Differential effects of transferrin receptor blockade on the cellular mechanisms involved in graft rejection

Transplant Immunology 1999; 7: 131-139

Differential effects of transferrin receptor blockade on the cellular mechanisms involved in graft rejection Allison L Bayer”, Prabhakar Baligab and Jennifer E Woodwardb Departments of “Microbiology and Immunology and bSurgery, Medical University of South Carolina, Charleston, South Carolina Received 18 May 1999, accepted 12 July 1999

Abstract: Since transfertin receptor (TfR) appears on activated T cells following the interaction of the antigen-major histocompatibility complex (MHC) with the T cell receptor (TCR) and the appearance of interlet&in (IL)-ZR, we therefore hypothesize that in viva blockade of TfR prolongs allograft survival by altering the cellular mechanisms involved in graft rejection. Previous results in our laboratory have demonstrated that anti-TfR monoclonal antibody (mAb) at 100 ug on days 0 and 1 of transplantation signihcantly prolonged allograft survival to 25.7 f 0.9 days in a murine heterotopic, nonvascularixed cardiac allogmft model. In the current studies, administration of anti-TfR mAb at the time of maximal TfR expression, on days 2 and 3 post-transplantation, failed to prolong allograft survival (13.0 + 0.0 days) compared to the &type controls (10.5 + 0.5 and 10.7 + 0.4 days) (p c 0.01, Wilcoxon rank sum). A 4-day course of anti-TfR mAb sign&antly prolonged allograft survival compared to the isotype controls, but was no more effective than a 2-day course of the mAb. Anti-TfR mAb suppressed the mixed lymphocyte response to donor-specific and third-party alloantigen by 78.7% @ < 0.05) and 80.8% @ < 0.05), respectively, while stimulating the CTL response to donor-speci6c (16.3%,p < 0.05) and third party (49.3%,p < 0.01) alloantigen. Anti-TfR mAb suppressed IL-15 and increased IL-4 intragraft mRNA expression when compared to the isotype controls. Examination of cell surface receptors important during T cell activation revealed alterations in expression

following anti-‘IYRmAb treatment. Anti-TfR mAb is an effective immunosuppressant prolonging allograft survival by altering cell-mediated immune responses and the intragraft cytokine micro-environment.

‘Ikansferrin receptor (TfR), a disulfide-linked transmembrane glycoprotein, is a widely distributed cell surface receptor expressed on rapidly proliferating normal cells, transformed cells, and specialized quiescent cells which have high requirements for iron.tS2 TfR expression is essential for continued growth3 and is closely linked to the proliferative status of a cell.’ Cell surface expression of TfR is decreased or absent on quiescent and differentiated cells.’ In addition to its crucial role in Address for correspondence:

JE Woodward, University of Pittsburgh, Thomas E Starxl ‘Bansplantation Institute, Section of Cellular ‘Bansplantation, El551 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261, USA. E-mail: [email protected] 0 Arnold 1999

iron uptake, TfR is involved in T cell activation.4J However, the roles and mechanisms of TfR in T cell activation remain illdefined. T cell activation is a two-signal process. The primary signal is provided by the interaction of the T cell receptor (TCR) with the antigen-major histocompatibility complex (MHC) on the antigen presenting cells (APCs).6Y7The co-stimulatoty or secondary signal is provided by a co-receptor-ligand interaction.a’O Only when both signals are received does this process result in proliferation, interleukin (IL)-2 production, and effector functions.10 The expression of IL-2 receptor (IL-2R) is upregulated following T cell activatio&’ for IL-2 binding and for promoting T cell proliferation.‘*” TfR expression increases subsequent to T cell activation and the expression of IL-2R.3V5,‘2 0966-3274(99)TI271OA

132 AL Bayer et al.

It is well established that T cells differentiate into two distinct types of helper cells, T helper-l (Thl) or Th2 with different functions and patterns of cytokine secretion. Thl cells are responsible for cell-mediated immune responses such as the activation of CDS+ cytotoxic T lymphocytes,13114which in transplantation, are one of the primary cells responsible for acute rejection. l5 On the contrary, Th2 cells regulate humoral immune responses and are associated with a suppressed immune state.13 Previous work with monoclonal antibodies (mAbs) against TfR have been primarily utilized in cancer immunotherapy,‘417 demonstrating a down-regulation in TfR expression and alterations in iron transport. In renal-cell carcinoma patients, absence of TfR on tumour infiltrating cells has been associated with poor proliferative responses.” In contrast, peripheral T cells from these same patients have normal levels of TfR and demonstrate strong proliferative responses.” In a murine leukaemia model, anti-TfR mAbs prolonged survival of tumour-bearing mice. l6 Nevertheless, the mechanisms responsible for these alterations in,immune function following TfR blockade have not been completely defined. These observations suggest that TfR may be a potential therapeutic target in transplantation. Previous results in our laboratory have demonstrated that in vivo administration of anti-TfR mAb prolongs allograft survival and suppresses in vitro cytotoxic and proliferative T cell responses to alloantigen.18Y’9Therefore, we hypothesize that in vivo blockade of TfR prolongs allograft survival by altering the cellular mechanisms involved in graft rejection.

