CD4+CD25+Foxp3+ indirect alloreactive T cells from renal transplant patients suppress both the direct and indirect pathways of allorecognition.

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)-10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)-DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA-DR peptides, dependent on IL-4 or IL-10, suggesting regulatory activity as part of the alloreactive T-cell repertoire. PBMC-derived indirect alloreactive T-cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T-cell lines which were able to inhibit both direct and indirect alloproliferation of another T-cell line from the same patient presented a CD4(+)CD25(+)Foxp3(+) T-cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.

CD4+CD25+Foxp3+ indirect alloreactive T cells from renal transplant patients suppress both the direct and indirect pathways of allorecognition

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)-10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)-DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA-DR peptides, dependent on IL-4 or IL-10, suggesting regulatory activity as part of the alloreactive T-cell repertoire. PBMC-derived indirect alloreactive T-cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T-cell lines which were able to inhibit both direct and indirect alloproliferation of another T-cell line from the same patient presented a CD4+CD25 +Foxp3+ T-cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.

Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graft-infiltrating responses.

Autoreactivity to heat shock protein 60 (Hsp60) has been implicated in the pathogenesis and regulation of chronic inflammation, especially in autoimmune diseases. In transplantation, there is a lack of information regarding the cytokine profile and specificity of cells that recognize self-Hsp60 as well as the kinetics of autoreactivity following transplantation. We studied the cellular reactivity of peripheral and graft-infiltrating lymphocytes against Hsp60 in renal transplant patients. Cytokine production induced by this protein in peripheral blood mononuclear cells indicated a predominance of interleukin (IL)-10 during the late post-transplantation period, mainly in response to intermediate and C-terminal peptides. Patients with chronic rejection presented reactivity to Hsp60 with a higher IL-10/interferon (IFN)-gamma ratio compared to long-term clinically stable patients. Graft-infiltrating T cell lines, cocultured with antigen-presenting cells, preferentially produced IL-10 after Hsp60 stimulation. These results suggest that, besides its proinflammatory activity, autoreactivity to Hsp60 in transplantation may also have a regulatory role.

T-cell response to self HSP60 peptides in renal transplant recipients: a regulatory role?

T-cell responses to heat shock proteins (Hsp) have been suggested to play a role not only in inflammatory conditions, but also in various human autoimmune diseases and in the allograft response. Previous data from our group suggested that during the early posttransplantation (post-Tx) period (<6 months post-Tx), the anti-Hsp60 T-cell repertoires in renal transplant recipients were predominantly proinflammatory. In the later period, they were predominantly regulatory. In agreement with our results, diversification of the T-cell responses toward the carboxy-terminal determinants of Hsp60, related to the resolution of the inflammatory process, was shown in an experimental model of adjuvant arthritis. It has not been clarified whether this diversification is also present in transplantation. In this context, our objective was to analyze cytokine production against autologous Hsp60 peptides from different regions of the protein, using peripheral blood mononuclear cells of 9 renal transplant recipients at 2 timepoints after transplantation: early (<6 months) and late (>1 year). IFN gamma production induced by Hsp60 peptides was observed in 71% and 75% of the patients in the early and late post-Tx periods, respectively. Interleukin (IL)-10 production induced by Hsp60 peptides was observed in 28% of the patients in the early period and in 62% in the late period. Interestingly, the production of IL-10 was induced mainly by peptides of the intermediate and the C-terminal regions. This suggests a predominance of autoreactive regulatory anti-Hsp T-cell repertoire in the late post-Tx period, which predominantly recognize peptides from the intermediate and C-terminal regions of the protein.

Predominant IL-10 production in indirect alloreactivity is not associated with rejection.

In a prospective study of indirect alloresponse in renal transplantation, we detected proliferation and cytokine production to donor and third-party HLA-DR peptides unrelated to rejection. Twenty of 28 patients (71%) presented proliferation, 29% before and 71% after transplantation. Half of the patients also presented proliferation to third-party peptides. Indirect alloresponse was also detected in 75% of healthy individuals (HI). Variability of response was observed in patients and HI for both proliferation and cytokine production. IL-10 predominance was observed in indirect alloresponses to donor peptides pre- and post-Tx, in contrast with more IFN-gamma and TGF-beta being detected in HI. IL-10 production was frequently detected without proliferation, in contrast with more frequent proliferation being found with IFN-gamma and TGF-beta production. The lack of association of either cytokine or proliferation with rejection, together with the predominance of IL-10 unrelated to proliferation, suggests that regulatory cells may be part of the T cell repertoire involved in indirect alloreactivity.

