Contributions of direct and indirect alloresponses to chronic rejection of kidney allografts in non-human primates

Abstract

The relative contribution of direct and indirect allorecognition pathways to chronic rejection of allogeneic organ transplants in primates remains unclear. In this study, we evaluated T and B cell alloresponses in cynomolgus monkeys which had received combined kidney/bone marrow allografts and myeloablative immunosuppressive treatments. We measured donor-specific direct and indirect T cell responses and alloantibody production in monkeys (n = 5) which did not reject their transplant acutely but developed chronic humoral rejection (CHR) and in tolerant recipients (n = 4) which never displayed signs of CHR. All CHR recipients exhibited high levels of anti-donor antibodies and mounted potent direct T cell alloresponses in vitro. Such direct alloreactivity could be detected for more than one year after transplantation. In contrast, only two out of five monkeys with CHR had a detectable indirect alloresponse. No indirect alloresponse by T cells and no alloantibody responses were found in any of the tolerant monkeys. Only one of four tolerant monkeys displayed a direct T cell alloresponse. These observations indicate that direct T cell alloresponses can be sustained for prolonged periods post transplantation and result in alloantibody production and chronic rejection of kidney transplants, even in the absence of detectable indirect alloreactivity.

Introduction

Enormous advances over the past four decades with the use of non-selective immunosuppressive agents have greatly improved the early survival of allogeneic transplants in patients. Nevertheless, longer term success rates remain unsatisfactory owing to treatment-related complications and chronic allograft rejection, a slow process involving perivascular inflammation, fibrosis and arteriosclerosis associated with intimal thickening and subsequent luminal occlusion of graft vessels (1-3).

Host pro-inflammatory T cells recognize donor antigens displayed on allogeneic transplants via two mechanisms: 1) direct allorecognition in which T cells interact with intact allo-MHC molecules displayed on donor cells (4-6) and, 2) indirect allorecognition in which T cells recognize donor peptides (from MHC and minor antigens) presented by self-MHC molecules on recipient APCs (7-11). The direct alloresponse is initiated in the recipient’s secondary lymphoid organs via alloantigen presentation by infiltrating donor MHC class II⁺ APCs (passenger leukocytes) (12, 13). Alternatively, the indirect alloresponse is oligoclonal in that it is mediated by a restricted set of T cells displaying selected TCR genes and recognizing a limited number of dominant determinants on donor antigens (14-16). While it has become clear that both allorecognition pathways contribute to the post-transplant alloimmune response, their respective contributions to chronic rejection remain controversial.

It is generally believed that donor “passenger leukocytes” such as dendritic cells infiltrate the recipient’s secondary lymphoid organs and present alloantigens to the host’s T cells immediately after transplantation but then rapidly vanish. Consequently, while this direct alloresponse is potent, it would presumably be short-lived. In contrast, the indirect alloresponse may be perpetuated via the continuous processing and presentation of donor antigens by recipient bone marrow-derived APCs. Based upon this principle, it has been postulated that the indirect allorecognition pathway plays an essential role in chronic transplant rejection (11, 17-19). In fact, there are a number of observations suggesting that indirect rather than direct type of alloreactivity represents the driving force behind chronic rejection of allografts. First, indirect alloreactivity is thought to govern the production of alloantibodies (4, 20, 21) that are known mediators of the chronic rejection process (22-26). Second, some correlation between the presence of indirect alloreactivity and chronic rejection of kidney and heart allotransplants has been reported in patients (27-31). Finally, some studies show that immunization with donor MHC peptides is sufficient to induce or accelerate the onset of chronic allograft vasculopathy in heart-transplanted mice and swine (32, 33). Collectively, these studies suggest that the indirect T cell alloresponse can mediate chronic allograft rejection. However, whether the direct alloresponse is truly short-lived and, therefore does not contribute to chronic allograft rejection has not been formally demonstrated.

In the present study, we investigated direct and indirect T cell alloresponses and alloantibody production in monkeys treated with various tolerance-inducing immunosuppressive regimens. Lack of alloantibodies and T cell alloresponses were regularly associated with transplant tolerance. Alternatively, sustained T cell alloreactivity mediated via both direct and indirect pathways or even the direct pathway alone was always detected along with the production of anti-donor antibodies in monkeys undergoing chronic allograft rejection.

