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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: J Immunol. 2013 Feb 25;190(7):3189–3196. doi: 10.4049/jimmunol.1203364

Constitutively CD40-activated B cells regulate CD8 T cell inflammatory response by IL-10 induction

Pandelakis A Koni *,§, Anna Bolduc §, Mayuko Takezaki §, Yutetsu Ametani §, Lei Huang †,§, Jeffrey R Lee ‡,§,, Stephen L Nutt ∥, Masahito Kamanaka #, Richard A Flavell #, Andrew L Mellor *,§, Takeshi Tsubata **, Michiko Shimoda ‡,§,††
PMCID: PMC3608804  NIHMSID: NIHMS441797  PMID: 23440421

Abstract

B cells are exposed to high levels of CD40 ligand (CD40L, CD154) in chronic inflammatory diseases. In addition, B cells expressing both CD40 and CD40L have been identified in human diseases such as autoimmune diseases and lymphoma. However, how such constitutively CD40-activated B cells under inflammation may impact on T cell response remains unknown. Using a mouse model in which B cells express a CD40 ligand transgene (CD40LTg) and receive autocrine CD40/CD40L signaling, we show that CD40LTg B cells stimulated memory-like CD4 and CD8 T cells to express IL-10. This IL-10 expression by CD8 T cells was dependent on IFN-I and Programmed cell death protein 1, and was critical for CD8 T cells to counter-regulate their over activation. Furthermore, adoptive transfer of naïve CD8 T cells in RAG-1−/− mice normally induces colitis in association with IL-17 and IFNγ cytokine production. Using this model, we show that adoptive co-transfer of CD40LTg B cells, but not wild type B cells, significantly reduced IL-17 response and regulated colitis in association with IL-10 induction in CD8 T cells. Thus, B cells expressing CD40L can be a therapeutic goal to regulate inflammatory CD8 T cell response by IL-10 induction. 194

Introduction

CD40-CD154 (CD40 ligand, CD40L) interaction delivers a critical co-stimulatory signal for B cell differentiation and function (1). CD40L is highly expressed by activated T cells as well as by platelets and various other cell types under chronic inflammatory diseases such as autoimmune diseases (2). CD40L derived from platelets has been shown to modulate adaptive immune response (3). In Multiple Sclerosis patients, B cells had a trait of CD40-activated B cells and stimulated CD8 T cells in vitro via IL-15 (4). Moreover, CD40L is functionally expressed on some B cells in patients with EBV-infection (5), autoimmune diseases (6-8) and lymphoma (9-11). In B cell lymphoma, this autonomous CD40/CD40L interaction has been shown to increase their survival through constitutive NF-kB and NFAT activation (12, 13). These findings support the hypothesis that the heightened B cell CD40/CD40L signaling due to elevated CD40L expression during chronic inflammatory diseases changes B cell functions and has an impact on on-going immune response through altered B cell reactivity.

In this study, we employed CD40L transgenic (CD40LTg) mice that express CD40L under the IgVH promoter specifically on B cells (14). Thus, CD40LTg mice serve as a model for human diseases in which B cells abnormally express CD40L and are exposed to excessive CD40/CD40L signaling under chronic inflammation. Based on their phenotype, B cells in CD40LBTg mice are not constitutively activated (14, 15). However, binding of CD40L or anti-CD40 antibody breaks up the CD40 and CD40L complex formed on the cell surface of B cells and triggers cis-activation of B cells (16), as evidenced by robust NFκB-1 activation (15), without triggering trans-activation of DCs (16). This augmented B cell specific CD40/CD40L signaling enhanced the magnitude of primary antigen-specific humoral response as a result of premature termination of on-going germinal center response (15, 16). Moreover, aged CD40LBTg mice have been shown to develop B cell-mediated lupus-like disease and colitis with autoantibody production (14, 17).

Here, we show that CD40LTg B cells stimulated memory-like CD4 and CD8 T cells to express IL-10. Furthermore, in a RAG-1−/− colitis model, adoptive co-transfer of CD40LTg B cells could suppress inflammatory CD8 T cell response by inducing IL-10 expression and regulated CD8 T cell-mediated colitis.

