Analysis of variable region of T-cell receptor beta chain usage in the human anti-porcine xenoresponse.

BACKGROUND: The human xenoreactive T-cell receptor (TCR) repertoire is not well documented. The aim of this study was to analyze the TCR repertoire in human anti-porcine xenoresponses. METHODS: Peripheral blood lymphocytes (PBLs) from healthy donors were used to generate human T-cell lines against two different haplotypes of inbred Yucatan miniature swine (y/y and z/z). The variable region of TCR beta-chain (Vbeta) gene usage was determined by fluorescence CDR3 spectrotyping. RESULTS: TCR Vbeta usage of an established human antiporcine T-cell line analyzed at weeks 5, 7, and 9 showed a sequential increase in Vbeta 1, 2, 6.2, 11, and 19 as compared to unprimed peripheral blood lymphocytes, whereas the usage of other Vbetas decreased. The selection of limited Vbeta genes correlated with the sequential increase in the specific lysis of the T-cell line, suggesting a non-random clonal selection and expansion of T-cell clones that recognized porcine targets. Different Vbeta restriction was found using the same peripheral blood lymphocytes against a different haplotype of swine, indicating this selection of Vbeta gene was swine leukocyte antigen-dependent. CONCLUSIONS: There is restricted TCR Vbeta usage in the human anti-porcine response, suggesting that a limited number of xenogeneic epitopes are recognized by human T cells. The selection of particular TCR Vbeta clonotypes depends on the swine leukocyte antigen background.     

Usage of T cell receptor variable segments of the beta-chain in IgA nephropathy

BACKGROUND: We previously reported that glomerulonephritis associated with Staphylococcus aureus infection (SAGN) showed an increased usage of T cell receptor Vbeta 5.3 and 8 in peripheral lymphocytes and mesangial IgA and IgG depositions. To elucidate the immunological mechanisms and pathogenesis of IgA nephropathy, we analyzed the usage of TCR Vbeta in both peripheral blood lymphocytes (PBLs) and renal infiltrating T cells from IgA-N patients. METHODS: In 38 patients with IgA nephropathy and controls, the usage of TCR Vbeta in PBLs were analyzed using monoclonal antibodies against Vbeta 3.1, Vbeta 5.1, Vbeta 5.2 + 5.3, Vbeta 5.3, Vbeta 6.7, Vbeta 8, Vbeta 12.1, and Vbeta 13.1 + 13.3 with three-color flow cytometry. Furthermore, we examined immunohistochemically renal biopsy specimens using antibodies against Vbeta 5.3 and Vbeta 8. RESULTS: The percentages of DR+CD4+CD8- cells, CD45RO+CD4+ cells, and CD45RO+CD4+DR+ cells in PBLs from IgA nephropathy were significantly higher than controls. The percentages of TCR Vbeta 5.3 positive cells and TCR Vbeta 8 positive cells in PBLs from patients were 1.3 +/- 0.1 and 3.1 +/- 0.2%, and both were significantly higher than controls. The percentage of renal interstitial TCR Vbeta 5.3 expression was significantly higher than that in PBLs. However, there was no significant difference between the TCR Vbeta 8 expression in the interstitium and that in PBLs. CONCLUSIONS: TCR Vbeta 5.3 and 8 usage and broad CD4+ T cell activation have occurred in IgA nephropathy. These changes were similar but weak compared with formerly reported SAGN.     