Materials and methods Animalsand

reagents

Female CBA/J (H-s), C57BU6J (H-2b), and BALB/c ByJ (H2d) mice (7-8 weeks of age) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). R17 217.1.3 rat IgG2, antimouse TfR hybridoma, a gift from Dr Jayne Lesley (Salk Institute),20 and the isotype-matched control El3 161-7 rat IgG2, anti-mouse stem cell antigen-l hybridoma (which fails to demonstrate complement-mediated cytotoxicity in isolated CBA/J splenocytes)21 (American Qpe Culture Collection, Rockville, MD, USA) were grown in culture and purified with a protein G affinity column.

were performed as previously described.z A standard 4-h ‘lCr release assay was performed with EL-4 (H-2b) or P815 (H-2d) targets. Controls included irrelevant CCL13 (H-2k) targets and were I5% specific lysis. Spontaneous release was 5-15% of total incorporated counts. CTL generated were CD8+, H-2 restricted, and alloantigen-specific with peak primary CTL responses noted after 7 days of culture. Results are expressed as: specific lysis =

(experimental-spontaneous) (100% lysis - spontaneous)

fSEM

The SEM was always 52%. Statistical significance for the CTL assay was performed with Student’s two-tailed t-test. Mixed iymphocyta reaction Responder splenocytes (2 x 16) were co-cultured with 2 x 16 1500 rad y-irradiated C57BU6 or BALB/c ByJ stimulator cells in triplicate for 5 days. The wells were pulsed with 0.5 pCi of 3H-thymidine 18 h prior to termination of the culture, harvested onto glass-fibre filters, and incorporation quantitated on a scintillation counter. Results are expressed as mean -C SEM. Statistical significance was performed with Student’s two-tailed t-test.” Flow cytometry Cell washes and antibody dilutions were performed in PBS plus 1% bovine serum albumin (BSA) at 4°C. TfR was stained with FITC-R17 217.1.3 rat anti-mouse TfR mAb (l:lOO), CD3 with FITC-145-2Cll hamster anti-mouse CD3 mAb (l:lOO), and B7 with FITC-CTLA4Ig. CD4 was stained with either FITC-rat anti-mouse CD4 or PE-rat anti-mouse CD4 (1:30), CD8 with FITC-rat anti-mouse CD8 (1:30), and CD28 with Tri-Colorhamster anti-mouse CD28 (1:30) (Caltag, Burlingame, CA, USA). CD2 was stained with Biotin-12-15 (1:30) followed by PE-SA (1:lOO) (TAG0 Immunologicals, Inc., Burlington, CA, USA). IL-2R was stained with 3C7 rat anti-mouse IL-2R mAb (50 &/ml) followed by FITC F(ab’)a goat anti-rat IgG (H+L) (TAG0 Immunologic&, Inc). The latter antibody was used alone as a negative control. Flow cytometric analysis was performed using a Coulter Epics Elite with Elite Work Station software (Hialeah, FL, USA). Results are expressed as the percentage of cells stained above background.

Cardiac transplantation

Donor neonatal C57BU6 mice were sacrificed, whole hearts removed, and placed into subcutaneous pockets iu the ear pinnae of adult CBA/J recipients as previously described.18Y22 Recipients received intravenous injections of anti-TfR or control mAbs at the indicated dose in 0.5 ml of phosphate buffered saline (PBS) at the specified time after grafting. Survival of the allografts was followed by EKG monitoring (Grass Instruments, Polygraph 78 series with preamplifer and filters) every other day. Rejection was determined by cessation of cardiac activity on three consecutive days. Statistical significance was determined by Wilcoxon rank sum. Alloantigcrrspeciflc cytotoxic T lymphocytes Spleens from naive CBA/J or allograft recipients 7 days posttransplantation”‘” were removed and gently dissociated into a single cell suspensions. Red blood cells were removed from the responder (CBA) and stimulator (CS7Bu6 or BALB/c ByJ) cell populations by tris-NH&l lysis. The CIX culture and assays Transplant Immunology 1999; 7: 131-139

lntragraft R’WCR

Cardiac allografts were removed from anti-TfR or isotypematched control mAb-treated mice on day 7 post-transplantation. RNA was isolated as previously described.” Briefly, cardiac allografts were homogenized with 1 ml Ultraspec (Chma Bio’Ibx, Houston, TX, USA), and RNA was extracted with 0.200 ml of chloroform for each 1 ml of lysed cells. Whole cell RNA was precipitated with an equal volume of isopropanol. Following precipitation, RNA was.washed with 0.800 ml of 75% ethanol and reprecipitated with 0.200 ml 0.2 M NaCl and 0.400 ml 100% ethanol. RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water. Total RNA concentration was determined by optical density and equal concentrations for each experimental group were confirmed on a 1% agarose gel containing 0.5 clg/ml ethidium bromide. Equal concentrations of undiluted RNA were reverse transcribed into cDNA for the experimental treatment groups as previously described.” The reverse transcription was performed for 10 min at room tem-

