H.A DePaz

Induction of Transplant Tolerance with Immunodominant Allopeptide-pulsed Host Lymphoid and Myeloid Dendritic Cells

We have studied the effects of adoptive transfer of host thymic dendritic cells pulsed with immunodominant WF Class I peptide 5 (residues 93–109) on cardiac allograft survival in the WF‐to‐ACI rat combination. Our results showed that, whereas intrathymic inoculation of WF peptide 5‐pulsed ACI thymic dendritic cells alone on day −7 did not prolong graft survival, similar treatment combined with 0.5 mL antilymphocyte serum (ALS) led to 100% permanent acceptance (> 200 d) of donor‐specific cardiac allografts. Extension of our study to systemic administration of peptide 5‐pulsed host thymic dendritic cells confirmed that intravenous injection of peptide 5‐pulsed self thymic dendritic cells combined with ALS transient immunosuppression resulted in 100% permanent donor‐specific graft survival (> 200 d). These results were reproducible in a clinically relevant model using intravenous injection of peptide‐pulsed host myeloid dendritic cells. In contrast, thymectomy prior to adoptive transfer of peptide‐pulsed host dendritic cells resulted in acute graft rejection at times equivalent to rejection in thymectomized controls. The long‐term unresponsive recipients challenged with second‐set grafts accepted permanently (> 100 d) donor‐type (WF) but not third party (Lewis) cardiac allografts. This study suggests that intravenous administration of genetically engineered dendritic cells expressing donor MHC molecules has the potential of inducing transplant tolerance.

Immature rat myeloid dendritic cells generated in low-dose granulocyte macrophage-colony stimulating factor prolong donor-specific rat cardiac allograft survival.

Background. Because the differential polarization of T cells in response to antigen presentation is dependent on the maturational state of dendritic cells (DCs), we hypothesized that the adoptive transfer of immature myeloid DCs (iMDCs) would prolong graft survival. Methods. To evaluate this hypothesis, we studied the effects of transfer of iMDCs and mature myeloid DCs (mMDCs) on rat cardiac allograft survival. Results. Whereas iMDCs that do not express costimulatory molecules induce allogeneic T-cell hyporesponsiveness in coculture studies, mMDCs that express high levels of major histocompatibility complex class II costimulatory and maturation molecules induce a robust allostimulatory T-cell response. Adoptive transfer of Wistar Furth iMDCs, unlike mMDCs, 7 days before cardiac transplantation significantly prolonged graft survival. It was important that adoptive transfer of iMDCs combined with 0.5 mL antilymphocyte serum (ALS) transient immunosuppression on day −7 led to donor-specific permanent graft survival in 50% of recipients. In contrast, adoptive transfer of mMDCs combined with ALS led to graft survival similar to that in recipients treated with ALS alone. Stimulation of CD4+ T cells isolated from the spleen of unresponsive allograft recipients with donor antigen resulted in donor-specific hyporesponsiveness and production of interleukin (IL)-10 and transforming growth factor-&bgr; but not IL-4 and interferon-&ggr;. The tolerant T-cell unresponsiveness was reversed by the addition of IL-2. Conclusion. Our data confirming the immunoregulatory effect of immature DCs indicate that induction of transplant tolerance by iMDCs is partly dependent on in vivo generation of regulatory T cells. This finding suggests that immunization with immature donor DCs has therapeutic potential for the induction of transplant tolerance and treatment of autoimmune diseases.

Role of reentry of in vivo alloMHC peptide-activated T cells into the adult thymus in acquired systemic tolerance.