Materials and Methods

Mice, immunization and inflammatory challenge

RAG-1−/−, C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) and C57BL/6-Tg(TcraTcrb)425Cbn/J (OT-II) mice were all on a C57BL6/J background and were purchased from The Jackson Laboratory as were C57BL6/J mice. All other mice used were on a C57BL6/J background (n>10) and were bred in our facility under specific pathogen-free conditions. CD40LTg mice (14), IL-10-GFP reporter mice (18), JH−/− mice (19) and Blimp-1-GFP reporter mice (20) were described before. IFNα/βR−/− mice and PD-1−/− mice were the kind gifts of Drs D. Moskofidis (GHSU) and T. Honjo (Kyoto University), respectively. Typical experiments used mice at 6-12 wk of age. For immunization and inflammatory challenge, mice 6-10 wk of age were given an intraperitoneal (i.p.) challenge with 100 μl of PBS containing 2 mg of alum (Sigma) with or without 200μg of OVA. All studies were reviewed and approved by the institutional animal care and use committee.

Antibodies and reagents

Antibodies used in this study were against IAb (AF6-120.1), CD4 (RM4-5), CD11c (HL3), CD8α (53-6.7), CD90.1 (OX-7), TCRβ (H57-597), CD19 (1D3), CD21 (7G6), CD23 (B3B4), CD44 (IM7), CD62L (MEL-14), CD103 (M290), CD122 (TMB1), CD127 (A7R34), IFNγ (XMG1.2), B7H-1 (PD-L1), B7-DC (PD-L2), CD80, CXCR5-biotin, CD16/CD32 (2.4G2) and H-2kd (SF1-1.1) from BD Biosciences. Streptavidin-eFluor780 and antibodies against Foxp3 (FJK-16a), CD8β (H35-17.2), MHC-II (M5/114.15.2), CD4 (GK5.1) and PD-1 (J43) were from eBioscience. PNA-FITC was from Vector Laboratories (Southfield, MI). Recombinant mouse IL-2 and IL-27 were from R &D Systems (Minneapolis, MN).

Flow cytometry and cell sorting

For analysis of DCs, spleens were diced, incubated in RPMI 1640/1% FCS containing 0.5 mg/ml collagenase type IV (Sigma) for 30 min at 37°C, single-cell suspensions prepared and red blood cells depleted with ACK lysing buffer (BioWhittaker). Spleen cells were then washed twice with PBS and filtered through nylon mesh in RPMI 1640/1% FCS. For general analysis, cells were pretreated with anti-CD16/CD32 antibody on ice for 15 min and then incubated at 4°C for 30 min with specific antibodies. For intracellular staining for IFNγ and granzyme B, cells were incubated with anti-CD3 antibody (100 ng/ml) for 3 hours in the presence of Brefeldin A (eBioscience), followed by fixation/permeabilization and staining with anti-IFNγ (BD Bioscience) and anti-granzyme B antibody (Invitrogen). For IL-10-GFP and granzyme B staining, cells were treated with 0.01% paraformaldehyde/PBS for 5 min and 0.05% Tween20/PBS for 5 min, followed by anti-granzyme B antibody.

Cells were analyzed on a FACS Canto (BD Biosciences) with FlowJo software (Ashland, OR). Singlet lymphocyte gates were set based on forward scatter and side scatter channels, and autofluorescent cells were gated out using a ‘dump’ channel. Positive gates were set using fluorescently-labeled isotype-control non-relevant antibodies, or internal non-relevant cellular populations. IL-10-GFP+ gates were set using non-IL-10-GFP reporter cells as a negative control. B cells, CD4 T cells, CD8 T cells and CD11c DCs were sorted from spleen cells using magnetic beads (Miltenyi Biotec), while CD23hiCD21lo follicular (FO) and CD23loCD21hi marginal zone (MZ) B cells, CD62Llo CD8 or CD4 T cells were purified using an Aria cell sorter (BD Biosciences).

Cell culture

For CFSE-dilution assays, sorted CD4 and CD8 T cells were labeled with 5 nM CFSE (Invitrogen) for 10 min at 37°C, followed by washing with cold PBS. Purified B cells (1 × 106) and DCs (2 × 104) were cultured with 105 CFSE-labeled OT-I, OT-II cells and CD8 T cells, or FACS ARIA-sorted CD62Llo CD8 T cells and CD62Llo CD4 T cells in 250 μl RPMI 1640/10% FCS containing various concentrations of OVA (Sigma) or 100 ng/ml of anti-CD3 antibody for 2-3 days. CFSE-dilution was analyzed on a FACS Canto (BD Biosciences) with FlowJo software (Ashland, OR). IL-10 in the culture supernatant was measured by IL-10 ELISA kit (R&D). For cytotoxic CD8 T cell induction, 10 ng/ml each of IL-2 and IL-27 were added to the culture.