Difienmtiol effects of transferrin receptor blockade on the cellular mechanisms involved in graft mjection

perature, 60 min at 42”C, and 10 min at 95°C. The amplification of DNA was performed using cytokine- and receptor-specific primers (Clonetech, Palo Alto, CA, USA) as previously described.” Amplification occurred under the following conditions: 95°C for 1 mm, 60°C for 2 min, and 72°C for 3 min for the indicated number of cycles, followed by a 7-min extension at 72°C. Once the number of cycles had been optimized to be on the linear portion of the curve for each cytokine and receptor, S-actin, IL-lo, and INF-ywere amplified for 30 cycles, IL-6 for 40 cycles, and IL-2 and IL-4 for 45 cycles. The amplified DNA was analysed on a 1% agarose gel containing 0.5 pg/ml ethidiurn bromide and photographed.26 The agarose gel was scanned using Snappy software (Play Inc., Ranch0 Cordova, CA, USA) and quantitated using GelPro Analyzer imaging software (Media Cybernetics, Silver Spring, MD, USA) according to the manufacturer’s instructions. Briefly, the scanned image was analysed with single band analysis. Using the ID-Gel toolbar, the scanned image was rotated, lanes and bands analysed, and data recorded as intensity of optical density (IOD). The mRNA IOD was normalized to the Sactin mRNA IOD and recorded as the normalized numbers. An alteration in the mRNA expression was reported if at least a two-fold change occurred, which was indicative of at least a 50% up-regulation or down-regulation of the RNA.

Results Anti-transferrin receptor mAb prolongs cardiac allograft survival

Previous studies in our laboratory have demonstrated that 100 pg of anti-TfR mAb administered on the day of transplantation (day 0) and on the following day (day 1) significantly prolonged allograft survival to 25.7 + 0.9 days compared to the isotype control (10.7 f 0.4 days).‘s” Since TfR is maximally expressed between 48 and 72 h following T cell activation, it was important to determine if TfR blockade at the time of maximal receptor expression was more effective than TfR blockade on days 0 and 1 during antigen presentation. Anti-TfR mAb at 100 ug on days 2 and 3 post-transplantation failed to prolong allograft survival compared to the isotype controls (13.0 + 0.0 days vs 10.5 f 0.5 days and 10.7 f 0.4 days) (Figure 1A). Therefore, anti-TfR mAb at 100 pg on days 0 and 119was a more effective regimen for prolonging allograft survival than anti-TfR mAb on days 2 and 3 (p < 0.01, Wilcoxon rank sum) (Figure 1A). Since administration of anti-TfR mAb on days 2 and 3 failed to prolong allograft survival, a 4-day course of anti-TfR mAb (100 pg) was administered on days &3. Anti-TfR mAb on days O-3 significantly (p = 0.051) prolonged allograft survival compared to the isotype controls (Figure 1B). However, the 4day course was no more effective than administering anti-TfR mAb on days 0 and 1 (Figure 1B). Since TfR is found on a variety of cell types and tissues, lT3high doses of anti-TfR mAb had the potential to be toxic. Previous results in our laboratory demonstrated no significant changes in body weight or haematocrits following high doses of anti-TfR mAb.” receptor mAb alters cytotoxicity in allograft recipients

Anti-tranafexrIn

To begin addressing the possible mechanisms involved in allograft prolongation, allogeneic CTL responses were determined on day 7 post-transplantation. Day 7 was chosen as the time to examine CTL responsiveness, since the animals were maintainTransplant Immunology 1999; 7: 131-139

133

ing their grafts during the initiation of acute rejection.uY24 Spleens from naive CBA/J or allograft recipients 7 days posttransplantation were removed and stimulated in culture with C57BU6 alloantigen for 7 days in the absence of anti-TfR or isotype control mAbs. A standard 4-h ‘tCr release assay was performed. In vivo treatment with anti-TfR mAb at 100 ug on days 0 and 1 significantly increased the CI’L response to donor-specific alloantigen by 16.8% @ < 0.05) compared to the isotype control (Figure 2A). The primary response of naive CDS+ T cells following administration of anti-TfR mAb was determined by a third-party CTL response. Anti-TfR mAb significantly increased the CTL response to third party alloantigen by 49.3% (p < 0.01) compared to the &type control (Figure 2B). Naive CBA/J splenocytes (from mice not receiving a transplant or antibody treatment) were used as positive controls for CI’L responsiveness. As shown in Figure 2A and B, CTL responsiveness to donor-specific and third party alloantigen following in vivo treatment with anti-TfR mAb reached the level of naive CBA/J splenocytes. Anti-transfenin receptor mAb suppresses T cell proliferation in allograft recipients SinceCD4+ Th cells proliferate extensive? following the recog-

nition of alloantigen and costimulation,‘9’ ’ a mixed lymphocyte response assay was performed to determine the immunosuppressive effects of anti-TfR mAb on proliferating T cells in our allografts recipients. Proliferation was examined in allograft recipients 7 days post-transplantation. Anti-TfR mAb signiflcantly suppressed the MLR to donor-specific alloantigen by 78.7% (p < 0.05) compared to the isotype control (Table 1). The specificity of the allogeneic immune response following administration of anti-TfR mAb was determined by a third party MLR. Anti-TfR mAb significantly suppressed the MLR to third party alloantigen by 80.8% (p c 0.05) compared to the isotype control (Table 1). receptor mAb alters receptors important in T cell activation

