James I. Kim

Therapeutic siRNA silencing in inflammatory monocytes in mice

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes—but not the noninflammatory subset—depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.

The Transcription Factor c-Maf Controls the Production of Interleukin-4 but Not Other Th2 Cytokines

IL-4 promotes the differentiation of naive CD4+ T cells into IL-4-producing T helper 2 (Th2) cells. Previous work provided suggestive but not conclusive evidence that the transcription factor c-Maf directed the tissue-specific expression of IL-4. It was not known whether c-Maf controlled the transcription of other Th2 cytokine genes. To elucidate the role of c-Maf in vivo, we examined cytokine production in mice lacking c-Maf (c-maf(-/-)). CD4+ T cells and NK T cells from c-maf(-/-) mice were markedly deficient in IL-4 production. However, the mice produced normal levels of IL-13 and IgE, and, when differentiated in the presence of exogenous IL-4, c-maf(-/-) T cells produced approximately normal levels of other Th2 cytokines. We conclude that c-Maf has a critical and selective function in IL-4 gene transcription in vivo.

Indefinite survival of MHC class I-deficient murine pancreatic islet allografts.

To examine the immune response to class I-deficient allogeneic tissue, we used beta 2-microglobulin-deficient mice as graft donors. These mice lack cell surface class I major histocompatibility complex antigen expression. Pancreatic islet allografts from class I-deficient donors survived indefinitely in a majority of fully allogeneic BALB/c recipients. In contrast, host recognition of graft class I antigen was unnecessary for prompt destruction of skin allografts of for autoimmune damage of transplanted pancreatic islet grafts in nonobese diabetic mice. These studies provide evidence that intentional genetic elimination of immunologically relevant donor antigens may prove an effective strategy for preventing allograft rejection.

Survival of MHC-deficient mouse heterotopic cardiac allografts

The immunologic mechanisms involved in the destruction of murine cardiac allografts were evaluated using MHC-deficient mice. Specifically, we examined the survival of immediately vascularized heterotopic adult cardiac grafts deficient in MHC class I, MHC class II, or both MHC class I and II antigens following transplantation to allogeneic hosts. We observed indefinite cardiac graft survival when donors lacked MHC class II or both MHC I and II antigens. In parallel experiments, we studied the survival of cardiac grafts harvested from normal donors in recipients severely depleted of either CD4 (class II-deficient mice) or CD8 (class I-deficient mice) T cells. Graft survival was dramatically prolonged in the absence of CD4 but not CD8 T cells. Collectively, our results demonstrate that the interaction of host CD4 T cells with donor class II antigens is critical to the rejection of murine cardiac grafts.

TGF-β-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance

Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti‐T cell immunoglobulin domain and mucin domain‐1 and anti‐CD45RB antibody treatment results in tolerance to full MHC‐mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen‐specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4+CD25− T cells than with naive B cells. We also show that Breg cells express the TGF‐β associated latency‐associated peptide and that Breg‐cell mediated graft prolongation post‐adoptive transfer is abrogated by neutralization of TGF‐β activity. Breg cells, like Treg cells, demonstrate preferential expression of both C‐C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody‐induced transplantation tolerance, Breg cells promote graft survival by promoting Treg‐cell development, possibly via TGF‐β production.

Cutting Edge: Transplant Tolerance Induced by Anti-CD45RB Requires B Lymphocytes

Selective interference with the CD45RB isoform by mAb (anti-CD45RB) reliably induces donor-specific tolerance. Although previous studies suggest participation of regulatory T cells, a mechanistic understanding of anti-CD45RB-induced tolerance is lacking. We report herein the unexpected finding that tolerance induced by this agent is not established in B cell-deficient mice but can be recovered by preemptive B lymphocyte transfer to B cell-deficient hosts. Using B cells from genetically modified donors to reconstitute B cell-deficient recipients, we evaluate the role of B lymphocyte-expressed CD45RB, T cell costimulatory molecules, and the production of Abs in this novel tolerance mechanism. Our data document an Ab-induced tolerance regimen that is uniquely B lymphocyte-dependent and suggest mechanistic contributions to tolerance development from the B cell compartment through interactions with T cells.

