Mel Garrovillo

Indirect allorecognition in acquired thymic tolerance: induction of donor-specific tolerance to rat cardiac allografts by allopeptide-pulsed host dendritic cells.

BACKGROUND Presentation of peptides either by recipient or donor MHC molecules displayed on the surface of antigen-presenting cells is an essential element in the induction of T cell responses to transplant antigens. The finding that intrathymic (IT) injection of an immunodominant peptide induces acquired thymic tolerance suggests an indirect pathway of allorecognition in the thymus. To address this theory, we studied the effects of IT injection of host bone marrow (BM)-derived dendritic cells (DC)-pulsed with the immunodominant Wistar Furth (WF) MHC class I (RT1.Au) peptide 5 (93-109) on cardiac allograft survival in the WF-to-ACI rat combination. METHODS DC were propagated from cultures of ACI (recipient) bone marrow (BM) maintained in a medium supplemented with granulocyte-macrophage colony-stimulating factor and IL-4. The BM-derived DC after 8 days of culture were pulsed in vitro with a single WF MHC class I peptide (Residue 93-109) with the dominant epitope, washed, and injected into the thymus of ACI rats. The ACI recipients received donor-type (WF) or 3rd party (Lewis) cardiac allografts 7 days after IT immunization with peptide-pulsed DC. RESULTS BM-derived DC cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 for 8 days have a strong allostimulatory ability and present peptide 5 to naive syngeneic T cells in mixed lymphocyte reaction. IT inoculation of 300 microg RT1.Au peptide 5 combined with transient antilymphocyte serum immunosuppressive therapy induced donor-specific tolerance to cardiac allografts. Extension of this finding to peptide-pulsed self DC showed that IT injection of peptide 5-pulsed host DC consistently led to permanent acceptance (>150 days) of donor-type (WF) cardiac allografts, whereas third-party (Lewis) grafts were acutely rejected. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (>100 days) donor-type(WF) grafts while rejecting third-party (Lewis) grafts without the rejection of the primary WF grafts. CONCLUSION This novel finding that allopeptide-pulsed host DC induces tolerance to cardiac allografts suggests that the induction of acquired tolerance is dependent on the indirect allorecognition pathway. The results further suggest that genetically engineered DC expressing donor MHC class I or II molecules or a peptide analogue might have therapeutic potential in the induction of transplant tolerance and in the treatment of autoimmune diseases.

Major histocompatibility complex class I peptide-pulsed host dendritic cells induce antigen-specific acquired thymic tolerance to islet cells.

BACKGROUND As T-cell receptor-major histocompatibility complex (MHC) class I/self peptide interaction regulates T-cell development in the thymus, we reasoned that presentation of peptides by self dendritic cells (DC) to developing T cells in the thymus might induce acquired thymic tolerance. This hypothesis is based on the finding that intrathymic injection of allopeptides in the adult animal induces acquired tolerance. To examine this hypothesis, we studied the effects of intrathymic (IT) injection of a single immunodominant Wistar-Furth (WF) MHC class I (RT1.Au) peptide-pulsed host DC on islet allograft survival in the WF-to-ACI rat combination. METHODS Bone marrow-derived ACI DC expressing MHC class I and II, OX62, and ED2 present allopeptides to naive and specifically peptide-primed syngeneic T cells in mixed lymphocyte reaction. Host DC pulsed with RT1.Au peptide 5 (residues 93-109) were injected into the thymus of streptozotocin-induced diabetic ACI that were transplanted 7 days later with donor-type (WF) or third-party (Brown Norway [BN]) islets. RESULTS Whereas IT injection of 300 microg of peptide 5 alone led to normoglycemia and permanent islet survival in three of six diabetic ACI recipients, similar treatment combined with simultaneous intraperitoneal injection of 0.5 ml of anti-lymphocyte serum (ALS) on day -7 led to 100% permanent islet allograft survival (>200 days) compared to a mean survival time of 15.0+/-2.3 days in controls treated with ALS alone. In contrast, similarly prepared animals rejected the third-party (BN) islets in an acute fashion. To address the question of indirect allorecognition in acquired thymic tolerance, we examined the effect of peptide-pulsed host DC on graft survival. Whereas IT injection of peptide-pulsed host DC alone resulted in permanent islet survival in two of five animals, IT injection of peptide-pulsed host DC combined with 0.5 ml of ALS induced 100% donor-specific permanent islet allograft survival in the WF-to-ACI rat combination. These results suggest that thymic DC take up, process, and present the administered peptide to the developing T cells by the indirect allorecognition pathway in the induction of acquired thymic tolerance. CONCLUSION We have demonstrated a novel approach to inducing transplant tolerance to islet allografts with IT injection of allopeptide-pulsed host DC. This finding suggests that immunization strategies using DC expressing MHC allopeptides or peptide analogue might be potentially useful in the treatment of autoimmune diabetes mellitus.

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.

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.

Mechanism of acquired thymic tolerance induced by a single major histocompatibility complex class I peptide with the dominant epitope : Differential analysis of regulatory cytokines in the lymphoid and intragraft compartments

BACKGROUND We have recently shown that intrathymic injection of a combination of immunogenic WAG-derived or Wistar-Furth (WF) (RT1.Au) major histocompatibility complex class I peptides induces acquired systemic tolerance to cardiac and islet allografts in the WF-to-ACI rat combination and therefore hypothesized that identification of the class I peptide dominance may play an important role in the induction of antigen (Ag)-specific tolerance. This study defined the peptide with the dominant epitope among the seven synthetic RT1.Au peptides and analyzed the immunoregulatory cytokines within the lymphoid and intragraft compartments associated with acquired thymic tolerance. METHODS ACI recipients were pretreated with intrathymic (IT) injection of 300 microg of the individual seven RT1.Au peptides 7 days before WF or Lewis cardiac transplantation. Cytokine profile of mixed lymphocyte reaction supernatants of T cells obtained from the thymus, mesenteric lymph nodes, spleen, peripheral blood leukocytes, and graft infiltrating cells after donor (WF) or third-party (Lewis) Ag stimulation were measured by enzyme-linked immunosorbent assay, whereas cytokine gene expression was determined by reverse transcription-polymerase chain reaction. RESULTS Only IT injection of peptide 5 (93-109) among the seven RT1.Au peptides induced donor-spe cific tolerance to cardiac allografts in the WF-to-ACI rat combination. In addition, intravenous injection of peptide 5 did not prolong WF graft survival in ACI recipients. Analysis of cytokine production by the tolerant recipients showed significant Ag-specific reduction in the production of interleukin (IL)-2 and interferon-gamma (IFN-gamma) in the thymus, mesenteric lymph nodes, spleen, and peripheral blood leukocytes, which was not associated with a concomitant Ag-specific increase in IL-4 and IL-10 production. Measurement of cytokine mRNA expression confirmed undetectable

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.