Clonotype analysis of human alloreactive T cells: a novel approach to studying peripheral tolerance in a transplant recipient.

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vbeta and Valpha genes and made clonotype-specific probes that allowed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction in direct pathway T cell clonotype mRNA in peripheral blood and the graft. Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.

The effect of tolerance to noninherited maternal HLA antigens on the survival of renal transplants from sibling donors.

BACKGROUND: During pregnancy and nursing, a baby's developing immune system is intimately exposed to the mother's antigens. To determine whether this exposure is of clinical benefit to patients who later receive an allograft as an adult, we analyzed the outcome of primary renal transplantations from sibling donors. METHODS: We retrospectively studied graft survival and rejection episodes in 205 patients who had received renal transplants at nine centers between 1966 and 1996 from sibling donors bearing maternal or paternal HLA antigens not inherited by the recipient. The sibling donors were categorized by analysis of family HLA-typing data. RESULTS: In the multicenter analysis, graft survival was higher at 5 years and at 10 years after transplantation in recipients of kidneys from siblings expressing maternal HLA antigens not inherited by the recipient than in recipients of kidneys from siblings expressing paternal HLA antigens not inherited by the recipient (86 percent vs. 67 percent at 5 years and 77 percent vs. 49 percent at 10 years, P=0.006 for both). Paradoxically, there was a higher incidence of early rejection in the former group, suggesting that fetal and neonatal exposure to maternal antigens results in immunologic priming. Pretransplantation transfusions of donor blood reduced the incidence of acute rejection while preserving the beneficial effect of tolerance to noninherited maternal antigens on graft survival. Since 1986, new immunosuppressive drugs have lessened the short-term, but not the long-term, survival advantage of grafts expressing maternal HLA antigens not inherited by the recipient. CONCLUSIONS: In the transplantation of a kidney from a sibling donor who is mismatched with the recipient for one HLA haplotype, graft survival is higher when the donor has maternal HLA antigens not inherited by the recipient than when the donor has paternal HLA antigens not inherited by the recipient.

Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient.

A patient was found to be functionally tolerant of a maternal kidney allograft as evidenced by good graft function 5 years after cessation of all immunosuppressive drug therapy. Despite normal in vitro proliferative and IL-2 responses, patient anti-donor 1 degree MLR cultures yielded little donor-specific CTL activity in either bulk or limiting dilution analysis (LDA) cultures. Using polymerase chain reaction, the patient's PBL and skin were found to contain donor-derived Bw6+ cells. Removal of Bw6+ donor cells from the patient PBL with mAb and immunomagnetic beads before stimulation with donor PBL on day 0 failed to restore donor-specific CTL in either bulk 1 degree MLR or LDA cultures. Restimulation of 1 degree cultures with donor stimulator cells plus exogenous IL-2, however, completely restored anti-donor HLA class I-specific CTL, indicating class I-specific CTL precursors were not clonally deleted. Fresh patient PBL, as well as donor cell-enriched fractions, when added at the initiation of 3 degrees MLR cultures, inhibited the generation of anti-donor CTL, whereas donor cell-depleted fractions did not. The inhibition was cell dose-dependent, was specific for the anti-donor response, and was radioresistant (1200 rad). Thus, the clinical tolerance observed in patients with microchimerism may be due to the presence of veto cells within the circulating donor cell pool.

Activation of HLA-A2-specific memory B cells in severe combined immunodeficient mice.