Absence of transcription factor c-maf causes abnormal terminal differentiation of hypertrophic chondrocytes during endochondral bone development.

In this study, we report that the transcription factor c-Maf is required for normal chondrocyte differentiation during endochondral bone development. c-maf is expressed in hypertrophic chondrocytes during fetal development (E14.5-E18.5), with maximal expression in the tibia occurring at E15.5 and E16.5, in terminally differentiated chondrocytes. In c-maf-null mice, fetal bone length is decreased approximately 10%, and hypertrophic chondrocyte differentiation is perturbed. There is an initial decrease in the number of mature hypertrophic chondrocytes at E15.5 in c-maf-null tibiae, with decreased expression domains of collagen X and osteopontin, markers of hypertrophic and terminal hypertrophic chondrocytes, respectively. By E16.5, there is an expanded domain of late hypertrophic, osteopontin-positive chondrocytes in the c-maf-/-. This accumulation of hypertrophic chondrocytes persists and is still observed at 4 weeks of age. These data suggest that c-Maf facilitates the initial chondrocyte terminal differentiation and influences the disappearance of hypertrophic chondrocytes. BrdU and TUNEL analyses show normal proliferation rate and apoptosis in the c-maf-null. There is a specific decrease in MMP-13 expression at E15.5 in the c-maf-null. MMP-13 is known to be regulated by AP-1 and may also be a target of c-Maf. Thus, cartilage is a novel system in which c-Maf acts during development, where c-Maf is required for normal chondrocyte differentiation.

Anti‐CD45RB/Anti‐TIM‐1‐Induced Tolerance Requires Regulatory B Cells

The role of B cells in transplant tolerance remains unclear. Although B‐cell depletion often prolongs graft survival, sometimes it results in more rapid rejection, suggesting that B cells may have regulatory activity. We previously demonstrated that tolerance induction by anti‐CD45RB antibody requires recipient B cells. Here, we show that anti‐CD45RB in combination with anti‐TIM‐1 antibody has a synergistic effect, inducing tolerance in all recipients in a mouse islet allograft model. This effect depends on the presence of recipient B cells, requires B‐cell IL‐10 activity, and is antigen‐specific. These data suggest the existence of a regulatory B‐cell population that promotes tolerance via an IL‐10‐dependent pathway.

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

Early T cell activation by recipient antigen-presenting cells cross-dressed with donor MHC antigens may initiate acute allograft rejection. Transplant ride along Despite the successful use of organ transplantation in the clinic, the mechanisms behind early rejection of transplants remain unclear. The passenger leukocyte theory suggests that graft leukocytes that express donor major histocompatibility complexes (MHCs) migrate to recipient lymphoid organs, where they activate host T cells. In multiple mouse models of transplantation, Marino et al. found few donor leukocytes in lymph nodes; rather high numbers of recipient antigen-presenting cells (APCs) were present that were cross-dressed with donor MHC. The donor MHC was derived from allogeneic exosomes, which could induce proinflammatory alloimmune responses even without transplantation. These data suggest that cross-dressed recipient APCs rather than passenger leukocytes may contribute to early T cell activation and transplant rejection. 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.

Deletion of donor-reactive T lymphocytes in adult mice after intrathymic inoculation with lymphoid cells

Clonal deletion of self antigen-reactive T lymphocytes is known to be a dominant mechanism of tolerance induction in the normal immune system. This report considers whether deletion of antigen-reactive T cells is also the immunologic basis for the recently described model of transplantation tolerance that follows intrathymic inoculation with allogeneic lymphoid cells. We found that the outcome of injecting Mlsa- hosts with lymphocytes from Mlsa+ donors was depletion of V beta 6+ T cells (which are known to be reactive with the Mlsa superantigen). The process was found to be specific in that a similar reduction was not seen in an irrelevant T cell population (V beta 8+) in IT injected hosts. Deletion was observed in this model only if immunosuppression with ALS or anti-CD4 accompanied intrathymic injection. When the inoculum of allogeneic lymphocytes was administered intravenously instead of intrathymically only minimal deletion was observed. The induction of transplantation tolerance by intrathymic injection of donor lymphoid cells may prove especially efficacious since it relies on deletion of only those T cells specifically reactive to donor antigens, a process analogous to tolerance induction to self antigens.