In vivo cytotoxicity

Spleen cells or B cells purified were pulsed with 2 μM OVA257-264 peptide for 2 hr at 37°C. OVA257-264 peptide pulsed cells and control, non-pulsed cells were labeled with 3 μM and 0.3 μM CFSE, respectively, for 3 min at 37°C. After washing, these two were mixed 1:1 and injected i.v. (107) into day 10-14 immunized mice. The ratios of CFSEhi versus CFSElo B cells in the host spleen were measured after 18 hr.

qRT-PCR

RNA was prepared using a QIAGEN MINI kit according to the manufacturer’s protocol. cDNA was prepared using a cDNA synthesis kit from SABioscience (QIAGEN). qRT-PCR analyses was performed in triplicate using primers and SYBR Green master mix from SABioscience with an iQ5 cycler (Bio-Rad). Gene expression levels in each sample were normalized against β-actin expression and statistically analyzed with software from SABioscience.

Adoptive transfer

Six week old sex-matched RAG-1−/− recipient mice received an intravenous injection in 100 μl PBS of either wild type or CD40LTg B cells (5 × 106), plus one or other combination of IL-10 deficient or sufficient (IL-10-GFP reporter) CD4 T cells (106) and CD8 T cells (106). Likewise, RAG-1−/− recipient mice received CD40LTg B cells with IL-10-GFP reporter CD4 T cells (106) and CD8 T cells (106) from either PD-1−/− or IFNα/βR−/− mice. Two to three weeks after transfer, spleen cells were analyzed by FACS.

Colitis model

Purified naïve CD62L+ CD44− CD8 T cells (106) by ARIA sort from IL-10-GFP wild type or IL-10−/− mice were intravenously transferred into RAG-1−/− mice (6 weeks old male) with or without B cells (5 × 106) from wild type or CD40LTg mice. The recipients were weighed weekly for clinical signs of disease. After 5 weeks of transfer, mice were sacrificed for tissue harvest. Each colon was graded by an experienced pathologist blinded to the treatment group using a 0-3 scoring system to evaluate acute and chronic inflammation, crypt damage and regeneration. The highest injury score could be as high as 12, and the lowest without injury 1 (physiological inflammation).

Statistics

P values were determined by applying the two-tailed, two-sample equal variance Student’s t test or Mann-Whitney U test.

Results

CD40LTg B cells activate CD8 T cells and induce augmented antigen-specific cytotoxic response

Previous studies have shown that in vitro CD40-activated B cells become potent antigen-presenting cells for CD8 T cells and generate augmented antigen-specific cytotoxic response (4, 21, 22). To test whether or not CD40LTg B cells can activate T cells, CD40LTg and wild type B cells were cultured with CFSE-labeled ovalbumin (OVA)-specific CD8 (OT-I), or CD4 (OT-II), TCR transgenic T cells in the presence of various amounts of soluble OVA. We found that CD40LTg, but not wild type, B cells induced proliferation of OT-I, but not OT-II, cells as evidenced by significantly higher percentage of CFSElo population in the OT-I cell culture, in a manner dependent on the amounts of OVA (Fig.1A). Also, at 2 weeks after immunization with OVA plus alum, CD40LTg mice generated significantly greater numbers of OVA-specific tetramer-binding CD8 T cells (Fig.1B) with augmented OVA-specific in vivo cytotoxicity compared to wild type control mice (Fig.1C). It should be noted that DCs in Tg mice were not functionally activated (16) and, in fact, up-regulated PD-L1 expression and suppressed CD8 T cell activation (P. Koni et al., a manuscript in preparation). These results indicate that like CD40-activated B cells, CD40LTg B cells are potent antigen-presenting cells for CD8 T cells.

Figure 1.

Figure 1

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CD40LTg B cells prime CD8 T cells in vitro and generate augmented in vivo antigen-specific cytotoxicity. (A) CFSE-labeled OT-I or OT-II cells were cultured with wild type (WT) or CD40LTg (Tg) B cells and with various concentrations of OVA protein for three days. Representative histogram overlays with percentages of CFSElo OT-I and OT-II cells co-cultured with B cells from WT (black) or Tg (red) mice are shown. (B) Averages with SDs of total OT-I tetramer-binding cell numbers in spleen of WT (n=3) or Tg mice (n=3) after day 10 immunization with OVA-alum are shown. (C) Representative histograms with percentages of CFSElo control (black) and CFSEhi OVA257-264 peptide loaded (red) B220+ donor cells in Tg or WT mice (n=3/group) pre-immunized with OVA plus alum. Averages with SDs of OVA257-264-specific lysis (%), determined as [CFSElo(%)-CFSEhi(%)]/ CFSElo(%) × 100. Experiments in (A-C) were repeated twice.