Anti-txan&xxIn

Since T cells require antigen-specific and co-stimulatory signals for complete activation, alterations in the expression of receptors important during T cell activation were determined by flow cytometry on day 7 post-transplantation. Splenocytes from antiTfR mAb-treated recipients failed to exhibit alterations in the expression of the T cell marker CD3 or in the T cell activation markers TfR and IL-2R compared to the isotype controls (Figure 3 and Table 2). Similarly, anti-TfR mAb had no effect on the co-stimulatory receptors CD2 or CD28 compared to the isotype controls (Figure 3 and Table 2). In contrast, anti-TfR mAb decreased the number of B7-expressing cells by 30% compared to the &type control (Figure 3 and Table 2). The in vitro environment was examined by flow cytometry in order to clarify the affects of anti-TfR mAb treatment on receptor expression, since the suppression of T cell proliferation in the MLR following anti-TfR mAb treatment suggests that TfR blockade inhibits complete activation of allo-reactive T cells and may inhibit the migration of T cells into the allograft site. CBA/J splenocytes were placed in culture for 7 days with C57BU6 alloantigen in the presence of anti-TfR or isotype control mAbs. Since CD4 and CD8 are important co-receptors involved in helper and cytotoxic T cell functions, respectively, it was important to determine if anti-TfR mAb alters their expression. AntiTfR mAb increased the population of CD4-expressing cells by 35%, and the individual number of CD4 co-receptors (demon-

134

AL Eayer et al.

6. _

Cardiac Allograftsurvival

Donor-SpecificCytotoxicityof Allogmftrecipients

lW’TTr

Naive CBA A Untreated control 0 lsotypecontrol V Anti-TfR n

lsotypecontrol 100 dO,l (n=6) V lsotypecontrol 300 d2,3 (n=4) A Anti-TfR 100 dO,l (n=6) 0 Anti-TfR n

O0

5

10

(A)

15 Days

20

25

30

0

1Oo:i

(A)

3O:l 1O:l Effector:Target ratio

3:l

Third party Cytotoxicityof Allogmftrecipients

60-

S Naive CBA A Untreated control 0 lsotypecontrol V Anti-TfR 0

(6)

4O-

10

20

30

Days

Figure 1 Cardiac allograft survivalin mice receiving anti-TfR mAb. CBA/J (H-2k) recipients received neonatal, donor CWBU6J (H-24 hearts subcutaneously in the ear pinnae. (A) Anti-TfR or isotypematched control mAbs were administered intravenously on days 0 and 1 or on days 2 and 3 at the indicated dose. (B) Anti-TfR or isotypcmatched control mAbs were administered intravenously at the indicated dose and time. n denotes the number of transplant recipients in each experimental group.

strated by an increase in the mean channel fluorescence) compared to the isotype control (Figure 4 and Table 3). In contrast, anti-TfR mAb decreased the population of CDS-expressing cells by S4%, while increasing the individual number of CD8 coreceptors when compared to the isotype control (Figure 4 and Table 3). Anti-TfR mAb had no effect on the activation marker IL-2R, while completely abrogating the population of TfRexpressing cells (Figure 4 and Table 3). To further define the affects of anti-TfR mAb on CD4 and CD8 positive T cells, dualcolour flow cytometry was utilized to determine their activation status. Despite the increase in the number of CDCexpressing cells following anti-TfR mAb treatment, the majority of these cells were inactivated, single positive CD4+ T cells as exhibited by the limited number of CD4+1L-2R+ T cells (50.8% CD4+ vs 8.5% CD4+IL-2R+) (Figure 5). Similarly, the remaining CD8+ cells were inactivated, since CDS+IL-2R+ cells were undetected (Figure S), supporting the hypothesis that anti-TfR mAb inhibits complete T cell activation. Anti-tranderrin receptor mAb alters intragraft cytokfne mFWA expression Since the cytokine micro-environment can play an important role in the development of an effective immune response, intra-

Tmnsplant Immunology 1999; 7: 131-139

20-

0

I

0 (6)

