the restoration of normal lymphocyte numbers, and may be associated, at least in part, with the immunologic characteristics of the infused peripheral stem cell ŽPSC. products w4–16x. We have previously shown that this is due to the frequency of monocytes within mobilized stem cell products w17x and the expression of Fas-L w18,19x. We reported recently that high levels of cellulardependent T cell inhibiting activity are present in both granulocyte macrophage colony-stimulating factor ŽGM-CSF.-mobilized PSC products and peripheral blood ŽPB. following autologous PSCT w5,6,17,18,20–23x. We also have found that this activity is associated with CD14q monocytes, which can suppress T cell function in vitro via cell-to-cell contact w17x. Since immune function is determined by a balance of positive and negative immunoregulatory cytokines, the CD14q cells observed at a high frequency Ž30–40%. in PSC products have the potential to inhibit immune recovery following HDT and PSCT w6x. Furthermore, monocyte-dependent T cell inhibitory ŽMDTI. activity has the potential to reduce therapeutic responses to adjuvant immunotherapy following autologous PSCT. Conversely, MDTI activity may prevent an increase in acute graft-vs.-host disease ŽGVHD. in allogeneic PSCT patients, despite the infusion of 100-fold more T cells than is infused in bone marrow transplant ŽBMT. patients, as observed by recent clinical studies w19,24,25x. Here we report the MDTI activity by CD14q monocytes isolated from mobilized PSC products for lymphocytes with or without mitogenic or interleukin ŽIL.-2 stimulation. Our results indicate that monocytes inhibit the proliferation of activated, but not steady-state, lymphocytes. Further, we provide preliminary data suggesting apoptosis as one mechanism of MDTI, and that MDTI is observed in mobi-
lized PSC products from both normal and cancer patients.
2. Materials and methods 2.1. Cell preparation PSC product from either the second or third apheresis was collected with a Cobe spectra ŽCobe BCT, Lakewood, CO. from 10 cancer patients Žsix with non-Hodgkin’s lymphoma and four with breast cancer. following administration of GM-CSF ŽImmunex, Seattle, WA. at a dose of 250 m grm2 to mobilize progenitor cells. Written informed consent for PSC collection and PSCT was obtained from each patient. All samples were obtained according to protocols approved by the Institutional Review Board of the University of Nebraska Medical Center ŽUNMC.. PSC products Ž2 ml. were diluted in Hanks Balanced Salt Solution ŽHBSS, Gibco, Grand Island, NY. with 10 mM HEPES ŽResearch Organics, Cleveland, OH. and peripheral blood mononuclear cells ŽPBMC. separated by centrifugation on a Ficoll-Hypaque ŽOrganon Teknika, Durham, NC. gradient. After washing, cells were resuspended in complete medium consisting of RPMI 1640 medium ŽGibco. supplemented with 10% fetal bovine serum ŽFBS, Gibco., 40 m grml gentamicin ŽGibco., 2 mM L-glutamine, and 10 mM HEPES. PBMC from the PB of healthy donors were also obtained following Ficoll-Hypaque gradient. 2.2. Isolation of CD14 q monocytic cells from PSC products [26–28] CD14q monocytic cells were isolated from PSC products using Percoll density gradients centrifugation. In brief, Ficoll-purified PSC were suspended in iso-osmotic complete medium adjusted to 285 mosM. The stock solution of Percoll ŽSigma, St. Louis, MO. was prepared by mixing 9.25 parts of concentrated Percoll and 0.75 parts of 10 = concentrated phosphate buffered saline ŽPBS.. The osmolarity of this solution was adjusted to 285 mosM. Iso-osmotic complete medium and Percoll solution were mixed to prepare seven different concentrations of Percoll
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Žvrv.: 38.6, 47.5, 52.1, 56.5, 61.1, 65.6, 70.1%. Gradients consisted of seven fractions layered in a discontinuous manner using 1.5 ml of each with fraction 7 Ž70.1%. at the bottom of a 15 ml conical tube. Thereafter, 3 ml of the PSC suspension Žup to 1 = 10 8 cells. were carefully placed on the top of the gradients, and centrifuged at 550 = g for 30 min at room temperature. Cells at each interface were collected and washed twice in HBSS and resuspended in complete medium. Following separation, most monocytes were contained in the low density fraction from the band at the 38.6–47.5% interface, whereas the majority of lymphocytes were in the higher density fractions from the bands at 47.5– 52.1% and 52.1–56.5%. To obtain further purified CD14q monocytic cells, the contaminated CD4q and CD8q T cells in the low density fraction were depleted by immunomagnetic bead cell isolation ŽMiniMACS, Miltenyi Biotec, Auburn, CA. using magnetic microbead-conjugated anti-CD4 and CD8 monoclonal antibodies ŽmAbs.. The monocyte purity was assessed by fluorescent cytometry using antiCD14 mAb Ždescribed below. and morphology using Wright-Giemsa stained smears. The CD4q and CD8q T cell-depleted low density PSC were 85% to 95% Ž89% on average. purified CD14q monocytic cells. Cell viability was determined by Trypan blue dye exclusion Žviability always exceeded 95%.. 2.3. Treatment of responder cells with anti-CD3 mAb and r or IL-2 To generate activated effector cells such as lymphokine-activated killer ŽLAK., natural killer ŽNK. cells and activated T cells, PBMC were treated as described in previous reports w29,30x. In brief, Ficoll-purified PBMC from normal donors were added at a concentration of 2 = 10 6 cellsrwell in complete medium including 100 Urml of recombinant human IL-2 ŽChiron, Emeryville, CA. in a 24-well culture plate. Cells were cultured for 72 h at 378C in a humidified 5% CO 2 atmosphere. The cells were then harvested by pipetting vigorously, washing once with HBSS, and resuspending in complete medium. In some experiments, cells were pretreated with anti-CD3 cross-linking mAb ŽOKT3, Ortho Biotech, Raritan, NJ. before the start of culture with