Renal transplant patients show variations in their self-reactive repertoires: a serial study.

We addressed the question of whether allo-transplantation (Tx) induces breakdown of tolerance to self-antigens or alteration of the autoreactive T cell repertoire in humans. The serial variation of T cell autoreactivity was studied in the peripheral blood of 12 renal transplant patients, by autologous limiting dilution assay and autologous mixed lymphocyte reaction. Ten of 12 patients presented a positive response in autologous peripheral blood mononuclear cells in the post-Tx period, in contrast to four of 12 patients before Tx (P = 0.038). Multi-hit kinetics was found in 57% of the assays analyzed, indicating frequent regulatory control of the autologous response. Quantitative analysis performed in eight patients showed an increase in precursor frequency at >1 year post-Tx in five patients. These data indicate that autoreactivity increases or develops following Tx, in humans. Post-Tx events such as alloreactivity, infections or immunosuppression could interfere with the balance of autoreactive and regulatory cells, leading to changes in the T cell repertoires to self-antigens and eventually breakdown of self-tolerance. Further investigation is needed to elucidate whether post-Tx autoreactivity contributes to rejection, plays a regulatory role over alloreactivity or both, at separate times.

Two novel anti-von Willebrand factor monoclonal antibodies.

Von Willebrand Factor is a multimer produced by endothelial cells and megakaryocytes, being stored in intracellular organelles, such as the Weibel-Palade bodies and alpha-granules in endothelial cells and platelets, respectively. This molecule acts as a carrier protein for factor VIIIc, involved in the intrinsic pathway of blood coagulation maintaining its stability in circulation. Von Willebrand Factor also plays an important role in platelet aggregation and adhesion to injured vessel wall. It interacts with platelets through two distinct glycoproteins, GPIb and GPIIb/IIIa. We raised two monoclonal antibodies, ECA-3 and ECA-4, against human umbilical vascular endothelial cells that recognize and immunoprecipitate von Willebrand Factor. Interestingly, ECA-4 monoclonal antibody is able to completely inhibit platelet agglutination induced by ristocetin, suggesting that it binds to von Willebrand Factor close to platelet GPIb binding site. The use of monoclonal antibodies to identify von Willebrand Factor binding regions to factor VIII or platelets has been reported by others. In pulmonary hypertension, abnormalities have been detected on the multimeric structure of the molecule as well as on its proteolytic fragments, by using monoclonal antibodies. Moreover, monoclonal antibodies raised against specific regions of von Willebrand Factor molecule may allow studies of functional abnormalities of this protein in inherited and acquired disorders like subtypes of von Willebrand's disease.

Evidence of indirect allorecognition in long-term human renal transplantation.

The purpose of this study was to investigate indirect alloreactivity in the peripheral blood of long-term renal transplanted patients. We evaluated the T cell proliferative response to a whole pool of donor cell-derived allopeptides, processed and presented by host antigen-presenting cells (APC), rather than to synthetic peptides. For the indirect pathway, proliferation assays were performed using APC-depleted donor cells. Indirect alloreactivity was detected in 57% (8/14) of the patients, 6 of whom presented no evidence of rejection, but 2 patients had a diagnosis of chronic rejection. In 4 of 8 positive cases (50%), proliferation was detected with 5 days of culture, and sometimes indirect alloresponse was the dominant route. We present evidence that the indirect alloproliferative response to a pool of naturally processed donor peptides is present in the peripheral blood of long-term renal transplanted patients irrespective of rejection.

Mechanisms of indirect allorecognition: characterization of MHC class II allopeptide-specific T helper cell clones from animals undergoing acute allograft rejection.

