The RMT1-10 monoclonal antibody reacts with mouse T cell immunoglobulin and mucin domain 1 (TIM-1) also known as CD365. TIM-1 is a type I cell-surface glycoprotein and member of the Ig superfamily. TIM-1 is preferentially expressed on TH2 cells and has been identified as a stimulatory molecule for T cell activation. The TIM gene family, plays critical roles in regulating the immune response to viral infection. TIM-1 is also involved in allergic responses, asthma, and transplant tolerance. The RMT1-10 antibody has been shown to neutralize TIM-1 in vivo and reduce the severity of EAE, in mice studies.
Yeung, M. Y., et al. (2015). "TIM-1 signaling is required for maintenance and induction of regulatory B cells" Am J Transplant 15(4): 942-953. PubMed
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-1(Deltamucin) ) exhibit decreased phosphatidylserine binding and are unable to produce IL-10 in response to ACs or by specific ligation with anti-TIM-1. TIM-1(Deltamucin) 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.
in vivo TIM-1 neutralization
Lee, K. M., et al. (2014). "TGF-beta-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance" Eur J Immunol 44(6): 1728-1736. PubMed
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-beta associated latency-associated peptide and that Breg-cell mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-beta 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-beta production.
in vivo TIM-1 neutralization
Tan, X., et al. (2014). "Tim-1 blockade with RMT1-10 increases T regulatory cells and prolongs the survival of high-risk corneal allografts in mice" Exp Eye Res 122: 86-93. PubMed
Anti-Tim-1 monoclonal antibody (mAb) RMT1-10 is effective in promoting allograft survival through blocking Tim-1. However, its role in corneal transplantation is unclear. This study aims to evaluate the effect of RMT1-10 on high-risk corneal transplantation. BALB/c mice were transplanted with corneal grafts from C57BL/6 mice and intraperitoneally injected with RMT1-10 or isotype IgG. The transparency of corneal graft was evaluated by slit lamp biomicroscopy. Flow cytometry was used to determine the phenotype of CD4(+) T cells, including CD154, Tim-3, CD25 and Foxp3, and to analyze the proliferation capacity of CD4(+) T cells and the suppressive capacity of T regulatory (Treg) cells. The levels of interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta1 (TGF-beta1) were investigated by intracellular staining and/or ELISA assay. The delayed-type hypersensitivity (DTH) response was evaluated by ear swelling assay. RMT1-10 therapy delayed the onset of rejection and significantly prolonged the survival of corneal allograft. In RMT1-10 treated mice, percentages of CD4(+)CD154(+) cells and CD4(+)Tim-3(+) cells were significantly decreased while the frequency of CD4(+)CD25(+)Foxp3(+) Treg cells was significantly up-regulated, compared with those of isotype IgG treated mice. And, in vitro proliferation of CD4(+) T cells was significantly inhibited by RMT1-10. In addition, percentage of intracellular expression of IFN-gamma and IL-4 in CD4(+) T cells isolated from RMT1-10 treated mice was significantly reduced. After co-culturing with RMT1-10 in vitro, CD4(+) T cells produced significantly decreased levels of IFN-gamma and IL-4 and significantly increased levels of TGF-beta1. Furthermore, RMT1-10 inhibited DTH response of recipient mice and enhanced the suppressive capacity of Treg cells isolated from RMT1-10 treated mice. Our data indicate that Tim-1 blockade with RMT1-10 could suppress immunological rejection and prolong the survival of corneal allograft through regulating T cell responses.
in vivo TIM-1 neutralization
Zhang, Y., et al. (2013). "Targeting TIM-1 on CD4 T cells depresses macrophage activation and overcomes ischemia-reperfusion injury in mouse orthotopic liver transplantation" Am J Transplant 13(1): 56-66. PubMed
Hepatic injury due to cold storage followed by reperfusion remains a major cause of morbidity and mortality after orthotopic liver transplantation (OLT). CD4 T cell TIM-1 signaling costimulates a variety of immune responses in allograft recipients. This study analyzes mechanisms by which TIM-1 affects liver ischemia-reperfusion injury (IRI) in a murine model of prolonged cold storage followed by OLT. Livers from C57BL/6 mice, preserved at 4 degrees C in the UW solution for 20 h, were transplanted to syngeneic recipients. There was an early (1 h) increased accumulation of TIM-1+ activated CD4 T cells in the ischemic OLTs. Disruption of TIM-1 signaling with a blocking mAb (RMT1-10) ameliorated liver damage, evidenced by reduced sALT levels and well-preserved architecture. Unlike in controls, TIM-1 blockade diminished OLT expression of Tbet/IFN-gamma, but amplified IL-4/IL-10/IL-22; abolished neutrophil and macrophage infiltration/activation and inhibited NF-kappaB while enhancing Bcl-2/Bcl-xl. Although adoptive transfer of CD4 T cells triggered liver damage in otherwise IR-resistant RAG(-/-) mice, adjunctive TIM-1 blockade reduced Tbet transcription and abolished macrophage activation, restoring homeostasis in IR-stressed livers. Further, transfer of TIM-1(Hi) CD4+, but not TIM-1(Lo) CD4+ T cells, recreated liver IRI in RAG(-/-) mice. Thus, TIM-1 expressing CD4 T cells are required in the mechanism of innate immune-mediated hepatic IRI in OLTs.
in vivo TIM-1 neutralization
Ding, Q., et al. (2011). "Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice" J Clin Invest 121(9): 3645-3656. PubMed
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.
Purinergic receptor-7 (P2X7R) signaling controls Th17 and Th1 generation/differentiation, while NOD-like receptor P3 (NLRP3) acts as a Th2 transcriptional factor. Here, we demonstrated the existence of a P2X7R/NLRP3 pathway in T cells that is dysregulated by a P2X7R intracellular region loss-of-function mutation, leading to NLRP3 displacement and to excessive Th17 generation due to abrogation of the NLRP3-mediated Th2 program. This ultimately resulted in poor outcomes in cardiac-transplanted patients carrying the mutant allele, who showed abnormal Th17 generation. Transient NLRP3 silencing in nonmutant T cells or overexpression in mutant T cells normalized the Th profile. Interestingly, IL-17 blockade reduced Th17 skewing of human T cells in vitro and abrogated the severe allograft vasculopathy and abnormal Th17 generation observed in preclinical models in which P2X7R was genetically deleted. This P2X7R intracellular region mutation thus impaired the modulatory effects of P2X7R on NLRP3 expression and function in T cells and led to NLRP3 dysregulation and Th17 skewing, delineating a high-risk group of cardiac-transplanted patients who may benefit from personalized therapy.
