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The Journal of Immunology, 2004, 172: 5456-5466.
Copyright © 2004 by The American Association of Immunologists

In Vivo Helper Functions of Alloreactive Memory CD4+ T Cells Remain Intact Despite Donor-Specific Transfusion and Anti-CD40 Ligand Therapy1

Yifa Chen*, Peter S. Heeger*,{dagger},{ddagger} and Anna Valujskikh2,*

* Department of Immunology and {dagger} Glickman Urologic Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; and {ddagger} Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Memory T cells have specific properties that are beneficial for rapid and efficient protection from pathogens previously encountered by a host. These same features of memory T cells may be deleterious in the context of a transplanted organ. Consistent with this contention is the accumulating evidence in experimental transplantation that previously sensitized animals are resistant to the effects of costimulatory blockade. Using a model of murine cardiac transplantation, we now demonstrate that alloreactive memory CD4+ T cells prevent long-term allograft survival induced through donor-specific cell transfusion in combination with anti-CD40 ligand Ab (DST/anti-CD40L). We show that memory donor-reactive CD4+ T cells responding through the direct or indirect pathways of allorecognition provide help for the induction of antidonor CD8+ T effector cells and for Ab isotype switching, despite DST/anti-CD40L. The induced pathogenic antidonor immunity functions in multiple ways to subsequently mediate graft destruction. Our findings show that the varied functions of alloreactive memory CD4+ T cells remain intact despite DST/anti-CD40L-based costimulatory blockade, a finding that will likely have important implications for designing approaches to induce tolerance in human transplant recipients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Memory T cells exhibit specific properties that permit rapid and effective control of infectious agents previously encountered by the host. These features, which include stable differentiated cytokine profiles, relatively low activation threshold and costimulatory requirements compared with naive T cells, the ability to rapidly engage effector functions, and the ability to migrate to peripheral tissues, are essential for host defense (1, 2, 3). However, in transplantation, the same properties are likely to be deleterious rather than beneficial. It is well known that animals that rejected one allograft reject a second graft from the same donor with accelerated kinetics (second set rejection) (4, 5). Similarly, human recipients of second or third transplants, following initial graft loss due to rejection, have poorer graft survival than recipients of first transplants (6).

In addition, the immune repertoire of many humans who have not been given transplanted organs contains alloreactive memory T cells (7, 8, 9). These cells were presumably primed through exposure to alloantigens via pregnancy or blood transfusions, or by exposure to an infection/immunization that happens to induce a cross-reactive response to alloantigens. Such memory T cells may have clinical relevance, as higher frequencies of alloreactive T cells before transplantation correlate with an increased risk of acute rejection posttransplantation (8, 9, 10, 11).

Induction of immunologic tolerance to an allograft is the ultimate goal of transplantation. Blockade of T cell costimulation is a well-studied and promising approach for achieving long-term graft survival and can lead to donor-specific tolerance (12, 13, 14, 15). The precise mechanisms by which costimulatory blockade causes donor-specific unresponsiveness are still only partially understood, but there is experimental support for T cell clonal deletion, anergy, cytokine deviation, and the induction of regulatory T cells, depending on the therapy used and model studied (12, 14, 16, 17, 18). Costimulatory blockade-based tolerance-inducing protocols are generally effective in naive animals with naive T cell repertoires. However, as memory T cells have lower activation thresholds and costimulatory requirements than naive T cells, memory T cells are likely to be resistant to the effects of costimulatory blockade (1, 2). Consistent with this hypothesis, memory T cells specific for OVA were shown to be able to mediate multiple effector functions despite costimulatory blockade (19).

There is accumulating evidence in transplantation models that previously sensitized animals are resistant to the effects of costimulatory blockade. Kupiec-Weglinski and colleagues (20) demonstrated that anti-CD40 ligand (CD40L)3 Ab treatment was ineffective in prolonging cardiac allograft survival in mice acutely sensitized with skin allograft from the same donor. Rossini and colleagues reported similar observations in another model of CTLA4-Ig-resistant transplant rejection (21, 22). These latter studies also showed that while naive donor-reactive CD8+ T cells underwent deletion in the context of costimulatory blockade, the presensitized CD8+ T cells did not. Others, as well as our group, have also reported that memory/effector CD8+ T cells, induced either by previous exposure to donor Ags or by cross-reactive antiviral immunity, prevented the beneficial effect of costimulatory blockade on prolonging heart allograft survival (23, 24, 25).

The effects of alloantigen-reactive memory CD4+ T cells on long-term graft survival induced through costimulatory blockade remain less clearly understood. Using murine models of heart and skin allotransplantation, our laboratory has previously shown that primed donor-reactive CD4+ T cells prevented long-term graft survival normally achieved in naive mice via donor-specific cell transfusion and anti-CD40L Ab (DST/anti-CD40L) (23, 26). The mechanisms of graft destruction under these conditions are not known, and we propose two nonmutually exclusive possibilities to account for the findings. First, it is possible that the memory CD4+ T cells, being resistant to the effects of costimulatory blockade, respond to the allograft by expanding and differentiating into CD4+ effector T cells that subsequently mediate graft injury. Alternatively or in addition, the memory CD4+ T cells could influence the remainder of the recipient’s immune repertoire, thereby preventing the tolerogenic effects of the therapy on naive alloreactive T cells. We performed a series of experiments to address these questions, and we now provide new mechanistic insight into how memory CD4+ T cells induce rejection despite costimulatory blockade. Our data indicate that memory CD4+ T cells responding through the direct or the indirect pathway of allorecognition are not only resistant to the tolerogenic effects of the therapy themselves, but also provide help for induction of donor-specific CD8+ and B cell immunity that in turn contribute to graft rejection. The information gained from this work will have important therapeutic implications for human transplant recipients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male and female C57BL/6 (H-2b), congenic B6.PL-Thy-1a (H-2b), C3H (H-2k), and BALB/c (H-2d) mice, aged 6–8 wk, were purchased from The Jackson Laboratory (Bar Harbor, ME). Male and female C57BL/10NA;- (Tg)TCR Marilyn-(KO) Rag2 N11, N2 mice (H-2b, Marilyn (Mar)), aged 6–8 wk, were obtained as a generous gift from P. Matzinger (National Institutes of Health) and O. Lantz (Institut National de la Santé et de la Recherche Médicale). All animals were maintained and bred in the pathogen-free animal facility at the Cleveland Clinic Foundation.