BACKGROUND T cell recognition of alloMHC peptide presented by self dendritic cells via the indirect pathway of allorecognition in the thymus induces T cell tolerance. Most recently we have shown that the i.v. administration of immunodominant Wistar Furth MHC class I (RT1.Au) peptide 5- (P5) pulsed myeloid or lymphoid dendritic cells induces operational tolerance to a fully MHC-mismatched cardiac allograft. This finding led us to hypothesize that circulation of peripheral P5-activated T cells to the thymus plays an important role in the induction of acquired tolerance. METHODS We used the adoptive transfer of 111Indium-oxine- (111In-oxine) labeled P5-pulsed syngeneic dendritic cells and in vivo P5-activated syngeneic T cells to study the role of their circulation to the thymus in the induction of transplantation tolerance. RESULTS Intravenously administered 111In-oxine-labeled naïve DC actively migrated to and localized in the liver and spleen but did not enter the lymph nodes, bone marrow, and thymus. In vitro peptide-pulsed dendritic cells had a similar pattern of tissue localization except for a modest number of myeloid but not lymphoid DC entering the thymus. The demonstration that adoptive transfer of in vivo peptide-primed T cells induces permanent graft survival in antilymphocyte serum transiently immunosuppressed syngeneic secondary hosts led us to examine the traffic of in vivo activated T cells. Whereas naïve syngeneic T cells preferentially homed to the peripheral lymphoid organs, they did not reenter the thymus. In contrast, in vivo peptide-activated peripheral T cells migrated to and accumulated in the thymus, thus confirming that reentry of T cells to the thymus is restricted to in vivo activated T cells. Although antilymphocyte serum immunosuppression significantly reduced circulation of primed T cells to the thymus, it did not completely abolish it, as seen with gamma-irradiated primed T cells. CONCLUSION These findings provide the first formal evidence directly linking reentry of in vivo alloMHC peptide-activated T cells to the thymus with the induction and possibly maintenance of acquired antigen-specific tolerance. Our results suggest that the thymus is open to a two-way traffic with the periphery and may function as a repository of immunological memory.

CD4+CD25+ regulatory T cells mediate acquired transplant tolerance

The Holy Grail of clinical organ transplantation is the safe induction of allograft tolerance. Transplant tolerance has been successfully induced in animal models. Since T cells play a pivotal role in graft rejection, modulating T cell function has been the primary focus of studies aimed at inducing transplant tolerance. Rodent models of transplant tolerance induction include central deletion and peripheral mechanisms involving activation-induced cell death (AICD), anergy, immune deviation, and production of regulatory T cells. These mechanisms are not mutually exclusive. Although clonal deletion and anergy limit self-reactive T cells in the thymus, these mechanisms alone are not sufficient for controlling self-reactive T cells in the periphery. There is now evidence that the adult animal harbors two functionally distinct populations of CD4(+) T cells; one mediates autoimmune disease and the other dominantly inhibits it. The latter cells express CD4, CD25 and CTLA-4. These thymus-derived T cells have recently been shown to mediate the induction and maintenance of transplant tolerance. These CD4(+)CD25(+) T cells are similar in origin, phenotype, and function to those that maintain natural self-tolerance and T cell homeostasis in the periphery. Against this background, is it possible that alloantigen specific regulatory T cells might be generated and expanded ex vivo before organ transplantation and then infused to induce long-term tolerance, perhaps without the need for chronic immunosuppression?

Role of CD4+CD25+ regulatory T cells from naive host thymus in the induction of acquired transplant tolerance by immunization with allo-major histocompatibility complex peptide.1

Background. Immunization with allo-major histocompatibility complex peptide induces operational tolerance, whereas thymectomy abrogates this effect. We hypothesized that recent thymic emigrants with regulatory function are important in the induction of acquired transplant tolerance in this system. Methods. In this study, we examined the possibility of restoring transplant tolerance to thymectomized (TMX) ACI recipients with concomitant adoptive transfer of syngeneic T cells indirectly primed with a single immunodominant Wistar Furth allo-major histocompatibility complex class I peptide (peptide 5, residues 93–109) and unmodified thymocytes or CD4+CD25+ thymic T cells. Results. Co-transfer of in vivo allopeptide-primed T cells and naive syngeneic thymic T cells on day −7 restored permanent acceptance of cardiac allografts to 70% of transiently antilymphocyte serum-immunosuppressed TMX recipients. Similarly, the adoptive transfer of allopeptide-primed T cells led to 100% donor-specific permanent graft acceptance among transiently antilymphocyte serum-immunosuppressed TMX recipients with renal subcapsular syngeneic thymic grafts. To demonstrate the role of regulatory T cells among new thymic emigrants in the induction of tolerance, we showed that the co-transfer of CD4+CD25+ but not CD4+CD25− thymic T cells with allopeptide-primed syngeneic T cells restored tolerance to TMX recipients. It seems that the induction of transplant tolerance in this system is dependent on the presence of CD4+CD25+ regulatory T cells among the recent thymic emigrants. Conclusions. This study suggests that CD4+CD25+ regulatory T cells specific for the induction of transplant tolerance are similar in origin, phenotype, and function to those involved in the maintenance of self-tolerance and the prevention of autoimmunity.

