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Characteristics of human EBV-specific cytotoxic T lymphocytes utilized for adoptive immunotherapy of EBV-induced lymphoproliferations in xenografted SCID mice
Annals of Oncology 8 (Suppl. 2): S137-S140, 1997. © 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Original article Characteristics of human EBV-specific cytotoxic T lymphocytes utilized for adoptive immunotherapy of EBV-induced Ivmphoproliterations in xenografted SCID mice J. F. Lacerda1'2 & R. J. O'Reilly1 x
Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, USA; 2Department of Hemalology and Bone Marrow Transplantation, University of Lisbon, Santa Maria Hospital, Lisbon, Portugal
Summary
Introduction
Epstein-Barr virus-specific cytototoxic T lymphocytes (EBV-CTLs) play a critical role in the control of B lymphocytes latently infected with EBV [1]. Immunocompromised patients such as recipients of allogeneic organ or marrow grafts are particularly at risk for the development of EBV-induced B-cell lymphoproliferative disorders (EBV-LPDs) [2-4]. Recently, we have shown that EBV-LPDs arising in recipients of T-cell depleted marrow transplants can be cured with infusions of donorderived unirradiated peripheral blood mononuclear cells (PBMCs) [5]. To characterize the nature of the effector cells mediating these clinical responses, we established animal models in the C.B.-17 scid/scid (severe combined immune deficiency, SCID) mouse for the growth of human EBVLPDs [6]. In these models, we have shown that EBVCTLs, but not PBMCs, IL-2-activated PBMCs, purified T cells or anti-CD3-activated T cells, significantly prolong the survival of SCID mice bearing the autologous, but not HLA-mismatched, human EBV-LPD [7]. We have also demonstrated that EBV-CTLs selectively migrate to the autologous tumor in SCID mice bearing both the autologous and either a fully HLA-mismatched or a haplotype-sharing subcutaneous EBV+ tumor [7],
Key words: bone marrow transplantation, cytotoxic T lymphocytes, Epstein-Barr virus, immunotherapy, lymphoproliferative disorders, severe combined immune deficiency mice
In the current study, we characterize the immunophenotype, cytotoxicity, and proliferative responses of unirradiated and irradiated human EBV-CTLs. We also show that the selective homing displayed by unirradiated EBVCTLs in vivo is correlated with both the in vivo and the in vitro HLA-restricted activity of these effector cells.
Materials and methods Generation of EBV-specific CTLs PBMCs and purified T cells were isolated from the peripheral blood of healthy EBV-seropositive individuals, as previously described [7]. For the generation of EBV-CTLs, standard stimulation techniques were used [7, 8]. Briefly, purified T cells (2 x 106 cells/well) were incubated in complete media (CM) (RPMI 1640 supplemented with 10% heat-inactivated human serum, L-glutamine, sodium pyruvate, penicillin and streptomycin, essential and nonessential amino acids, and anti-PPLO agent; all purchased from Gibco, Long Island, New York) in 24-well plates (Costar, Cambridge, MA, USA) with irradiated (9000 rads) autologous EBV-LCLs (5 x 105 cells/well) in the presence of irradiated (3000 rads) autologous PBMCs (1 x lo 6 cells/well). On days 3, 6, and 9, the cell cultures received 1 ml of CM containing 10-20 IU IL-2. On day 12, responder cells were replated in 24-well plates (1.5-2.0 x 106 cells/well) in the presence of autologous irradiated EBV-LCLs (responder/stimulator ratio 5/1) and 106 irradiated PBMCs [7]. Thereafter, cultures were fed every 3 to 4 days with CM containing 10 IU/ml IL-2 and restimulated weekly with the autologous EBV-LCLs. For the experiments depicted in
Downloaded from http://annonc.oxfordjournals.org/ at Northern Arizona University on June 8, 2015
Human Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) prolong the survival of mice with severe combined immune deficiency bearing the autologous, but not HLA-mismatched, human EBV-induced lymphoproliferative disorders (EBV-LPDs). In the present study, we demonstrate that the HLA-restricted activity displayed by EBV-CTLs both in vitro and in vivo correlates with their in vivo homing pattern, and further characterize these effectors. EBV-CTLs were CD3+, CD16/56-, TCR a/p+, predominantly CD8+ and CD4-, and had a high expression of T-cell activation antigens. EBV-CTLs were positive for CDlla/CD18, CD54, CD58, CD44, CD49d, CD28, and CD45RO, and negative for CD45RA, CDllb, CDllc. After 26 days in culture, EBV-CTLs displayed strong cytotoxicity against the autologous EBV-transformed B-cell line
(EBV-LCL), which was inhibited by the addition of anti-CD3 MoAb and mostly HLA class I-restricted. Unirradiated and irradiated EBV-CTLs in the absence of IL-2 failed to proliferate after more than 2 days in culture with the autologous EBVLCLs, while unirradiated EBV-CTLs with IL-2 formed large colonies and had a high thymidine incorporation both on days 5 and 8. The cytotoxicity of irradiated EBV-CTLs against the autologous EBV-LCLs was conserved. It remains to be determined whether irradiated EBV-CTLs are capable of homing to EBV-LPDs in vivo and to mediate a therapeutic response comparable to that observed with unirradiated EBV-CTLs.