1OO:l

I

-I

3O:i IO:1 Effector:Target ratio

T

3:l

FIgwe 2 Anti-TfR mAb alters alloantigen-specific cytotoxic T lymphocyte responses in al&raft recipients. Naive CBA, or recipients bearing CVBU6 aflografts 7 days post-transplantation were used as a source of responders. Recipients received intravenous injections of anti-Tfp or control mAbs at 100 pg on the day of transplantation and on the following day. Four animals were used in each treatment group. (A) Stimulators were naive donor-specitk C57BU6 splenocytes. (B) Stimulators were naive third-party BALB/c ByJ splenocytes. After 7 days in culture, CTL responses were determined. P values are compared to the isotype-matched controls. Data are representative of at least three complete experiments.

graft cytokine mRNA expression was examined in anti-TfR mAb-treated recipients. On day 7 post-transplantation, intragraft cytokine mRNA was examined b a well-established method of semi-quantitative RTPCR. 199 * As shown in Figure 6, anti-TfR mAb suppressed the IL-15 mRNA by 70% compared to the isotype control, while having no effect on INF-y mRNA expression (Figure 6A, B). There were no detectable levels of IL-2 mRNA expression within the allografts (Figure 6A, B). In contrast, anti-TfR mAb increased the Th2 cytokine IL-4 mRNA expression by 93.0% compared to the isotype control (Figure 6A, B). Anti-TfR mAb had an immunosuppressive effect on IL-10 mRNA expression, however, the suppression was less than 50% when compared to the isotype control (Figure

Difierentiol effects of transfertin receptor blockade on the cellular mechanisms involved in grajt rejection

6A, B). Anti-TfR mAb failed to alter the ARC cytokines IL-6, IL-12 ~35, and IL-12 ~4.0 mRNA expression (Figure 6A, B).

Discussion Previous work in our laboratory has demonstrated that anti-TfR mAb administered at 100 pg on days 0 and 1 of transplantation prolonged cardiac allograft survival in our murine mode1.‘8~‘9 Even though maximal expression of TfR following T cell activation occurs between 48 and 72 h after stimulation, anti-TfR mAb administered on days 2 and 3 failed to prolong allograft survival when compared to anti-TfR mAb on days 0 and 1 or the isotype controls. An extended, 4-day course of anti-TfR mAb during the time of antigen presentation and maximal TfR expression was no more effective than a 2-day course. of the mAb. Therefore, anti-TfR mAb administered during the time of antigen presentation was the most effective regimen for prolonging allograft survival in our model. Since the mAb used in these studies does not interfere with iron transport in proliferating T cells,” the effects of anti-TfR mAb on allograft survival were the result of alterations in immune function rather than the lack of sufficient iron availability. TfR blockade resulted in marginal allograft prolongation, therefore, a more effective immunosuppressive regimen may be achieved by combining anti-TfR mAb with other immunosuppressants that interfere with T cell activation and co-stimulation. Since the expression of TfR follows the appearance of IL-2R, this receptor is a potential target for immunosuppression. Future studies will assess the potency of simultaneous blockade of TfR and IL-2R for allograft survival. One of the mechanisms involved in allograft survival following anti-TfR mAb treatment may be the lack of an effective immune response. Our MLR studies revealed a significant suppression to donor-specific and third party alloantigen following anti-TfR mAb treatment in the allograft recipients, while sup-

CD3

135

Table 1 Mixed lymphocyte response Stimulators (cpm) Allograft recipients

Donor-specific

Third party

Naive CBA

14102 * 13767 + 15025 f 3198 f

24491 + 3217 22866 + 1831 19698 &lo18 3783 f 623*

Untreated control Isotypecontrol Anti-TfR

1456 933 2009 215*

Cardiac recipientsreceivedintravenousinjectionsof 100 ug of anti-TfB or control mAbs on the day of transplantationand on the followingday. Splenocytesfrom naive CBA or recipientsbearingC57BL/6 allografts7 dayspost-transplantationwere stimulatedwithdonor-specificC57BL/6 or third-partyBALB/c alloantigen.proliferationwas determinedon day 5 followingstimulation.Results are expressedas a mean 2 SEM. Data are representativeof at least three complete experiments. *p < 0.05, Student’stwo-tailedr-test.

porting our previous in vitro studies, which demonstrated

that anti-TfR mAb suppresses proliferating T cells to alloantigen and that TfR plays a pivotal role in T cell activation and proliferation. lEJ9 CDS+ Cl% are one of the primary cells reslpnsible for acute Previous reports allograft rejection following transplantation. in our laboratory have revealed that in vitro blockade of TfR completely abrogates the CTL response to alloantigen,18Y’9indirectly suggesting that CTL suppression may be involved in allograft prolongation. In our allograft recipients, anti-TfR mAb unexpectedly failed to suppress the CTL response to donor-specific or third party alloantigen upon in vitro restimulation, while demonstrating alterations in T cell activation (flow cytometry) and suppression in proliferation (MLR) following anti-TfR mAb treatment. These data suggest that the increase in CD., responsiveness following anti-TfR mAb treatment may be due to a failure in T cell activation and subsequent recruitment of CTLs to the site of alloantigen rather than to an actual increase in CTL function, thus leading to a strong CTL response on in vitro res-

IL12R

Figure 3 Anti-TfRmAb decreasescell surfaceexpressionof the costimulatoryligandsB7 in our allograftrecipients.Flow cytomettywas performed on splenocytesfrom recipientsbearing C57BU6 allograftson day 7 post-transplantation.Recipientsreceived intravenousinjectionsof anti-TfRor isotypecontrol mAbs at 100 ug on the day of transplantationand on the followingday. Thin line = background,dotted tine = isotypecontrol mAb, and thick line = anti-TfRmAb. Four animalswere used in each treatment group. Data are representativeof least three complete experiments.

