Topical therapy for regression and melanoma prevention of congenital giant nevi.

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.

On the elusive TCR specificity of thymic regulatory T cells.

Therapies using thymus-derived regulatory T cells (Tregs) are promising strategies for preventing autoimmunity or graft rejection. The efficacy of these approaches is, however, contingent on a better understanding of Treg mode of action, especially about factors controlling their activation in vivo. Although key parameters of Treg suppression have been identified, little information is available on Treg activation in vivo via the TCR. In light of recent studies using TCR transgenic mouse models as well as unpublished data, we discuss evidence in support of the view that Treg TCR specificities are not necessarily highly diverse, that the accessibility of Treg selective antigens control Treg development, and that peptides derived from MHC class II (MHC-II) could be prevailing antigens involved in Treg selection. This novel perspective provides insights on Treg development as well as a conceptual basis to a significant contribution of MHC-II derived peptides in the shaping of the Treg TCR repertoire.

Primary vascularization of allografts governs their immunogenicity and susceptibility to tolerogenesis.

We investigated the influence of allograft primary vascularization on alloimmunity, rejection, and tolerance in mice. First, we showed that fully allogeneic primarily vascularized and conventional skin transplants were rejected at the same pace. Remarkably, however, short-term treatment of mice with anti-CD40L Abs achieved long-term survival of vascularized skin and cardiac transplants but not conventional skin grafts. Nonvascularized skin transplants triggered vigorous direct and indirect proinflammatory type 1 T cell responses (IL-2 and IFN-γ), whereas primarily vascularized skin allografts failed to trigger a significant indirect alloresponse. A similar lack of indirect alloreactivity was also observed after placement of different vascularized organ transplants, including hearts and kidneys, whereas hearts placed under the skin (nonvascularized) triggered potent indirect alloresponses. Altogether, these results suggest that primary vascularization of allografts is associated with a lack of indirect T cell alloreactivity. Finally, we show that long-term survival of vascularized skin allografts induced by anti-CD40L Abs was associated with a combined lack of indirect alloresponse and a shift of the direct alloresponse toward a type 2 cytokine (IL-4, IL-10)-secretion pattern but no activation/expansion of Foxp3(+) regulatory T cells. Therefore, primary vascularization of allografts governs their immunogenicity and tolerogenicity.

Intracellular MHC class II controls regulatory tolerance to allogeneic transplants.

MHC class II (MHCII) genes have been implicated in the regulation of T lymphocyte responses. However, the mechanism of MHCII-driven regulation remains unknown. Matching for MHCII between donors and recipients of allografts favors regulatory T cell tolerance to transplants and provides a unique opportunity to study this regulation. In this study, we investigated MHCII regulation using transfer of donor MHCII genes in recipients of cardiac allografts. Transfer of MHCII IA(b) genes in the bone marrow of CBA mice (H-2(k)) prior to the grafting of IA(b+) fully allogeneic C57BL/6 (B6, H-2(b)) heart transplants resulted in donor-specific tolerance associated with long-term survival of B6, but not third-party, allografts without sustained immunosuppression. Strikingly, the majority of accepted heart transplants (>170 d) were devoid of allograft vasculopathy. Further studies indicated that intracellular IA(b) initiated the tolerogenic process, which was mediated by regulatory T cells (Tregs) that polarized antigraft responses to Th2 cytokine producers. This mechanism seems to be unique to MHCII genes, because previous MHC class I gene-based therapies failed to produce Tregs. These results demonstrate the key role of MHCII in the induction of Tregs. They also underscore a potential mechanism of specific inactivation of T cells in this model; when activated by IA(b+) grafts, IA(b)-specific Tregs repress the entire alloresponse to C57BL/6 transplants (including MHC I and minor Ags), thus mediating T cell tolerance.

Differential effects of 2-deoxy-D-glucose on in vitro expanded human regulatory T cell subsets.