138 "•" Unirrad. 1000 R • 2000R
incorporated thymidine was evaluated by liquid scintillation spectrometry in a beta counter (Betaplate, Wallac Inc., Gaithersburg, MD, USA). The results are presented as mean values of triplicate cultures performed in parallel.
•3000R
Flow cytometry Purified T cells, EBV-CTLs and single-cell suspensions obtained from subcutaneous EBV+ tumors were stained with the indicated conjugated MoAb (purchased from Becton-Dickinson, San Jose, CA, and AMAC Westbrook, ME, USA). The cells were prepared following standard protocols described in detail elsewhere [6, 7], and analyzed in a FACS scan flow cytometer utilizing LYSIS II software (Becton Dickinson) in the Institution's Flow Cytometry Core Facility. 2.5:1 0.6:1 Effector:Target Ratio
0.15:1
In vivo studies
b)
ElOOO R 01OOOR + IL-2 02000 R • 2000 R + IL-2 Q3000R B3000R + IL-2
100
80
60
40 I
20
D2
D5
C.B-17 scid/scid (SCID) mice, 5 to 8 weeks old, purchased from Taconic Farms (Germantown, New York), were treated with rabbit anti-asialo GM1 antiserum (Wako Chemicals, Richmond, VA, USA) for depletion of endogenous NK-cell function, as previously described [6]. SCID mice were inoculated subcutaneously in each flank with 2 x 107 EBV-LCLs, one autologous and the other HLA-mismatched to the EBV-specific CTL donor. At the time of tumor development on both flanks (day 36), mice received 107 autologous EBV-CTLs, stimulated in vitro with the autologous EBV-LCLs for 26 days, by the intravenous route, after which they were followed to evaluate tumor response. Animals also received 5000 IU IL-2 intraperitoneally every other day for a total of 4 doses. Seven to 14 days following T-cell adoptive transfer, SCID mice were sacrificed and tumor single-cell suspensions analyzed by flow cytometry for the presence of human T cells.
08
Figure 1. (A) Cytotoxicity of unirradiated and irradiated (1000, 2000, and 3000 rads [R]) EBV-CTLs against the autologous EBV-LCLs, at the indicated effector to target ratios. (B) Thymidine uptake of unirradiated and irradiated (1000, 2000, and 3000 rads [R]) EBV-speciflc CTLs after 2, 5, or 8 days in culture with the autologous EBV-LCLs, in the presence and absence of IL-2. Figure 1, the EBV-CTLs were irradiated after 26 days in culture with the autologous EBV-LCLs (i.e., immediately prior to the cytotoxicity and proliferation assays).
Cytotoxicity assay Cytotoxicity of unirradiated and irradiated (1000, 2000, and 3000 rads) EBV-CTLs was assessed using standard 4-hour chromium release assays, as previously described [6, 7]. Target cells included the autologous EBVLCLs, EBV-LCLs sharing one or more HLA class I and/or class II antigens with autologous EBV-LCLs, fully HLA-mismatched EBVLCLs, autologous EBV-activated B cells (stimulated with 10 ng/ml PMA and 1 uM/ml ionomycin), and the NK-sensitive erythroleukemia cell line K562.