Adjuvant induces activation of natural memory-like CD8 T cells in CD40LBTg mice

During the immunization experiment described above, we found that, unlike wild type control mice, CD40LTg mice exhibited spontaneous global activation of CD8 T cells in their spleen with accumulation of CD62LloCD44hi/int CD8 T cells. This was substantially augmented after intraperitoneal alum (2mg) injection even without antigen (Fig.2A), although the accumulation of CD62LloCD44hi/int CD4 T cells was not so obvious (data not shown). At 2 weeks after alum injection, significantly higher levels of CD4 and CD8 T cells in CD40LTg mice were still in cell cycle as judged by 24hr-BrdU labeling compared to those in wild type mice (Fig.2B). The proliferating BrdU+ CD8 T cells were CXCR5+CD62LloCD44hi/int with effecter memory (TEM) phenotype (Fig.2B), which are similar to those found in the B cell mantle zone of human tonsils (23). This accumulation of CD62LloCD44hi/int CD8 TEM cells after alum injection did not occur in CD40LTg mice on a B cell deficient Jh−/− background (data not shown).

Figure 2.

Figure 2

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CD40LTg mice trigger activation of CD8 TEM cells during alum-mediated inflammatory response. Wild type (WT) and CD40LTg (Tg) mice were given alum and their spleen cells were analyzed 2 weeks later. (A) Average frequencies with SDs of naïve (N), memory (M), effecter memory (EM) and effecter (E) CD8 T cells (as gated on the left) in WT and Tg mice with (alum)(n=9/group) or without (naïve)(n=6/group) alum injection are shown. (B) Average percentages with SDs of BrdU+ CD4 and CD8 T cells in naïve and alum-treated wild type (white) and Tg (black) mice are shown (n=3/group). Representative histograms for BrdU staining of CD8 T cells in Tg (solid) and WT mice (dash) with isotype control (shaded) and for CD62L, CD44 and CXCR5 expression of BrdU+ cells are shown. (C) Representative CD62L vs CD44 FACS profiles of CD8 T cells in wild type or Tg Blimp-1-GFP reporter mice and gating strategy of CD62Llo CD44hi/int CD8 TEM cell population with percentages are shown (left). Representative histograms of Blimp-1-GFP, PD-1, and IL-10-GFP expression in CD62Llo CD44hi/int CD8 TEM cell population in Tg (solid) and WT mice (dash) with isotype control (shaded) are shown. (D) ELISA IL-10 concentration in triplicate 4-day culture supernatants for anti-CD3-stimulated CD62Llo CD8 and CD4 T cells of Tg mice with B cells from WT or Tg mice.

Total numbers of CD4 and CD8 T cells and the frequency of Foxp3+ regulatory CD4 and CD8 T cells were not significantly different between wild type and CD40LTg mice 2 weeks after alum injection although Foxp3+ CD4 T cells from CD40LTg mice had a more activated phenotype with down-regulated CD62L expression compared to those from wild type mice (data not shown).

Activated natural effecter memory CD8 T cells express IL-10

The CD62LloCD44hi/int CD8 T cells in CD40LTg did not express IFN-γ by intracellular staining. By gene expression analysis, we found that CD8 T cells in CD40LTg mice expressed significantly higher levels (67.4-fold) of IL-10 compared to those in wild type mice. To better track IL-10 differentiation in CD8 T cells, IL-10-GFP reporter mice (18) and Blimp-1-GFP reporter mice (20, 24) were crossed onto a CD40LTg background. As shown in Fig.2C, the CD62Llo CD44int/lo population has a phenotype consistent with effecter/effecter memory CD8 T cells defined by Blimp-1-GFP reporter expression (20, 24). This population in CD40LTg/IL-10-GFP mice also expressed elevated PD-1 and IL-10-GFP reporter expression. This population barely contained Foxp3-GFP+ cells (data not shown).

In IL-10-GFP reporter mice, the level of GFP expression correlates with the amount of IL-10 mRNA (18). Since the mean fluorescent intensity of IL-10-GFP reporter expression in GFP+ CD8 T cells was consistently lower than that in GFP+ CD4 T cells (data not shown), we tested whether or not IL-10-GFP+ CD8 T cells in CD40LTg mice produce IL-10. Thus, CD62Llo CD8 and CD4 T cells enriched with IL-10-GFP+ cells were purified from CD40LTg and re-stimulated with anti-CD3 antibody in the presence of wild type or CD40LTg B cells. As expected and shown in Fig.2D, the amount of IL-10 produced by CD62Llo CD8 T cells was significantly lower than that produced by CD4 T cells from these mice.