Peptide

HYDby peptide (NAGFNSNRANSSRSS) was synthesized by Research Genetics (Huntsville, AL) at >90% purity.

Placement and evaluation of cardiac allografts

Vascularized heterotopic cardiac allografts were placed in the abdomen using standard techniques (15) and palpated daily for evidence of a heartbeat. Rejection was defined as a loss of palpable heartbeat. All grafts were harvested at the time of rejection or at predetermined time points, embedded in paraffin, stained with H&E, and examined by microscopy. A portion of each graft was frozen in OCT compound (Sakura Finetek USA, Torrance, CA) for further immunohistochemical staining. Frozen sections of cardiac tissue were acetone fixed, hydrated in PBS for 10 min, blocked with the Biotin Blocking System (DAKO, Carpinteria, CA), washed with PBS three times, and incubated for 60 min at room temperature with biotinylated anti-CD4 or anti-CD8 (BD PharMingen, San Diego, CA; 1/50 dilution in PBS-1% BSA). After three additional PBS washes, the sections were incubated with peroxidase-conjugated streptavidin (stock concentration; DAKO) and developed using the Novared Substrate Kit (Vector Laboratories, Burlingame, CA). Sections were dehydrated with ethanol and mounted for analysis.

Costimulatory blockade

DST and costimulatory blockade were performed, as described by Hancock et al. (15). A total of 10 x 106 donor spleen cells plus 0.4 mg of anti-CD40L Ab MR1 (BioExpress, West Lebanon, NH) was administered i.v. 1 day before the heart graft placement.

T cell subset isolation

Subsets of CD4+ and CD8+ T cells were isolated using commercially available murine T cell isolation columns from R&D Systems (Minneapolis, MN), following the instructions supplied by the manufacturer. In particular, naive CD4+ T cells were purified from the spleens of the naive B6 mice using CD4+ T cell enrichment columns (>94% pure by flow cytometry; data not shown); memory CD4+CD44highCD62low T cells were purified from the spleens of the B6 recipients that rejected C3H skin allografts 6 wk after rejection using CD4+CD62L–CD44high memory T cell enrichment columns (>65% enriched by flow cytometry; data not shown); and CD8+ T cells were isolated from the spleens of the experimental animals using CD8+ T cell enrichment columns (>92% pure by flow cytometry; data not shown). Resultant cells were washed in HBSS, assessed for viability by trypan blue exclusion, and resuspended at appropriate concentrations for use in the various assays and for adoptive transfers.

Flow cytometry

PE-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD25, FITC-conjugated anti-mouse CD44, FITC-conjugated anti-mouse CD62L, and FITC-conjugated anti-mouse CD69 Abs were purchased from BD PharMingen. Spleen cells from the heart graft recipients or naive mice were labeled on ice for 30 min with appropriate Ab, followed by three washes in PBS. The labeled cells were fixed with 1% paraformaldehyde and analyzed on a BD Biosciences (Bedford, MA) FACScan using CellQuest software.

Adoptive transfer experiments

Naive B6 female mice were adoptively transferred with 5 x 106 naive or memory CD4+ T cells via tail vein injection. Two to three days later, animals were treated with DST plus anti-CD40L Ab or left untreated and transplanted with C3H male cardiac allografts. In other experiments, naive B6 females were adoptively transferred with 5 x 106 naive or in vitro primed (4 days in culture with 2 µM HYDby peptide) Mar T cells. Animals were rested for 4 wk, and then given costimulatory blockade treatment and C3H male heart grafts.

ELISPOT assay

Assays were performed, as outlined previously in detail (23). Briefly, ELISPOT plates (Millipore, Bedford, MA) were coated overnight with the anti-IFN-{gamma} capture Ab (BD PharMingen) in sterile PBS, blocked with sterile 1% BSA in PBS, and washed three times with sterile PBS. Spleen cells (0.05–0.4 x 106 per well) or purified CD8+ T cells (0.025–0.2 x 106 per well) were plated in HL-1 medium (BioWhittaker, Walkersville, MD) with or without mitomycin C-treated C3H or control BALB/c stimulator cells (0.4 x 106 per well) and then incubated at 37°C, 5% CO2 for 24 h (or for other defined time periods, as outlined). In some experiments, anti-CD8 Ab YTS169 (BioExpress) was added to the selected wells at final concentration 50 µg/ml. After washing with PBS, followed by PBS/0.025% Tween (PBST), biotinylated anti-IFN-{gamma} detection Ab (BD PharMingen) was added overnight. After washing with PBST, alkaline phosphatase-conjugated anti-biotin Ab (Vector Laboratories) diluted 1/2000 in PBST was added for 2 h at room temperature. The plates were developed, as previously described. The resulting spots were analyzed using an ImmunoSpot Series 1 Analyzer (Cellular Technology, Cleveland, OH).

In vivo CTL assay

Naive B6 (control) and C3H (experimental) spleen cells were isolated, and RBC were removed by osmotic lysis. The control B6 cells were labeled with a high concentration of CFSE (6.25 µM) (CFSEhigh cells), and the C3H cells were labeled with low concentration of CFSE (1 µM) (CFSElow cells). Labeled B6 and C3H cells were mixed at 1:1 ratio and injected into the tail vein (10 x 106 cells in 400 ml of PBS per mouse) of experimental animals. Concurrently, target cells were injected into naive B6 females as controls. Twelve hours later, the animals were sacrificed and the spleen cells were analyzed by flow cytometry, collecting up to a total of 500,000 events per sample. Transferred cell populations were detected by differential CFSE fluorescence intensities. A ratio of the number of CFSElow cells to the total number of CFSElow plus CFSEhigh cells was calculated for each animal. Specific lysis was calculated as ((ratio in naive B6 mice – ratio in experimental mouse)/(ratio in naive mice)) x 100.