Materials and Methods

Animals, conditioning and transplantations

Eighteen cynomolgus monkeys weighing 3 to 5 kg were used in this study (Charles River Primates, Wilmington, Massachusetts). Details of recipient/donor pair selection were previously reported (34). All the 9 recipients were conditioned using our “standard regimen” consisting of total body irradiation (TBI) at day −6 and −5 (1.5 Gy) followed by thymic irradiation at day −1 and −2, (7 Gy) and three injections of ATG (day −2, −1 and 0) pre-donor cell infusion. In addition to the standard conditioning, the recipients were treated as follows: M1601 received donor splenocytes (200 × 10⁶ cells/kg) as well as two injections of anti-CD40L mAbs (5c8, 20 mg/kg) ; M1501 was splenectomized at the time of transplantation and received two injections of anti-CD40L mAbs (20 mg/kg) ; M1900 and M200 were treated with two injections of anti-CD40L mAbs (20 mg/kg) ; M2800 was treated with anti-CD8 (x8, 1mg/kg) and anti-CD40L (x6, 20mg/kg) mAbs, the kidney transplant was removed at day 12 due to thrombosis and a second kidney allograft from the same donor was placed at day 77 and followed for rejection thereafter ; M4102 and M2702 were conditioned at d-1 (instead of d-6) and were treated with anti-CD8 (x6, 1mg/kg) and anti-CD40L mAbs (x6, 20 mg/kg); M6601 and 1902 were first transplanted with an allogeneic kidney whose rejection was prevented with FK506, MMF and prednisone, they were then conditioned (d112 and d126, respectively), injected with donor bone marrow cells (300 × 10⁸ cells/kg) and treated with anti-CD40L mAbs (x6, 20 mg/kg).

Blood samples

Heparinized blood was obtained from all recipient monkeys prior to transplant and at monthly intervals after POD 100 when they no longer showed therapeutic levels of circulating cyclosporine. Peripheral blood and/or spleen cells from the donors were collected, processed and frozen in 10% DMSO and stored in liquid nitrogen.

PBMC isolation

Peripheral blood mononuclear cells (PBMC) were isolated from whole heparinized blood using a percoll density gradient method. Briefly, whole blood was first centrifuged at 2000 rpm for 10 minutes to obtain an interphase layer of enriched PBMC just below the upper plasma layer. The interphase layer was harvested and diluted 15 fold in 1xPBS and layered onto a 60% percoll gradient (Amersham, Sweden) at a blood to percoll ratio of 2:1. After a 30-minute spin at 2000 rpm, the PBMC-rich buffy layer was harvested and contaminating red blood cells were removed by water shock treatment. PBMC were washed and viable cells counted based on eosin-dye exclusion. PBMC were either frozen at −80°C and stored in liquid nitrogen or used immediately for assays.

Cell sonication

The method for cell sonication was adapted for the monkey system from established protocols previously reported for murine studies (35). Briefly, 6 to 12 million PBMC (adjusted to 3×10⁶ cells per ml) in 50 ml culture tubes were sonicated over ice for 2-3 minutes (Microson Cell Disruptor, Misonix, Inc) until whole cells were no longer visible under the microscope. The contents were further centrifuged at 1700 rpm for another 10 minutes and supernatants harvested. The supernatants’ volumes were further adjusted to deliver the desired cell equivalent amounts of PBMC sonicate to be used in the assay.

ELISPOT assays of direct and indirect pathway alloresponses

Direct and indirect T cell alloresponses were measured as previously described in mice and monkeys (35-37). Briefly, ELISPOT plates were pre-coated with 5ug/ml of capture antibodies against γIFN (Mabtech, Sweden) and stored overnight at 4°C. The plates were blocked for 1 hr with PBS containing 1% BSA-fraction V (A1933, Sigma) followed by 3 washes in PBS. Three x10⁵ responding PBMC cells were added to each well of a 96-well ELISPOT plate in 100ul RPMI 1640 supplemented with 10% pooled naïve monkey serum and L-glutamine, penicillin/streptomycin and Hepes buffer. The responding cells were co-cultured with an equal number of irradiated stimulating cells (for direct allostimulation), or the cell equivalent number of sonicated cells (for indirect allostimulation), or nonstimulated (in medium alone) (Supplemental Fig. 1), or with PHA at 1 ug/ml (Sigma). After a 48 hours incubation at 37°C, the plates were washed and botinylated detection antibodies (Mabtech, Sweden) were added, and the plates were maintained at 4°C for an additional overnight incubation. After 4 washes with PBS/Tween, streptavidin horseradish peroxidase conjugate in PBS/BSA (Dako #PO397, Glostrup, Denmark) was added for at least 2 hr at room temperature, followed by an additional 6 washes. Development was done with aminoethylcarbazole (10 mg/ml in N,N-dimethylformamide) freshly prepared in 0.1 M sodium acetate buffer (pH 5.0) mixed with 30% H202. The resulting spots were counted with a computer-assisted ELISPOT image analyzer (Cellular Technology, Cleveland OH).