1309
IL-2 to upregulate T cell receptors on CD3q T cells. PBMC were incubated with 100 ngrml of anti-CD3 mAb for 30 min in a 15 ml conical tube in a 378C water bath with gentle agitation. These anti-CD3 mAb coated cells were then cultured with 100 Urml IL-2 for 72 h. Both activated effector cell populations were used as responder cells for a subsequent MDTI assay. 2.4. Immunofluorescent staining and flow cytometric analysis Three-color fluorescent cytometry analysis was used for cell surface immunophenotyping of responder lymphocytes. Cells were counted and adjusted to 1 = 10 6rml in PBS containing 2% FBS. Aliquots of 1 = 10 5 cells were stained at 48C for 30 min with mAbs specific for the following cell surface markers: Fluorescein isothiocyanate ŽFITC.-labeled anti-CD3 ŽBecton Dickinson, San Jose, CA., phycoerythrin ŽPE.-labeled anti-CD4, CD56, CD25 ŽBecton Dickinson., and biotin-conjugated anti-CD8 ŽBecton Dickinson. with CD19 ŽCoulter, Hialeah, FL. added with streptavidian allophycocyanin ŽAPC. as the third fluorochrome. To assess the purity of CD14q monocytes isolated from PSC products, we also used biotin-conjugated anti-CD14 mAb ŽCoulter.. Background staining using isotype controls was done to determine thresholds for positive cells. All three-color data were acquired on a FACStar Plus ŽBecton Dickinson. and analyzed using the Cellquest software ŽBecton Dickinson.. 2.5. MDTI actiÕity assay The methodology for the co-culture assay to measure MDTI activity has been previously described w5,6,17x. Briefly, freshly purified normal PBMC or IL-2-cultured cells Žwith or without treatment with anti-CD3. as responder cells Ž1 = 10 5 . were co-cultured with varying numbers of irradiated Ž500 cGy. inhibitor cells Žisolated CD14q cells. at inhibitor to responder ŽI:R. ratios of 4:1, 2:1, 1:1, and 0.5:1. Cells were cultured in complete medium with or without an optimal concentration of phytohemagglutinin ŽPHA. Ž0.1 m grml. or IL-2 Ž100 Urml. in 96-well flat bottom microplates for 72 h at 378C in a
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1310
humidified 5% CO 2 atmosphere. In some wells, responder cells alone were cultured with PHA or IL-2 as a control. The proliferative response of responder cells was determined by 3 H-thymidine incorporation over the final 18 h of culture. All experiments were performed in triplicate. The percent MDTI activity was calculated using the following formula Žcpm s counts per minute.:
The mean and standard error of the mean ŽS.E.M.. are shown for all data. The unpaired Student’s t-test was used to compare groups. A p value of less than 0.05 was considered significant.
% inhibition
3. Results
s 1 y Ž mean cpm in tested wells with co ycultured cells . r Ž mean cpm in control wells without co ycultured cells . = 100. 2.6. Measurement of apoptosis by flow cytometry Ficoll-purified PBL Ž6 = 10 5rwell. were co-cultured with irradiated CD14q inhibitor cells Ž1.2 = 10 6rwell. in 24-well culture plates in the presence of 0.1 m grml PHA. In some wells, only responder PBL were cultured with the same concentration of PHA as a control. After culture for 2, 4, 12, 24, and 48 h, non-adherent cells Žlymphocytes. were collected by repeated pipetting and washing in PBS twice. To measure the percentage of apoptotic cells, cell cycle analysis was carried out as reported by Telford et al. w31x. In brief, cells Ž2 = 10 6 . were resuspended in 1 ml of ice-cold 70% ethanol with vigorous mixing. The cells were incubated at 48C for 1 h. The ethanol-fixed cells were then washed in ice-cold PBS and resuspended in 1 ml of a staining reagent consisting of 0.1% Triton X-100, 0.1 mM EDTA, 50 m grml RNase A Ž50 Urmg. and 50 m grml propidium iodide ŽPI, Sigma.. Samples were stored in the dark at 48C overnight. Samples were acquired on a FACStar Plus ŽBecton Dickinson. using the 488 nm argon laser and analyzed by Cellquest and Modifit software ŽBecton Dickinson.. Only lymphocytes were gated and evaluated for apoptosis. Contaminating CD14q monocytic cells were removed by gating based on differences in forward and side scatter intensity. Apoptotic cells were determined by the hypodiploid areas Žbelow the G0rG1 region. in the PI-staining profiles.
2.7. Statistical analysis
3.1. MDTI actiÕity against PHA or IL-2-induced lymphocyte proliferation We evaluated the MDTI activity of CD14q cells purified from PSC products for lymphoproliferative responses to either PHA or IL-2 using fresh Žsteadystate. or activated lymphocyte responders. The PHA-induced mitogenesis of normal allogeneic PBMC was significantly Ž p F 0.05. inhibited compared to control Žwithout monocyte co-culture. in a dose-dependent manner when co-cultured with irradiated CD14q inhibitor cells for 72 h at inhibitor to
Fig. 1. Monocyte-dependent T cell inhibitory ŽMDTI. activity of peripheral stem cell ŽPSC.-derived CD14q cells against phytohemagglutinin ŽPHA. or interleukin ŽIL.-2-induced proliferation of fresh peripheral blood mononuclear cells ŽPBMC.. Normal peripheral blood mononuclear cells ŽPBMC. were co-cultured with irradiated monocytes for 72 h at the indicated inhibitor:response ŽI:R. ratios with phytohemagglutinin ŽPHA. or IL-2, and percent monocyte-dependent T cell inhibitory ŽMDTI. activity was calculated. Points, mean"standard error of the mean ŽS.E.M... ) Significant difference compared to the IL-2 stimulation group Ž pF 0.05.. aSignificant difference compared to control Žwithout co-culture. Ž pF 0.05..
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1311
Table 1 Effect of activation on PHA-induced proliferation of PBMC in the presence or absence of CD14q monocytes Responder peripheral blood mononuclear cells ŽPBMC. were untreated or pretreated with interleukin ŽIL.-2 alone or anti-CD3 and IL-2 for 72 h. The responder cells were then co-cultured with irradiated CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 72 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. Mitogenic proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M.. for the 3 H-thymidine incorporation expressed as counts per minute Žcpm.. Responder cells PBMC Ž n s 6.
IL-2-pretreated Ž n s 3. Anti-CD3q IL-2-pretreated Ž n s 3.
cpm PI b % Inhibition cpm PI b % Inhibition cpm PI b % Inhibition
Control Žwithout co-culture.
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1
I:R s 1:1
I:R s 0.5:1
89,839 " 2713 – – 91,771 " 6006 – – 87,085 " 1004 – –
3653 " 1247 a 0.04 " 0.01c 96 " 1%c 13,188 " 2534 a 0.15 " 0.05 85 " 5% 7147 " 3325a 0.08 " 0.04 92 " 4%
55,489 " 6915a 0.62 " 0.08 d 38 " 8%d 88,098 " 16,138 0.96 " 0.15 4 " 15% 85,964 " 1008 0.98 " 0.01 2 " 1%
77,953 " 4295a 0.88 " 0.06 d 12 " 6%d 113,613 " 10,904 1.26 " 0.14 y26 " 14% 105,371 " 11,241 1.21 " 0.13 y21 " 13%
31,264 " 6333 a 0.35 " 0.07 d 65 " 7%d 58,804 " 11,070 a 0.67 " 0.15 33 " 15% 48,970 " 2003 a 0.56 " 0.03 44 " 3%
a
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s cpm with co-cultured inhibitor cellsxrwcpm without co-cultured inhibitor cellsx. c Significant difference in PI and % monocyte-dependent T cell inhibitory ŽMDTI. activity compared to IL-2-pretreated group Ž p - 0.05.. d Significant difference in PI and % MDTI activity compared to IL-2-pretreated and anti-CD3 and IL-2-pretreated groups Ž p - 0.05.. b
response ratios ŽI:R. of 4:1 to 0.5:1 ŽFig. 1 and Table 1.. The proliferation of PBL to IL-2 stimulation was also significantly Ž p F 0.05. inhibited by co-culture
at I:R of 4:1 and 2:1 ŽTable 2., although inhibition was significantly greater with PHA stimulation than with IL-2 stimulation ŽFig. 1..
Table 2 Effect of activation on IL-2-induced proliferation of PBMC in the presence or absence of CD14q monocytes Responder peripheral blood mononuclear cells ŽPBMC. were untreated or pretreated with interleukin ŽIL.-2 alone or anti-CD3 and IL-2 for 72 h. The responder cells were then co-cultured with irradiated CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 72 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. IL-2-induced proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M... Responder cells PBLs Ž n s 6.
IL-2-pretreated Ž n s 3. Anti-CD3q IL-2-pretreated Ž n s 3. a b
Control Žwithout co-culture. cpm PI b % Inhibition cpm PI b % Inhibition cpm PI b % Inhibition
18,003 " 1868 – – 47,156 "20,896 – – 87,085 " 1004 – –
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1 a
3042 " 659 0.16 " 0.03 84 " 3% 8734 " 2774a 0.30 " 0.17 70 " 17% 11,900 " 7528 a 0.15 " 0.07 85 " 7%
a
10,407 " 1288 0.57 " 0.03 43 " 3% 18,208 " 2417 0.58 " 0.25 42 " 25% 45,591 " 22,528 0.70 " 0.27 30 " 27%
I:R s 1:1
I:R s 0.5:1
16,985 " 2688 0.94 " 0.10 6 " 10% 36,638 " 4935 1.00 " 0.27 0 " 27% 62,088 " 25,160 0.99 " 0.36 1 " 36%
20,309 " 2505 1.13 " 0.08 y13 " 8% 50,773 " 8685 1.34 " 0.30 y34 " 30% 74,476 " 25,400 1.19 " 0.34 y19 " 34%
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s wcounts per minute Žcpm. with co-cultured monocytesxrwcpm without co-cultured monocytesx.
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1312
The PSC-derived inhibitory cells also inhibited the PHA- or IL-2-induced proliferation of activated responder cells. Following preculture of PBMC for 72 h with IL-2 alone, CD56q NK cells were significantly increased to 69.62 " 12.3% on average Ž n s 4. as compared to unstimulated PBMC Ž7.0 " 1.9%., while CD3q CD25q activated T cells were significantly increased to 61.1 " 0.52% on average Ž n s 3. following anti-CD3 and IL-2 treatment as compared to unstimulated PBMC Ž2.11 " 0.75%.. The proliferation of the IL-2-activated NK cell predominant population was significantly Ž p F 0.05. inhibited when co-cultured with monocytes at I:R of 4:1 and 2:1 with PHA stimulation and at 4:1 with IL-2 stimulation. Similarly, the proliferative response to PHA and IL-2 of anti-CD3- and IL-2-activated T cells was significantly Ž p F 0.05. inhibited at the same I:R. However, both types of activated cells were significantly less sensitive to inhibition with PHA stimulation as compared to steady-state PBMC ŽTable 1., while there was no difference in sensitivity to inhibition among the three responders with IL-2 stimulation ŽTable 2.. In contrast to the significantly inhibited PHA or IL-2 mitogenic response, no MDTI activity was observed to any responder cells Žwhether previously
activated or not. when cells were co-cultured with monocytes and without mitogenic or IL-2 stimulation ŽTable 3.. These results indicate that PSC-derived cells can exert their MDTI activity against T cells and NK cells undergoing activation, but have less activity against activated cells and no activity against steady-state cells in the absence of mitogenic stimulation. 3.2. MDTI actiÕity from PBMC and PSC products from normal donors MDTI activity could be associated not only with the frequency of monocytes, but also the mobilizing cytokine, the presence of neoplastic disease, and the number of prior chemotherapy cycles w22x. In previous studies, we reported that there is a direct association between MDTI activity and the frequency of monocytes contained within the product w17x and the number of cycles of prior chemotherapy w22x. In the studies shown in Fig. 2, we compared at an I:R of 1:4 and 1:2 the MDTI activity of G-CSF-mobilized PSC products from metastatic breast cancer patients to G-CSF-mobilized PSC products and non-mobilized, apheresis products obtained from volunteer normal donors and normal PBMC products. The
Table 3 Effect of activation on steady state proliferation of PBMC in the presence or absence of CD14q monocytes Responder peripheral blood mononuclear cells ŽPBMC. were untreated or pretreated with interleukin ŽIL.-2 alone or anti-CD3 and IL-2 for 72 h. The responder cells were co-cultured with irradiated CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 72 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. Proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M... Responder cells PBLs Ž n s 6.