CD40LTg B cells induce IL-10-GFP expression in T cells during lymphopenic proliferation

Based on the CD8 T cell IL-10 expression in CD40LTg mice during alum-induced inflammatory response, we hypothesized that CD40LTg B cells stimulate memory-like CD8 T cells to express IL-10. To test the hypothesis, we performed adoptive transfer experiments in RAG-1−/− mice. CD4 and CD8 T cells transferred into the lymphopenic environment of RAG-1−/− mice undergo spontaneous proliferation and generate natural memory/effecter memory T cells (25). When RAG-1 −/− mice were reconstituted with CD4 and CD8 T cells (1 × 106 each) from IL-10-GFP reporter mice and B cells (5 × 106) either from wild type or CD40LBTg mice, the recipients of CD40LTg B cells had significantly greater numbers of CD8 T cells (8.7-fold) with a higher frequency of CD62LloCD44lo effecters (2.8-fold) compared to the recipients of wild type B cells at two weeks post-transfer (Fig.3A). In addition, their CD4 and CD8 T cells had significantly higher frequencies of IL-10-GFP expression compared to those in the recipients of wild type B cells (Fig.3B). These results confirm that CD40LTg B cells stimulate CD4 and CD8 T cells during lymphopenic proliferation to express IL-10.

Figure 3.

Figure 3

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CD40LTg B cells induce IL-10 expression from CD8 T cells during lymphopenic response in a manner dependent on IFN-I and PD-1 to counter regulate over activation. Wild type (WT) or CD40LTg (Tg) B cells and T cells from IL-10-GFP or IL-10−/− mice were transferred into RAG-1−/− mice. Their spleen cells were analyzed after 2 weeks. (A) Representative CD62L vs CD44 FACS profiles for CD4 and CD8 T cells of RAG-1−/− recipients of WT or Tg B cells. Average frequencies with SDs of naïve (N), memory (M), effecter memory (EM), and effecter (E) cells among CD8 T cells are shown (n=3/group). Repeated twice. (B) Representative histograms for IL-10-GFP expression of CD4 and CD8 T cells in RAG-1−/− recipients of WT (dash) or Tg (solid) B cells. Average percentages with SDs of IL-10-GFP+ cells are shown (n=7/group). Repeated twice. (C) RAG-1−/− mice received B cells from WT (n=3) or CD40LBTg (n=5) mice together with CD4 and CD8 T cells (IL-10−/− or +/+). Representative granzyme B expression FACS profiles of CD8 T cells in the recipients at 5 weeks post-cell transfer, with percentages for the gated granzyme B+ CD8 T cells. Average percentages with SDs of granzyme B+ CD8 T cells (n=3/group) are shown. Repeated once. (D) RAG-1−/− mice received CD8 T cells from WT, PD-1−/− or IFNα/βR−/− mice together with WT CD4 T cells and Tg B cells. Representative granzyme B vs IFNγ FACS profiles of CD8 T cells in RAG-1−/− recipients, with granzyme B+ percentages. Average percentages with SDs of granzyme B+ CD8 T cells and fold changes of IL-10 mRNA level in CD8 T cells compared to that in control WT CD8 T cells are shown (n=3/group). Repeated once.

Autocrine IL-10 expression is essential for CD8 T cells to counter-regulate their over-activation during lymphopenic proliferation

Using this adoptive transfer model, we further addressed the role of IL-10 expression in activated CD8 T cells by CD40LTg B cells. When RAG-1−/− mice were reconstituted with CD40LTg or wild type B cells and different combinations of IL-10 deficient or sufficient CD4 and CD8 T cells, the frequency of CD62LloCD44lo effector CD8 T cells (data not shown) and granzyme B+ CD8 T cells significantly increased when CD8 T cells lacked IL-10, and this was not further increased when CD4 T cells were also IL-10-deficient (Fig.3C). These results indicate that autonomous IL-10 expression in CD8 T cells is critical to counter-regulate their over activation and granzyme B expression when CD40LTg B cells stimulate CD8 T cells.