Regulatory T cells (Tregs) are required for the maintenance of immune tolerance and adoptive Treg infusion therapy has become a promising approach to suppress immune responses in diseases such as autoimmunity and transplant rejection. However, one critical challenge of Treg therapy is the requirement of in vitro expansion of functionally stable Tregs while preventing either the contamination of T effector and/or emergence of unstable pathogenic Tregs. Recent studies showing distinct metabolic requirements of T effectors and Tregs suggest that manipulation of cell metabolism may be an attractive strategy to achieve this goal. Here we show that human thymically derived Tregs (tTregs) and in vitro induced Tregs (iTregs) from naive T cells engage glycolysis equivalently upon activation. However, inhibiting glucose metabolism via 2-deoxy-D-glucose (2DG) has distinct effects on each of these subsets. While 2DG treatment at the onset of activation significantly reduced the proliferation and expression of suppressive molecules such as ICOS and CTLA-4 in tTregs, its effect on FOXP3 expression was small. In contrast, 2DG treatment during iTreg induction modestly decreased their proliferation but strongly reduced both ICOS and FOXP3 expression. Importantly, both Treg subsets became insensitive to 2DG after day 3 post activation with little effect on either proliferation or FOXP3 expression while T conventional Th0 cells showed reduced proliferation under the same conditions. Moreover, 2DG treatment at day 3 did not impair the suppressive capabilities of Treg subsets. Collectively, these findings suggest that there is a distinct temporal requirement of glycolysis in each of the activated human Treg subsets and T conventional cells. Furthermore, 2DG treatment at the onset as a strategy to impair contaminating T effector cell proliferation is unfavorable for optimal Treg generation as well.

Differential Roles of IL-2 Signaling in Developing versus Mature Tregs.

Although Foxp3+ regulatory T cells (Tregs) require interleukin-2 (IL-2) for their development, it has been unclear whether continuing IL-2 signals are needed to maintain lineage stability, survival, and suppressor function in mature Tregs. We generated mice in which CD25, the main ligand-binding subunit of the IL-2 receptor, can be inducibly deleted from Tregs after thymic development. In contrast to Treg development, we find that IL-2 is dispensable for maintaining lineage stability in mature Tregs. Although continuous IL-2 signaling is needed for long-term Treg survival, CD25-deleted Tregs may persist for several weeks in vivo using IL-7. We also observe defects in glycolytic metabolism and suppressor function following CD25 deletion. Thus, unlike developing Tregs in which the primary role of IL-2 is to initiate Foxp3 expression, mature Tregs require continuous IL-2 signaling to maintain survival and suppressor function, but not to maintain lineage stability.

Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation.

Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host's lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.

Diphtheria-toxin based anti-human CCR4 immunotoxin for targeting human CCR4(+) cells in vivo.

CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4(+) tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 immunotoxins bound to the human CCR4(+) tumor cell line as well as CCR4(+) human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4(+) tumor cells compared to the monovalent anti-human CCR4 immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4(+) tumor-bearing mouse model. The immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with human CCR4(+) acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 immunotoxin is a promising drug candidate for targeting human CCR4(+) tumor cells and Tregs in vivo.

Expression of MHC class II DQ alpha/beta heterodimers from recombinant polycistronic retroviral genomes.

PURPOSE: In the swine model, the transfer of the polymorphic DRbeta or DQalpha/beta cDNAs in vivo via double copy retroviruses led to a prolonged survival or tolerance of subsequent kidney grafts which were matched for DR or DQ, respectively. However, DQ-induced tolerance required the expression of the alpha/beta heterodimers in the same target cell, a task not reproducibly achieved with double copy vectors. Therefore, the present study was designed to evaluate the ability of polycistronic proviral constructs to express class II DQ alpha/beta heterodimers in transduced cells. METHODS: A swine class II DQ recombinant polycistronic construct (JAB) was developed to contain two internal ribosomal entry sites (IRES) for sequential translation of the DQalpha and DQbeta chains. RESULTS: Although a genomic recombination occurred between the two identical IRES, flow cytometry analyses of JAB-transfected virus-packaging cells demonstrated the cell surface expression of DQalpha/DQbeta heterodimers, indicative of a correct transcription, translation, and transport of swine class II. CONCLUSION: JAB-transfected virus-packaging cells demonstrated the cell surface expression of DQalpha/DQbeta heterodimers. We believe that our study represents an essential step in the design of efficient protocols to transfer graft-matched class II molecules in recipient bone marrow cells and thereby induce transplantation tolerance.