IL-2-pretreated Ž n s 3. anti-CD3q IL-2-pretreated Ž n s 3. a
Control Žwithout co-culture. cpm PI b % stimulation cpm PI b % stimulation cpm PI b % stimulation
559 " 16 – – 14,199 " 8188 – – 11,694 " 5020 – –
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1 508 " 85 0.98 " 0.10 9.1 " 1.5 c 12,718 " 6288 0.92 " 0.17 10.4 " 5.1 10,656 " 4469 0.90 " 0.05 8.9 " 3.7
1944 " 517 a 2.12 " 0.67 247.8 " 66 21,384 " 7110 a 3.50 " 0.97 50.6 " 16.8 28,425 " 13,240 a 2.25 " 0.21 143 " 66.6
I:R s 1:1
I:R s 0.5:1
2220 " 484 a 2.64 " 0.96 297.1 " 64.8 30,090 " 14,998 a 3.96 " 0.89 111.9 " 55.8 36,782 " 18,914 a 2.60 " 0.64 214 " 54
2425 " 406 a 3.17 " 0.82 333.8 " 55.9 38,133 " 19,385a 4.30 " 0.70 168.6 " 85.7 43,995 " 24,864 a 3.09 " 0.82 276.2 " 156.1
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s wcounts per minute Žcpm. with co-cultured monocytesxrwcpm without co-cultured monocytesx. c Percent stimulations wŽcpm with co-culture monocytes. y Žcpm without co-culture monocytes.xrcpm without co-culture monocyte. b
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
1313
Fig. 2. Monocyte-dependent T cell inhibitor ŽMDTI. activity of various peripheral blood mononuclear cells ŽPBMC. source. This includes peripheral stem cell ŽPSC. obtained following granulocyte-colony stimulating factor ŽG-CSF. mobilization of autologous cancer patients Žauto Mob PSC., G-CSF mobilized normal PSC Žnorm Mob PSC. and normal non-mobilized apheresis cells Žnorm non-Mob PSC.. In these studies, normal PBMC were co-cultured with irradiated sources of cellular inhibitors for 72 h at the indicated inhibitor:responder ŽI:R. ratio with phytohemagglutinin ŽPHA. and percent MDTI activity calculated and expressed as the mean " standard error of the mean ŽS.E.M... In these studies, there was an N of 19 for auto Mob PSC, 18 for norm Mob PSC, 9 for normal non-MOB PSC and 13 for norm PBL. The insert graphic shows the percent of monocytes contained within the various products" S.E.M. ) Significantly different from normal peripheral blood leukocytes Žnorm PBL.. aSignificantly different from the auto Mob PSC, p F 0.05 by Student’s t-test.
normal PBMC cells and normal, non-mobilized PSC cells had minimal MDTI activity. However, normal, mobilized PSC products, used to transplant allodonors, and mobilized PSC cells from autologous donors demonstrated significant levels of MDTI activity. As shown in the insert to Fig. 2, there was a direct relationship between the frequency of monocytes within these products and the extent of MDTI activity, verifying these results and previous reports on the frequency of monocytes and MDTI activity w17x. Further, as shown in Table 1, pure populations of monocytes from G-CSF-mobilized products are capable of high levels of MDTI activity, suggesting the need for a high I:R ratio. In contrast to mobilized products from normal donors and cancer patients, normal PMNC have minimal MDTI activity. Nonmobilized PSC products have a slight, but significant, increase in monocyte frequency, but not a significant increase in MDTI levels. These results also suggest a relationship between MDTI activity
and percent monocytes. Despite minimal MDTI activity by normal PBMC if the monocytes are enriched by Percoll isolation w17x, they also demonstrate MDTI activity ŽTable 4.. In these studies we examined the MDTI of enriched monocytes on the PHA response of autologous and allogeneic lymphocytes. As can be seen in Table 4, there was a significant inhibition of both allogeneic and autologous lymphocyte response to PHA mitogenesis ranging from 49% to 56%. Although not as extensive as that observed with purified monocytes from mobilized apheresis products from cancer patients, it does suggest that normal monocytes are capable of MDTI activity in response to PHA mitogenesis. 3.3. Induction of T cell apoptosis by monocytes To determine the possible mechanism of MDTI activity, a cell cycle study was undertaken to deter-
1314
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Table 4 Effect of PHA induced proliferation of autologous and allogeneic PBMC in the presence or absence of isolated, normal CD14q monocytes Responder, autologous or allogenic normal peripheral blood mononuclear cells ŽPBMC. were co-cultured in the presence or absence of irradiated, normal CD14q monocytes at the indicated inhibition:response ŽI:R. ratios for 96 h in the presence of 0.1 m grml phytohemagglutinin ŽPHA.. Mitogenic proliferation of responder cells was determined by a 3 H-thymidine incorporation assay, and compared with controls. Results are expressed as mean " standard error of the mean ŽS.E.M.. for the 3 H-thymidine incorporation assay, and compared with controls. Responder cells Autologous lymphocytes Ž n s 6. Allogeneic lymphocytes Ž n s 9. a b
cpm PI b % stimulation cpm PI b % stimulation
Control Žwithout monocyte co-culture.