IFNα/βR and PD-1 signaling is essential for effecter memory CD8 T cells to express IL-10

Type I IFN signaling is critical for CD8 down-regulation (26) and PD-1 up-regulation on CD8 T cells (27) upon T cell receptor engagement. PD-1 engagement has been shown to induce IL-10 expression in T cells (28). As shown in Fig.2C, CD62Llo CD44hi/int CD8 T cells in CD40LTg mice, enriched with IL-10-GFP expression, expressed elevated levels of PD-1. Therefore, we hypothesize that CD8 T cell IL-10 expression is triggered as a regulatory mechanism down stream of Type-I IFN and PD-1 pathway. To test the hypothesis, CD8 T cells from IFNα/βR−/− or PD-1−/− mice were transferred into RAG-1−/− along with CD40LTg B cells. At 2 weeks after transfer, CD8 T cells were recovered from the recipient mice, and their granzyme B expression and IL-10 mRNA expression level was examined. As shown in Fig.3D, the frequency of granzyme B+ CD8 T cells in the recipients of CD40LTg B cells significantly increased when CD8 T cells lacked IFNα/βR or PD-1. Thus, signals from IFNα/βR and PD-1 are important to block the activation of CD8 T cells. Furthermore, whereas IL-10 mRNA level in CD8 T cells increased by about 54,000-fold after transfer into RAG-1−/− recipients together with CD40LTg B cells, it increased only 4- and 170-fold when CD8 T cells lacked IFNα/βR and PD-1, respectively. Thus, IFNα/βR deficient CD8 T cells almost completely failed to up-regulate IL-10 mRNA expression upon stimulation with CD40LTg B cells. These results indicate that the signal from IFNα/βR triggers IL-10 expression in CD8 T cells and this IL-10 expression is further enhanced by PD-1 signal delivered by PD-1/PD-L1 and PD-L2 interaction during CD8 T cell activation.

CD40LTg B cells induce IL-10 expression and suppress intestinal inflammation caused by spontaneous proliferation of CD8 T cells

Adoptive transfer of naïve CD62L+ CD44− CD8 T cells into RAG-1−/− mice causes colitis in association with MHC/antigen-driven rapid spontaneous proliferation and differentiation of inflammatory effector CD8 T cells producing IL-17 and IFNγ (29). Using this model, we tested whether or not co-transfer of CD40LTg B cells could suppress this CD8 T cell-mediated inflammatory response with induction of IL-10. As previously reported, RAG-1−/− recipients of naïve CD8 T cells significantly lost body weight (Fig.4A) in association with the differentiation of IL-17+ and IFNγ+ effector CD8 T cells in their mesenteric lymph nodes (MLNs) and spleen (Fig.4B). As shown in Fig.4A, co-transfer of CD40LTg, but not wild type, B cells could suppress inflammation and maintained the body weight of recipient RAG-1−/− mice during the period studied. Also, the frequency of IL-17+, but not IFNγ+, CD8 T cells was significantly reduced in the spleen (data not shown) and MLNs (Fig.4B) of the recipients of CD40LTg B cells compared to recipients of wild type B cells or without B cell transfer. As expected, CD8 T cells of the recipients of CD40LTg B cells had a significantly higher frequency of IL-10-GFP expression compared to those in the recipients of wild type B cells (Fig.4C). By histology, colon pathology was significantly improved in the recipients of CD8 T cells with B cell transfer compared to the recipients of CD8 T cells alone, and this was even more significant in the recipients with CD40LTg B cell co-transfer, compared to wild type B cell co-transfer, in association with reduced lymphocyte infiltration in the colon (Fig.4D).

Figure 4.

Figure 4

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CD40LTg B cells regulate naïve CD8 T cell-mediated inflammatory colitis by IL-10 induction. (A-D) RAG-1−/− mice received CD62L+ CD44− CD8 T cells from IL-10-GFP reporter mice with B cells from wild type or Tg mice (n=4 each), without B cells (n=4 each) or left untreated (n=3 each). (E-G) RAG-1−/− mice received CD62L+ CD44− CD8 T cells from IL-10-GFP reporter or IL-10−/− mice with Tg B cells (n=4 each). Repeated once. (A, E) Average percentages with SDs of body mass changes (%) for each recipient group are shown. (B, F) Representative intracellular staining IL-17 vs IFNγ FACS profiles for spleen or mesenteric lymphonode (MLN) CD8 T cells in the recipients with percentages for each quadrant. Average frequencies with SDs of IL-17+ (B, F) or IL-17+ and IFNγ+ (F) CD8 T cells in the MLN are shown (n=4/group). (C) Representative histograms for IL-10-GFP expression of spleen and MLN CD8 T cells in the recipients of WT (dash) or Tg (solid) B cells, with percentages of IL-10-GFP+ cells. (D, G) Representative H&E staining of colon histology are shown (100x). Average clinical scores with SDs are shown (n=4/group) to the right.

