Soji F. Oluwole

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 transplantation tolerance to rat cardiac allografts by intrathymic inoculation of allogeneic soluble peptides

Since intrathymic (i.t.) injection of UV-B-irradiated spleen cells (SC) or purified resting allogeneic T cells, but not resting B cells, dendritic cells, or macrophages induces specific tolerance in transiently immunosuppressed recipients, we hypothesized that presentation of donor MHC peptide Ag by the host thymic APCs may convey a tolerogenic signal to the recipient. This study examined if i.t. inoculation of allogeneic soluble Ag obtained from 3 M KCl extracts of purified resting T cells can induce specific tolerance to cardiac allografts in transiently immunomodulated recipients. We have now shown that i.t. inoculation of donor soluble Ag on day -7 combined with 1 ml ALS on days -7 and 0 leads to indefinite WF cardiac allograft survival (> 200 days) in Lewis recipients. This finding was reproducible in sublethally irradiated (200 rads TBI) ACI recipients of i.t. Lewis soluble Ag. In contrast, ACI cardiac allografts were promptly rejected in ALS-treated Lewis recipients of i.t. WF soluble Ag, confirming the donor specificity of such immunologic manipulation. Extrathymic inoculation of WF soluble Ag via the intravenous route in controls failed to prevent normal graft rejection in ALS-treated recipients. The long-term unresponsive recipients specifically and permanently accepted donor-type, second-set cardiac allografts. The observation that thymectomy performed 7 days after i.t. Ag injection led to graft rejection strongly suggests that the early phase of induction of donor-specific tolerance is dependent on the presence of donor alloantigens in the host thymus. This approach may have important clinical therapeutic potential in the induction of transplantation tolerance.

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.

EFFECTIVENESS OF INTRATHYMIC INOCULATION OF SOLUBLE ANTIGENS IN THE INDUCTION OF SPECIFIC UNRESPONSIVENESS TO RAT ISLET ALLOGRAFTS WITHOUT TRANSIENT RECIPIENT IMMUNOSUPPRESSION

Our finding that intrathymic inoculation of resting T cells but not dendritic cells induces donor-specific unresponsiveness to organ allografts led us to hypothesize that presentation of MHC class I alloantigens by thymic antigen-presenting cells to T cell precursors during their ontogeny may convey a tolerogenic signal to the recipient. In this study, we examined whether intrathymic inoculation of soluble antigen obtained from 3M KCl extracts of allogeneic T cells could induce donor-specific unresponsiveness to islet allografts in the Lewis-to-WF rat combination. Our results showed that while intrathymic injection of 0.5 mg soluble antigen on day -7 relative to islet transplantation caused acute graft rejection, intrathymic inoculation of 1.0 mg soluble antigen significantly prolonged the survival of Lewis islet allografts from 10.3 +/- 1.1 days in controls to 53.5 +/- 15.6 days (P < 0.001) in naive (nonimmunosuppressed) STZ (streptozotocin)-induced diabetic WF recipients. In contrast, intrathymic inoculation of 2.0 or 4.0 mg soluble Ag on day -7 led to indefinite Lewis islet survival (> 150 days) in all naive diabetic WF recipients; a finding that suggests that 2.0 mg soluble Ag is the optimal effective dose of intrathymic inoculum required to induce donor-specific unresponsiveness in naive recipients in this model. This finding could not be reproduced by intravenous injection of 2.0 mg soluble Ag, thus confirming the privileged position of the thymus in the induction of Ag-specific unresponsiveness. Third-party (BN) islet allografts were rejected in an acute fashion in similarly prepared recipients. Our results suggest that (1) intrathymic inoculation of soluble Ag, unlike cellular Ag, induces donor-specific unresponsiveness to islet allografts without the use of transient recipient immunosuppression; (2) induction of specific unresponsiveness appears to be dose dependent; and (3) the tolerogenic effect of soluble Ag is dependent on the indirect pathway of Ag-presentation in the thymus in the absence of donor antigen-presenting cells in the inoculum. This novel approach of thymic reeducation of adult animals by the deliberate intrathymic inoculation of soluble major histocompatibility complex Ag without the use of recipient immunosuppression may have therapeutic potential in the induction of transplantation tolerance.

Acquired systemic tolerance to rat cardiac allografts induced by intrathymic inoculation of synthetic polymorphic MHC class I allopeptides.