Co-culture with CD14q monocytes I:R s 4:1 I:R s 2:1
I:R s 1:1
109,983 " 21,173 – – 118,026 " 23,043 – –
44,384 a " 11,944 0.44 " 0.11 56.4 " 11.2 62,985a " 16,618 0.51 " 0.11 49.2 " 11.1
105,752 " 17,545 1.00 " 0.05 0.1 " 5.4 105,206 " 18,850 0.92 " 0.04 8.1 " 4.4
88,703 a " 14,985 0.83 " 0.07 16.6 " 6.9 93,304 a " 20,274 0.78 " 0.09 21.6 " 9.1
Significant difference compared to control Žwithout co-culture. Ž p - 0.05.. PI: Proliferation index s cpm with co-cultured inhibitor cellsrcpm without co-cultured inhibitor cells.
mine the induction of apoptosis by flow cytometry using PI-stained cells. After PBMC were co-cultured with irradiated CD14q cells in the presence of PHA,
only non-adherent responder cells were collected and evaluated. As shown in Fig. 3, the fragmented DNA Žhypodiploid areas. of responder lymphocytes were
Fig. 3. DNA histograms by flow cytometry using propidium iodide ŽPI.-stained lymphocytes. Peripheral blood mononuclear cells ŽPBMC. were co-cultured with CD14q monocytes with phytohemagglutinin ŽPHA. stimulation at an inhibition:responder ŽI:R. ratio of 2:1 for 0 Žcontrol. ŽA., 4 ŽB., 24 ŽC., or 48 h ŽD.. Then only lymphocytes were gated and evaluated for DNA histograms by flow cytometric analysis as described in Materials and methods. Percent apoptotic cells were determined as the cells below the G0rG1 peak ŽM1 region.. In this representative sample, % apoptotic cells in A, B, C, and D were 0.4%, 2.1%, 21.0%, and 32.2%, respectively.
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Fig. 4. T cell apoptosis induced by peripheral blood stem cell ŽPSC.-derived monocytes. Peripheral blood mononuclear cells ŽPBMC. were cultured with or without CD14q monocytes with phytohemagglutinin ŽPHA. stimulation at a inhibition:response ŽI:R. ratio of 2:1 for the indicated time and non-adherent cells were collected and stained with propidium iodide ŽPI.. Then only lymphocytes were gated and evaluated for DNA histograms by flow cytometric analysis. Percent apoptotic cells were determined as the cells below the G0 rG1 peak. Points, mean"standard error of the mean ŽS.E.M.. Ž ns 4.. ) Significant difference compared to control Žwithout co-culture. Ž pF 0.01..
clearly observed after co-culture for 24 and 48 h. Since we previously found that irradiated CD14q cells alone Ž89% purity., at least within the irradiation dose used in this study, showed no apoptosis at any culture time Ž2–48 h. Ždata not shown., this result demonstrates the apoptosis of responder T cells during PHA stimulation. The frequency of apoptotic cells was increased by co-culture in a time-dependent manner and significantly increased Ž p F 0.01. as compared to control Žwithout co-culture. at 24- and 48-h incubation ŽFig. 4..
4. Discussion Cells with T cell inhibitory activity have been found in sites of intense hematopoiesis w32–39x and can be increased by tumor secretion or exogenous administration of hematopoietic growth factors such as GM-CSF or IL-3 w37–39x. However, the cellular lineage and mechanism of the MDTI action remains controversial w32–39x and few studies focus
1315
on human cells. Schmidt-Wolf et al. found one cellular subset in human bone marrow that had a CD4y CD8y ab TCRq phenotype w33x while Pak et al. reported that CD34q progenitor cells inhibited immune function in patients with a GM-CSF-secreting cancer w37x. In contrast, several studies demonstrated the immune inhibitor is from the macrophagermonocyte lineage w35,39–41x. In our earlier studies w5,6x. we found, for the first time, that growth factor-mobilized human PSC products from cancer patients contained high levels of MDTI activity, which is associated with GM-CSF mobilization andror leukapheresis. This profound inhibition of T cell function is observed not only in the PSC product itself but also in the PB of patients post-transplantation w5,18x. More recently, we isolated and characterized the origin of this inhibitor cell activity in PSC products and found that the MDTI activity is associated with a CD14q monocytic cell w17,18,21–23x. Since about 40% of GM-CSF-mobilized and apheresed PSC and more than 10% of PB leukocytes up to day 30 post-PSCT are CD14q monocytes w5,6x, this high frequency of inhibitor cells could have a significant effect on immune recovery following PSCT. Increased levels of T cell apoptosis are observed following allogeneic PSCT and may occur by similar mechanisms w9,10,42x. Further, the MDTI activity may reduce the efficiency of adjuvant immunotherapy, including IL-2 administration posttransplant or adoptive immunotherapy using ex vivo expanded and activated effector cells. Thus, in this paper we examine the MDTI activity of PSC-derived CD14q cells for proliferative response of steady-state PBL as well as activated T cells, NK and LAK cells. The data show that PSC-derived cells responsible for MDTI activity can significantly inhibit the proliferation of responder lymphocytes, although previously activated T cells and NKrLAK cells are less sensitive to MDTI activity as compared to the strong inhibition observed in PBMC undergoing PHA blastogenesis. Further, no MDTI activity was found for any responder cells when cells were co-cultured without mitogenic or IL-2 stimulation. Indeed, we observed significantly increased 3 H-thymidine uptake in the PBMC rather than inhibition when co-cultured without stimulation at I:R of 2:1 to 0.5:1, although counts per minute values were very low Žless than 3000.. These results suggest that the cells