Ab detection by flow cytometry

Serum samples were collected from the experimental animals. Donor C3H or third-party BALB/c thymocytes were isolated, and live cells were counted by trypan blue exclusion and divided into aliquots (1 x 106 cells per sample). Cells were pelleted by centrifugation and resuspended in 100 µl of diluted serum (serial dilutions were made from 1/10 through 1/2430 in PBS/5% FCS/0.02% NaN3), followed by 1-h incubation on ice and three washes with PBS/5% FCS/0.02% NaN3. Detecting FITC-conjugated goat anti-mouse IgG Ab (BD PharMingen) was diluted 1/100 in PBS/5% goat serum/0.02% NaN3 and added to the pelleted cells (100 µl per sample). Cells were incubated 1 h on ice in the dark, washed three times, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry.

CD8+ T cell depletion

Two groups of B6 female mice were adoptively transferred with 5 x 106 memory CD4+ T cells, followed by C3H DST/anti-CD40L treatment and placement of C3H male cardiac allografts. One group was given a mixture of anti-CD8-depleting mAbs YTS169.4 (rat IgG2b) and TIB105 (rat IgG2b) purchased from BioExpress (0.2 mg of each/mouse/day on days –3, –2, and –1 with relevance to the graft placement, and then 0.2 mg of each/mouse every 5 days); the control group was treated with rat IgG (Southern Biotechnology Associates, Birmingham, AL).

Statistical analysis

Statistical analysis to determine differences between groups for recall immune responses was performed using the Student’s t test for equal or unequal variances. A value of p < 0.05 was considered statistically significant. Kaplan Meier survival analysis was performed to determine the difference in median graft survival between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antidonor memory CD4+ T cells prevent DST/anti-CD40L-induced graft prolongation

We previously reported that adoptive transfer of splenic effector/memory CD4+ T cells from mice sensitized with skin allografts into naive recipients of cardiac allograft from the same donor specifically interfered with our ability to achieve long-term heart graft survival (23). To test whether memory CD4+ T cells, as opposed to effector T cells, were the mediators of costimulatory blockade resistance, we generated B6 anti-C3H memory T cells in vivo by placing C3H male skin grafts onto B6 female recipients. Six weeks after rejection (8 wk posttransplant), splenic CD4+CD44highCD62Llow T cells were isolated by negative selection and analyzed by flow cytometry. The resulting population was enriched for CD44highCD62LlowCD4+ T cells (>65%), but did not express markers of ongoing T cell activation (CD25lowCD69low), consistent with memory phenotype (Fig. 1A). Importantly, unlike naive cells, the memory CD4+ T cells were capable of rapidly secreting the effector cytokine, IFN-{gamma}, as early as 4 h after restimulation with C3H alloantigens. The memory CD4+ T cells also secreted more IFN-{gamma} per cell compared with naive cells, as determined by the average ELISPOT size (Fig. 1B).



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  		FIGURE 1. Memory CD4+ T cells precipitate cardiac allograft rejection despite DST/anti-CD40L treatment. A, Surface phenotype of naive and memory CD4+ T cells used in adoptive transfer experiments. Naive CD4+ T cells were isolated from the spleens of naive B6 female mice, and memory CD4+ T cells were enriched from the spleens of B6 recipients of C3H male skin allografts 6 wk after rejection, and analyzed by flow cytometry for expression of activation markers. The histograms were obtained after gating on CD4+ cells for each isolated population. The numbers next to each histogram show the percentage of positive cells and the mean fluorescence intensity. The results are representative of two individual experiments. B, Kinetics of IFN-{gamma} production by naive and memory CD4+ T cells after in vitro stimulation. Purified naive CD4+ T cells and enriched memory CD4+ T cells were tested in a recall IFN-{gamma} ELISPOT assay against donor C3H stimulator cells. The assay plates were incubated for 4, 8, 18, and 26 h, and IFN-{gamma} spots were further developed, as described in Materials and Methods. The average spot sizes were calculated using ImmunoSpot Series 1 Analyzer software (Cellular Technology, Cleveland, OH). The experiment was performed twice with similar results. C, Cardiac allograft survival. B6 female mice were adoptively transferred with 5 x 106 naive ({circ}) or 5 x 106 memory (•) CD4+ T cells, treated with C3H DST/anti-CD40L, and transplanted with C3H male cardiac allografts. Control naive B6 recipients received no cell transfers and no treatment before placement of C3H male heart grafts (triangles). n = 3–5 per group. *, p < 0.05 vs mice transferred with naive CD4+ T cells by Kaplan Meier survival analysis. Transfer of memory CD4+ T cells primed to third-party SJL alloantigens did not interfere with long-term graft survival induced through C3H DST/anti-CD40L (MST >60 days; n = 6; data not shown).

 
Five million enriched memory CD44highCD62LlowCD4+ T cells were transferred into naive B6 female recipients via tail vein injection. Control animals were adoptively transferred with 5 x 106 naive CD4+ T cells isolated from the spleens of naive B6 female mice (CD25lowCD44lowCD62LhighCD69low; Fig. 1A). Both groups received DST/anti-CD40L treatment, followed by placement of C3H male cardiac transplants. Consistent with previous reports (12, 15, 23), DST/anti-CD40L resulted in significantly prolonged heart allograft survival in the animals transferred with naive CD4+ T cells (three of four grafts survived longer than 70 days; Fig. 1C). In stark contrast, animals containing donor-reactive memory CD4+ T cells all rejected their heart grafts by day 13 (median survival time (MST) of 9.6 ± 0.9 days; Fig. 1C) despite the DST/anti-CD40L therapy. The kinetics of the rejection in this latter group was similar to the rejection of C3H male heart allografts by naive nontreated B6 female recipients (MST of 7.0 ± 0.3 days; Fig. 1C). These results corroborate our previous findings and indicate that enriched memory CD4+ T cells are capable of inducing cardiac allograft rejection even under conditions of costimulatory blockade.