T-cell proliferation assay An aliquot of 105 unirradiated or irradiated (1000, 2000, and 3000 rads) EBV-CTLs, generated in culture in the presence of the autologous EBVLCLs for 26 days, were incubated in CM with 5 x 104 autologous EBVLCLs, in the presence and absence of 10 IU/ml IL-2, at 37 °C and 5% CO 2 . The cell cultures were fed with fresh CM with or without 10 IU IL-2 on days 3 and 7. After 2, 5, and 8 days of incubation, the cell cultures were pulsed with 4 uCi/ml of tritiated thymidine for a period of 6 hours, after which the cells were harvested on filter paper and the
Results Immunophenotype and cytotoxicity of EBV-CTLs EBV-CTLs were generated in vitro from purified T cells derived from EBV-seropositive individuals upon stimulation for 26 or more days with the autologous EBV-LCLs. The immunophenotype of both cell populations is displayed in Table 1. The responding cells were CD3+, TCR
Downloaded from http://annonc.oxfordjournals.org/ at Northern Arizona University on June 8, 2015
• Unirrad. • Unirrad. + IL-2
139 Proliferation and cytotoxicity of unirradiated and irradiated EBV-CTLs Since the administration of allogeneic leukocytes is hampered by the emergence of graft versus host disease, we investigated the cytotoxicity and proliferative responses of unirradiated and irradiated EBV-specific CTLs, upon stimulation with the autologous EBV-LCLs. As shown in Figure 1 A, the in vitro cytotoxicity of EBV-CTLs was not significantly affected by irradiation with 1000, 2000, or 3000 rads. However, as depicted in the lower panel (Figure IB), only unirradiated EBV-CTLs stimulated with the autologous EBV-LCLs in the presence of exogenous IL-2 proliferated after 4 days in culture, while as few as 1000 rads completely inhibited the proliferation of EBV-CTLs, even in the presence of IL-2.
After intravenous adoptive transfer into SCID mice bearing both the autologous and an HLA-mismatched EBV+ tumor (n = 6), against which there was no cytotoxicity in vitro, only the autologous tumors decreased in size over the next 10 to 14 days (25%-50% reduction), while HLAmismatched tumors continued to grow causing the death of the animals. In this experiment, in two animals tested, flow cytometry analyses of both tumors from the same mouse disclosed a preferential migration of EBV-CTLs into the autologous tumor, which were predominantly of CD8 immunophenotype, while virtually no T cells were detected infiltrating the HLA-mismatched EBV+ tumor (Figure 2). The tumor cells were CD20+.
Discussion Patients with severe defects of cell-mediated immunity are at risk for the development of EBV-LPDs [2]. Recip-
Allogenic EBV* Tumor
CD3 (%)
TCR a/p (%) CD4 (%) CD8 (%) CD16/56(%) CD25 (%) CDlla/CD18(%) CD54 (%) CD58 (%) CD44 (%) CD49d (%) CD28 (%) CD45RO (%) CD45RA (%) CDllb(%) CDllc(%) SL (%) Untreated SL (%) Anti-CD3 SL (%) Anti-HLA class I SL (SL) Anti-HLA class II
Purified T cells
EBV-CTLs
87.3-95.9 73.2-86.8 51.9-69.0 25.1-35.4 1.5-5.1 2.3-16.0 32.1-34.5 15.6-22.1 35.7-38.2 ND ND 77.3-85.4 ND ND ND ND ND ND ND ND
94.3-98.8 95.0-97.4 1.1-21.3 75.7-98.6 <2.1 48.1-83.2 >90 >90 >90 >90 >90 >90 >90 0 <1 <1 34.4-56.4 8.2-12.5 12.3-28.1 24.0-46.3
Abbreviations: ND - not done; SL - specific lysis.
ients of allogeneic marrow grafts, particularly in the setting of HLA-mismatched and/or unrelated transplants, are especially prone to developing this complication. EBVLPDs arising in BMT recipients frequently present as oligoclonal or monoclonal lymphomas, which have uniformly been refractory to conventional antineoplastic or antiviral treatment regimens [3, 4]. Recently, we have shown that adoptive transfer of donor PBMCs induce durable complete remissions of donor-type EBV-LPDs arising in recipients of marrow transplants [5]. Subsequently, our group has demonstrated that these PBMC infusions result in a dramatic increase in the number of EBV-specific CTL precursors in the peripheral blood of the patients [9], suggesting that EBV-specific T cells might be the effector cells involved in such clinical responses. Autologous EBV+ Tumor
Figure 2. Flow cytometry analysis of single-cell suspensions of the autologous and HLA-mismatched EBV+ tumors from one mouse sacrificed 14 days after adoptive EBV-CTL transfer.
