Depletion of CD8 memory T cells for induction of tolerance of a previously transplanted kidney allograft

Abstract

Heterologous immunologic memory has been considered a potent barrier to tolerance induction in primates. Induction of such tolerance for a previously transplanted organ may be more difficult, because specific memory cells can be induced and activated by a transplanted organ. In the current study, we attempted to induce tolerance to a previously transplanted kidney allograft in nonhuman primates. The conditioning regimen consisted of low dose total body irradiation, thymic irradiation, antithymocyte globulin, and anti-CD154 antibody followed by a brief course of a calcineurin inhibitor. This regimen had been shown to induce mixed chimerism and allograft tolerance when kidney transplantation (KTx) and donor bone marrow transplantation (DBMT) were simultaneously performed. However, the same regimen failed to induce mixed chimerism when delayed DBMT was performed after KTx. We found that significant levels of memory T cells remained after conditioning, despite effective depletion of naïve T cells. By adding humanized anti-CD8 monoclonal antibody (cM-T807), CD8 memory T cells were effectively depleted and these recipients successfully achieved mixed chimerism and tolerance. The current studies provide 'proof of principle' that the mixed chimerism approach can induce renal allograft tolerance, even late after organ transplantation if memory T-cell function is adequately controlled.

Figure 1. Posttransplant course (serum creatinine levels) of recipients in Groups SKBMT, A, B and C

Red solid lines and black dashed lines indicate creatinine changes in the tolerant recipients and rejectors, respectively. SKBMT: All eight recipients of SKBMT developed mixed chimerism and 7 of 8 survived long term without acute rejection with four of them acquired allograft tolerance. Group A: All four recipients in Group A failed to develop mixed chimerism and rejected their kidney allografts. Group B: One recipient that developed limited chimerism survived long term, but eventually developed chronic rejection and was euthanized due to kidney failure on day 703 after DBMT. Two recipients failed to develop chimerism and rejected their kidney allograft on days 64 and 16. Group C: Three recipients (red lines) in Group C successfully developed mixed chimerism and have never developed rejection with two recipients currently surviving at 1.8 and 2.8 years after DBMT with normal kidney function. The other (red squares) died on day 67 due to refractory ascites caused by right atrial thrombus, which may be attributed to side effects of anti-CD154 antibody at the time of DBMT. Two recipients failed to develop mixed chimerism and rejected their allograft on days 61 and 35.

Figure 2. Renal allograft biopsies obtained from recipients in Groups B and C

(A) Renal allograft from autopsy of a Group B long-term survivor (M6601) on day 702 after DBMT. An enlarged glomerulus shows mesangial and endocapillary hypercellularity with duplication of the basement membranes (arrows), PAS stain, 400×. (B) Renal allograft (M6601 on day 702) showing capillary staining for C4d, 400×. (C) Biopsy taken on day 629 after DBMT from M 1902 (Group C) showing a normal glomerulus without hypercellularity and with a normal basement membrane, PAS stain, 400×. (D) C4d staining of the biopsy taken from M 1902 on day 629. No C4d staining of the peritubular capillaries, 400×.

Figure 3. Lymphocyte subsets after DBMT

After DBMT, there is no significant difference observed in the absolute counts of CD3+CD4+, CD3-CD16+ (NK cell) and CD3-CD20+ (B cell) cells in the peripheral blood in Groups A–C. The CD8+CD3+ cells were significantly suppressed (p < 0.05) for 3 weeks in Group C. Statistical analysis was performed by comparing the average of cell counts between days 0–16 in Groups A (n = 4), B (n = 3) and C (n = 5) using Kruskal–Wallace test. N.S.; statistically not significant.

Figure 4. Absolute counts of naïve and memory T-cell subsets after DBMT in Group B (n = 2) and Group C recipients (n = 3)

(A) CD4 naïve T cells were effectively depleted and remained suppressed. (B) The absolute counts of CD4 effector memory T cells (CD95+CD28 & minus; CD4T_em remained low in the peripheral blood. (C) CD4 central memory T cells (CD95+CD28+, CD4T_cm were resistant to the conditioning regimen and their absolute counts remained >200/mm³ in both Groups B and C. (D) CD8 naïve T cells (CD95−) were effectively depleted and remained suppressed after DBMT in both Groups B and C, regardless of cM-T807 treatment. (E) CD8 effector memory T cells (CD95+CD28−, CD8 T_EM) were effectively depleted until day 5 in both groups. However, rapid expansion of CD8 T_EM ensued thereafter in Group B, while such rapid expansion of CD8 T_EM was effectively inhibited by cM-T807 treatment in Group C. (F) Absolute counts of CD8 T_CM were also more suppressed in Group C than those in Group B.