To test whether or not CD8 T cell IL-10 expression is essential for the regulation of inflammatory response by CD40LTg B cells, IL-10+/+ or IL-10−/− CD8 T cells were transferred into RAG-1−/− mice along with CD40LTg B cells. As shown in Fig.4E, the therapeutic effect of CD40LTg B cells was largely dependent on IL-10 expression by CD8 T cells since the recipients of IL-10−/− CD8 T cells with CD40LTg B cell co-transfer exhibited systemic inflammation, as evidenced by significant weight loss, along with significantly elevated frequency of IL-17+ and IFNγ+ CD8 T cells in their spleens and MLNs (Fig.4F). The recipients with IL-10−/− CD8 T cells also exhibited enhanced lymphocyte infiltration into the colon tissues, although the clinical scoring of this effect was not statistically significant (Fig.4G). These results demonstrate that CD40LTg B cells are capable of suppressing systemic as well as intestinal inflammation in spontaneously proliferating CD8 T cells under lymphopenic conditions by inducing IL-10 expression.

Discussion

Using CD40LTg B cells as a model for B cells in human inflammatory diseases which abnormally express CD40L or constitutively receive CD40L, this study presents the possibility that such B cells induce suppressive mechanisms in memory-like T cells with IL-10 expression. Furthermore, this study demonstrates in a mouse model that adoptive transfer of CD40LTg B cells can regulate inflammatory CD8 T cell response under lymphopenia, indicating a potential therapeutic use of CD40L-expressing B cells in CD8 T cell-mediated inflammatory diseases.

Lymphopenia, a condition characterized by reduced numbers of lymphocytes, is a critical co-factor of autoimmunity (30-32). Under lymphopenic conditions, residual a low numbers of CD8 T cells proliferate in response to cytokines and self- and commensal bacterial antigens presented by DCs (25, 29), and quickly form memory-like cells. Such memory-like cells are functionally indistinguishable from adaptive memory T cells to provide immediate protection (33), but may cause tissue damage. In the absence or failure of regulatory mechanisms, self- and microbial reactive T cells are abnormally activated and differentiated into inflammatory IL-17 and IFNγ effector cells to cause autoimmune diseases (31, 32). Lymphopenia-induced proliferation of autoreactive CD8 T cells closely correlates with the onset of diabetes in NOD mice (34). Furthermore, memory-like CD8 and CD4 T cells cooperate to break peripheral tolerance under lymphopenic conditions in an autoimmune diabetes model (35). These studies collectively support the notion that regulating effector differentiation of CD8 T cells during lymphopenic proliferation is a critical therapeutic target for autoimmune diseases.

In the autoimmune NOD mouse diabetes model, a nanoparticle vaccine coated with peptide-MHC complex could prevent and cure diabetes by selective expansion of low-affinity memory-like autoregulatory CD8 T cells, in an epitope-specific manner, to blunt the activation and recruitment of CD8 T cells with other specificities to the islets (36). In this context, CD40LTg B cells induced the expansion of effector memory-like CD8 T cells under lymphopenia (Fig.3A) and substantially reduced recruitment of CD8 T cells to the colon compared to wild type B cells (Fig.4D). It is interesting to speculate that CD40L-expressing B cells have a capacity similar to the nanoparticle vaccine and may be used for an adoptive cell transfer therapy to treat CD8 T cell-mediated inflammatory autoimmune diseases.

The current study identified CD40LTg B cells to be potent antigen-presenting cells for CD8 T cells, which can be used for a therapeutic target. In fact, accumulating evidence indicates a potential physical interaction between memory-like CD8 T cells and B cells under physiological settings. In a human/SCID rheumatoid arthritis model, IFNγ+ CD40L+ CD8 T cells in the mantle zone were required for the maintenance of ectopic germinal centers (37). In tonsils, CXCR5+ CD44hi memory-like CD8 T cells found in the mantle zone supported B cell antibody production (23). In a viral infection model, long-lived memory CD8 T cells were mainly found in B cell follicles (38). More recently, it was shown that MZ B cell numbers are determined by perforin-mediated CD8 T cell cytotoxicity (39). Further studies are needed to examine the role and significance of interactions between CD40-activated B cells and memory-like CD8 T cells in immune response.