Statistical methods

SD of the ratio was estimated by the Delta Method.

Results

Chronic rejection vs. tolerance in recipients of an allogeneic kidney transplant

All recipient monkeys exhibited low levels (< 5%) and short-term mixed lymphocyte chimerism (< 2 months) (data not shown). None of the 9 monkeys selected in this study underwent acute rejection. Kidney transplant biopsies were performed at different time points after transplantation and analyzed for the presence of chronic humoral rejection (CHR). Five recipients developed CHR detected from d122 to d386 post-transplantation (CHR Monkeys: M1501, M1900, M1601, M6601, M4102) while the remaining 4 recipients never developed any pathological signs of chronic rejection and were subsequently referred to as tolerant (TOL Monkeys: M200, M2800, M1902, M2702) (Table 1 and Figure 1). Additionally, the creatinine levels were serially monitored after transplantation in each of the recipients. As shown in Figure 2A, four of the five recipients developing CHR on allograft biopsy displayed elevated creatinine blood levels (> 2) which became detectable between 1 and 9 months after the histopathological diagnosis of CHR (Table 1). A slight but not significant increase of creatinine level was detected at day 400 in the fifth recipient M1900 (Fig. 2A). In contrast, none of the tolerant monkeys exhibited any increase of their creatinine levels (Fig. 2B).

Table 1. Monkeys used in this study.

Nine recipient/donor pairs were studied. Five recipients of allogeneic kidney transplants developed chronic rejection (CHR) and displayed an elevation of their serum creatinine levels as indicated in the table. Four recipients never exhibited signs of chronic rejection and had normal creatinine levels throughout the study and were referred to as tolerant (TOL). The third column shows the protocols used to treat each recipient. STD: standard regimen (see Methods section) ; SPLX: splenectomy ; Post-Tx: post-transplantation. Each color corresponds to one particular regimen.

Recipient	Donor	Treatment	REJECTION	CHR detection (day)	Creatinine elevation
1501	301	STD, SPLX, anti-CD40L	CHR	d 386	d 487
1900	3099	STD, anti-CD40L	CHR	d 369	none
1601	2501	Donor splenocytes, anti-CD40L	CHR	d 231	d 274
6601	5801	Post-Tx BMT, anti-CD40L, anti-CD8	CHR	d 122	d 369
4102	4202	STD (d-1), anti-CD40L, anti-CD8	CHR	d 196	d 280
200	1799	STD, anti-CD40L	TOL	-	none
2800	3800	STD, anti-CD40L, anti-CD8	TOL	-	none
1902	2102	Post-Tx BMT, anti-CD40L, anti-CD8	TOL	-	none
2702	1502	STD (d-1), anti-CD40L, anti-CD8	TOL	-	none

Figure 1. Histology in CHR and tolerant monkeys.

M1500, M1900, M1601, M6601, and M4102 show enlarged glomeruli with endocapillary mononuclear cells and glomerular basement duplication. The C4d was positive in all cases (data not shown). M1501, M6601, and M4102 also show marked glomerular inflammation. M200, M2800, M1902, and M2702 show normal looking glomeruli without interstitial inflammation.

Figure 2. creatinine levels in CHR and tolerant monkeys.

Serial serum creatinine levels were determined pre- (d0) and post-kidney transplantation in the peripheral blood of recipients which developed CHR (top panel, A) or tolerance (TOL) (lower panel, B).

Direct and indirect pathway T cell alloresponses in tolerant vs. CHR recipients

Next, we evaluated the alloresponses by T cells collected from each of the tolerant and CHR monkey. Direct and indirect T cell alloresponses were tested as previously described (35, 37, 38). Serial measurements of the pre- and post-transplant anti-donor responses recorded in CHR and tolerant monkeys are shown in Figure 3 and 4 and summarized in Table 2. In each panel, the X axis represents the mean delta gIFN spot per million T cells (delta spm), corresponding to the mean spm ± SD obtained with T cells stimulated with donor cells/sonicates minus the mean spm obtained with T cells stimulated with medium. In each panel, the Y axis represents the mean stimulation index (SI) ± SD corresponding to the mean spm obtained with T cells stimulated with donor cells/sonicates divided by the mean spm obtained with T cells stimulated with medium. The pre-transplant valued are represented by open symbols while the post-translant values are displayed as closed symbols. This type of scattered representation provides an accurate picture of the response which takes in account both the actual spm and the SI. Each value corresponds to one time point measured post-transplantation (as indicated in the figure). We considered as positive all the values which were higher than the mean pre-transplant values by more than three times SI and provided a mean delta cpm more than 3 x the SD obtained with T cells stimulated with medium alone.