MHC alloantigens elicit secondary, but not primary, indirect in vitro proliferative responses.

The relative contributions of direct and indirect pathways of allorecognition to graft rejection remain controversial. Recent reports suggest that the indirect pathway may play a prominent role in both acute and chronic allograft rejection. Such studies suggest that MHC-derived allopeptides are more immunogenic than those derived from minor histocompatibility or other nominal Ags. The aim of this study was to characterize the immunogenicity of MHC alloantigens in MHC-defined miniature swine via primary and secondary MLR culture assays. APCs were selectively depleted from either responder or stimulator cell populations to specifically analyze direct and indirect proliferative responses, respectively. Radio-resistant cytokine secretion and subsequent backstimulation of responder cells was eliminated by using stimulators that were either lysed or unresponsive to the responder MHC haplotypes. When the effect of backstimulation was eliminated from MLR culture assays, indirect proliferative responses were not observed among naive responders. Only after in vivo priming of responder animals could indirect proliferation be detected. These data do not refute the potential importance of indirect allorecognition in graft rejection. However, they suggest that MHC-derived alloantigens behave similarly in vitro to minor histocompatibility Ags, with comparable immunogenicity. These data also suggest that the MLR culture assay does not accurately reflect the importance of indirect mechanisms that have previously been reported in experimental models of graft rejection. A greater understanding of the indirect pathway and the associated immunogenicity of MHC allopeptides has the potential benefit of enabling the development of therapeutic interventions to prevent or halt allograft rejection.

Expression of xenogeneic MHC class II molecules in HLA-DR(+) and -DR(-) cells: influence of retrovirus vector design and cellular context.

We recently established that molecular chimeras of major histocompatibility complex (MHC) class II molecules, created via retroviral transfer of allogeneic class II cDNAs into bone marrow cells (BMCs), alleviated complications associated with mixed BMC chimeras while leading to T cell tolerance to renal grafts sharing the transferred class II. Initially demonstrated for allogeneic transplants in miniature swine, this concept was extended to T-dependent antibody (Ab) responses to xenogeneic antigens (Ags) in the pig --> baboon combination. Successful down-regulation of T cell responses appeared, however, to be contingent on a tight lineage-specific expression of transferred class II molecules. The present studies were, therefore, designed to evaluate the influence of construct design and cellular environment on expression of retrovirally transferred xenogeneic class II cDNAs. Proviral genomes for pig class II SLA-DR expression, differing only at the marker neo(r) or enhanced green fluorescent protein (EGFP) gene, showed increased membrane SLA-DR density on HLA-DR(-) fibroblasts as well as HLA-DR(+), TF-1 erythroleukemia cells. More importantly, HLA-DR(+) human B cell lines, although efficiently transduced with pig DR retroviruses, exhibited unstable surface pig DR. Surface pig DR- B cells, nevertheless, stimulated autologous human T cells pre-sensitized to pig Ags, a proliferation likely occurring through presentation of class II-derived peptides. Collectively, these data suggest that surface expression of transferred class II molecules is not related to the ability of recipient cells to synthesize xenogeneic class II molecules but rather to their Ag processing capacities.

Engraftment of quiescent progenitors and conversion to full chimerism after nonmyelosuppressive conditioning and hematopoietic cell transplantation in miniature swine.

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.