Transplant Immunology 1999; 7: 131-139

136 AL Bayer et al.

‘able 2 Cell surface expression of T cell markers on day 7 post-transplantation Receptor In vivo treatment

CD3

TfR

IL-2R

CD2

CD28

B7

Isotype control Anti-TfR

51.9 (3.82) 49.0 (3.48)

9.7 (6.19) 10.7 (6.82)

11.7 (2.45) 12.0 (2.39)

41.0 (0.88) 32.1 (0.83)

7.5 (1.25) 6.1 (1.23)

29.5 (2.65) 20.6 (2.47)

Animals were treated with either anti-TfR or isotype control mAbs. Flow cytometry was performed on day 7 post-transplantation. Values represent total percent positive staining above background for each of the cell surface markers. Data are representative of at least three experiments. Numbers in parentheses are mean channel fluorescence for each group.

Figure 4 Anti-TfR mAb alters the expression of cell surface receptors important during T cell activation. Flow cytometry was performed on CBA/J splenocytes cultured for 7 days with C57BU6 alloantigen in the presence of anti-TfR or isotypc control mAbs at 5 ug/ml. Thin line = background, dotted line = isotype control mAb, and thick line = anti-TfR mAb. Four animals were used in each treatment group. Data are a representative of at least three complete experiments.

-cDI-

CD8

Eirpve 5 CD4 and CD8 expressing T cells remain inactive following in v&v treatment with anti-TfR mAb. Flow cytometry was performed on CBA/J splenocytes cultured for 7 days with C57BU6 alloantigen in the presence of anti-TfR or isotype contml mAbs. Values represent total percent positive staining minus background staining for each of the cell surface markers CD4, CD8, and IL-2R.

Transplant Immunology 1999; 7: 131-139

Differential effects of transfertin receptor blockade on the cellular mechanisms inuolued in graft rejection

137

I helper 2 MW IC aTtR

T helper 1 MW IC aTtR

IL-12p35

Thelper 1 1000

Thelper 2

L 11c 1k 0.600

0.750 0.100

0.400

0.500 i

0.050

IL?

0.000 IL-2

0.400 0.300

0.200 0.100 0.000 i

u INF-1

IL-6

0.500

0.600

0.200

0.400

0.000

0.600

0.000

i

0.300~

0.100.

IL-15

0.200

IL4

0.400

(W

0.800

0.150

0.200

1

o.ooo

IL-10

m

lsolypecontrol

0

An&TtR

IL-12p40

6 Anti-TfR rdb alters intragraft cytokine mRNA expression. Cardiac al&raft recipients received intravenous injections of anti-TfR or control mAbs at 100 mg on the day of transplantation and on the following day. Ahografts from anti-TfR or isotype-matched control mAb_treated mice were harvested on day 7 post-transplantation and RNA isolated. Equal concentrations of RNA were determined by optical density and continned by gel electrophoresis. RIP-PCRwas performed. (A) Amplified DNA was am@sed on a 1% agarose gel comaining 0.5 &nl ethidium bromide and photographed. Lane 1 s molecular weight markers, lane 2 = isotype-matched control mAb, lane 3 = anti-TfR mAb. Three cardiac allografts were used in each treatment group. (B) Semi-quantitation of the cytokine mRNA expression. The cytokine mRNA IOD was normalized to the g-a&in mRNA IOD and recorded as the normalized IOD. Mm

Tmnsplant Immunology 1999; 7: 131-139

138

AL Bayer et al.

‘able 3

Cell surface expression of T cell markers

Receptor In vitm treatment

CD4

CD8

TfR

IL-2R

Isotype control Anti-TfR

38.9 (7.5) 59.0 (9.3)

39.9 (8.9) 18.4 (12.7)

29.2 (8.2) 0 (2.9)

12.0 (2.0) 19.7 (2.9)

Flow cytometrywas performed on day 7 after in vitro treatment with anti-TfRor isotypecontrol mAbs at 5 pg/ml.values representtotal percentage positivestainingabove backgroundfor each of the cell surface markers.Data are representativeof at Ieast three experiments.Numbers in parenthesesare mean channel ftuorescence for each group.