1316
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
responsible for MDTI activity can exert their inhibitory activity against activated lymphocytes, but not against steady-state, unstimulated lymphocytes. Our data also suggest that monocytes exert their greatest effect on the initial step of T cell activation from resting cells to activated cells with primary stimulation, while exerting less effect on previously activated cells. Proliferation of T and NK cells is dependent on the interaction of IL-2 with its receptors w29,30,43,44x. Monocytes can negatively control T cell activation and proliferation w39,45–49x by various mechanisms, including prostaglandins w39,46x, nitric oxide w46–49x and tumor necrosis factor ŽTNF. w49x. However, in earlier studies, we found that inhibition by PSC-derived monocytes requires direct cell-to-cell contact and is not abrogated by the anti-TNF neutralizing antibody w17x. More recently, macrophage- or antigen-presenting cell ŽAPC.-induced T cell apoptosis has been proposed w50–52x. Munn et al. w52x suggested that macrophages exposed to macrophage-CSF ŽM-CSF. can induce the selective deletion of mature T cells at a point before their clonal expansion. In their study, a form of activation-induced T cell death Žapoptosis. was proposed. They found that T cell proliferation was inhibited by M-CSF-derived macrophages in a contact-dependent manner. The macrophage-induced apoptosis was characterized as affecting resting T cells before blastogenesis and cell division and preventing functional activation and clonal expansion. We demonstrate herein that GM-CSF-mobilizedPSC-derived monocytes inhibited T cell proliferation upon stimulation. Interestingly, the results from our cell cycle studies clearly show that PSC-derived MDTI activity-induced T cell apoptosis in the coculture during PHA stimulation, which is consistent with the activation-induced apoptosis reported by Munn et al. w52x. Recent studies suggest that activation-induced apoptosis is mainly involved in FasrFas ligand ŽFasL. interactions w51,53–55x. Suss ¨ et al. report that some APCrdendritic cells that also express high levels of FasL can kill T cells via FasrFasL-induced apoptosis w51x. Flow cytometry studies from our laboratory revealed that Fas was expressed on more than 70% of CD3q T cells after 24–48 h co-culture with PHA stimulation, while FasL was expressed on less than 10% of CD3q T cells and on 10–20% of CD14q monocytes Žunpub-
lished data.. Furthermore, we have demonstrated that MDTI activity was significantly reduced by the addition of anti-FasL neutralizing antibody at concentrations of 1–10 m grml w19x. We, therefore, believe that T cell apoptosis by PSC-derived cells occurs, at least in part, by FasrFasL interactions between T cells and monocytes, or T cells and T cells, resulting in a profound inhibition of T cell function and peripheral tolerance post-transplant w18x. Further studies are being undertaken to clarify the detailed mechanism for T cell apoptosis induced by PSC-derived monocytes. Clinically, PSC-derived MDTI activity may have a significant regulatory role in the immune function following SCT. While the highest MDTI activity is seen in mobilized apheresis products from tumorbearing patients, it is also found in mobilized apheresis products from normal donors used in allotransplantation. In contrast, apheresis products from normal, non-mobilized apheresis products and normal PBMC do not have significant MDTI activity. It should be noted that when monocytes from normal PBMC or normal apheresis products are enriched for andror isolated, MDTI activity is observed, suggesting that the lack of activity in normal products is due to a low percentage of monocytes. Based on these results, and our prior studies, there is a clear relationship between the frequency of monocytes in a product and MDTI activity w17x. Furthermore, there is a direct link between the number of cycles of prior chemotherapy and MDTI activity w22x. There is also an association between MDTI activity and the activating effects of G-CSF-mobilization on monocytes and the increased frequency of monocytes available for enrichment by apheresis. Therefore, we suggest that MDTI activity is associated with mobilization, the frequency of monocytes, FasL expression, and the extent of prior chemotherapy that the donor has received. Thus, it appears that one possible therapeutic strategy to induce immune tolerance following autologous PSCT is to increase the frequency of CD14q FasLq cells from apheresed PSC products by T cell depletion with retention of monocytes before infusion to patients. Additionally, the existence of MDTI in PSC products may be associated with a decrease in acute GVHD after allogeneic PSCT, as reported by recent clinical studies w9,10,24,25,42x.
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
Acknowledgements We would like to thank the individuals in the Cell Processing Laboratory at UNMC for collecting PSC samples. Our thanks also to Tina Winekauf for secretarial assistance in the preparation of this manuscript and Julie Sweeney for technical assistance in the flow cytometric study.
