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Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs., Introduction Regulatory [CD4.sup.+] T cells expressing FOXP3 (Tregs) play a critical role in tolerance by modulating immune responses to both endogenous and exogenous antigens (1, 2). FOXP3 is required for [...]

Maturation of T cell-activating APCs directly links innate and adaptive immunity and is typically triggered by microbial infection. Transplantation of allografts, which are sterile, generates strong T cell responses; however, it is unclear how grafts induce APC maturation in the absence of microbial-derived signals. A widely accepted hypothesis is that dying cells in the graft release 'danger' molecules that induce APC maturation and initiate the adaptive alloimmune response. Here, we demonstrated that danger signals associated with dying cells are not sufficient to initiate alloimmunity, but that recognition of allogeneic non-self by the innate immune system is required. In WT as well as in T cell-, B cell-, and innate lymphoid cell-deficient mice, allogeneic grafts elicited persistent differentiation of monocytes into mature DCs that expressed IL-12 and stimulated T cell proliferation and IFN-γ production. In contrast, syngeneic grafts in the same mice elicited transient and less pronounced differentiation of monocytes into DCs, which neither expressed IL-12 nor stimulated IFN-γ production. In a model in which T cell recognition is restricted to a single foreign antigen on the graft, rejection occurred only if the allogeneic non-self signal was also sensed by the host's innate immune system. These findings underscore the importance of innate recognition of allogeneic non-self by monocytes in initiating graft rejection., Introduction The mammalian innate immune system recognizes non-self molecules unique to microbial organisms (1,2). This recognition step triggers a rapid inflammatory response and, of paramount importance to the survival of [...]

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved [...]

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD[4.sup.+] 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 CD1[d.sup.hi]CD[5.sup.+]. 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.sup.+] 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.sup.+] 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.sup.+] 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., Introduction T cell Ig domain and mucin domain (TIM) proteins constitute a family of costimulatory molecules that play an important role in effector differentiation of CD[4.sup.+] cells (1). The 8 [...]

T cell Ig mucin 1 (TIM-1) plays an important role in regulating immune responses in autoimmune and asthma models, and it is expressed on both Th1 and Th2 cells. Using an antagonistic TIM-1-specific antibody, we studied the role of TIM-1 in alloimmunity. A short course of TIM-1-specific antibody monotherapy prolonged survival of fully MHC-mismatched vascularized mouse cardiac allografts. This prolongation was associated with inhibition of alloreactive Th1 responses and preservation of Th2 responses. TIM-1-specific antibody treatment was more effective in Th1-type cytokine--deficient Stat4-/- recipients as compared with Th2-type cytokine--deficient Stat6-/- recipients. Subtherapeutic doses of rapamycin plus TIM-1-specific antibody resulted in allograft acceptance and prevented the development of chronic allograft vasculopathy. Allograft survival via this treatment was accompanied by a Th1- to Th2-type cytokine switch. Depletion of natural Tregs abrogated the graft-protecting effect of the TIM-1-specific antibody. Importantly, [CD4.sup.+][CD25.sup.+] Tregs obtained from long-term survivors had enhanced regulatory activity as compared with naive [CD4.sup.+][CD25.sup.+] Tregs. Consistent with this, TIM-1-specific antibody treatment both preserved Tregs and prevented the expansion of alloreactive effector Th1 cells in an alloreactive TCR transgenic adoptive transfer model. These studies define previously unknown functions of TIM-1 in regulating alloimmune responses in vivo and may provide a novel approach to promoting transplantation tolerance., Introduction The T cell Ig mucin (TIM) family of genes encodes proteins that are expressed by T cells and contain an IgV-like and a mucin-like domain (1). The TIM family [...]

Author(s): Junko Irie-Sasaki [1, 2, 3]; Takehiko Sasaki (corresponding author) [1, 2, 3]; Wataru Matsumoto [4]; Anne Opavsky [5]; Mary Cheng [1]; Grant Welstead [1]; Emily Griffiths [1]; Connie Krawczyk [...]

B cells can be polarized to express various cytokines. The roles of IFNγ and IL-10, expressed respectively by B effector 1 (Be1) and Bregs, have been established in pathogen clearance, tumor growth, autoimmunity and allograft rejection. However, the in vivo role of B cell IL-4, produced by Be2 cells, remains to be established. We developed B-IL-4/13 iKO mice carrying a tamoxifen-inducible B cell-specific deletion of IL-4 and IL-13. After alloimmunization, B-IL-4/13 iKO mice exhibited decreased IL-4+ Th2 cells and IL-10+ Bregs without impact on Th1, Tregs, or CD8 T cell responses. B-IL-4/13 iKO mice rejected islet allografts more rapidly, even when treated with tolerogenic anti-TIM-1 mAb. In ovalbumin-induced allergic airway disease (AAD), B-IL-4/13 iKO mice had reduced inflammatory cells in BAL, and preserved lung histology with markedly decreased infiltration by IL-4+ and IL-5+ CD4+ T cells. Hence, B cell IL-4 is a major driver of Th2 responses in vivo which promotes allograft survival, and conversely, worsens AAD. [ABSTRACT FROM AUTHOR]