timulation with alloantigen compared to the control allograft recipients. To begin to further understand the immunosuppressive effects of anti-TfR mAb, important receptors involved in T cell activation were examined. In our transplant recipients, anti-TfR mAb had no effect on the T cell marker CD3, the T cell activation markers IL-ZR and TfR, or the costimulatory receptors CD2 and CD28. However, anti-TfR mAb suppressed the expression of the B7 costimulatory ligands, suggesting inadequate costimulation following TfR blockade. Thompson ei al. have suggested that limited B7 availability results in a competitive blockade of CD28 binding by CTLA4, thereby producing inhibitory signals that can prevent T cell activation by interfering with costimulatory signals generated through the CD28 and TCR.” Suppressed proliferation (MLR) and B7 expression in our allograft recipients following treatment with anti-TfR mAb support this hypothesis, since the examination of splenic receptor expression in our allograft recipients following anti-TfR mAb treatment was in comparison to non-responsive isotype control splenocytes. Therefore, receptor expression was examined in a more controlled in vitro environment. In vitro administration of anti-TfR mAb strongly influences the population of CD4+ and CD@ T cells by increasing the CD4 and decreasing the CD8 expressing cells, which may explain why no alterations in the number of CD3+ T cells were observed in our anti-TfR mAbtreated allograft recipients. Despite these changes in the CD4 and CD8 populations, the cells remained inactivated, indirectly supporting that anti-TfR mAb prohibits complete T cell activation and subsequent T cell recruitment, which may be contributing to the allograft prolongation and suppression of T cell proliferation. These effects on T cells demonstrate an important role for TfR in the generation of cell-mediated immune responses. Since the cytokine micro-environment can influence the development of an effective immune response, intragraft cytokine mRNA expression was examined in cardiac allograft recipients receiving anti-TfR mAb 7 days post-transplantation by a well established method of semi-quantitative RTPCR.‘g928 Several researchers have suggested that Th2 cytokines are associated with graft survival. CILA4Ig in a rat renal allograft model inhibited Thl and increased Th2 cytokine expression.30 Murine cardiac allografts considered tolerant expressed lower levels of IL-2 and INF-y mRNA and higher levels of IL-4 and IL-10 mRNA.31 In our studies, anti-TfR mAb demonstrated a decrease in IL-15 mRNA, while increasing the Th2 cytokine IL4 intragraft mRNA expression. IL-2 mRNA expression was undetectable in all the treatment groups. Since the stimulatory cytokine IL-15 mRNA was expressed, IL-15 appears to be a late acting cytokine, unlike IL-2 expression, which occurs within the first 24 h after activation, thereby suggesting the window of Tmnsplant Immunology

1999; 7: 131-139

detection had been missed. Despite the increase in IL-4 and the decrease in IL-15 intragraft mRNA expression, allograft prolongation was moderate and may be associated with the strong presence of INFy mRNA expression. The presence of exogenous IL-12 during a primary T cell stimulus leads to a predominant Thl phenotype. Hence, intragraft IL-12 ~35 and p40 mRNA expression were not altered by anti-TfR mAb and may account for the presence of INF-y mRNA expression. Even though IL-4 mRNA was strongly enhanced following anti-TfR mAb treatment, the presence of INF-y mRNA expression suggests that cell-mediated immune responses are still ongoing, resulting in the lack of long-term allograft survival and further supporting the need of combining anti-TfR mAb with other immunosuppressants that interfere with T cell activation and costimulation. These studies are the first to explore the T cell responsiveness of allograft recipients following anti-TfR mAb administration. Our data confirm previous reports that TfR is important in T cell activation, independent of its role in iron transport. TfR blockade at the time of antigen presentation was more effective than at the time of maximal receptor expression, suggesting an early role for TfR in T cell responsiveness. Furthermore, antiTfR mAb suppressed T cell proliferation at the time of antigen presentation, while down-regulating IL-15 and up-regulating IL-4 intragraft mRNA expression. Hence, anti-TfR mAb may be an effective immunosuppressant in prolonging allograft survival by shifting the Th cytokine profile and altering the T cell responses to alloantigen. Since anti-TfR mAb moderately prolongs allograft survival, this mAb in combination with other immunosuppressant may provide a more effective regimen for achieving long-term allograft survival. Future studies will improve our understanding of TfR in T cell activation and will provide the knowledge necessary for utilizing TfR blockade as a novel therapeutic strategy for clinical transplantation.