Mechanisms of acquired thymic tolerance: induction of transplant tolerance by adoptive transfer of in vivo allomhc peptide activated syngeneic T cells.

Background. Our most recent observation that i.v. injection ofWistar-Furth (WF) major histocompatibility complex Class I peptide 5(P5)-pulsed self-myeloid or lymphoid dendritic cells (DC) inducestransplantation tolerance suggests that adoptive transfer of in vivoallopeptide-primed host T cells might induce acquired tolerance through theirinteraction with thymicDC. Methods. To examine this hypothesis, host myeloid DC cultured in ratgranulocyte/macrophage colony stimulating factor and interleukin 4 were pulsedin vitro with P5 and injected intravenously into syngeneic ACI rats. The Tcells primed to P5 via the indirect pathway of allorecognition were harvested7 days later and administered by either intravenously or intrathymically intosyngeneic ACI recipients of WF cardiacallografts. Results. Syngeneic T cells obtained from the spleen of P5-primedrats had a high mixed lymphocyte reaction proliferative response to P5presented by self-DC. I.v. administration of2×10 7 P5-primed alloreactive purified hostsplenic T cells alone on day −7 significantly(P <0.001) prolonged cardiacallograft survival from 10.5±1.0 days to 18.6±1.8 days in theWF-to-ACI rat combination. I.v. injection of P5-activated host T cellscombined with 0.5 ml antilymphocyte serum (ALS)-transient immunosuppression onday −7 led to 100% donor-specific permanent graft survival (>200days). Thymectomy before i.v. injection of P5-activated syngeneic T cells ledto acute graft rejection, suggesting that the homing of in vivo activated Tcells to the host thymus might play a role in the induction of tolerance. Tofurther define the role of the recipient thymus in this model, we examined theeffects of intrathymic (i.t.) injection of P5-primed alloreactive T cells ongraft survival and found that i.t. administration of the P5-primed T cells onday −7 alone significantly prolonged graft survival (15.0±0.7days) and when combined with 0.5 ml ALS led to donor-specific permanent graftsurvival. The long-term unresponsive recipients permanently (>100 days)accepted second-set donor-specific cardiac allografts but not third-party(Lewis)grafts. Conclusions. These findings demonstrate that the adoptive transfer ofsplenic T cells primed to an indirectly presented donor peptide inducestransplantation tolerance in a transiently immunosuppressed secondarysyngeneic recipient. Our data suggest that the interaction of thymic DC withactivated peripheral T cells induces alloantigen (Ag)-specific T-celltolerance by either inactivation or deletion of alloreactive T cells in thethymus. This observation provides the first formal evidence that theinteraction between thymic DC and activated peripheral T cells thatcontinuously circulate through the thymus plays an important role in theinduction and maintenance of Ag-specifictolerance.

Breast Cancer in Women with Human Immunodeficiency Virus Infection: Pathological, Clinical, and Prognostic Implications.

BACKGROUND AIDS and breast cancer have become two important public health issues for women. Of interest is the prolonged survival of patients diagnosed with HIV infection as a result of the use of highly active antiretroviral therapy (HAART). With improved survival, we are likely to see more HIV-infected patients with breast cancer. METHODS This study, which is a review of our experience at Harlem Hospital Center, New York, between 2000 and 2008, compared HIV-positive with HIV-negative breast cancer patients, with attention to tumor size, stage, grade, molecular markers and lymphovascular invasion, treatment, and patient survival. RESULTS Only 63 of 370 patients with breast carcinoma were tested for HIV, and 6 of the 63 women tested positive for HIV. We, therefore, compared the clinical features and tumor characteristics seen in the 6 HIV-infected women with those of the 57 HIV-seronegative breast cancer patients. We found no differences in presentation, median age, and tumor morphology in the two groups of patients. When the patients in our previous report on 5 HIV-positive breast cancer patients were added to the present group, the overall 5-year survival rate among the 11 HIV-infected patients was 75%. Of note is the finding that HIV infection in premenopausal women was not associated with aggressive breast cancer subtypes with poor survival outcome. CONCLUSIONS These results demonstrate that histological subgroups and 5-year survival appear similar among HIV-positive breast cancer patients.