BACKGROUND: Recent evidence indicates that T cells primed via the indirect pathway of allorecognition play an important role in allograft rejection, although the effector mechanisms remain unknown. The purpose of this study was to characterize and study the in vivo function of self-restricted MHC allopeptide-specific T-cell clones generated from animals undergoing allograft rejection. METHODS AND RESULTS: We generated self-restricted class II MHC allopeptide-specific T-cell clones from the spleen and kidney of Lewis (LEW; RT1l) rats undergoing acute rejection of MHC-incompatible Wistar Furth (WF; RT1u) renal allografts. RT1.Du/beta20-44 peptide-specific CD4+ T helper 1 clones from the spleen and kidney of rejecting animals expressed a restricted T cell receptor (TCR) Vbeta repertoire: Vbeta4, 8.2, or 9. In comparison, clones generated from RT1.Dubeta20-44 immunized LEW rats all expressed TCR Vbeta9. The amino acid sequence of RT1.Dl (LEW) and RT1.Du (WF) residues 20-44 differ only at positions 30 and 38. T-cell clones expressing TCR Vbeta9 preferentially proliferated to the peptide fragment RT1.Dubeta20-33. T-cell clones expressing TCR Vbeta4 proliferated weakly to peptide fragments RT1.Dubeta20-33 and 31-44, whereas those expressing TCR Vbeta8.2 proliferated preferentially to the peptide fragment 31-44. Adoptive transfer of T-cell clones expressing TCR Vbeta9 or Vbeta8.2, but not Vbeta4, to naive LEW animals elicited significant delayed-type hypersensitivity responses after challenge with the RT1.Dubeta20-44 peptide or allogeneic WF (RT1u) splenocytes. CONCLUSION: This is the first report on the cellular, molecular, and functional characterization of self-restricted MHC allopeptide-specific T-cell clones from animals undergoing acute rejection. Our data provide support for a biologically significant role of indirect allorecognition in allograft rejection.

Indirect allorecognition of major histocompatibility complex allopeptides in human renal transplant recipients with chronic graft dysfunction.

BACKGROUND: It has been suggested that T cells primed by processed donor major histocompatibility complex antigen (the "indirect" pathway of allorecognition) may be responsible for mediating chronic allograft rejection. The purpose of this study was to develop a clinically useful assay to study the occurrence of indirect allorecognition during chronic rejection in humans. METHODS: A panel of 20 mer peptides corresponding to the hypervariable regions of HLA-DRB1*0101, DRB1*1501, and DRB1*0301 were synthesized. Lymphocytes obtained from renal allograft recipients were cocultured with these peptides. Proliferation was assayed by DNA incorporation of [3H]thymidine, and positive proliferation was defined by a statistically significant increase in counts per minute over background with a minimum stimulation index of 2. The precursor frequency of allopeptide reactive T cells was determined by limiting dilution analysis. RESULTS: Lymphocytes from 82% of patients who were mismatched for at least one of the three DR molecules and had chronic allograft dysfunction specifically proliferated to the mismatched allopeptides (n=11). Proliferation was seen in only 6% of control subjects (2/33, P<0.0001). The proliferative response was low grade and was best detected on day 7-8 of culture in vitro. The precursor frequency of peptide-specific T cells was more than 10-fold higher compared with controls (P<0.001). CONCLUSIONS: These data demonstrate for the first time that T cells of patients with chronic graft dysfunction are primed to recognize and respond to specific donor-derived major histocompatibility complex allopeptides. Our results support the hypothesis that T cells primed via the indirect pathway of allorecognition may be important mediators of chronic rejection and provide the rationale to develop specific therapeutic strategies to prevent or interrupt this process.

Reactivity of renal transplant sera against a 17 kD mononuclear cell antigen.

In recent years, studies have shown that non-HLA antigens can be involved in renal graft rejection. The so called minor antigens have been found to be expressed on a variety of cells, including endothelial cells. With the aim of understanding better the role of minor antigens in graft rejection, we undertook a Western blot screening of sera from patients waiting for or having received grafts from living-related or cadaveric donors. In this study we described the reactivity of these sera against a 17 kD antigen with expression in many different cell types.