Downloaded from http://annonc.oxfordjournals.org/ at Northern Arizona University on June 8, 2015
In vivo HLA-restricted homing of unirradiated EBV-CTLs
Table 1. Characteristics of EBV-specific CTLs.
140 References 1. Rickinson AB, Moss DJ, Wallace LE et al. Long-term cell-mediated immunity to Epstein-Barr virus. Cancer Res 1981; 41:4216-21. 2. Cohen JI. Epstein-Barr virus lymphoproliferative disease associated with acquired immunodeficiency. Medicine 1991; 70:137-60. 3. Shapiro RS, MaClain K, Frizzera G et al. Epstein-Barr virus associated B-cell lymphoproliferative disorders following bone marrow transplantation. Blood 1988; 71:1234-43. 4. Zutter MM, Martin PJ, Sale GE et al. Epstein-Barr virus lymphoproliferation after bone marrow transplantation. Blood 1988; 72: 520-9. 5. Papadopoulos EB, Ladanyi M, Emanuel D et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders complicating allogeneic bone marrow transplantation. N Engl J Med 1994; 330:1185-91. 6. Lacerda JF, Ladanyi M, Jagiello C et al. Administration of rabbit anti-asialo GM1 antiserum facilitates the development of human EBV-induced lymphoproliferations in xenografted C.B-17 scid/scid mice. Transplantation 1996; 61: 492-7. 7. Lacerda JF, Ladanyi M, Louie DC et al. Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced lymphoproliferations in xenografted C.B-17 scid/scid mice. J Exp Med 1996; 183:1215-28. 8. Bourgault I, Gomez A, Gomard E et al. Limiting-dilution analysis of the HLA restriction of anti-Epstein-Barr virus-specific cytolytic T lymphocytes. Clin Exp Immunol 1991; 84: 501-7. 9. Lucas K, Small T, Heller G et al. The development of Epstein-Barr virus specific cellular immunity following allogeneic marrow transplantation. Blood 1996; 87: 2594-603. 10. Rooney CM, Smith CA, Ng CYC et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet 1995; 345: 9-13. Correspondence to: Dr. Joao Forjaz de Lacerda Hospital de Santa Maria Av. Prof. Egas Moniz 1600 Lisbon Portugal
Downloaded from http://annonc.oxfordjournals.org/ at Northern Arizona University on June 8, 2015
Indeed, our xenografted animal models have confirmed that EBV-CTLs are the effector cells mediating the therapeutic responses observed [7]. In the present study, we show that these putative EBVspecific and HLA-restricted effectors are predominantly HLA class I-restricted, potentially reflecting the predominance of CD8+ HLA class I-restricted EBV-CTLs, naturally selected in culture by EBV stimulation. These cells also express the majority of adhesion molecules tested, which may be crucial for homing in vivo in the SCID mouse model and, probably, in man. As shown in Figure 2, and in more detail elsewhere [7], unirradiated EBVCTLs selectively migrate to EBV+ tumors bearing the appropriate HLA antigens in order to induce a therapeutic response. Although Rooney et al. [10] have not encountered higher GVHD incidence in recipients of marrow transplants treated with in vitro -generated donor-derived EBVCTLs for the prevention of EBV-LPDs, and the frequency of alloreactive T cells in the EBV-CTL population is certainly lower than that encountered in unstimulated PBMC, we evaluated the in vitro cytotoxicity and proliferation of irradiated EBV-CTLs, since this approach might further reduce the risk of GVHD in recipients of allogeneic marrow grafts. As shown in Figure 1, nonproliferating irradiated EBV-CTLs maintained their in vitro cytotoxicity against the autologous EBV-LCLs. However, it remains to be determined whether these cells are capable of homing selectively to EBV+ tumors and inducing an HLA-restricted therapeutic effect such as we have documented with the infusion of unirradiated EBVCTLs [7].