References 1 May WS, Jr., CuatrecasasI? ‘Bansferrinreceptor: its biologicalsignificance.JMembr Bioll985; 88: 205-15. 2 Huebers HA, Finch CA The physiologyof transferrinand transferrin receptors. Physiol Rev 1987; 67: 520-82. 3 NeckersLM. Regulationof transferrinreceptor expressionand control of cell growth.Pathobiology 1991; 59: 11-18. 4 PattanapanyasatK, Hoy TG. Expressionof cell surface transferrin receptor and intracxlhrlarferritinafter invitro stimulationof peripheral blood T lymphocytes.Eur J Haematoll991; 47: 14&45. 5 GalbraithRM, WernerP,Amaud P, GalbraithGM. ‘Ransferrinbmding to peripheralblood lymphocytesactivated by phytohemagglutinin involvesa specificreceptor. Ligand interaction J Clin Invest 1980; 66: 1135-43. 6 MarrackP,KapplerJ. The T cellreceptor.Science1987;23% 1073-79. 7 Bierer BE, Burakoff ST. T-lymphocyteactivation: the biology and functionof CD2 and CD4. ImmtuwlRev 1989; 111: 267-94. 8 Mueller DL, Jenkins MK, SchwartzRH. Clonal expansionversus functional clonal inactivation: a costimulatorysignallingpathway determinesthe outcome of T cell antigenreceptor occupancy.dnnu Rev Immud 1989; 7: 445-80. 9 WeissA. Structureand functionof the T cell antigenreceptor.JClin Invest1990,86: 1015-22. 10 Weiss A, I&man DR. Signaltransductionby lymphocyteantigen receptors. Cell 1994; 76: 263-74. 11 Ledbetter JA, ImbodenJB, S&even GL et al. CD28 ligationin T cell activation:evidencefor two signaltransductionpathways.Blood 1990: 75: 1531-39.

Differential

efjects of transfertin

receptorblockadeon the cellular mechanisms involved in graft rejection

12 Teixeira S, Kuhn LC. Post-transcriptional regulation of the transferrin receptor and 4F2 antigen heavy chain mRNA during growth activation of spleen cells Eur J Biochem 1991; Un: 819-26. 13 MosmarmTR, Cherwinski H, Bond Mw, Giedlin MA, Coffman RL. ‘Bvo types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136: 2348-57. 14 Romagnani S. The Thlm paradigm. 2mnuuwl Todny 1997; 18: 263-66. 15 Halloran PF, Broski Ap Batiuk TD, Madrenas J. The molecular immunology of acute rejection: an overview. Tramp1Immunoll993; 1: 3-27. 16 Sauvage CA, Mendelsohn JC, Lesley JF, ‘Bowbridge IS. Effects of monoclonal antibodies that block transfetrin receptor function on the in vivo growth of a syngeneic murine leukemia. CancerRes 1987; 47: 747-53. 17 Kudoh S, Stanley J, Edinger MG et al. T lymphocytes iniiltrating renal cell carcinoma have a reduced expression of transferrin receptor. Int J Cancer 1994; 58: 36%75. 18 Woodward JE, Bayer AL, Chavin KD, Boleza KA, Baliga I! Antitransferrin receptor monoclonal antibody: a novel immunosuppressant. Pansplantation 1998; 65: 6-9. 19 Bayer AL, Baliga P, Woodward JE. ‘Bansferrin receptor in T cell activation and transplantation. JLeulcocyte Bill 1998; 64: 19-24. 20 Lesley JF, Schulte RJ. Inhibition of cell growth by monoclonal antitransferrin receptor antibodies Mol Cell Bioll985; 5: 1814-21. 21 Aihara Y, Buhring HJ, Aihara M, Klein J. An attempt to produce ‘pre-T’ cell hybridomas and to identity their antigens. Eur J Immunol1986; 16: 1391-99. 22 Judd Kp, Trentin JJ. Cardiac transplantation in mice. I. Factors intluencing the take and survival of heterotopic grafts. Transplantation 1971; 11: 298-302.

Tmnsplant Immunology

1999; 7: 131-139

139

23 Carlquist JF, Edelman LS, White W, Shelby J, Anderson JL. Cytokines and rejection of mouse cardiac allografts. Transplantation 1996; 62: 1160-66. 24 Larsen CP, Elwood ET, Alexander DZ et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 1996; 381: 434-38. 25 Finberg R, Burakoff ST, Benacerraf B, Greene MI. The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. II. Evidence for antigen-specific suppressor T cells.J Zmmunol 1979; 123: 1210-14. 26 Arrigo SJ, Weitsman S, Rosenblatt JD, Chen IS. Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method. J Vhl1989; 63: 4875-81. 27 Baliga P, Chavin KD, Qin L et al. CTLA4Ig prolongs allograft survival while suppressing cell-mediated immunity. Tmnsplantation 1994,58: 1082-90. 28 Woodward JE, Bayer AL, Chavin KD, Blue ML, Baliga P. T-cell alterations in cardiac allograft recipients after B7 (CD80 and CD86) blockade. Tmnsphntation 1998; 66: 14-20. 29 Thompson CB, Allison JP The emerging role of CI’LA-4 as an immune attenuator. immunity 1997; 7: 445-50. 30 Sayegh MH, Akalin E, Hancock WW et al. CD28-B7 blockade after alloantigenic challenge in vivo inhibits Thl cytokines but spares Th2. J Exp Med 1995; 181: 1869-74. 31 Takeuchi T, Lowry Rp, Koniecxny B. Heart allografts in murine systems. The differential activation of Th2-like effector cells in peripheral tolerance. Tmnsplantation 1992; 53: 1281-94.