T cell repertoire expression in murine recipients of bone marrow transplant after LF 08-0299 (Tresperimus) administration

LF 08-0299 (Tresperimus), a novel immunosuppressive compound, has been previously shown to prevent graft-versus-host disease in murine models. In this study, we investigated the influence of LF 08-0299 on the TCR Vbeta repertoire of irradiated F1 recipient mice reconstituted with either syngeneic or parental bone marrow cells. We showed that a partial blockade of thymic differentiation occurred in normal mice under treatment at the transition CD4-/CD8- to CD4+/CD8+, and that this blockade was fully reversible. Despite the effect on the thymus, normal T cell repertoire negative selection was preserved following syngeneic bone marrow transplantation. We further assessed whether LF 08-0299 administration could modify Vbeta T cell expression in irradiated recipients reconstituted with parental bone marrow cells. In our murine parental to F1 transplant model, abnormal TCR Vbeta3, Vbeta5, Vbeta6 and Vbeta11 expression was demonstrated in peripheral lymph nodes of irradiated recipients. Moreover, Vbeta6 and Vbeta3 T cell populations were overexpressed. Administration of LF 08-0299 modified the pattern of Vbeta T cell expression. The expansion of Vbeta6 T cells was selectively inhibited under LF 08-0299 therapy and, in contrast, Vbeta5 T cells were overexpressed. Lymph node histological analysis showed that LF 08-0299 administration fully prevented the graft-versus-host reaction occurring in untreated recipient mice.     

Pretransplant donor peripheral blood mononuclear cells infusion induces transplantation tolerance by generating regulatory T cells

BACKGROUND: It was suggested that maintenance of tolerance to organ transplantation may depend on the formation of T regulatory cells. METHODS: Lewis (LW) rats were made tolerant to a Brown Norway kidney by pretransplant donor peripheral blood mononuclear cells (PBMC) infusion. At greater than 90 days after transplantation, lymph node cells (LN) and graft-infiltrating leukocytes (GIL) alloreactivity was tested in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype was analyzed by FACS. mRNA expression of cytokines and other markers was analyzed on CD4+ T cells from LN. The tolerogenic potential of tolerant cells in vivo was evaluated by adoptive transfer. RESULTS: Tolerant LN cells showed a reduced proliferation against donor stimulators but a normal anti-third-party alloreactivity. In coculture, these cells inhibited antidonor but not antithird-party reactivity of na?ve LN cells. Interleukin (IL)-10 and FasL mRNA expression was up-regulated in tolerant CD4+ T cells, but an anti-IL-10 monoclonal antibody (mAb) only partially reversed their inhibitory effect. Immunoregulatory activity was concentrated in the CD4+ CD25+ T-cell subset. In a transwell system, tolerant T cells inhibited a na?ve MLR to a lesser extent than in a standard coculture. Regulatory cells transferred tolerance after infusion into na?ve LW recipients. CD4+ T cells isolated from tolerized grafts were hyporesponsive to donor stimulators and suppressed a na?ve MLR against donor antigens. CONCLUSIONS: Donor-specific regulatory T cells play a role in tolerance induction by donor PBMC infusion. Regulatory activity is concentrated in the CD4+ CD25+ subset and requires cell-to-cell contact. Regulatory CD4+ T cells accumulate in tolerized kidney grafts where they could exert a protective function against host immune response.     

SLE nephritis is associated with an oligoclonal expansion of intrarenal T cells

Autoimmune systemic lupus erythematosus (SLE) nephritis is characterized by the influx of mononuclear inflammatory cell infiltrates within the glomeruli and renal interstitium. To evaluate the possibility that intrarenal T cells result from the expansion of lymphocytes using limited T-cell receptor (TCR) genes, we analyzed the TCR Vbeta gene expression among infiltrating lymphocytes in renal tissue compared with simultaneous peripheral blood lymphocytes of four children with new-onset SLE nephritis. The TCR Vbeta gene expression in peripheral blood T cells from patients with SLE nephritis, when compared with normal controls, showed no preferential expansion or deletion of select Vbeta gene families. In contrast, when paired peripheral blood and renal tissue were analyzed, intrarenal lymphocytes in SLE nephritis demonstrated evidence of expansion of select Vbeta gene families. Sequence analysis of the V(D)J joining regions of the TCRbeta with the expanded families demonstrated a striking oligoclonality. These observations suggest that infiltrating T cells within renal tissue may use TCR Vbeta genes targeted toward nephritogenic antigens.     