Original article Characteristics of human EBV-specific cytotoxic T lymphocytes utilized for adoptive immunotherapy of EBV-induced Ivmphoproliterations in xenografted SCID mice J. F. Lacerda1'2 & R. J. O'Reilly1 x
Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, USA; 2Department of Hemalology and Bone Marrow Transplantation, University of Lisbon, Santa Maria Hospital, Lisbon, Portugal
Summary
Introduction
Epstein-Barr virus-specific cytototoxic T lymphocytes (EBV-CTLs) play a critical role in the control of B lymphocytes latently infected with EBV [1]. Immunocompromised patients such as recipients of allogeneic organ or marrow grafts are particularly at risk for the development of EBV-induced B-cell lymphoproliferative disorders (EBV-LPDs) [2-4]. Recently, we have shown that EBV-LPDs arising in recipients of T-cell depleted marrow transplants can be cured with infusions of donorderived unirradiated peripheral blood mononuclear cells (PBMCs) [5]. To characterize the nature of the effector cells mediating these clinical responses, we established animal models in the C.B.-17 scid/scid (severe combined immune deficiency, SCID) mouse for the growth of human EBVLPDs [6]. In these models, we have shown that EBVCTLs, but not PBMCs, IL-2-activated PBMCs, purified T cells or anti-CD3-activated T cells, significantly prolong the survival of SCID mice bearing the autologous, but not HLA-mismatched, human EBV-LPD [7]. We have also demonstrated that EBV-CTLs selectively migrate to the autologous tumor in SCID mice bearing both the autologous and either a fully HLA-mismatched or a haplotype-sharing subcutaneous EBV+ tumor [7],
Key words: bone marrow transplantation, cytotoxic T lymphocytes, Epstein-Barr virus, immunotherapy, lymphoproliferative disorders, severe combined immune deficiency mice
In the current study, we characterize the immunophenotype, cytotoxicity, and proliferative responses of unirradiated and irradiated human EBV-CTLs. We also show that the selective homing displayed by unirradiated EBVCTLs in vivo is correlated with both the in vivo and the in vitro HLA-restricted activity of these effector cells.
Materials and methods Generation of EBV-specific CTLs PBMCs and purified T cells were isolated from the peripheral blood of healthy EBV-seropositive individuals, as previously described [7]. For the generation of EBV-CTLs, standard stimulation techniques were used [7, 8]. Briefly, purified T cells (2 x 106 cells/well) were incubated in complete media (CM) (RPMI 1640 supplemented with 10% heat-inactivated human serum, L-glutamine, sodium pyruvate, penicillin and streptomycin, essential and nonessential amino acids, and anti-PPLO agent; all purchased from Gibco, Long Island, New York) in 24-well plates (Costar, Cambridge, MA, USA) with irradiated (9000 rads) autologous EBV-LCLs (5 x 105 cells/well) in the presence of irradiated (3000 rads) autologous PBMCs (1 x lo 6 cells/well). On days 3, 6, and 9, the cell cultures received 1 ml of CM containing 10-20 IU IL-2. On day 12, responder cells were replated in 24-well plates (1.5-2.0 x 106 cells/well) in the presence of autologous irradiated EBV-LCLs (responder/stimulator ratio 5/1) and 106 irradiated PBMCs [7]. Thereafter, cultures were fed every 3 to 4 days with CM containing 10 IU/ml IL-2 and restimulated weekly with the autologous EBV-LCLs. For the experiments depicted in
Downloaded from http://annonc.oxfordjournals.org/ at Northern Arizona University on June 8, 2015
Human Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) prolong the survival of mice with severe combined immune deficiency bearing the autologous, but not HLA-mismatched, human EBV-induced lymphoproliferative disorders (EBV-LPDs). In the present study, we demonstrate that the HLA-restricted activity displayed by EBV-CTLs both in vitro and in vivo correlates with their in vivo homing pattern, and further characterize these effectors. EBV-CTLs were CD3+, CD16/56-, TCR a/p+, predominantly CD8+ and CD4-, and had a high expression of T-cell activation antigens. EBV-CTLs were positive for CDlla/CD18, CD54, CD58, CD44, CD49d, CD28, and CD45RO, and negative for CD45RA, CDllb, CDllc. After 26 days in culture, EBV-CTLs displayed strong cytotoxicity against the autologous EBV-transformed B-cell line
(EBV-LCL), which was inhibited by the addition of anti-CD3 MoAb and mostly HLA class I-restricted. Unirradiated and irradiated EBV-CTLs in the absence of IL-2 failed to proliferate after more than 2 days in culture with the autologous EBVLCLs, while unirradiated EBV-CTLs with IL-2 formed large colonies and had a high thymidine incorporation both on days 5 and 8. The cytotoxicity of irradiated EBV-CTLs against the autologous EBV-LCLs was conserved. It remains to be determined whether irradiated EBV-CTLs are capable of homing to EBV-LPDs in vivo and to mediate a therapeutic response comparable to that observed with unirradiated EBV-CTLs.