Stromal cells in training immunity: Immune responses to pathogens are heightened in individuals previously exposed to pathogen-associated stimuli, a phenomenon known as innate immune training. Innate immune training is accomplished through metabolic reprogramming and epigenetic changes, but the underlying mechanisms are not fully understood. In this study, Wu et al. investigate the functional requirement of IL-17 receptor signaling in fibroblastic reticular cells (FRCs) in the development of protective antibody responses against gut infection and studied how innate immune training affects these responses. Mild gut inflammatory training before C. rodentium infection enhanced gut-protective antibody production and reduced colon inflammation. Inflammatory training required IL-17 signaling in FRCs and IL-10 production by B cells. These findings reveal a mechanism of innate immune training in FRCs for protection against gut infection. Prior experience of pathogen-associated stimuli reduces morbidity and mortality to newly encountered infections through innate immune training, which can be enhanced by childhood vaccination. Fibroblastic reticular cells (FRCs) are stromal cells in lymphoid organs that support lymphocyte localization and survival and modulate adaptive immune responses. IL-17 signaling is important for FRC metabolism and proliferation during inflammatory responses. Here, we show that FRC-intrinsic IL-17 signaling was required for protective antibody-mediated immunity to the gut bacterial pathogen Citrobacter rodentium. We asked whether prior activation of FRC through nonspecific inflammatory "training" of the gut would alter subsequent immune response to C. rodentium. Inflammatory training increased the number of activated FRC in mesenteric LN (MLN) and enhanced the antibody response to C. rodentium in an IL-17–dependent manner. FRC demonstrated cardinal features of innate immune training, including increased epigenetic markers of activation and increased metabolic response to infection. Enhanced responses were still evident 6 weeks after training. The kinetics of bacterial infection were not changed by inflammatory training, but colon inflammation was paradoxically reduced. Mechanistically, IL-10 production by activated B cells was required for colon protective effects of inflammatory training. Enhancing tissue protective B cell responses thus led to increased production of antibody and IL-10, allowing clearance of infection with reduced tissue inflammation. These data identify a new mode of immune training through FRC to modulate future adaptive responses and better preserve host health. [ABSTRACT FROM AUTHOR]

A ratio to predict rejection: Current strategies to identify rejection of transplanted kidneys rely on invasive surveillance biopsies or have limited predictive value. Thus, identification of an early and noninvasive biomarker to predict rejection is required. Here, Cherukuri et al. characterized a ratio of interleukin-10 (IL-10) to tumor necrosis factor–α (TNFα) expressed by transitional B cells as a biomarker of early T cell–mediated allograft rejection. The authors found that the ratio of IL-10 to TNFα produced by transitional B cells predicted rejection at 3 months after transplantation in three patient cohorts. These findings suggest that the IL-10/TNFα ratio may be an effective biomarker that can be used clinically to tailor therapy based on risk of rejection. Early immunological biomarkers that predict rejection and chronic allograft loss are needed to inform preemptive therapy and improve long-term outcomes. Here, we prospectively examined the ratio of interleukin-10 (IL-10) to tumor necrosis factor–α (TNFα) produced by transitional-1 B cells (T1B) 3 months after transplantation as a predictive biomarker for clinical and subclinical renal allograft rejection and subsequent clinical course. In both Training (n = 162) and Internal Validation (n = 82) Sets, the T1B IL-10/TNFα ratio 3 months after transplantation predicted both clinical and subclinical rejection anytime in the first year. The biomarker also predicted subsequent late rejection with a lead time averaging 8 months. Among biomarker high-risk patients, 60% had early rejection, of which 48% recurred later in the first posttransplant year. Among high-risk patients without early rejection, 74% developed rejection later in the first year. In contrast, only 5% of low-risk patients had early and 5% late rejection. The biomarker also predicted rejection in an External Validation Set (n = 95) and in key patient subgroups, confirming generalizability. Biomarker high-risk patients exhibited progressively worse renal function and decreased 5-year graft survival compared to low-risk patients. Treatment of B cells with anti-TNFα in vitro augmented the IL-10/TNFα ratio, restored regulatory activity, and inhibited plasmablast differentiation. To conclude, the T1B IL-10/TNFα ratio was validated as a strong predictive biomarker of renal allograft outcomes and provides a rationale for preemptive therapeutic intervention with TNF blockade. [ABSTRACT FROM AUTHOR]

Regulatory B cells (Bregs) ameliorate autoimmune disease and prevent allograft rejection. Conversely, they hinder effective clearance of pathogens and malignancies. Breg activity is mainly attributed to IL‐10 expression, but also utilizes additional regulatory mechanisms such as TGF‐β, FasL, IL‐35, and TIGIT. Although Bregs are present in various subsets defined by phenotypic markers (including canonical B cell subsets), our understanding of Bregs has been limited by the lack of a broadly inclusive and specific phenotypic or transcriptional marker. TIM‐1, a broad marker for Bregs first identified in transplant models, plays a major role in Breg maintenance and induction. Here, we expand on the role of TIM‐1+ Bregs in immune tolerance and propose TIM‐1 as a unifying marker for Bregs that utilize various inhibitory mechanisms in addition to IL‐10. Further, this review provides an in‐depth assessment of our understanding of Bregs in transplantation as elucidated in murine models and clinical studies. These studies highlight the major contribution of Bregs in preventing allograft rejection, and their ability to serve as highly predictive biomarkers for clinical transplant outcomes. [ABSTRACT FROM AUTHOR]

Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2−/−γc−/− mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein α (SIRPα) is a key modulator of the recipient’s innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRPα to recipient CD47 and was modulated by the strength of the SIRPα-CD47 interaction. Therefore, sensing SIRPα polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells. [ABSTRACT FROM AUTHOR]

Background: A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3+Treg and IL-10-producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell-associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent. Methodology/Principal Findings: Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3+Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4+IL-10+IL-4- T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4+IL-10+IL-4- T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4+IL-10+IL-4- T cells. Conclusions/Significance: The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic. [ABSTRACT FROM AUTHOR]