Peripheral blood leukocytes of all five HLA-A2-sensitized patients produced significant levels of human IgG (> or = 0.25 micrograms/ml) following polyclonal activation in vitro, but PBLs from only one patient (K.H.) who had been transfused recently (< 4 weeks) produced detectable anti-HLA-A2 IgG. PBLs from this in vitro responder and from one in vitro nonresponder (L.G.) were transferred intraperitoneally into SCID mice. A low level (range, 5-40 ng/ml) of human anti-HLA-A2 IgG was detected in the serum of the mice without additional stimulation. This anti-HLA-A2 IgG response was boosted (range, 40-200 ng/ml) when mice received a human skin xenograft or an early challenge with x-irradiated human leukocytes intraperitoneally. Although the anti-HLA antibodies produced were specific for HLA-A2, the boosting of anti-HLA-A2 IgG production did not require the expression of the HLA-A2 protein, since either HLA-A2-negative skin xenografts or HLA-identical x-irradiated PBLs enhanced the production of anti-HLA-A2 IgG. Dose-response of transferred PBLs and kappa:lambda composition of individual mouse anti-HLA-A2 production suggested that low-frequency human memory B-cell clones were stimulated to proliferate and/or triggered to become high Ab secretors by skin graft or PBL boost.

Donor-specific cytotoxic T lymphocyte hyporesponsiveness following renal transplantation in patients pretreated with donor-specific transfusions.

Our purpose was to investigate the mechanism of the continuing beneficial effect of donor-specific transfusions in the cyclosporine era. We describe the development of donor-specific cytotoxic T lymphocyte hyporesponsiveness in peripheral blood lymphocytes obtained up to 2 years posttransplant in patients preconditioned with 3 DST plus azathioprine. In a group of 12 such patients, hyporesponsiveness developed gradually, becoming detectable in some patients as early as 1 month posttransplant and becoming statistically significant for the entire group at 9-12 months posttransplant. A complete specificity for donor alloantigens was seen in the hyporesponsiveness of some patients; in others, partial suppression of the response to a third party HLA-mismatched control was also seen. Although slight suppression of the mixed lymphocyte culture response was seen in some patients, overall there were no statistically significant differences in MLC responses to control or donor stimulators at any time point posttransplant as compared with pretransplant, pre-DST. The mechanism of donor-specific CTL hyporesponsiveness 2 years posttransplant was explored in one patient (HLA A1, 2, B 57, 60; DR 3, 6) who had received a 2-HLA haplotype-mismatched kidney transplant from her husband (HLA A2,--; B5, 8; DR4,--) following DST plus AZA pretreatment. Bulk culture CTL analysis showed specific nonresponsiveness to donor stimulators; however in the presence of exogenous recombinant IL-2, the antidonor response was restored to the level of pretransplant PBL. Limiting dilution analysis using recombinant IL-2 revealed equivalent precursor frequency of antidonor CTL in pre- and posttransplant PBL. These data suggest that the hyporesponsive PBL contained donor-specific CTL precursors but were deficient in helper function necessary for CTL maturation.

Inhibition of human antigen-specific memory B cell response in vitro by a diphtheria toxin-related interleukin 2 fusion protein.

Recombinant diphtheria toxin-related interleukin-2 fusion protein (DAB486IL-2) is specifically cytotoxic for cells bearing the high-affinity IL-2 receptor (p55/75). We evaluated the effects of DAB486IL-2 on the generation of tetanus toxoid (TT)-specific IgG antibody-forming cells in 6-day cocultures of human splenocytes and TT-coupled Sepharose beads. The results indicate that a significant portion (30-75%) of the anti-tetanus toxoid IgG response in vitro was susceptible to inhibition by 10(-10) M DAB486IL-2. The inhibition required both the IL-2 portion of the fusion protein and an active toxin moiety and was greater when the IL-2 toxin was added on Day 3 as compared with Day 0 of culture. The induction of the p55 (Tac) subunit of the IL-2R was demonstrable by two-color flow cytometry on a small percentage (5%) of B cells and on a higher percentage (10%) of non-B cells 3 days after exposure to TT-coupled Sepharose. Short-term (2 hr) treatment of T and B cell subpopulations separated on Day 3 of culture followed by remixing indicated that while activated T helper cells were most strongly inhibited by DAB486IL-2, up to 50% of the TT-specific IgG response was inhibited by treatment of B cells alone with DAB486IL-2. Our results suggest that a strategy of eliminating human memory B cells by a combination of antigen activation and properly timed administration of a recombinant lymphokine-toxin fusion protein is feasible.