w11x
w12x
References w1x Kessinger A, Bierman PJ, Vose JM, Armitage JO. High-dose cyclophosphamide, carmustine, and etoposide followed by autologous peripheral stem cell transplantation for patients with relapsed Hodgkin’s disease wpublished erratum appears in Blood 1991 Dec 15; 78 Ž12. 3330x. Blood 1991;77:2322–5. w2x Vose JM, Anderson JR, Kessinger A, Bierman PJ, Coccia P, Reed EC, et al. High-dose chemotherapy and autologous hematopoietic stem-cell transplantation for aggressive nonHodgkin’s lymphoma. J Clin Oncol 1993;11:1846–51. w3x Chao NJ, Schriber JR, Grimes K, Long GD, Negrin RS, Raimondi CM, et al. Granulocyte colony-stimulating factor AmobilizedB peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy. Blood 1993;81:2031–5. w4x Roberts MM, To LB, Gillis D, Mundy J, Rawling C, Ng K, et al. Immune reconstitution following peripheral blood stem cell transplantation, autologous bone marrow transplantation and allogeneic bone marrow transplantation. Bone Marrow Transplant 1993;12:469–75. w5x Talmadge JE, Reed EC, Kessinger A, Kuszynski CA, Perry GA, Gordy CL, et al. Immunologic attributes of cytokine mobilized peripheral blood stem cells and recovery following transplantation. Bone Marrow Transplant 1996;17:101–9. w6x Mills KC, Gross TG, Varney ML, Heimann DG, Reed EC, Kessinger A, et al. Immunologic phenotype and function in human bone marrow, blood stem cells and umbilical cord blood. Bone Marrow Transplant 1996;18:53–61. w7x Peters BG, Adkins DR, Harrison BR, Velasquez WS, Dunphy FR, Petruska PJ, et al. Antifungal effects of yeast-derived rhu-GM-CSF in patients receiving high-dose chemotherapy given with or without autologous stem cell transplantation: a retrospective analysis. Bone Marrow Transplant 1996;18:93–102. w8x Avigan D, Wu Z, Joyce R, Elias A, Richardson P, McDermott D, et al. Immune reconstitution following high-dose chemotherapy with stem cell rescue in patients with advanced breast cancer. Bone Marrow Transplant 2000;26: 169–76. w9x Lin MT, Tseng LH, Frangoul H, Gooley T, Pei J, Barsoukov A, et al. Increased apoptosis of peripheral blood T cells following allogeneic hematopoietic cell transplantation. Blood 2000;95:3832–9. w10x Brugnoni D, Airo P, Pennacchio M, Carella G, Malagoli A,
w13x
w14x
w15x
w16x
w17x
w18x
w19x
w20x
w21x
w22x
1317
Ugazio AG, et al. Immune reconstitution after bone marrow transplantation for combined immunodeficiencies: downmodulation of Bcl-2 and high expression of CD95rFas account for increased susceptibility to spontaneous and activation-induced lymphocyte cell death. Bone Marrow Transplant 1999;23:451–7. Mackall CL, Stein D, Fleisher TA, Brown MR, Hakim FT, Bare CV, et al. Prolonged CD4 depletion after sequential autologous peripheral blood progenitor cell infusions in children and young adults. Blood 2000;96:754–62. Verfuerth S, Peggs K, Vyas P, Barnett L, O’Reilly RJ, Mackinnon S. Longitudinal monitoring of immune reconstitution by CDR3 size spectratyping after T-cell-depleted allogeneic bone marrow transplant and the effect of donor lymphocyte infusions on T-cell repertoire. Blood 2000;95: 3990–5. Talmadge JE, Reed E, Ino K, Kessinger A, Kuszynski C, Heimann D, et al. Rapid immunologic reconstitution following transplantation with mobilized peripheral blood stem cells as compared to bone marrow. Bone Marrow Transplant 1997;19:161–72. Kiesel S, Pezzutto A, Korbling M, Haas R, Schulz R, Hunstein W, et al. Autologous peripheral blood stem cell transplantation: analysis of autografted cells and lymphocyte recovery. Transplant Proc 1989;21:3084–8. Scambia G, Panici PB, Pierelli L, Baiocchi G, Rumi C, Menichella G, et al. Immunological reconstitution after high dose chemotherapy and autologous blood stem cell transplantation for advanced ovarian cancer. Eur J Cancer 1993;29A: 1518–22. Olsen GA, Gockerman JP, Bast Jr. RC, Borowitz M, Peters WP. Altered immunologic reconstitution after standard-dose chemotherapy or high-dose chemotherapy with autologous bone marrow support. Transplantation 1988;46:57–60. Ino K, Singh RK, Talmadge JE. Monocytes from mobilized stem cells inhibit T cell function. J Leukocyte Biol 1997;61: 583–91. Singh RK, Varney ML, Buyukberber S, Ino K, Ageitos AG, Reed E, et al. Fas–FasL-mediated CD4q T-cell apoptosis following stem cell transplantation. Cancer Res 1999;59: 3107–11. Talmadge JE, Singh RK, Kazuhiko I, Ageitos A, Suleyman B. Potential for cytokine and product manipulation to improve the results of autologous transplantation for rheumatoid arthritis. J Rheumatol 2000, in press. Ageitos AG, Ino K, Ozerol I, Tarantolo S, Heimann DG, Talmadge JE. Restoration of T and NK cell function in GM-CSF mobilized stem cell products from breast cancer patients by monocyte depletion. Bone Marrow Transplant 1997;20:117–23. Ageitos AG, Singh RK, Ino K, Ozerol I, Tarantolo S, Reed EK, et al. IL-2 expansion of T and NK cells from growth factor-mobilized peripheral blood stem cell products: monocyte inhibition. J Immunother 1998;21:409–17. Ageitos AG, Varney ML, Bierman PJ, Vose JM, Warkentin PI, Talmadge JE. Comparison of monocyte-dependent T cell inhibitory activity in GM-CSF vs. G-CSF mobilized PSC products. Bone Marrow Transplant 1999;23:63–9.