Memory CD4+ T cells provide help for induction of donor-reactive CTLs despite costimulatory blockade

To assess the effects of memory CD4+ T cells on donor-reactive immunity in the context of costimulatory blockade, we next performed in vivo cytotoxicity assays on day 7 posttransplant. In this approach, the ability to mediate cytotoxicity directly in vivo is determined by transferring specifically labeled allogeneic target cells along with a control syngeneic target population into the allograft primed or naive control recipients, and then determining the relative number of specific target and control cells detectable in the mice by flow cytometry at a later time point. Syngeneic B6 control target cells or specific C3H target cells are labeled with different concentrations of CFSE so that they can be differentiated by flow cytometry (a 5-fold concentration difference permits distinguishing between the two populations). If performed simultaneously in experimental and naive animals, one can calculate the percentage of killing in vivo based on relative numbers of detectable target cells in each recipient. Comparative side-by-side experiments showed that in vivo CTL activity detected by analysis of spleen cells is fully reflective of CTL activity in peripheral lymph nodes and peripheral blood (data not shown). As shown in Fig. 2A, animals transferred with naive CD4+ T cells and treated with DST/anti-CD40L exhibited minimal in vivo killing (21.5 ± 3.5% specific lysis; Fig. 2A), and the results did not significantly differ from that found in naive nontransplanted B6 females. In contrast, animals transferred with memory CD4+ T cells efficiently killed C3H target cells with or without DST/anti-CD40L treatment (95.0 ± 1.4% and 91.0 ± 9.0% specific lysis respectively; Fig. 2A).



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  		FIGURE 2. Memory, but not naive CD4+ T cells provide help for the induction of antidonor CD8+ T effector cells despite costimulatory blockade. Groups of B6 recipients of C3H cardiac allografts (transferred with naive CD4+ T cells and given DST/anti-CD40L, or transferred with memory CD4+ T cells with or without DST/anti-CD40L) were sacrificed, and recall immune responses were evaluated on day 7 posttransplant. A, In vivo cytotoxicity. Donor C3H and control B6 spleen cell targets were labeled with different concentrations of CFSE, mixed at 1:1 ratio, and i.v. injected into experimental animals and into control naive B6 mice. Twelve hours later, recipient’s spleen cells were isolated and analyzed by flow cytometry gating on CFSE+ cells. Representative flow cytometry histograms for CFSE staining are shown. Numbers next to each histogram indicate the mean percent specific lysis for two to three animals per group. B, Ex vivo IFN-{gamma} production. Spleen CD8+ T cells were purified and tested in a recall IFN-{gamma} ELISPOT assay against donor C3H or third-party BALB/c stimulator cells. {circ}, Depict IFN-{gamma} production by CD8+ T cells isolated from naive nontransplanted B6 female mice. The response to BALB/c stimulator cells was <100 IFN-{gamma} spots per 200,000 CD8+ T cells in all groups (data not shown). The experiment was repeated twice with similar results.

 
As cytotoxicity is generally considered a characteristic of effector CD8+ T lymphocytes, the results of in vivo CTL assays suggested that efficient donor-specific CD8+ T cell immunity was induced in the presence of memory CD4+ T cells despite DST/anti-CD40L therapy. To specifically evaluate whether donor-reactive CD8+ T lymphocytes were activated under these conditions, we next purified CD8+ T cells from the spleens of the C3H heart graft recipients on day 7 posttransplant, and tested them in recall IFN-{gamma} ELISPOT assays against donor C3H or third-party BALB/c stimulator cells. As shown in Fig. 2B, splenic CD8+ T cells from the recipients given naive CD4+ T cells and treated with DST/anti-CD40L responded weakly to donor Ags (130 ± 15 IFN-{gamma} producers per 200,000 CD8+ T cells vs 78 ± 6 IFN-{gamma} spots in naive nontransplanted B6 mice). In contrast, CD8+ T cells isolated from animals transferred with memory CD4+ T cells responded strongly to C3H stimulators, but not to third-party BALB/c stimulators, by producing IFN-{gamma} (410 ± 38/200,000 CD8+ T cells vs 124 ± 18/200,000 CD8+ T cells; Fig. 2B). The magnitude of anti-C3H response in this group was not significantly different from the response in control animals transferred with memory CD4+ T cells and transplanted with C3H heart allografts in the absence of DST/anti-CD40L treatment (390 ± 44/200,000 CD8+ T cells; Fig. 2B). Taken together, these results suggest that memory CD4+ T cells can provide help for generation of donor-reactive CD8+ effector cells even under conditions of costimulatory blockade.

Donor-reactive CD8+ T cells participate in costimulatory blockade-resistant rejection initiated by memory CD4+ T cells

We next analyzed the pathology and cellular composition of the T cell infiltrate in the rejecting heart grafts. For this purpose, immunohistochemical staining for CD4+ and CD8+ was performed on frozen sections of the grafts harvested at the time of rejection (or on day 60 in cases when rejection did not occur earlier). The infiltrate in C3H heart grafts undergoing rejection in untreated B6 female recipients was predominantly comprised of CD8+ T cells with very few CD4+ T cells (Fig. 3, B and C). Similarly, the infiltrate found in the costimulatory blockade-resistant heart grafts (rejection induced by memory CD4+ T cells) also consisted mainly of CD8+ T cells with minimal numbers of CD4+ T cells (Fig. 3, H and I). In contrast, the long-term surviving C3H grafts in mice given naive CD4+ T cells and treated with DST/anti-CD40L had a normal histologic appearance, and there were essentially no CD4+ or CD8+ T cells detected by immunohistochemistry (Fig. 3, D–F). Overall, these findings suggest that endogenous effector CD8+ T cells mediate the costimulatory blockade-resistant rejection induced by memory CD4+ T cells.



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  		FIGURE 3. CD8+ T cells infiltrate cardiac allografts despite costimulatory blockade. Heart grafts were harvested from naive nontreated B6 recipients at the time of rejection (day 7, A–C), from B6 recipients transferred with naive CD4+ T cells and treated with DST/anti-CD40L on day 60 posttransplant (D–F), from B6 recipients transferred with memory CD4+ T cells and treated with DST/anti-CD40L at the time of rejection (day 8, G–I), and from CD8-depleted B6 recipients transferred with memory CD4+ T cells and given DST/anti-CD40L at the time of rejection (day 22, J–L). Paraffin sections were stained with H&E (A, D, G, J); immunohistochemical staining for CD4+ (B, E, H, K) and CD8+ (C, F, I, L) was performed on frozen sections; brown color indicates positively stained cells. Magnification x40. The sections are representative of three to five recipients analyzed per each group.