138 "•" Unirrad. 1000 R • 2000R
incorporated thymidine was evaluated by liquid scintillation spectrometry in a beta counter (Betaplate, Wallac Inc., Gaithersburg, MD, USA). The results are presented as mean values of triplicate cultures performed in parallel.
•3000R
Flow cytometry Purified T cells, EBV-CTLs and single-cell suspensions obtained from subcutaneous EBV+ tumors were stained with the indicated conjugated MoAb (purchased from Becton-Dickinson, San Jose, CA, and AMAC Westbrook, ME, USA). The cells were prepared following standard protocols described in detail elsewhere [6, 7], and analyzed in a FACS scan flow cytometer utilizing LYSIS II software (Becton Dickinson) in the Institution's Flow Cytometry Core Facility. 2.5:1 0.6:1 Effector:Target Ratio
0.15:1
In vivo studies
b)
ElOOO R 01OOOR + IL-2 02000 R • 2000 R + IL-2 Q3000R B3000R + IL-2
100
80
60
40 I
20
D2
D5
C.B-17 scid/scid (SCID) mice, 5 to 8 weeks old, purchased from Taconic Farms (Germantown, New York), were treated with rabbit anti-asialo GM1 antiserum (Wako Chemicals, Richmond, VA, USA) for depletion of endogenous NK-cell function, as previously described [6]. SCID mice were inoculated subcutaneously in each flank with 2 x 107 EBV-LCLs, one autologous and the other HLA-mismatched to the EBV-specific CTL donor. At the time of tumor development on both flanks (day 36), mice received 107 autologous EBV-CTLs, stimulated in vitro with the autologous EBV-LCLs for 26 days, by the intravenous route, after which they were followed to evaluate tumor response. Animals also received 5000 IU IL-2 intraperitoneally every other day for a total of 4 doses. Seven to 14 days following T-cell adoptive transfer, SCID mice were sacrificed and tumor single-cell suspensions analyzed by flow cytometry for the presence of human T cells.
08
Figure 1. (A) Cytotoxicity of unirradiated and irradiated (1000, 2000, and 3000 rads [R]) EBV-CTLs against the autologous EBV-LCLs, at the indicated effector to target ratios. (B) Thymidine uptake of unirradiated and irradiated (1000, 2000, and 3000 rads [R]) EBV-speciflc CTLs after 2, 5, or 8 days in culture with the autologous EBV-LCLs, in the presence and absence of IL-2. Figure 1, the EBV-CTLs were irradiated after 26 days in culture with the autologous EBV-LCLs (i.e., immediately prior to the cytotoxicity and proliferation assays).
Cytotoxicity assay Cytotoxicity of unirradiated and irradiated (1000, 2000, and 3000 rads) EBV-CTLs was assessed using standard 4-hour chromium release assays, as previously described [6, 7]. Target cells included the autologous EBVLCLs, EBV-LCLs sharing one or more HLA class I and/or class II antigens with autologous EBV-LCLs, fully HLA-mismatched EBVLCLs, autologous EBV-activated B cells (stimulated with 10 ng/ml PMA and 1 uM/ml ionomycin), and the NK-sensitive erythroleukemia cell line K562.