Deletion of donor‐reactive T cell clones after human liver transplant

We recently developed a high throughput T cell receptor β chain (TCRβ) sequencing‐based approach to identifying and tracking donor‐reactive T cells. To address the role of clonal deletion in liver allograft tolerance, we applied this method in samples from a recent randomized study, ITN030ST, in which immunosuppression withdrawal was attempted within 2 years of liver transplantation. We identified donor‐reactive T cell clones via TCRβ sequencing following a pre‐transplant mixed lymphocyte reaction and tracked these clones in the circulation following transplantation in 3 tolerant and 5 non‐tolerant subjects. All subjects showed a downward trend and significant reductions in donor‐reactive TCRβ sequences were detected post‐transplant in 6 of 8 subjects, including 2 tolerant and 4 non‐tolerant recipients. Reductions in donor‐reactive TCRβ sequences were greater than those of all other TCRβ sequences, including 3rd party‐reactive sequences, in all 8 subjects, demonstrating an impact of the liver allograft after accounting for repertoire turnover. Although limited by patient number and heterogeneity, our results suggest that partial deletion of donor‐reactive T cell clones may be a consequence of liver transplantation and does not correlate with success or failure of early immunosuppression withdrawal. These observations underscore the organ‐ and/or protocol‐specific nature of tolerance mechanisms in humans.     

Tolerance to rat heart grafts induced by intrathymic immunomodulation is mediated by indirect recognition primed CD4+CD25+ Treg cells

BACKGROUND: In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. METHODS: Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. RESULTS: Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. CONCLUSIONS: Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.     

Flt3-L augments the engraftment of donor-derived bone marrow cells when combined with sublethal irradiation and costimulatory (CD28/B7 and CD40/CD40L) blockade

T-cell costimulatory blockade as a constituent for recipient conditioning prior to bone marrow transplantation has led to the development of less toxic protocols for the establishment of donor cell chimerism. We therefore hypothesized that the addition of the hematopoietic growth factor, Flt3-ligand (Flt3-L), to the perioperative inhibition of the CD28/B7 and CD40/CD40 ligand costimulatory pathways would enhance the engraftment of allogeneic bone marrow. Recipient BALB/c ByJ (H-2(d), Mls(c), Vbeta6+/Vbeta8+ TCR) received a single sublethal dose of total body irradiation (300 rad) 6 h prior to transplantation IV with unfractionated donor CBA/J (H-2(k), Mls(d), Vbeta6-/Vbeta8+ TCR) bone marrow cells. CTLA4-Ig and/or MRI were administered at 500 microg IP on days 0, 2, 4, and 6 posttransplantation. Flt3-L was administered at 10 microg IP on days 0-6. Donor cell chimerism was determined on days 30-90 by flow cytometric analysis. Donor-specific tolerance was assessed by skin grafting. In vitro TCR cross-linking assays and flow cytometry were utilized to explore the deletion of donor-reactive T cells. Recipients receiving CTLA4-Ig and MRI engrafted allogeneic bone marrow cells in the peripheral blood (3/6; 50%) with chimerism being detected at 2-31%. Addition of Flt3-L to this preconditioning regimen enhanced the incidence of engraftment of donor bone marrow cells (10/13; 3-70%). Long-term survival of donor but not third-party-specific skin grafts demonstrated that donor-specific tolerance had been achieved in the chimeric recipients. Deletion of the donor-reactive T cells within the chimeric recipients was also observed. The addition of hematopoietic growth factors and cytokines to the nonmyeloablative regimen of sublethal irradiation and T-cell costimulatory blockade provides a novel strategy for the establishment of donor cell chimerism and for the induction of stable and robust donor-specific tolerance. The deletion of donor-reactive T cells using this protocol suggests the reliability and feasibility of this protocol for clinical transplantation.     