 
If CD8+ T cells are relevant effector cells under these circumstances, then depletion of CD8+ T cells should prolong graft survival. To test this hypothesis, groups of B6 mice were given adoptive transfers of polyclonal anti-C3H memory CD4+ T cells (generated as outlined previously), treated with C3H DST/anti-CD40L, and transplanted with C3H heart grafts. Depleting anti-CD8 mAb or an isotype control was administered, as outlined in Materials and Methods, before the cardiac graft placement and throughout the whole duration of the experiment. As in the previous experiments, recipients in the control Ab group rejected the heart grafts by day 12 with MST of 9.6 ± 0.9 days (Fig. 4A). Depletion of CD8+ T cells resulted in significant prolongation of heart graft survival (MST of 21.6 ± 0.7 days; Fig. 4A), although all grafts were ultimately rejected. The effectiveness of CD8+ T cell depletion was confirmed by flow cytometric analysis of recipient spleen cells (Fig. 4B).



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  		FIGURE 4. CD8+ T cell depletion prolongs cardiac allograft survival in the recipients transferred with memory CD4+ T cells and treated with DST/anti-CD40L. Two groups of female B6 recipients were adoptively transferred with memory CD4+ T cells and treated with DST/anti-CD40L. Recipients in one group received anti-CD8-depleting Ab before treatment with DST/anti-CD40L and throughout the duration of the experiment. The animals in the control group were given rat IgG. A, Cardiac allograft survival. n = 5 per group. *, p < 0.05 vs nondepleted mice by Kaplan Meier survival analysis. B, Effectiveness of CD8+ T cell depletion. Spleen cells were isolated from the recipients at the time of rejection and analyzed by flow cytometry for the expression of CD8. The histograms are representative of five animals tested per group. Less than 0.05% CD8+ T cells were detected in all recipients from the CD8-depleted group. C, Donor-specific IFN-{gamma} production. Spleen cells were isolated from all recipients at the time of rejection and tested in recall IFN-{gamma} ELISPOT assay against donor C3H or third-party BALB/c stimulator cells. Anti-CD8 Ab YTS169 was added to selected wells at a concentration of 50 µg/ml. The response to BALB/c stimulator cells was <80 IFN-{gamma} spots per 1 x 106 spleen cells in both groups. Experiment was performed twice with similar results.

 
Recall ELISPOT assays in the recipients treated with control Ab revealed a high frequency of donor-specific IFN-{gamma}-producing T cells (1270 ± 180 spots per million spleen cells) that was blocked by addition of anti-CD8 Ab to the assay wells (405 ± 32 spots per million spleen cells) (Fig. 4C). In contrast, spleen cells from recipients depleted of CD8+ T cells contained a low frequency of antidonor IFN-{gamma} cells (200 ± 21 spots per million spleen cells). This response was not significantly affected by addition of anti-CD8 Ab (174 ± 16 spots per million spleen cells), suggesting that cells other than CD8+ T cells may mediate graft rejection in these animals. Consistent with this interpretation, the rejected grafts in the anti-CD8-treated recipients were infiltrated with CD4+ T cells, but not CD8+ T cells (Fig. 3, K and L). Moreover, serum obtained from both anti-CD8- and control-treated recipients (previously given memory CD4+ cells and DST/anti-CD40L treatment) contained antidonor alloantibodies (Fig. 5). C3H-reactive, but not third-party BALB/c-reactive IgG alloantibodies were detected in both groups, although higher titers were found in the anti-CD8-depleted mice. This difference in Ab titers may be at least partially explained by the longer time to rejection in CD8-depleted mice (20–25 days vs 7–13 days in nondepleted group). Overall, these results indicate that DST/anti-CD40L treatment did not affect the ability of memory CD4+ T cells to mediate helper functions. Both the ability to induce CD8+ effector T cells and the ability to induce an alloantibody isotype switch remained intact despite the costimulatory blockade.



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  		FIGURE 5. Memory CD4+ T cells provide help for production of donor-reactive alloantibody despite DST/anti-CD40 treatment. Serum samples were obtained at the time of rejection from groups of CD8-depleted and nondepleted B6 recipients transferred with memory CD4+ T cells and treated with DST/anti-CD40L. Serial dilutions of the serum were tested for binding to donor C3H or third-party BALB/c thymocytes and detected with FITC-conjugated goat anti-mouse IgG by flow cytometry. A, Representative histograms of 1:90 titer of serum binding to thymocytes (C3H, filled line, or control BALB/c, dotted line). B, Percentage of donor C3H thymocytes stained by serum from individual CD8-depleted (filled symbols) or nondepleted (open symbols) recipients.

 
Monoclonal memory CD4+ T cells responding through the indirect pathway induce costimulatory blockade-resistant rejection

The memory CD4+ T cells used in the above studies were polyclonal and could potentially respond to donor Ags expressed directly on graft cells (direct pathway) or to donor Ags that were processed and presented by recipient APCs through the indirect pathway (27, 28). To specifically address whether and how memory CD4+ T cells responding through the indirect pathway alone interfered with the effect of DST/anti-CD40L on long-term cardiac graft survival, we performed additional experiments in a second model system. Mar TCR transgenic mice (B6 recombination-activating gene 2–/– background) contain only CD4+ T cells specific for the male Ag (HY)-derived peptide HYDby plus I-Ab. Mar T cells do not cross-react with H-2k alloantigens in vitro or in vivo (29). Therefore, upon adoptive transfer into wild-type B6 female recipients of C3H male heart allografts, Mar T cells can only respond to the donor-derived HYDby peptide processed by the recipient’s APC and presented in the context of I-Ab through the indirect pathway (Fig. 6). This model system therefore allows us to compare the effects of memory vs naive CD4+ T cells only capable of responding through the indirect pathway with a known peptide within the context of wild-type alloresponse. Furthermore, unlike in the experiments with polyclonal CD4+ T cell population, we could precisely control the numbers of transferred donor-specific T cells.