This study extends the finding that intrathymic (IT) injection of 3M KC1 extracts of T cells induces transplant tolerance to the use of well defined polymorphic MHC class I allopeptides derived from the hypervariable domain of RT1.Au (WF MHC class I). While three of the six synthetic RT1.Au peptides were immunogenic, three others were nonimmunogenic when tested in ACI responders. In our initial studies, we examined the effects of IT injection of a mixture of equal concentrations of the three nonimmunogenic RT1.Au peptides on WF cardiac allograft survival in ACI recipients. The results showed that a single IT injection of 100 and 300 microg class I MHC allopeptides on day -7 relative to cardiac transplant did not significantly prolong graft survival in naive ACI recipients (MST of 9.8, and 12.3 days vs. 10.5 days in controls). In contrast, 600 microg allopeptides injected IT resulted in modest prolongation of graft to an MST of 19.5 days. However, IT injection of 600 microg allopeptides combined with 0.5 ml ALS on day -7 led to permanent acceptance (>200 days) of cardiac allografts in 7/9 ACI recipients compared with survival of 24.2 days in ALS alone treated controls. In contrast, similar treatment led to acute rejection of third party (Lewis) cardiac allografts. Intravenous injection of 600 microg allopeptides combined with ALS did not result in prolonged graft survival (26.8 days). The long-term unresponsive ACI recipients (>100 days) challenged with second-set cardiac grafts accepted permanently donor-type (WF) grafts while rejecting the third party (Lewis) grafts, a finding that confirms acquired systemic tolerance. These findings confirm the role of IT injection of synthetic polymorphic allopeptides in the induction of acquired thymic tolerance and provide the rationale for testing this strategy in large animals and eventually in man.

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.

Humoral immunity in allograft rejection. The role of cytotoxic alloantibody in hyperacute rejection and enhancement of rat cardiac allografts.

The role of humoral immunity in graft rejection in the rat model remains controversial. Passive transfer of cytotoxic alloantibody (CAA) has resulted either in hyperacute rejection or in graft enhancement. This study examines the effect of transfer of CAA on cardiac allograft survival in three rat strain combinations that are fully mismatched at the major histocompatibility (MHC) loci. Strain-specific immune responsiveness in donorrecipient pairs varied from low (Lewis-to-ACI) to high (ACI-to-Lewis) as measured by mixed lymphocyte reactions. CAA was obtained from rats sensitized by three successive skin grafts at weekly intervals. Group 1 (high responder recipients), which consisted of Lewis rats presensitized to ACI and had a lymphocytotoxicity titer of 1:512 to 1:2048, rejected ACI cardiac allografts in 10.8±7.2 hr compared with 6.5±0.5 days in naive controls (P<0.001). Injection of 1 ml of high-titer CAA into naive Lewis rats immediately after ACI cardiac grafting led to hyperacute rejection of ACI hearts in 2.1±0.8 hr while 1 ml of CAA followed by 2 ml of guinea pig complement (GPC) resulted in even faster rejection (mean survival time (MST) of 23.8±4.7 min). Injection of 2 ml GPC alone or in combination with 1 ml naive Lewis serum had no effect on graft survival. Multiple pretransplant injections of 1 ml of CAA on days —3, —2, —1, and 0 relative to transplantation resulted in significant prolongation of allograft survival (MST of 10.3± 0.3 days; P <0.01). In group 2 (intermediate responder recipients), where Lewis rats were presensitized to WF

Interstitial class II-positive cell depletion by donor pretreatment with gamma irradiation. Evidence of differential immunogenicity between vascularized cardiac allografts and islets.

We used an allograft pretreatment regimen involving lethal total-body gamma irradiation of donor rats eight days prior to transplantation to compare directly the immunogenicty of immediately-vascularized heart allografts with isolated, neovascularized pancreatic islet allografts. TBI pretreatment of heart donors (IHT) caused a modest prolongation in cardiac allograft survival compared with controls in a low-responding fully-allogeneic strain combination, Lewis-to-ACI. The addition of 10(5) donor-type dendritic cells (DCs) at the time of transplantation reversed the prolongation of IHT in this strain combination. Donor pretreatment with TBI did not lead to prolonged cardiac allograft survival in either a high-responding combination, ACI-to-Lewis, or in an MHC class II-compatible strain combination, F344-to-Lewis. In contrast, islets from irradiated donors (IIT) showed markedly prolonged survival in both low-responding (Lewis-to-ACI) and high-responding (W/F-to-Lewis) strain combinations. The addition of 10(5) donor-type DCs to the islets at the time of transplantation caused the rejection of only two of six IIT in the Lewis-to-ACI combination. Combined pretreatment regimens involving TBI-pretreated cardiac allografts--such as recipient immunosuppression with peritransplant cyclosporine (10 mg/kg on days 0, 1, and 2) or the combination of TBI (1000 cGy on day -3) and cyclophosphamide (300 mg/kg on day -5) in a pretreatment regimen--did not lead to synergistic graft prolongation in the low-responding Lewis-to-ACI combination. Immunohistologic studies showed that eight days after TBI rat hearts and islet were both greater than 90% depleted of class II (0X6+) interstitial dendritic cells while class I (0X18+) expression was unchanged. IIT are able to increase class II expression when donors are treated with recombinant gamma interferon (4000 U/day on days -3, -2, and -1). MLR data showed that ACI recipients of Lewis IIT reacted normally to donor (Lewis) and third-party (W/F) stimulator cells greater than 100 days after transplantation. The apparent greater immunogenicity of heart allografts as compared with islets in this model could be due to (1) a quantitative difference between the number of residual class II-positive cells in hearts and islets or a greater nnon-MHC antigenic load in the heart grafts given its larger size, or (2) a quanlitative difference between hearts and islets due to the presence of a vascular endothelium in the immediately vascularized heart that can present alloantigen and provide a major stimulus to rejection.