Melissa Y. Yeung

Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.

The Link between the PDL1 Costimulatory Pathway and Th17 in Fetomaternal Tolerance

Fetomaternal tolerance has been shown to depend both on regulatory T cells (Tregs) and negative signals from the PD1–PDL1 costimulatory pathway. More recently, IL-17–producing T cells (Th17) have been recognized as a barrier in inducing tolerance in transplantation. In this study, we investigate the mechanisms of PDL1-mediated regulation of fetomaternal tolerance using an alloantigen-specific CD4+ TCR transgenic mouse model system (ABM-tg mouse). PDL1 blockade led to an increase in embryo resorption and a reduction in litter size. This was associated with a decrease in Tregs, leading to a lower Treg/effector T cell ratio. Moreover, PDL1 blockade inhibited Ag-specific alloreactive T cell apoptosis and induced apoptosis of Tregs and a shift toward higher frequency of Th17 cells, breaking fetomaternal tolerance. These Th17 cells arose predominantly from CD4+Foxp3− cells, rather than from conversion of Tregs. Locally in the placenta, similar decrease in regulatory and apoptotic markers was observed by real-time PCR. Neutralization of IL-17 abrogated the anti-PDL1 effect on fetal survival rate and restored Treg numbers. Finally, the adoptive transfer of Tregs was also able to improve fetal survival in the setting of PDL1 blockade. This is to our knowledge the first report using an alloantigen-specific model that establishes a link between PDL1, Th17 cells, and fetomaternal tolerance.

KIM-1–mediated phagocytosis reduces acute injury to the kidney

Kidney injury molecule 1 (KIM-1, also known as TIM-1) is markedly upregulated in the proximal tubule after injury and is maladaptive when chronically expressed. Here, we determined that early in the injury process, however, KIM-1 expression is antiinflammatory due to its mediation of phagocytic processes in tubule cells. Using various models of acute kidney injury (AKI) and mice expressing mutant forms of KIM-1, we demonstrated a mucin domain-dependent protective effect of epithelial KIM-1 expression that involves downregulation of innate immunity. Deletion of the mucin domain markedly impaired KIM-1-mediated phagocytic function, resulting in increased proinflammatory cytokine production, decreased antiinflammatory growth factor secretion by proximal epithelial cells, and a subsequent increase in tissue macrophages. Mice expressing KIM-1Δmucin had greater functional impairment, inflammatory responses, and mortality in response to ischemia- and cisplatin-induced AKI. Compared with primary renal proximal tubule cells isolated from KIM-1Δmucin mice, those from WT mice had reduced proinflammatory cytokine secretion and impaired macrophage activation. The antiinflammatory effect of KIM-1 expression was due to the interaction of KIM-1 with p85 and subsequent PI3K-dependent downmodulation of NF-κB. Hence, KIM-1-mediated epithelial cell phagocytosis of apoptotic cells protects the kidney after acute injury by downregulating innate immunity and inflammation.

Tubular Expression of KIM-1 Does not Predict Delayed Function After Transplantation

Injured epithelial cells of the proximal tubule upregulate the glycoprotein kidney injury molecule 1 (KIM-1), suggesting its potential as a biomarker of incipient kidney allograft injury. It is unknown whether KIM-1 expression changes in kidney allografts with delayed graft function (DGF), which often follows ischemia-reperfusion injury. Here, we prospectively measured KIM-1 RNA and protein expression in preperfusion biopsies of 30 living- and 85 deceased-donor kidneys and correlated the results with histologic and clinical outcomes after transplantation. We detected KIM-1 expression in 62% of deceased-donor kidneys and only 13% of living-donor kidneys (P < 0.0001). The level of KIM-1 expression before reperfusion correlated inversely with renal function at the time of procurement and correlated directly with the degree of interstitial fibrosis. Surprising, however, we did not detect a significant correlation between KIM-1 staining intensity and the occurrence of DGF. Our findings are consistent with a role for KIM-1 as an early indicator of tubular injury but do not support tissue KIM-1 measurement before transplantation to identify kidneys at risk for DGF.

TIM-3: A Novel Regulatory Molecule of Alloimmune Activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4+FoxP3− and CD8+ T cells in acutely rejecting graft recipients. A blocking anti–TIM-3 mAb accelerated allograft rejection only in the presence of host CD4+ T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ–, IL-6–, and IL-17–producing splenocytes, enhanced CD8+ cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4+ T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4+ T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance.