1318
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319
w23x Ozerol I, Ageitos A, Heimann DG, Talmadge JE. Impaired T and NK cell response of bone marrow and peripheral blood stem cell products to interleukin ŽIL.-2. Int J Immunopharmacol 1999;21:509–21. w24x Bensinger WI, Weaver CH, Appelbaum FR, Rowley S, Demirer T, Sanders J, et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. wsee commentsx Blood 1995;85:1655–8. w25x Korbling M, Przepiorka D, Huh YO, Engel H, van Besien K, Giralt S, et al. Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 1995;85:1659–65. w26x Osborne MP, Rosen PP. Detection and management of bone marrow micrometastases in breast cancer. Oncology 1994;8: 25–31. w27x Segre M, Tomei E, Segre D. Cyclophosphamide-induced suppressor cells in mice: suppression of the antibody response in vitro and characterization of the effector cells. Cell Immunol 1985;91:443. w28x Hock H, Dorsch M, Diamantstein T, Blankenstein T. Interleukin 7 induces CD4q T cell-dependent tumor rejection. J Exp Med 1991;174:1291–8. w29x Talmadge JE, Wiltrout RH, Counts DF, Herberman RB, McDonald T, Ortaldo TR. Proliferation of human peripheral blood lymphocytes induced by recombinant human interleukin-2: contribution of large granular lymphocytes and T lymphocytes. Cell Immunol 1986;102:261–72. w30x Yannelli JR, Crumpacker DB, Good RW, Friddell CD, Poston R, Horton S, et al. Use of anti-CD3 monoclonal antibody in the generation of effector cells from human solid tumors for use in cancer biotherapy. J Immunol Methods 1990;130: 91–100. w31x Telford WG, King LE, Fraker PJ. Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Proliferation 1991;24:447–59. w32x Maier T, Holda JH, Claman HN. Natural suppressor ŽNS. cells, members of the LGL regulatory family. Immunol Today 1986;7:312–8. w33x Schmidt-Wolf IG, Dejbakhsh-Jones S, Ginzton N, Greenberg P, Strober S. T-cell subsets and suppressor cells in human bone marrow. Blood 1992;80:3242–50. w34x Palathumpat V, Dejbakhsh-Jones S, Holm B, Wang H, Liang O, Strober S. Studies of CD4-CD8-alpharbeta bone marrow T-cells with suppressor activity. J Immunol 1992;148:373– 80. w35x Noga SJ, Wagner JE, Horwitz LR, Donnenberg AD, Santos GW, Hess AD. Characterization of natural suppressor cell populations in adult rat bone marrow. J Leukocyte Biol 1988;43:279–87. w36x Sykes M, Eisenthal A, Sachs DH. Mechanism of protection from graft-vs.-host disease in murine mixed allogeneic chimeras: I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component. J Immunol 1988;140:2903–11. w37x Pak AS, Wright MA, Matthews JP, Collins SL, Petruzzelli
w38x
w39x
w40x
w41x
w42x
w43x
w44x
w45x
w46x
w47x
w48x
w49x
w50x
GJ, Young MRI. Mechanisms of immune suppression in patients with head and neck cancer: Presence of immune suppressive CD34q cells in cancers that secrete granulocytermacrophage colony-stimulating factor. Clin Cancer Res 1995;1:95–103. Young MRI, Young ME, Wright MA. Stimulation of immune-suppressive bone marrow cells by colony-stimulating factors. Exp Hematol 1990;18:806–11. Oghiso Y, Yamada Y, Ando K, Ishihara H, Shibata Y. Differential induction of prostaglandin E2-dependent and-independent immune suppressor cells by tumor-derived GMCSF and M-CSF. J Leukocyte Biol 1993;53:86. Wanebo HJ, Riley T, Katz D, Pace RC, Johns ME, Cantrell RW. Indomethacin sensitive suppressor-cell activity in head and neck cancer patients. The role of the adherent mononuclear cell. Cancer 1988;61:462–74. Farinas MC, Rodriguez-Valverde V, Zarrabeitia MT, ParraBlanco JA, Sanz-Ortiz J. Contribution of monocytes to the decreased lymphoproliferative response to phytohemagglutinin in patients with lung cancer. Cancer 1991;68:1279–84. Hebib NC, Deas O, Rouleau M, Durrbach A, Charpentier B, Beaujean F, et al. Peripheral blood T cells generated after allogeneic bone marrow transplantation: lower levels of bcl-2 protein and enhanced sensitivity to spontaneous and CD95mediated apoptosis in vitro. Abrogation of the apoptotic phenotype coincides with the recovery of normal naiverprimed T-cell profiles. Blood 1999;94:1803–13. Caligiuri MA, Zmuidzinas A, Manley TJ, Levin H, Smith KA, Ritz J. Functional consequences of IL-2-receptor expression on resting human lymphocytes: identificaiton of a novel NK-cell sub-set with high-affinity receptors. J Exp Med 1990;171:1509–26. Minami Y, Kono T, Miyazaki T, Taniguchi T. The IL-2 receptor complex: its structure, function, and target genes. Annu Rev Immunol 1993;11:245–67. Krakauer T. A macrophage-derived factor that inhibits the production and action of interleukin-2. J Leukocyte Biol 1985;38:429–39. Schleifer KW, Mansfield JM. Suppressor macrophages in African trypanosomiasis inhibit T cell proliferative responses by nitric oxide and prostaglandins. J Immunol 1993;151: 5492–503. Mills CD. Molecular basis of AsuppressorB macrophages. Arginine metabolism via the nitric oxide synthetase pathway. J Immunol 1991;146:2719–23. Albina JE, Abate JA, Henry Jr. WL. Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway. J Immunol 1991;147:144–8. Tomioka H, Sato K, Maw WW, Saito H. The role of tumor necrosis factor, interferon-gamma, transforming growth factor-beta, and nitric oxide in the expression of immunosuppressive functions of splenic macrophages induced by Mycobacterium aÕium complex infection. J Leukocyte Biol 1995;58:704–12. Wu MX, Daley JF, Rasmussen RA, Schlossman SF. Mono-
K. Ino et al.r International Immunopharmacology 1 (2001) 1307–1319 cytes are required to prime peripheral blood T cells to undergo apoptosis. Proc Natl Acad Sci U S A 1995;92:1525– 9. w51x Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via FasrFas-ligand-induced apoptosis. J Exp Med 1996;183:1789–96. w52x Munn DH, Pressey J, Beall AC, Hudes R, Alderson MR. Selective activation-induced apoptosis of peripheral T cells imposed by macrophages: a potential mechanism of antigenspecific peripheral lymphocyte deletion. J Immunol 1996; 156:523–32.
1319
w53x Scott DW, Grdina T, Shi Y. T cells commit suicide, but B cells are murdered! J Immunol 1996;156:2352–6. w54x Ju ST, Panka DJ, Cui H, Ettinger R, el Khatib M, Sherr DH, et al. Fas ŽCD95.rFasL interactions required for programmed cell death after T-cell activation. Nature 1995;373: 444–8. w55x Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley KA, et al. Fas ligand mediates activation-induced cell death in human T lymphocytes. J Exp Med 1995;181: 71–7.