Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates

Abstract

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such "delayed tolerance" can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T-cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long-term survival without immunosuppression could be achieved in at least 6 of these 11 animals.

Figure 1. Conventional Immunosuppression after KTx and nonmyeloablative conditioning regimen for DBMT

All recipients initially underwent KTx alone with a conventional triple drug immunosuppressive regimen consisting of tacrolimus, mycophenolate mofetil and prednisone. The recipients then underwent conditioning and DBMT one (Group C) to four months later (Groups A and B). The standard conditioning regimen consisted of low-dose total body irradiation (1.5 GyX2) on days -6 and -5, thymic irradiation (TI, 7 Gy) on day-1, equine ATG (hATG) and anti-CD154 mAb. In Groups B and C, additional anti-CD8 mAb was also administered. After DBMT, the recipient was treated with cyclosporine for 28 days, after which no further immunosuppression was administered. KTx: Kidney Transplantation, TBI: Total Body Irradiation, TI: Thymic Irradiation, DBMT: Donor Bone Marrow Transplantation, CNI: Calcineurin Inhibitor, hATG: equine Anti-Thymocyte Globulin

Figure 2. Lymphocyte subsets after DBMT

There was no significant difference observed in CD4 T cell subsets among Groups A, B and C. In Group A, although naïve CD8+ T cells were easily deleted, both CD8+ TEM and CD8+ TCM started to recover rapidly by day 5. In Groups B, by adding humanized anti-CD8 mAb (cMT807), prolonged deletion of both CD8+ TEM and CD8+ TCM were achieved for 4 weeks. There was no difference observed in T cell subsets between Groups B and C. There were significant differences in CD8+TEM and CD8+TCM between Group A vs. Groups B or C (*p<0.05, **p<0.01).

Figure 3. Mixed chimerism after DBMT in Groups A, B and C

Chimerism (%) in the myeloid (A) and the lymphoid (B) lineages: In Group B (n=5), 4/5 failed to develop mixed chimerism. One monkey in Group A developed limited chimerism only in the myeloid lineage. By adding humanized anti-CD8 mAb (cMT807) to the conditioning regimen in Group B (n=13), 11/13 recipients successfully developed multilineage mixed chimerism. In Group C (n=7), DBMT was performed at 1 month after KTx with same conditioning used in Group B. Excellent chimerism was also induced in both myeloid and lymphoid lineages in Group C.

Figure 4. Serum Creatinine after donor bone marrow transplantation (DBMT)

In Group A, none of the recipients achieved renal allograft tolerance. One of the 5 recipients developed limited hematopoietic chimerism and the transplanted kidney survived long-term (703 days) but with chronic rejection detected by day 100 after DBMT. In Group B, 11 recipients developed chimerism and six of them achieved long-term renal allograft survival without maintenance immunosuppression. In Group C, none of recipients achieved long-term allograft survival.

Figure 5. Histopathology of kidney allografts in long-term survivors (Group B)

Renal biopsies from recipients with functioning allografts 9–24 months post-transplant. A: M1902 Day 1825 Autopsy showed transplant glomerulopathy and focal segmental glomerulosclerosis (X40). Although alloantibody and C4d deposition were negative, this monkey started to develop transplant glomerulopathy after day 1100. B: M4403 Day 852 Autopsy showed no rejection (X20). Euthanized due to nonimmunological reason with normal kidney function. C: M8907 at two years post transplant shows normal histopathology. The capsule is at the upper right. D: M3208 at one year post-transplant shows a moderate diffuse mononuclear infiltrate with mild glomerulitis and segmental GBM duplication (transplant glomerulopathy) not well seen at this magnification. The insert shows a focal nodular infiltrate present at 8 months. E: M2108 at nine months shows a focal mononuclear aggregrate. The glomeruli are normal and there is no evidence of rejection. F: M6007 at two years shows hypertrophied glomeruli with GBM duplication and a minimal interstitial infiltrate. All PAS stains; original magnifications x100. C4d stains were negative on all the biopsies (not shown).

Figure 6. Renal allograft survival and MHC matching

To evaluate immunological outcome alone, death due to virus infection or PTLD were censored. A. Renal allograft survival in Groups A, B and C: Kaplan-Meier analysis showed significantly superior renal allograft survival in Group B versus Group A (p=0.0199) and Group C (p=0.0338). B. MHC mismatch and renal allograft survival: Genotypes of MHC class I and class II of all recipients and donors were identified by the microsatellite assay (17, 18). Recipients in Groups B and C were grouped by the number of total MHC mismatches (A, B, DR and DP) with donor MHC. No correlation was found between the number of MHC mismatches and the renal allograft survival. mis: number of MHC mismatch. Group B 4 mis (n=4), Group B 5mis (n=3), Group B 6–8 mis (n=4), Group C 4 mis (n=2) and Group C 6–8 mis (n=2).

Figure 7

A. Anti-donor Tmem responses after KTx IFNγ spots in ELISPOT by either CD95+ sorted cells or bulk peripheral blood cells were measured sequentially after KTx in a representative recipient. Significantly high anti-donor Tmem responses were detectable until 2 months after KTx. However they waned thereafter and became significantly lower by 4 months. B. Anti-donor Tmem responses before KTx and DBMT Frequencies of Tmem (CD95+ cells) secreting IFNγ (left panel) and IL-2 (right panel) were measured by ELISPOT before KTx and immediately before DBMT. Anti-donor Tmem responses were not different between Groups B and C before KTx (Gray column). However, anti-donor Tmem responses waned after KTx in time-dependent fashion and decreased to almost undetectable levels by 4 months. Therefore, Tmem responses before DBMT was significantly lower (IFNγ p=0.028) in Group B (DBMT at 4 months) than in Group C (DBMT at 1 month). IL-2 Tmem responses were also not detectable in Group B immediately before DBMT. C. Anti-3^rd Party Tmem responses before KTx and DBMT In contrast to anti-donor Tmem responses, anti-3^rd party Tmem responses were relatively preserved after KTx (black column) and significant numbers of IFNγ and IL-2 spots were detectable even at 4 months after KTx (Group B).

Figure 8. Tmem responses after DBMT

Serial monitoring of Tmem responses were performed after DBMT in four Group B recipients who survived long-term without immunosuppression. Tmem responses in all long-term survivors remained unresponsive against the donor, while vigorous responses were observed against the third party cells (Figure 8A). In contrast, three rejectors in Group C showed vigorous anti-donor Tmem responses after DBMT (Figure 8B).

Figure 9. Inflammatory responses after KTx

A. Messenger RNA (RT-PCR): Peripheral blood samples were obtained from 10 recipients in Group B and 6 recipients in Group C before DBMT. Total RNA was extracted from recipient whole peripheral blood collected in PaxGene tubes and qt-RT-PCR was performed using cytokine specific primers and probes. 18S was used as a house keeping gene and copies of cytokine transcripts were normalized by copies of 18S. Results are expressed as ratio of cytokine copy number to 18S. Among various cytokines tested, mRNA for inflammatory cytokines IL-6, IL-17F, IL-1β and Granzyme B (GZMB) were significantly higher before BMT in Group C compared to those in Group B (p=0.008, 0.0167, 0.0167 and 0.009 respectively). The value in Y axis was expressed as ratio of copy number to 18s. B. Serum cytokine levels (Luminex): Among various serum cytokines measured by Luminex, only serum IL-6 levels were statistically higher in Group C immediately before DBMT conditioning (p=0.004). The serum levels of IL-17 were also higher in Group C but did not reach statistical significance.