The proinflammatory cytokine IL‐6 plays an important role in controlling T‐cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL‐6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild‐type or IL‐6‐deficient C57BL/6 mice. We observed that production of IL‐6 and IFN‐γ was upregulated during allograft rejection in untreated wild‐type mice. In IL‐6‐deficient mice, IFN‐γ production was greater than that observed in wild‐type controls, suggesting that IL‐6 production affects Th1/Th2 balance during allograft rejection. CD28‐B7 blockade by CTLA4‐Ig inhibited IFN‐γ production in C57BL/6 recipients, but had no effect on the production of IL‐6. Although wild‐type C57BL/6 recipients treated with CTLA4‐Ig rejected fully MHC‐mismatched BALB/c heart transplants, treatment of IL‐6‐deficient mice with CTLA4‐Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL‐6‐deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL‐6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL‐6 production may represent an important part of costimulation blockade‐based strategies to promote allograft acceptance and tolerance.

TIM‐1 Signaling Is Required for Maintenance and Induction of Regulatory B Cells

Apart from their role in humoral immunity, B cells can exhibit IL‐10‐dependent regulatory activity (Bregs). These regulatory subpopulations have been shown to inhibit inflammation and allograft rejection. However, our understanding of Bregs has been hampered by their rarity, lack of a specific marker, and poor insight into their induction and maintenance. We previously demonstrated that T cell immunoglobulin mucin domain‐1 (TIM‐1) identifies over 70% of IL‐10‐producing B cells, irrespective of other markers. We now show that TIM‐1 is the primary receptor responsible for Breg induction by apoptotic cells (ACs). However, B cells that express a mutant form of TIM‐1 lacking the mucin domain (TIM‐1Δmucin) exhibit decreased phosphatidylserine binding and are unable to produce IL‐10 in response to ACs or by specific ligation with anti‐TIM‐1. TIM‐1Δmucin mice also exhibit accelerated allograft rejection, which appears to be due in part to their defect in both baseline and induced IL‐10+ Bregs, since a single transfer of WT TIM‐1+ B cells can restore long‐term graft survival. These data suggest that TIM‐1 signaling plays a direct role in Breg maintenance and induction both under physiological conditions (in response to ACs) and in response to therapy through TIM‐1 ligation. Moreover, they directly demonstrate that the mucin domain regulates TIM‐1 signaling.

The Emerging Role of the TIM Molecules in Transplantation

Since their discovery in 2001, the T‐cell immunoglobulin mucin (TIM) family members have been shown to play important roles in the regulation of immune responses. The TIM family comprises of eight genes in the mouse, three of which are conserved in humans (TIM‐1, TIM‐3 and TIM‐4). Initially, TIM‐1 and TIM‐3 were thought to be expressed solely on T cells. However, emerging data suggest a much broader expression pattern where their presence on APCs confers differing functions, including the ability to mediate phagocytosis. In contrast, TIM‐4 is exclusively expressed on APCs. Together, the TIM molecules provide a functional repertoire for determining the fate of T‐cell activation and differentiation. To date, much of the knowledge about the TIM family members has been garnered from the models of asthma, allergy and autoimmunity. More recently, data from experimental models of transplantation demonstrate that TIM family members also have a key role in alloimmunity. This review will serve to highlight the emerging data regarding this unique family of molecules and to identify their potential in transplantation tolerance.

KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation

Phagocytosis of apoptotic cells by both professional and semi‐professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM‐1/TIM‐1 is a phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM‐1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi‐membrane organelles. KIM‐1‐mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM‐1‐mediated phagocytosis leads to pro‐tolerogenic antigen presentation, which suppresses CD4 T‐cell proliferation and increases the percentage of regulatory T cells in an autophagy gene‐dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response.

Essential role of PDL1 expression on nonhematopoietic donor cells in acquired tolerance to vascularized cardiac allografts.

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen‐presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC‐mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4‐Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long‐term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1‐deficient endothelium hearts had significant higher frequency of IFN‐γ‐producing alloreactive cells as well as higher frequency of CD8+ effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance.