TIM-1 signaling is required for maintenance and induction of regulatory B cells

Abstract

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(Δmucin) ) 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(Δmucin) 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.

Keywords: Animal models; B cell biology; basic (laboratory) research/science; cell death: apoptosis; immunobiology; tolerance: experimental.

Figure 1. TIM-1-mediated PS-binding requires an intact mucin domain

A) Flow-sorted CD19+TIM1+ or CD19+TIM1- B cells from WT mice were incubated with green fluorescence-labeled phosphatidylserine-coated liposomes (PS-liposomes). After 12h, cells were fixed and stained with DAPI. Random fields (n=7-10) were imaged by confocal microscopy (20×), and co-localization between liposomes and B cells determined using Volocity software. The frequency of FITC+ B cells in each field is shown. Cells were considered positive using a cut-off of >350 MFI (similar to the MFI of phosphatidylcholine (PC)-coated “control” liposomes). Data shown is from a single experiment, representative of 3 experiments, in duplicate wells. B) Flow-sorted CD19+TIM-1+ or CD19+TIM-1- B cells from wt and TIM-1^Δmucin mice were co-incubated with green fluorescence-labeled (PS-liposomes), or control phosphatidylcholine (PC)-liposomes, as above. Co-localization between liposomes and B cells, as defined by the mean fluorescence intensity (MFI) of FITC+ liposomes on each interacting CD19+ B cell, was determined using Volocity software. Data expressed as mean±SEM (*p<0.05; **p<0.01; ***p<0.001) from images of 12-20 random fields. Representative of 3 independent experiments, in triplicate wells. C) Representative images (100× magnification). Green=PS-liposomes, Blue=DAPI. C) 3D reconstruction of confocal z-stack images of wt CD19+TIM1+ B cells incubated with PS-liposomes as in A. Green=PS-liposomes, Red=TIM-1, Blue=DAPI.

Figure 2. Induction of IL-10 in B cells by apoptotic cells (ACs) is defective in TIM-1^Δmucin mice

A) Representative flow plots showing IL-10 expression on CD19⁺ and TIM-1⁺CD19⁺ B cells from wt vs. TIM-1^Δmucin mice 7d after administering 10⁷ ACs (iv). B) Cumulative data showing % of IL-10+ B cells derived from flow plots in A. C) Isolated splenic 10⁶ B cells from wt and TIM-1^Δmucin mice were co-incubated with naïve OTII T cells (5×10⁵/ml), Ovalbumin, and increasing numbers of AC for 48h, followed by intracellular staining for IL-10 on CD19⁺B cells. D) Flow-sorted CD19⁺TIM-1⁺ or CD19+TIM-1- B cells from wt and TIM-1^Δmucin mice were incubated with ACs for 48h followed by intracellular staining for IL-10 on CD19⁺B cells. Graphed data expressed as mean±SEM (*p<0.05; **p<0.01). Representative of ≥2 independent experiments, in triplicate wells or 3 mice/group.

Figure 3. Neither PS-coated liposomes nor apoptotic cells (AC) are internalized by isolated CD19+TIM-1+ splenic B cells

PS-coated liposomes or ACs were generated as described in methods, and labeled with pHRodo succinimidyl ester, a pH-sensitive red fluorophore whose emission increases dramatically at pH<6.0 (i.e. upon entry into phagolysosomes). Liposomes or ACs were then diluted in serum-free media and incubated with total or sorted TIM-1+ B cells from WT mice. Still images taken at t=16h from time-lapse video of images captured in 15 minute intervals is shown. Time-lapse videos are available in supplemental materials. Left panels: splenic B cells incubated with labeled PS-liposomes; right panels: splenic B cells incubated with ACs. Top row: WT CD19+ B cells; second row: WT CD19+TIM-1+ B cells; third row: WT retroperitoneal macrophages (positive control); bottom row: TIM-1+ (KIM-1+) WT kidney tubular epithelial cells. Red=pHRodo. Images show merged brightfield and TRITC channels.

Figure 4. The TIM-1 mucin domain is also critical for induction of IL-10+ Bregs by anti-TIM-1

A) Representative flow cytometry plots showing anti-TIM-1 mAb RMT1-10 binding to TIM-1^Δmucin vs. WT B cells, as assessed by indirect staining with RMT1-10 followed by PE-conjugated anti-rat Ig secondary mAb. B) Frequency of TIM-1 expression on CD19⁺ B cells in wt or TIM-1^Δmucin mice that were naive, allosensitized, or allosensitized and treated with anti-TIM-1 (RMT1-10) (*p<0.05, **p<0.01; n=4/group). Error bars indicate SEM. Representative of 3 independent experiments, with 3 mice per condition. C) Frequency of IL-10 and IL-4 expression on CD19⁺, CD19⁺TIM-1⁺ and CD19⁺TIM-1^- B cells in mice as in B. D) TIM-1^Δmucin B cells fail to promote allograft survival. Naïve B cells from wt or TIM-1^Δmucin C57BL/6 mice were transferred into B cell deficient μMT (C57BL/6) recipients of BALB/c islets, and treated with or without anti-TIM-1 (**p<0.01vs. other groups). n=5-6 mice per group.

Figure 5. TIM-1^Δmucin mice exhibit accelerated allograft rejection

A) bm12 hearts were transplanted into wt or TIM-1^Δmucin C57BL/6 mice. Left: Kaplan-Meir plots of graft survival (MST>100d vs. 29d; **p<0.01). Right: Representative H&E staining of allografts 30d post-transplant (100× magnification). n=5-6 mice per group. B) Grafts, from A, were scored for rejection using ISHLT guidelines: Grade 1, mild (interstitial or perivascular infiltrate with < 1 focus of myocyte damage; Grade 2, moderate (>2 foci of myocyte damage); Grade 3 severe (diffuse infiltrate with multiple foci of myocyte damage, vasculitis, edema) in a blinded fashion. *p<0.05; n=5 in each group. Error bars indicate SEM. C) Splenic CD4 cells from recipients 21d post-transplantation were restimulated in vitro with irradiated bm12 splenocytes for 48h. Cytokine concentration in culture supernatants was determined by Luminex (*p<0.05, **p<0.01). Error bars indicate SEM. Representative of 2 independent experiments, each with 3 mice per group. D) Kaplan-Meier plots of graft survival of TIM-1^Δmucin recipients of bm12 cardiac allografts after receiving no treatment vs. 5×10⁶ TIM-1⁺CD19⁺ or TIM-1^-CD19⁺ B cells from (d14) alloimmunized wt C57BL/6 mice (*p=0.01, **p=0.0025 vs. all other groups). n= 4-8 mice per group.