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  		FIGURE 6. Schematic representation of indirect allorecognition by Mar T cells transferred into a wild-type B6 female recipient of a C3H male cardiac allograft. The donor HY Ag found in the male C3H heart graft can be indirectly processed and presented by recipient female B6 APCs. Mar T cells are specific for the male peptide HYDby ({blacksquare}) expressed in the context of I-Ab. Mar T cells cannot recognize the male Ag on C3H cells (H-2k) because the C3H cells express I-Ak and I-Ek, neither of which forms complexes with HYDby. Other recipient T cells (non-Mar T cells) can directly recognize other donor C3H alloantigens.

 
We first tested whether we could generate a memory population of Mar CD4+ T cells. Mar T cells (Thy-1.2) isolated from the spleens of young naive Mar females have a naive phenotype: CD25lowCD44lowCD62LhighCD69low (Fig. 7A). After 4 days of stimulation with HYDby peptide and several rounds of cell division in vitro, Mar cells enlarged in size; up-regulated surface expression of CD25, CD69, and CD44; down-regulated CD62L; and rapidly produced IFN-{gamma} upon restimulation (Fig. 7A and data not shown), consistent with an effector cell phenotype. We then transferred in vitro primed Mar T cells into naive Thy-1.1 B6 female recipients and 4 wk later reisolated spleen T cells and characterized the population of transferred Thy-1.2+ Mar cells by flow cytometry. At this time, Mar T cells were detectable in spleen and lymph nodes of an adoptive host and did not express cell surface markers characteristic of ongoing T cell activation, CD25 and CD69, consistent with memory phenotype (Fig. 7A). In agreement with the results from other investigators (2, 30, 31), these data indicate that 4 wk in vivo in the absence of ongoing antigenic stimulation is sufficient to convert primary effector CD4+ T cells into memory T cells.



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  		FIGURE 7. Memory Mar T cells induce cardiac allograft rejection despite DST/anti-CD40L therapy. A, Surface phenotype of Mar T cells. Top, T cells were isolated from naive Mar female mice. Middle, Naive Mar T cells were stimulated in vitro with HYDby peptide for 4 days. Bottom, in vitro activated Thy-1.2+ Mar T cells were transferred into naive Thy-1.1+ B6 female recipients, and reisolated from spleen and lymph nodes 4 wk later. Cells were tested for activation marker expression by flow cytometry. The numbers next to each histogram show the percentage of positive cells and the mean fluorescence intensity. B, Cardiac allograft survival. B6 female mice were adoptively transferred with 5 x 106 of naive ({circ}) or memory (•) Mar T cells, treated with C3H DST/anti-CD40L, and transplanted with C3H male cardiac allografts. Control nontransferred naive B6 recipients received C3H male heart grafts without DST/anti-CD40L treatment (triangles). n = 3–4 per group. *, p < 0.05 vs mice transferred with naive CD4+ T cells by Kaplan Meier survival analysis.

 
To determine whether memory vs naive Mar T cells responding via the indirect pathway mediate costimulatory blockade resistance, we transferred 5 x 106 naive or in vitro primed Mar T cells into groups of naive B6 female recipients. Four weeks later, animals received C3H male DST/anti-CD40L treatment, followed by placement of C3H cardiac allografts. Analogous to the results obtained using polyclonal CD4+ T cells, adoptive transfer of naive Mar T cells did not influence the effectiveness of DST/anti-CD40L: all animals maintained their grafts for >40 days. By contrast, the effect of DST/anti-CD40L was completely abolished in the recipients containing memory Mar T cells in that all C3H male heart allografts were rejected by day 12 (MST of 9.3 ± 1.1 days; Fig. 7B). Thus, the presence of memory CD4+ T cells responding to alloantigens solely through the indirect pathway was sufficient to confer resistance to costimulatory blockade and precipitate allograft rejection. Importantly, to exhibit such an effect, the memory CD4+ T cells must be specific for donor-derived Ag, as B6 female recipients containing memory Mar T cells and treated with C3H DST/anti-CD40L did not reject heart allografts from C3H female donors (no HY Ag in donor graft, MST >35 days; n = 3; data not shown). This finding is consistent with our previously published data for polyclonal CD4+ T cells (23, 26).

Memory Mar T cells provide help for donor-reactive CD8+ T cells despite DST/anti-CD40L treatment

Recall immune responses were performed in groups of recipients on day 7 posttransplant. The animals transferred with naive Mar T cells and given DST/anti-CD40L exhibited essentially no in vivo antidonor cytotoxic activity (2 ± 2% specific lysis; Fig. 8A) and spleen cells contained only a low frequency of donor-reactive, IFN-{gamma}-producing CD8+ T cells (42 ± 7 IFN-{gamma} spots per 200,000 CD8+ T cells vs 36 ± 10 spots per 200,000 CD8+ T cells in naive nontransplanted B6 females; Fig. 8B). In addition, these recipients had no detectable C3H-specific alloantibody in their serum (data not shown). In striking contrast, recipients given adoptive transfers of memory Mar T cells (that rejected the grafts despite DST/anti-CD40L) developed a high frequency of C3H-reactive CD8+ T cells as measured by in vivo CTL activity and by IFN-{gamma} production (84 ± 14% specific lysis, Fig. 8A; 390 ± 62 IFN-{gamma} spots/200,000 CD8+ T cells, Fig. 8B). Serum from the recipients in this group revealed high titer of alloantibody specific to donor C3H, but not third-party alloantigens (data not shown). Analogous to the experiments using polyclonal memory CD4+ T cells, rejecting heart allografts in the recipients transferred with memory Mar cells and treated with DST/anti-CD40L were predominantly infiltrated by CD8+ T cells with minimal infiltrate of CD4+ T cells (data not shown). Overall, these results indicate that memory CD4+ T cells capable of recognizing donor Ags solely through the indirect pathway mediate costimulatory blockade-resistant graft rejection through provision of help for the induction of a potent, direct antidonor CD8+ T cell effector response.