Tissue-specific peptides influence human T cell repertoire to porcine xenoantigens

BACKGROUND: Human CD8+ T cells elicit a vigorous response to allo- or xenogeneic MHC class I molecules. However, the influence of a given MHC-bound peptide to the responding allo- or xenoreactive T cell repertoire is not clear. METHODS: In this study, we analyzed individual T cell responses to unique tissue epitopes presented on syngeneic porcine endothelial and lymphoblastoid cells by limiting dilution analysis and analyzed the responding T cell repertoire by T cell receptor beta (TCR Vbeta) chain spectrotyping. RESULTS: Both porcine endothelial and lymphoblastoid cells were able to elicit swine leukocyte antigen (SLA) class I restricted and peptide-dependent cytotoxic T lymphocyte (CTL) responses. The responding human CD8+ T cells showed a heterogenous but limited TCR Vbeta gene usage. Interestingly, although a large portion of the selected TCR Vbeta gene usage in response to endothelial and lymphoblastoid cells were shared (i.e., Vbeta-1, 2, 6.1, 13), unique Vbeta usage was noted in T cells that respond to either endothelial (Vbeta-5.3) or lymphoblastoid cells (Vbeta-5.1, 11), suggesting that porcine tissue-specific epitopes play a role in modulating the responding T cell repertoire. Limiting dilution cloning analysis revealed that a majority (89%) of the CTL clones stimulated by porcine endothelial cells recognized shared peptides presented by both endothelial cells and syngeneic lymphoblastoid cells. However, a significant portion (11%) of the CTL clones recognized unique peptides presented only in the context of SLA class I molecules on endothelial cells. CONCLUSION: These results provide evidence for the first time that tissue-specific peptides can directly influence T cell repertoire in response to the xenogeneic stimulus.     

Transient Depletion of Dividing T Lymphocytes in Mice Induces the Emergence of Regulatory T Cells and Dominant Tolerance to Islet Allografts

We previously showed that transient depletion of dividing T cells at the time of an allogeneic transplantation induces long-term tolerance to the allograft. Here we investigated the role of homeostatic perturbation and regulatory T cells (Treg) in such tolerance. Transient depletion of dividing T cells was induced at the time of an allogeneic pancreatic islets graft, by administration of ganciclovir for 14 days, into diabetic transgenic mice expressing a thymidine kinase (TK) conditional suicide gene in T cells. Allograft tolerance was obtained in 63% of treated mice. It was not due to global immunosuppression, permanent deletion or anergy of donor-alloantigens specific T cells but to a dominant tolerance process since lymphocytes from tolerant mice could transfer tolerance to naïve allografted recipients. The transient depletion of dividing T cells induces a 2- to 3-fold increase in the proportion of CD4⁺CD25⁺Foxp3⁺ Treg, within 3 weeks that persisted only in allograft-bearing mice but not in nongrafted mice. Tolerance with similar increased proportion of Treg cells was also obtained after a cytostatic hydroxyurea treatment in normal mice. Thus, the transient depletion of dividing T cells represents a novel means of immuno-intervention based on disturbance of T-cell homeostasis and subsequent increase in Treg proportion.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

Study of T cell receptor in patients with acute rejection after renal transplantation

Objective To evaluate the immune status of acute rejection recipients, and to improve the short-term and long-term survival rate of renal transplant recipients and grafts, and to investigate dynamically the changes in the immune repertoire of patients with acute rejection. Methods Combined multiplex PCR amplification technique and high throughput sequencing technique, the TCR β chain complementarity determining region 3 (CDR3) diversity and repertoire characteristics at different time points during renal transplantation were analyzed, in order to reveal the immunological characteristics of T lymphocytes in patients with acute rejection. Results The diversity of TCR CDR3 in acute rejection patients was reduced to the lowest one day after surgery. The diversity of TCR CDR3 before acute rejection was higher than before. The acute rejection-related up-regulated TCR CDR3 amino acid sequences were screened out. In addition, TCR beta chain V and J subfamily showed the phenomenon of advantage usage in pre-acute rejection, which may be due to T cell recognition of transplanted kidney antigens in vivo. Conclusions The immune diversity of patients with acute rejection is significantly lower. In addition, TCR beta chain V and J subfamily show the phenomenon of advantage usage.     