In a line with the mechanisms of CD8 T cell activation by B cells, previous studies showed that anti-CD40 activated B cells modulated T cell response via overproduction of the cytokines IL-6, IL-10 and IL-15 (4, 40, 41). Also, injection of agonistic anti-CD40 Abs into mice induced bystander proliferation of memory-like CD8 T cells in a manner dependent on CD40 expression on antigen-presenting cells (i.e. B cells and DCs) and IL-15 (42). In our model, naïve CD40LTg B cells expressed relatively higher levels of IL-6, IL-10 and IL-15 mRNAs (2.5-fold, 2.0-fold and 2.0-fold, respectively) compared to wild type B cells. Also, CD21hi CD23lo marginal zone (MZ) B cells from CD40LTg mice produced greater amounts of IL-6 and IL-10 in response to various stimuli compared to those from control mice (P. Koni et al., manuscript in preparation). Thus, over production of certain cytokines could also be a mechanism of augmented CD8 T cell response in CD40LTg mice. Another potential mechanism for memory CD8 T cell activation by CD40LTg B cells could be through CD40 expressed on memory CD8 T cells (43) although this was not the case in bystander proliferation of memory-like CD8 T cells induced by agonistic anti-CD40 Abs (42). Further studies are needed to understand the molecular mechanisms of CD8 T cell activation by CD40L-expressing B cells for therapeutic use.

IL-10 expression has been found in virus specific exhausted CD8 T cells (44, 45) under chronic infection and was implicated as a regulatory mechanism to prevent over activation. In this context, the current study demonstrates that the absence of IL-10 expression in CD8 T cells increased granzyme B expression (Fig. 3C), inflammatory cytokine production (Fig. 4E) and lymphocyte infiltration in the colon (Fig. 4F). Thus, autonomous IL-10 expression is a critical safeguard mechanism in activated memory CD8 T cells to counter regulate over activation and block inflammatory response. In a viral infection model, IL-10 production by cytotoxic T cells was amplified by IL-2 derived from CD4+ TH cells with innate-derived IL-27 through a Blimp-1 dependent mechanism (46). Like in this chronic viral infection model, Blimp-1 and PD-1 expression coincided with IL-10 reporter expression in CD8 TEM cells in our model (Fig.2C). Furthermore, based on the data presented in Fig. 3D, we propose IL-10 expression in CD8 T cells to proceed at least in two steps. First, the IL-10 expression is induced under IFN-I signaling as a part of IFN-I-mediated regulatory mechanisms such as down-regulation of CD8α to desensitize T-cell receptor (TCR) signaling (26). Next, the IL-10 expression is dramatically up-regulated through PD-1 engagement. In CD8 T cells, IFN-I induces IRF9 that directly enhances PD-1 expression at the transcriptional level (27). PD-1 and the PD-L1/PD-L2 interaction critically regulates self-reactive CD8 T cell activation in various autoimmune disease models (47) via induction of CD8 T cell ‘anergy’ (unresponsive) by limiting their autonomous IL-2 production (48) as well as by causing ‘exhaustion’ in fully differentiated effector CD8 T cells to block cytotoxicity in association with up-regulation of various negative regulators (49, 50). In this context, we show that IL-10 expression also is one of the critical regulatory mechanisms mediated by PD-1 engagement.

Finally, our finding may be related to regulatory B cell functions, which are induced by anti-CD40 activation (51-53). Regulatory functions of B cells have been mainly associated with their own over production of IL-10 (41, 52, 54, 55). In addition, the current study suggests that the regulatory action of anti-CD40 activated B cells may, in fact, involve induction of IL-10 in the memory-like CD4 and CD8 T cell compartment. Whereas such a mechanism is certainly important to prevent excessive tissue damage in infections (45), colitis and other autoimmune diseases, it might also play an adverse role in chronic infections and malignancies by creating an immune suppressive environment that impairs overall T cell function. Thus, our findings may be helpful to treat patients with chronic inflammatory diseases.

Acknowledgments

We thank Dr David Gray for critical comments, Domenica Powell, Denise Gamble, Taejin Lee and Danielle O’connell for technical assistance, Dr Dimitris Moskophidis for FACS Canto, Dr Shuhua Han for Blimp-1 GFP mice. We also thank Drs D. Moskophidis and T. Honjo for IFNα/βR−/− and PD-1−/− mice, respectively.

Funding sources:

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR052470 (M.Shimoda), National Institute of Allergy and Infectious Diseases Grant AI064752 (M.Shimoda), and Georgia Health Sciences University PSRP grant (M.Shimoda).

Abbreviations

CD40L

CD40 ligand

PNA

peanut agglutinin

GVHD

graft versus host disease

CD8 TEM

CD8 effector memory T cell

FO

follicular

MZ

marginal zone

PD-1

Programmed cell death protein 1

PD-L1

Programmed cell death 1 ligand 1

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