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  		FIGURE 8. Memory, but not naive Mar T cells provide help for the induction of antidonor CD8+ effector T cells despite costimulatory blockade. Groups of B6 recipients of C3H cardiac allografts containing naive or memory CD4+ T cells and given DST/anti-CD40L were sacrificed, and recall immune responses were assessed on day 7 posttransplant. Control B6 female recipients containing memory Mar T cells were given cardiac transplants without DST/anti-CD40L treatment. A, In vivo cytotoxicity. The assay was performed as outlined in Materials and Methods. Each symbol represents the percent specific lysis for an individual animal (two to three animals in each group). B, Ex vivo IFN-{gamma} production. Spleen CD8+ T cells were purified and tested in a recall IFN-{gamma} ELISPOT assay against donor C3H or third-party BALB/c stimulators. {circ}, Depict IFN-{gamma} production by CD8+ T cells isolated from naive nontransplanted B6 female mice. The response to third-party BALB/c stimulator cells was <50 IFN-{gamma} spots per 200,000 CD8+ T cells in all groups (data not shown). The experiment was repeated twice with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has become increasingly apparent that sensitized, donor-reactive CD4+ and CD8+ T cells are significant barriers to short- and long-term allograft survival. Graft rejection is accelerated, and tolerance is more difficult to induce, in hosts whose immune repertoires contain primed donor-reactive T cells (20, 21, 22, 23, 24, 26). Our data extend these concepts, providing the first conclusive evidence that donor-reactive memory CD4+ T cells confer resistance to the graft-prolonging effects of costimulatory blockade. More importantly, the data in this work provide insight into the cellular mechanisms behind this observation.

When untreated, naive animals are challenged with an allograft, recipient naive CD4+ T cells respond to donor Ags through the direct and indirect allorecognition pathways generally differentiating into proinflammatory Th1 cytokine-producing effector T cells. These activated CD4+ T cells are central mediators of rejection and can contribute to tissue destruction through direct cytotoxicity and delayed-type hypersensitivity reactions (29, 32, 33, 34). Moreover, these CD4+ T cells can provide helper signals for the activation of donor-reactive CD8+ effector T cells and for the isotype switching of donor-reactive Ab-producing B cells (35, 36) that, in turn, function together to destroy the graft.

In contrast, following DST/anti-CD40L therapy of naive allograft recipients, CD4+ T cells acquire a regulatory phenotype and play important roles in the induction and maintenance phases of the donor-specific unresponsiveness (12). These regulatory T cells function in part via secretion of tolerogenic and anti-inflammatory cytokines such as TGF-{beta} (37). In conjunction with induced deletion of a portion of the donor-reactive T cell repertoire (38), the regulatory T cells are thought to inhibit activation and differentiation of other donor-reactive CD4+ T cells, CD8+ T cells, and B cells, subsequently preventing downstream immune injury to the transplanted organ.

Our data now demonstrate that memory CD4+ T cells behave differently than naive CD4+ cells in the context of DST/anti-CD40L-based costimulatory blockade and an allograft. The memory CD4+ T cells are not depleted and do not develop into a regulatory phenotype. To the contrary, the memory T cells maintain their Th1 cytokine phenotype characterized by IFN-{gamma} production despite the costimulatory blockade. It is quite possible that the early onset of proinflammatory environment may prevent generation of endogenous regulatory T cells, normally induced by DST/anti-CD40L, or may inhibit the regulation at the effector stage. It should be noted that while treatment with anti-CD40L Ab was shown to induce immune regulation rather than clonal deletion (39, 40), DST alone has been associated with both (12, 37, 41). The combination therapy with DST plus anti-CD40L does complicate our ability to interpret the effects of memory CD4+ T cells on various potential mechanisms of hyporesponsiveness. However, we chose to use this combination therapy because it is generally more efficient than monotherapy with anti-CD40L Ab (41). Regardless, our data show that the memory T cells maintain their ability to elicit helper functions, leading to the development of a pathogenic antidonor immune response despite this stringent protocol.

The memory donor-reactive CD4+ T cells provided costimulatory blockade-resistant help for the differentiation of donor-reactive CD8+ T cells. In contrast to naive mice in which DST/anti-CD40L treatment leads to depletion of donor-reactive CD8+ T cells, the presence of memory CD4+ T cells permitted the antidonor CD8+ T cell precursors to become full-fledged effectors. The induced CD8+ effector cells mediated CTL activity in vivo, produced IFN-{gamma} in response to donor Ags presented through the direct pathway, and were found in high numbers in the graft. Confirming their contribution to the graft destruction, depletion of CD8+ T cells in animals treated with DST/anti-CD40L plus an allograft prolonged the graft survival despite the presence of donor-reactive memory CD4+ T cells.

CD4+ T cell help for activation of CD8+ T cells is commonly thought to occur via activation or conditioning of APCs (42, 43). In this model, CD40L on the CD4+ T cell interacts with CD40 on the APC, resulting in up-regulation of CD80/86 and thereby providing sufficient costimulation to prime naive CD8+ T cells (44, 45, 46). In the absence of this conditioning signal (for example, following DST/anti-CD40L treatment), the TCRs on naive CD8+ T cells are thought to interact with the Ag on the APCs without appropriate costimulation, leading to CD8+ T cell deletion. Our data show that memory CD4+ cells can provide the requisite helper signal despite costimulatory blockade, but the molecular basis of the helper function remains to be further elucidated. One possibility is that the anti-CD40L Ab treatment only blocks CD40/CD40L interactions for a short, time-limited period. Because memory CD4+ T cell phenotypes are stable in vivo and are resistant to deletion, they could still mediate APC conditioning via CD40/CD40L once the blocking Ab has been cleared. Alternatively or in addition, memory CD4+ T cells may efficiently condition APCs through secretion of soluble factors such as TNF-{alpha} and/or IFN-{gamma} or via alternate costimulatory pathways such as ICOS/B7RP-1 or OX40/OX40L (47, 48, 49, 50).