T-cell proliferation assay An aliquot of 105 unirradiated or irradiated (1000, 2000, and 3000 rads) EBV-CTLs, generated in culture in the presence of the autologous EBVLCLs for 26 days, were incubated in CM with 5 x 104 autologous EBVLCLs, in the presence and absence of 10 IU/ml IL-2, at 37 °C and 5% CO 2 . The cell cultures were fed with fresh CM with or without 10 IU IL-2 on days 3 and 7. After 2, 5, and 8 days of incubation, the cell cultures were pulsed with 4 uCi/ml of tritiated thymidine for a period of 6 hours, after which the cells were harvested on filter paper and the
Results Immunophenotype and cytotoxicity of EBV-CTLs EBV-CTLs were generated in vitro from purified T cells derived from EBV-seropositive individuals upon stimulation for 26 or more days with the autologous EBV-LCLs. The immunophenotype of both cell populations is displayed in Table 1. The responding cells were CD3+, TCR
Downloaded from http://annonc.oxfordjournals.org/ at Northern Arizona University on June 8, 2015
• Unirrad. • Unirrad. + IL-2
139 Proliferation and cytotoxicity of unirradiated and irradiated EBV-CTLs Since the administration of allogeneic leukocytes is hampered by the emergence of graft versus host disease, we investigated the cytotoxicity and proliferative responses of unirradiated and irradiated EBV-specific CTLs, upon stimulation with the autologous EBV-LCLs. As shown in Figure 1 A, the in vitro cytotoxicity of EBV-CTLs was not significantly affected by irradiation with 1000, 2000, or 3000 rads. However, as depicted in the lower panel (Figure IB), only unirradiated EBV-CTLs stimulated with the autologous EBV-LCLs in the presence of exogenous IL-2 proliferated after 4 days in culture, while as few as 1000 rads completely inhibited the proliferation of EBV-CTLs, even in the presence of IL-2.
After intravenous adoptive transfer into SCID mice bearing both the autologous and an HLA-mismatched EBV+ tumor (n = 6), against which there was no cytotoxicity in vitro, only the autologous tumors decreased in size over the next 10 to 14 days (25%-50% reduction), while HLAmismatched tumors continued to grow causing the death of the animals. In this experiment, in two animals tested, flow cytometry analyses of both tumors from the same mouse disclosed a preferential migration of EBV-CTLs into the autologous tumor, which were predominantly of CD8 immunophenotype, while virtually no T cells were detected infiltrating the HLA-mismatched EBV+ tumor (Figure 2). The tumor cells were CD20+.
Discussion Patients with severe defects of cell-mediated immunity are at risk for the development of EBV-LPDs [2]. Recip-
Allogenic EBV* Tumor
CD3 (%)
TCR a/p (%) CD4 (%) CD8 (%) CD16/56(%) CD25 (%) CDlla/CD18(%) CD54 (%) CD58 (%) CD44 (%) CD49d (%) CD28 (%) CD45RO (%) CD45RA (%) CDllb(%) CDllc(%) SL (%) Untreated SL (%) Anti-CD3 SL (%) Anti-HLA class I SL (SL) Anti-HLA class II
Purified T cells
EBV-CTLs
87.3-95.9 73.2-86.8 51.9-69.0 25.1-35.4 1.5-5.1 2.3-16.0 32.1-34.5 15.6-22.1 35.7-38.2 ND ND 77.3-85.4 ND ND ND ND ND ND ND ND
94.3-98.8 95.0-97.4 1.1-21.3 75.7-98.6 <2.1 48.1-83.2 >90 >90 >90 >90 >90 >90 >90 0 <1 <1 34.4-56.4 8.2-12.5 12.3-28.1 24.0-46.3
Abbreviations: ND - not done; SL - specific lysis.
ients of allogeneic marrow grafts, particularly in the setting of HLA-mismatched and/or unrelated transplants, are especially prone to developing this complication. EBVLPDs arising in BMT recipients frequently present as oligoclonal or monoclonal lymphomas, which have uniformly been refractory to conventional antineoplastic or antiviral treatment regimens [3, 4]. Recently, we have shown that adoptive transfer of donor PBMCs induce durable complete remissions of donor-type EBV-LPDs arising in recipients of marrow transplants [5]. Subsequently, our group has demonstrated that these PBMC infusions result in a dramatic increase in the number of EBV-specific CTL precursors in the peripheral blood of the patients [9], suggesting that EBV-specific T cells might be the effector cells involved in such clinical responses. Autologous EBV+ Tumor
Figure 2. Flow cytometry analysis of single-cell suspensions of the autologous and HLA-mismatched EBV+ tumors from one mouse sacrificed 14 days after adoptive EBV-CTL transfer.