Liver allografts are toleragenic in rats conditioned with posttransplant total lymphoid irradiation

BACKGROUND: Posttransplant total lymphoid irradiation (TLI) treatment has been applied to tolerance induction protocols in heart and kidney transplantation models. METHODS: We examined the efficacy and mechanism of posttransplant TLI treatment in the induction and maintenance of tolerance in a rat orthotopic liver transplantation model. RESULTS: Posttransplant TLI prolonged ACI (RT1(a)) liver allograft survival in Lewis (RT1(b)) hosts, with 50% long-term engraftment without immunosuppression and without evidence of chronic rejection. Injection of donor-type liver mononuclear cells (LMCs) facilitated the prolongation of graft survival, with more than 70% of grafts in LMC recipients surviving more than 100 days without chronic rejection. Recipients with long-term liver allograft survival accepted ACI but not PVG skin grafts. In TLI-conditioned recipients with accepted grafts, apoptosis occurred predominantly in graft-infiltrating leukocytes. In contrast, there were few apoptotic leukocytes in rejecting grafts. Recipients with long-term graft acceptance (>100 days of survival) demonstrated evidence of immune deviation; mixed lymphocyte reaction to ACI stimulator cells was vigorous, but secretion of interferon-gamma and interleukin-2 was reduced. In tolerant recipients, the number of Foxp3(+) CD25(+) CD4(+) regulatory T cells was increased in the liver allograft as well as in the peripheral blood. CONCLUSION: We conclude that posttransplant TLI induces tolerance to liver allografts via a mechanism involving apoptotic cell-deletion and immunoregulation.     

Allochimeric therapy induces unique regulatory T cells that mitigate chronic rejection

Induction of tolerance to an allogeneic graft without the need for nonspecific immunosuppression is a major goal of transplantation therapy. We have shown that treatment with molecularly engineered, allochimeric [alpha(1h)(1/u)]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.A(u)) or Lewis (LEW, RT1A(1)) amino acid substitutions for host-type ACI (RTI.A(a)) sequences in the alpha(1)-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. The mechanisms involved in the establishment and maintenance of specific allograft tolerance are still not fully understood. It is now clear that acquisition of transplantation tolerance is an active, highly regulated, multistep process. According to the pool size model of allograft tolerance, the allograft outcome, rejection, or tolerance often depend on the balance between cytopathic and regulatory T cells (T-regs). This study examined mechanisms of chronic rejection (CR) development on a model of cardiac transplant tolerance after adoptive transfer of T-regs followed by allochimeric therapy. Generation of T-regs was demonstrated in vitro by MLR coculture and confirmed by adoptive transfer of T cells from primary recipients to secondary hosts. To confirm the true nature of regulatory cells, we performed a second transfer into tertiary recipients. Unlike T-regs from tolerant hosts, T cells from naive rats did not prolong graft survival. Histological evaluation of T-regs-transfected groups showed absence of visible CR. In contrast, T-regs generated in recipients after high-dose cyclosporine treatment failed to inhibit CR in transferred singeneic recipients. Allochimeric therapy triggers generation of unique regulatory lymphocytes that mitigate development of chronic rejection through regulation of anti-inflammatory mechanisms and down-regulation of alloantibody response.     

Anti-CD28 antibody-induced kidney allograft tolerance related to tryptophan degradation and TCR class II B7 regulatory cells.

B7/CTLA-4 interactions negatively regulate T-cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short-term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti-MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third-party cells. The depletion of B7+, non-T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose-dependently inhibited alloreactivity. PBMC anti-donor reactivity could be partially restored in vitro by blocking indoleamine-2,3-dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7+ non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.