Another function of CD4+ T cells is to provide help for B cells to induce Ab isotype switching and secretion (36). Our data clearly indicate that alloreactive memory CD4+ T cells can provide help for the induction of donor-specific IgG alloantibodies despite treatment with DST/anti-CD40L. Moreover, high titers of donor-specific alloantibodies in the serum of CD8-depleted recipients suggest that alloantibody-mediated damage to the donor tissue may participate in costimulatory blockade-resistant graft rejection under these conditions.

Our data also show that in CD8-depleted animals transferred with memory CD4+ T cells and treated with DST/anti-CD40L, the rejecting grafts were heavily infiltrated with CD4+ T cells. It still remains to be determined whether these infiltrating CD4+ cells were direct descendants of transferred memory CD4+ T cells, or they were generated from the endogenous naive antidonor CD4+ T cell repertoire of the host. Regardless, the data show that the memory CD4+ T cells can initiate and contribute to the diverse antidonor pathogenic immune response, thereby overcoming the effects of costimulatory blockade.

The results additionally provide the first look at how memory CD4+ T cells responding through the indirect pathway affect the ability of costimulatory blockade to prolong graft survival. It is well established from classic studies using murine skin allografts by Auchincloss and colleagues (35) that CD4+ T cells responding to indirectly presented Ag can provide help for induction of direct antidonor CD8+ T cell responses. Importantly, indirect priming of naive T cells is dependent on proper costimulation. We have previously reported that naive animals treated with DST/anti-CD40L and enjoying long-term cardiac allograft survival had no detectable responses to indirectly presented donor Ags (23). The results of this study clearly indicate, however, that memory CD4+ T cells responding through the indirect pathway can provide help for CD8+ T cells and induce allograft rejection regardless of DST/anti-CD40L therapy. This issue is of high clinical relevance, as CD4+ T cells responding through the indirect pathway can be activated late posttransplant, and could therefore potentially initiate a CD8+ response and precipitate rejection overcoming tolerance at later time points. Another important lesson from the experiments using Mar cells is that memory CD4+ T cells specific for the single donor-derived determinant are sufficient to overcome the effects of DST/anti-CD40L on the entire (direct and indirect) alloreactive T cell repertoire (that would be tolerized by the DST/anti-CD40L in the absence of memory CD4+ T cells), thus precipitating rejection. This situation may be relevant to human transplant patients whose pretransplant, donor-reactive memory CD4+ T cells are presumably primed through the environmental exposure, and may respond via the indirect pathway to a subsequent transplanted organ.

The molecular mechanisms by which CD4+ T cells resist the effects of DST/anti-CD40L therapy still remain to be elucidated. It is known that memory T cells have precommitted cytokine profiles, and require less Ag and less costimulation than naive T cells to become reactivated (1, 2, 3). Furthermore, once activated, memory T cells produce cytokines, proliferate, and differentiate into effector T cells within hours, thereby preventing the tolerizing effect of costimulatory blockade on the rest of the immune repertoire. Memory T cells are also more resistant to apoptotic cell death (and therefore, more resistant to clonal deletion) than naive T cells (1, 3, 51), a feature that can be attributed to the up-regulated expression of antiapoptotic genes such as Bcl-xL and/or Bcl-x{gamma} (52, 53). Finally, it has been recently demonstrated that tolerance induction through costimulatory blockade is dependent on the ability of T cells to migrate to secondary lymphoid tissues (54). As certain populations of memory T cells tend to accumulate in the peripheral nonlymphoid tissues, these peripheral memory T cells may avoid tolerizing effect of costimulatory blockade, and instead give rise to potent effector cells.

Our data indicate that designing therapies aimed at preventing effector and helper functions of memory CD4+ T cells might be essential for inducing tolerance in large animals and humans. There is a possibility that higher doses or prolonged therapy with anti-CD40L Ab may be more efficient in inducing long-term allograft survival in the presence of memory CD4+ T cells. Although this needs to be formally tested, this approach is unlikely to be efficacious in vivo, as in vitro experiments revealed that increasing concentration of anti-CD40L Ab up to 100 µg/ml had little effect on effector functions of memory CD4+ T cells (data not shown). Depletion strategies might be more successful, but the obvious disadvantage of such an approach is that depletion is generally nonspecific and could result in loss of protective antiviral immunity, leading to significant complications. Novel strategies, including the use of reagents that are known to synergize with costimulatory blockade in prolonging allograft survival (for example, rapamycin and 15-deoxyspergualin) (24, 41, 55, 56), are attractive, but their specific effect on memory T cells needs to be evaluated in detail. Finally, recent studies have provided evidence that certain newly recognized costimulatory pathways, such as ICOS/B7RP-1 and OX40/OX40L (CD134/CD134L), may be involved in reactivation of memory T cells, but have little effect on naive T cells (57, 58, 59, 60, 61). Targeting these novel pathways (in combination with CD40/CD40L blockade) is a promising, although still unproven approach for inhibiting alloreactive memory CD4+ T cell function in vivo.

In summary, we showed that alloreactive memory CD4+ T cells are resistant to costimulatory blockade and remain able to provide help for induction of CD8+ T effector differentiation and alloantibody isotype switching. We have developed a defined model system that will permit further mechanistic studies of resistance to costimulatory blockade mediated by memory CD4+ T cells. Our findings emphasize that the varied functions of alloreactive memory CD4+ T cells represent significant hurdles for tolerance induction. The studies have important implications for studies of tolerogenesis in human transplant patients whose peripheral immune repertoires often contain alloreactive memory T cells.


   Acknowledgments
 
We thank Earla Biekert and Alla Gomer for their superb technical assistance.


   Footnotes
 
1 This work was supported by a Scientist Development Grant from the American Heart Association (to A.V.), an American Society of Transplantation Women’s and Minority Faculty Grant (to A.V.), and National Institutes of Health Grant R01AI-43578-02 (to P.S.H.). Back

2 Address correspondence and reprint requests to Dr. Anna Valujskikh, Department of Immunology, NB-30, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: valujsa{at}ccf.org Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; DST, donor-specific cell transfusion; Mar, Marilyn; MST, median survival time. Back

Received for publication August 5, 2003. Accepted for publication February 26, 2004.


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 Discussion
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