Downloaded from http://annonc.oxfordjournals.org/ at Northern Arizona University on June 8, 2015
In vivo HLA-restricted homing of unirradiated EBV-CTLs
Table 1. Characteristics of EBV-specific CTLs.
140 References 1. Rickinson AB, Moss DJ, Wallace LE et al. Long-term cell-mediated immunity to Epstein-Barr virus. Cancer Res 1981; 41:4216-21. 2. Cohen JI. Epstein-Barr virus lymphoproliferative disease associated with acquired immunodeficiency. Medicine 1991; 70:137-60. 3. Shapiro RS, MaClain K, Frizzera G et al. Epstein-Barr virus associated B-cell lymphoproliferative disorders following bone marrow transplantation. Blood 1988; 71:1234-43. 4. Zutter MM, Martin PJ, Sale GE et al. Epstein-Barr virus lymphoproliferation after bone marrow transplantation. Blood 1988; 72: 520-9. 5. Papadopoulos EB, Ladanyi M, Emanuel D et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders complicating allogeneic bone marrow transplantation. N Engl J Med 1994; 330:1185-91. 6. Lacerda JF, Ladanyi M, Jagiello C et al. Administration of rabbit anti-asialo GM1 antiserum facilitates the development of human EBV-induced lymphoproliferations in xenografted C.B-17 scid/scid mice. Transplantation 1996; 61: 492-7. 7. Lacerda JF, Ladanyi M, Louie DC et al. Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced lymphoproliferations in xenografted C.B-17 scid/scid mice. J Exp Med 1996; 183:1215-28. 8. Bourgault I, Gomez A, Gomard E et al. Limiting-dilution analysis of the HLA restriction of anti-Epstein-Barr virus-specific cytolytic T lymphocytes. Clin Exp Immunol 1991; 84: 501-7. 9. Lucas K, Small T, Heller G et al. The development of Epstein-Barr virus specific cellular immunity following allogeneic marrow transplantation. Blood 1996; 87: 2594-603. 10. Rooney CM, Smith CA, Ng CYC et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet 1995; 345: 9-13. Correspondence to: Dr. Joao Forjaz de Lacerda Hospital de Santa Maria Av. Prof. Egas Moniz 1600 Lisbon Portugal
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Indeed, our xenografted animal models have confirmed that EBV-CTLs are the effector cells mediating the therapeutic responses observed [7]. In the present study, we show that these putative EBVspecific and HLA-restricted effectors are predominantly HLA class I-restricted, potentially reflecting the predominance of CD8+ HLA class I-restricted EBV-CTLs, naturally selected in culture by EBV stimulation. These cells also express the majority of adhesion molecules tested, which may be crucial for homing in vivo in the SCID mouse model and, probably, in man. As shown in Figure 2, and in more detail elsewhere [7], unirradiated EBVCTLs selectively migrate to EBV+ tumors bearing the appropriate HLA antigens in order to induce a therapeutic response. Although Rooney et al. [10] have not encountered higher GVHD incidence in recipients of marrow transplants treated with in vitro -generated donor-derived EBVCTLs for the prevention of EBV-LPDs, and the frequency of alloreactive T cells in the EBV-CTL population is certainly lower than that encountered in unstimulated PBMC, we evaluated the in vitro cytotoxicity and proliferation of irradiated EBV-CTLs, since this approach might further reduce the risk of GVHD in recipients of allogeneic marrow grafts. As shown in Figure 1, nonproliferating irradiated EBV-CTLs maintained their in vitro cytotoxicity against the autologous EBV-LCLs. However, it remains to be determined whether these cells are capable of homing selectively to EBV+ tumors and inducing an HLA-restricted therapeutic effect such as we have documented with the infusion of unirradiated EBVCTLs [7].