Figure 3. Kinetics of direct alloresponses in CHR and tolerant monkeys.

The direct alloresponses were measured using recipient PBMCS collected pre-transplantation (d0) and at different time points post-transplantation in a series of monkeys developing CHR (left panel) and tolerant monkeys (right panel). To measure direct alloreactivity, recipient T cells were stimulated in vitro with donor APCs as decribed in the Methods section. The X axis represents the mean Δ spm (γIFN spots per million T cells) ± SD corresponding to the mean numbers of spm obtained with T cells exposed to donor cells ± SD minus the mean numbers of spm ± SD obtained with T cells exposed to medium. The Y axis represents the mean SI (stimulation index) ± SD corresponding to the mean numbers of spm obtained with T cells exposed to donor cells ± SD divided by the mean numbers of spm ± SD obtained with T cells exposed to medium. Closed symbols correspond to the values measured post-transplantion. Open symbols correspond to the values measured pre-transplantation. For values considered as positive i.e. higher than pre-transplant values and at least 3 times higher that both the Δspm and SI, the time point is indicated.

Figure 4. Kinetics of indirect alloresponses in CHR and tolerant monkeys.

The indirect alloresponses were measured using recipient PBMCS harvested pre-transplantation (d0) and at different time points post-transplantation in a series of monkeys developing CHR (left panel) and tolerant monkeys (right panel). To assess the indirect alloresponse, recipient T cells were stimulated with donor-derived sonicates as described in the Methods section. The X and Y axes represent the mean Δspm ± SD and mean SI ± SD established as decribed in the legends of Figure 3. For values considered as positive i.e. higher than pre-transplant values and at least 3 times higher that both the Δspm and SI, the time point is indicated.

Table 2. Summary of the results obtained in CHR and tolerant monkeys.

The T cell responses, antibody responses and MHC matching results for each of the transplanted monkey pairs displaying CHR or tolerance are shown. The first two subcolumns of the MHC matching column indicate the number of MHC class I and II alleles shared by the donor and the recipient. The third subcolumn indicates the presence (+) or absence (−) of haplomatching. The last three columns show the presence (+) or absence (−) of a direct response, an indirect response and anti-donor antibodies.

Recipient	Donor	REJECTION	MHC matching			Direct response	Indirect response	Anti-donor antibodies
			MHC I	MHC II	Haplo
1501	301	CHR	1	2	−	+	−	+
1900	3099	CHR	2	2	+	+	+	+
1601	2501	CHR	0/1	2	−	++	+	+
6601	5801	CHR	2/3	2	+	+	−	+
4102	4202	CHR	2	2	+	++	−	+
200	1799	TOL	2	2	+	−	−	−
2800	3800	TOL	1	0	−	−	−	−
1902	2102	TOL	2	2	+	+	−	−
2702	1502	TOL	0/1	2	−	−	−	−

Prior to transplantation, some recipients exhibited a substantial direct response against their donor, a phenomenon that is associated with the presence of alloreactive memory T cells in these monkeys (36). These cells have been shown to derive from prior crossreactive expansion/differentiation following exposure to microbes (39-41). The levels of post-transplant direct alloreactivity increased in these monkeys as well as the other monkeys which subsequently developed CHR (Figure 3, left panel). Conversely, no post-transplant direct alloreactivity was detected in three of the four tolerant monkeys. Only M1902 displayed a high and sustained direct alloresponse for up to 700 days post-transplantation (Figure 3, right panel). On the other hand, no indirect alloresponse was detected pre-transplantation in any of the nine monkeys tested (Figure 4), a result consistent with previous observations in mice and swine (35, 42). Post-transplantation, two of the five CHR monkeys, M1900 and M1601, mounted a vigorous indirect alloresponse, first detectable 200-300 days after placement of allogeneic kidneys (Fig. 4, left panel). In contrast, no indirect alloreactivity was detected in the three other CHR recipients (M1501, M6601 and M4102) (Fig. 4, left panel) or in any of the four tolerant monkeys (Fig. 4, right panel).