New strategies in immune tolerance induction

Induction of tolerance in clinical organ transplantation that will obviate the use of chronic immunosuppression and preserve host immune response to other antigens remains the goal of transplant research. The thymus plays a critical role in the ability of the immune system to discriminate between self- and nonself-antigens or harmful and harmless alloantigens. We now know that multiple factors determine how the immune system responds to a self-antigen or foreign antigen. These determinants include developmental stage of the host, stage of T-cell maturity, site of antigen encounter, type and maturity of antigen-presenting cells, and presence and type of costimulatory molecules. Our understanding of the mechanisms of T-cell interactions with peptide/major histocompatibility complex in peripheral lymphoid organs has led to experiments that translate into peripheral T-cell tolerance. The induction of high-avidity peripheral alloreactive T cells in the early phase of organ transplantation makes it difficult to achieve long-term alloantigen-specific tolerance without the use of transient perioperative immunosuppression. Therefore, protocols that induce robust tolerance in rodent and nonhuman primate models involve the use of donor antigen combined with a short course of perioperative immunosuppression. These studies suggest that the underlying mechanisms of peripheral tolerance include deletion, anergy, immune deviation, and regulatory T cells. This review focuses on recent advances in tolerance induction in experimental animal models and discusses their relevance to the development of protocols for the induction and maintenance of clinical transplant tolerance.

Peritransplant streptavidin recipient treatment prolongs rat cardiac allograft survival

Background. Because streptavidin shows high localization in inflamed tissues, it might also interfere with the proliferation of cells involved in allograft rejection. Methods and Results. Treatment of naïve ACI recipients with 20 mg/kg streptavidin i.p. alone significantly prolonged Lewis cardiac allografts from a mean survival time of 9.8±0.7 days in controls to 19.8±6.5 days, with one recipient accepting the graft permanently (>250 days). Peritransplant streptavidin treatment combined with 0.5 ml of antilymphocyte serum (ALS) transient immunosuppression led to permanent graft survival (>250 days) in 6 of 10 recipients. Second-set skin grafts performed 60 days after the primary cardiac allograft were prolonged to 45 days, whereas the third party Wistar-Furth (WF) skin grafts were rejected in 15 days without the rejection of the primary Lewis cardiac allografts. Pathology of transplanted cardiac allografts at 100 days showed no mononuclear cell infiltration or chronic allograft vasculopathy. Streptavidin given for 5 days at 20 mg/kg caused a moderate initial weight loss but had no effect on hematologic, biochemical, and histologic parameters in the treated recipients. Conclusion. This study demonstrates that peritransplant recipient treatment with streptavidin combined with peritransplant ALS induces prolonged cardiac and second-set skin allograft survival. We conclude that recipient peritransplant streptavidin treatment may provide a new strategy for the induction of transplant tolerance.

EFFECT OF STREPTAVIDIN ON CARDIAC ALLOGRAFT PROLONGATION IS DUE TO HOST T CELL SUPPRESSION.

AIM The aim of this study was to evaluate the effectiveness of streptavidin immunomodulation in the high-responder WF-to-Lewis combination. METHODS/RESULTS We examined the effects of streptavidin on the proliferative response of T cells in coculture studies. Two to 200 microg/mL streptavidin significantly (P < .001) suppressed the proliferation of Lewis T cells to WF by 76%-83% compared with untreated responders. Next, we studied the survival of WF cardiac allografts in Lewis recipients pretreated with streptavidin. A 5-day course of peritransplantation recipient treatment with streptavidin doses of 8, 12, 20, 40, and 60 mg/kg combined with single dose of 0.5 mL antilymphocyte serum (ALS) significantly (P < .001) prolonged cardiac allograft survival from MST of 7 +/- 0.5 and 8 +/- 0.5 days in naive and ALS-treated controls to 15 +/- 1, 20 +/- 3, 16 +/- 3, 17 +/- 3, and 23 +/- 2 days, respectively. In contrast, posttransplantation administration of 80 mg/kg streptavidin resulted in animal death, suggesting toxicity of this dose. Additionally, 10 mg/kg or 20 mg/kg streptavidin administration for 10 consecutive days resulted in significant graft prolongation (MST of 18 +/- 1 and 21 +/- 1 days, respectively; P < .001). CONCLUSION Although peritransplantation streptavidin treatment is effective in prolonging rat cardiac allografts in the high-responder WF-to-Lewis combination, it does not induce permanent graft survival as observed in the low-responder combination of Lewis-to-ACI. Our finding of in vitro immunomodulatory effect of streptavidin on T-cell proliferation suggests that its in vivo effect is partly due to prevention of T-cell activation following antigen exposure.