Alloantibody responses in tolerant vs. rejecting monkeys

Next, we investigated the presence of donor-specific alloantibodies in serum samples serially collected from CHR and tolerant recipients. A mean fluorescence intensity (MFI) greater than 55 was considered positive as described elsewhere (43). The results of these experiments are shown in Figure 5 and summarized in Table 2. No alloantibodies were detected pre-transplantation in any of the nine recipients. All the monkeys developing CHR mounted a significant donor-specific antibody response, which usually became detectable around 200 days post-transplantation (Fig. 5A). In contrast, three out of four tolerant monkeys displayed no alloantibodies, while the fourth recipient, M200, exhibited a slightly elevated level (MFI = 52) only on day 450 post-transplantation Fig. 5B).

Figure 5. Anti-donor antibodies in CHR versus tolerant monkeys.

The presence of anti-donor antibodies was detected by FACS in a series of serum samples collected pre-transplantation and at different time points post-transplantation from monkeys with CHR (top panel, A) and tolerant monkeys (lower panel, B). The results are expressed as mean fluorescence intensity (MFI).

Influence of MHC matching on chronic rejection vs. tolerance of kidney allografts

MHC gene disparity between donors and recipients represents an essential element in the acute rejection of allogeneic transplants. However, the influence of MHC gene matching/mismatching on chronic allograft rejection and susceptibility to tolerogenesis is less understood. To address this question, we determined the MHC class I and II genes inferred to be expressed by each of the recipient/donor monkey pairs used in this study. The predicted MHC class I (A and B) and II (DP, DQ, and DR) alleles expressed by our Mauritian-origin cynomolgus monkeys were characterized using a microsatellite-based genetic technique (44). The results of the genotyping for each donor-recipient monkey pair are shown in Figure 6. Each color represents a particular haplotype.

Figure 6. MHC gene expression in donors and recipients.

The MHC class I (A and B) and class II (DR, DQ and DP) genes expressed by each donor and recipient was determined using molecular techniques as described in the Methods section. The MHC genes expressed by each chromosome of each pair of monkeys are shown side by side (left panel: CHR and right panel: tolerant). Each color corresponds to one haplotype.

The degree of haplotype matching between donors and recipients are shown in Table 2. For MHC class I, we individually considered the A and B regions on each chromosome in a given recipient/donor pair, assigning a value for degree of MHC class I matching of 0-4, with 0 indicating sharing of no A or B regions and 4 indicating sharing of both A and both B regions between recipient and donor. MHC class II DR and DQ genes were considered as a whole given that none of the monkeys displayed any recombination between these two genes. Thus, values for degree of MHC class II matching range from 0-4. Three instances of recombination in the MHC class I region (class I A in M5801, class I B in M2501 and M2702) make it impossible to assign one specific value for degree of matching, so both possible values are shown in Table 2. It should also be noted that occasionally two distinct haplotypes share specific MHC alleles; most notably, the MHC class I A allele complement is identical for both the black and red haplotypes, and is highly similar to the allele complement of the blue haplotype, so functionally black, red, and blue are treated as the same haplotype in the MHC class I A region (Budde et al. in submission).

Complete haploidentical pairs, defined as a recipient/donor combination sharing all of the same MHC class I and II regions on one chromosome, are also indicated in Table 2. In the CHR group, 3 recipient/donor pairs (M1900/3099, M6601/5801 and M4102/4202) were completely haploidentical for the black haplotype. The remaining two recipient/donor pairs are haplo-matched for the MHC class II region. In the group of tolerant monkeys, all recipients except M2800 are MHC class II haplo-matched with their donors, and two recipient donor pairs (M200/1799 and M1902/2102) are completely haploidentical for the red haplotype. One combination (M2800/3800) are only haplo-matched for the MHC class I A region since as previously stated, black and red are functionally the same haplotype in that region. There was no apparent influence of MHC matching on CHR vs. tolerance. However, it is noteworthy that among the nine donor/recipient pairs tested, eight were haplo-matched for the MHC class II region and five were completely haplo-matched through the whole MHC, a series of features which may account for the lack of acute rejection in these recipients.

Discussion

In this paper, we report our investigations of the T and B cell alloresponses in nine cynomolgus renal allograft recipients, which had received donor cells and peri-transplant immunosuppressive treatments. None of these monkeys developed durable mixed chimerism (data not shown). Despite this, they all displayed allograft survival with no signs of chronic rejection for at least 3 months following withdrawal of immunosuppression. Four recipients never displayed signs of chronic rejection and were considered tolerant. Several monkeys had a very low direct allo response pre-transplantation which is indicative of the absence of significant numbers of donor-specific memory T cells, a feature presumably associated with lack of acute rejection (36, 37, 45-49). In turn, M1902 (tolerant) displayed a fairly high direct alloresponse pre-transplantation (> 400 spots/million cells). This direct response remained high and even increased around 200 days post-transplantation. Unlike the 3 tolerant monkeys that received conditioning and DBM in conjunction with the kidney transplant, M1902 had received conditioning and DBM 4 months after transplantation. It is possible that some memory T cells recognizing alloantigens directly had been generated or expanded during this interval. The fact that this monkey subsequently remained tolerant i.e. devoid of acute and chronic rejection for years supports the hypothesis that regulatory mechanisms appear to develop and suppress these allospecific T cells.

All the recipients, which developed CHR, mounted an anti-donor alloantibody response, a result consistent with previous observations in the same model (43). The presence of anti-donor antibodies was always detected prior to the onset of CHR which itself preceded elevation in serum creatinine levels (Table 3). Strikingly, all the CHR monkeys displayed a direct T cell alloresponse detectable in some instances for more than two years after transplantation. This shows that the ability to mount a direct response can be sustained in recipients and even increase overtime after transplantation. Unlike naïve T cells, these memory T cells could be activated by alloMHC antigens presented by donor non-professional APCs such as endothelial cells. In addition, presentation of captured allo-MHC molecules by recipient dendritic cells (semi-direct pathway) may also contribute to the maintenance of such direct alloresponsiveness long after donor passenger leukocytes have vanished (50-52).

Table 3. Sequence of the events detected in CHR monkeys.

The columns 3, 4 and 5 show the earliest time point (day post-transplant) at which alloantibodies, CHR and creatinine increases were detected, respectively, for each recipient of a kidney allograft having developed chronic rejection (CHR).

Recipient	Donor	REJECTION	Allo Ab (day)	CHR (day)	Creatinine raise (day)
1501	301	CHR	236	386	487
1900	3099	CHR	168	369	None
1601	2501	CHR	225	231	274
6601	5801	CHR	114	122	369
4102	4202	CHR	114	196	280

Only 2 (M1900 and M1601) of the 5 recipients developing CHR and alloantibodies displayed an indirect alloresponse. It is at first glance surprising that 3 out of 5 CHR monkeys (M1501, M6601 and M4102) had no dectable indirect alloreactivity which is traditionally considered the driving force behind alloantibody production and CHR (22-26). This is supporting by the view that cognate T-B cell cooperation requires antigen recognition on self-MHC class II molecules. In our study, it is important to note that the 3 CHR monkeys with direct but no indirect alloreactivity shared MHC class II genes with their donors (Fig. 6). It is possible that sharing MHC class II between donors and recipients my allow cognate interactions between recipient B cells and direcly activated T cells recognizing peptides on the shared MHC class II molecules. In this scenario, it is conceivable that direct T cell allorecognition can trigger and/or perpetuate an alloantibody response by host B cells and contribute to the chronic rejection process.

At first glance, our observations of persistence of direct alloreactivity and its involvement in the chronic rejection process, in the absence of indirect alloresponses, may appear to be in disagreement with a previous report by R. Lechler et al. (19). It should be emphasized, however, that loss of direct and maintenance of indirect alloresponses were observed in that study in chronically immunosuppressed patients who developed allograft nephropathy. In contrast, in our allograft recipients, all immunosuppressive treatment had been discontinued 28 days after transplantation.

In summary, this study shows that, unlike the traditionally accepted mechanistic basis for long-term alloresponsiveness, the direct pathway can be long-lived, presumably perpetuated by the presence of the allograft. This response could be maintained by recipient memory T cells recognizing allogeneic MHC molecules on donor non-professional APCs. Most importantly, in some monkeys, this direct response was associated with the production of donor-specific alloantibodies presumably due to T-B cognate interactions rendered possible by sharing of MHC class II genes between donor and recipients and it correlated with the presence of chronic allograft rejection of kidney-transplants. This suggests that while MHC class II gene matching between donor and recpients is likely to reduce the risk of acute rejection and presumably favor the activation/expansion of some Tregs (53, 54), it could increase the risk of chronic rejection.