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[Preprint]. 2024 Oct 3:2023.09.22.558524. Originally published 2023 Sep 23. [Version 3] doi: 10.1101/2023.09.22.558524

TIM-4 Identifies Effector B Cells Expressing a RORγt-Driven Proinflammatory Cytokine Module That Promotes Immune Responsiveness

Qing Ding 1, Yufan Wu 2, Elena Torlai Triglia 2, Jennifer L Gommerman 3, Ayshwarya Subramanian 2,4, Vijay K Kuchroo 2,4,5, David M Rothstein 1,6,*
PMCID: PMC10542535  PMID: 37790513

Abstract

B cells can express pro-inflammatory cytokines that promote a wide variety of immune responses. Here we show that B cells expressing the phosphatidylserine receptor TIM-4, preferentially express IL-17A, as well as IL-22, IL-6, IL-1β, and GM-CSF - a collection of cytokines reminiscent of pathogenic Th17 cells. Expression of this proinflammatory module requires IL-23R signaling and selective expression of RORγt and IL-17A by TIM-4+ B cells. TIM-4+ B cell-derived-IL-17A not only enhances the severity of experimental autoimmune encephalomyelitis (EAE) and promotes allograft rejection, but also acts in an autocrine manner to prevent their conversion into IL-10-expressing B cells with regulatory function. Thus, IL-17A acts as an inflammatory mediator and also enforces the proinflammatory activity of TIM-4+ B cells. Thus, TIM-4 serves as a broad marker for RORγt+ effector B cells (Beff) and allows further study of the signals regulating Beff differentiation and effector molecule expression.

INTRODUCTION

In addition to a primary role in humoral immunity, B cells are potent regulators of the immune response through antigen presentation, co-receptor engagement and production of cytokines1–6. Regulatory B cells (Bregs) inhibit immune responses through the expression of suppressive cytokines and coinhibitory molecules4–7. The phosphatidylserine receptor, TIM-1, is both a broad and functional marker for Bregs that play an essential role in restraining tissue inflammation and maintaining self-tolerance7,8. In addition to being a marker of Breg identity, TIM-1 signaling regulates the expression of a “regulatory module” that includes IL-10 and various coinhibitory molecules including TIGIT7. As such, specific deletion of TIM-1 in B cells results in spontaneous systemic autoimmunity characterized by inflammatory infiltration of multiple organs and accompanied by dermatitis, weight loss/rectal prolapse, and EAE-like paralytic disease.

In contrast to Bregs, effector B cells (Beffs), express proinflammatory cytokines that promote anti-microbial responses, autoimmunity, and allograft and tumor rejection2,5,6,9. For example, B cell-derived IL-2 and TNFα are required for clearance of H. polygyrus infection10. B cell-derived IL-6 enhances Th1 and Th17 responses and increases the severity of EAE11,12 (a model of human multiple sclerosis), and B cell-derived IFNγ promotes Th1 and inhibits Treg responses essential for generation of proteoglycan-induced arthritis11,12. During T. Cruzii infection, a pathogen-associated trans-sialidase was reported to specifically induce IL-17A expression in B cells through a RORγt-independent mechanism, promoting pathogen clearance13. However, a broader characterization and role for IL-17A in Beff function has not been elucidated. In humans, GM-CSF produced by B cells has been implicated in multiple sclerosis14,15. B cell depletion with anti-CD20 reduces T cell hyperreactivity and is now the treatment of choice for relapsing multiple sclerosis.

Unlike Bregs, the phenotype of Beffs has not been well-studied and this has impeded our understanding of their generation and effector functions. We previously showed that TIM-4+ B cells are enriched for IFNγ and enhance tumor and allograft rejection in vivo – activity opposite to that of TIM-1+ Bregs9. However, no more broadly unifying marker for Beffs has been identified and it remains unclear whether B cells expressing different inflammatory cytokines are related in phenotype or are regulated by common signaling mechanisms. It is also unknown whether Bregs and Beffs bear any functional relationship. Here, we characterize the role of TIM-4+ B cells as Beffs using high-throughput sequencing and murine models of autoimmunity and transplantation. We show that in addition to IFNγ, TIM-4+ B cells express a unique pro-inflammatory module driven by RORγt expression that includes IL-17A and additional proinflammatory cytokines in a pattern resembling pathogenic Th17 cells. B cell-derived IL-17A is not only a potent driver of inflammatory responses but is required as an autocrine factor to enforce expression of the proinflammatory module by TIM-4+ Beffs and prevent their conversion into IL-10-expressing B cells with regulatory function. Thus, TIM-4 is a marker that integrates B cells with many of the inflammatory functions previously ascribed to Beffs, and we identify a dual role for IL-17A as both a proinflammatory cytokine and regulator of Beff versus Breg function.

RESULTS

TIM-4+ B cells express IL-17A

As previously shown, TIM-4 is expressed on a subset of splenic B cells similar in size but largely distinct from TIM-1+ B cells (Fig. 1A)9. In addition to IFNγ production, we now show that TIM-4+ B cells from alloimmunized mice are also enriched for IL-17A production compared to total (CD19+) B cells or TIM-1+ B cells (Fig. 1B–C), and given their relatively low frequency of IL-10 expression, TIM-4+ B cells have a markedly higher IL-17A:IL-10 ratio than TIM-1+ B cells (Fig. 1B–D). In contrast, most B cells lack both TIM-1 and TIM-4 (double negative; DN) and produce minimal IL-10, IL-17A or IFNγ (Fig. 1B and 9). Most B cell-derived IL-17A is produced by the TIM-4+ subset, as confirmed by staining B cells from alloimmunized IL-17A GFP-reporter mice for TIM-4 in the absence or presence of in vitro stimulation with PMA and ionomycin plus brefeldin A (“PIB”) for 5 hours to enhance cytokine expression (Fig. 1E; Supplementary Fig. 1A shows B cell gating strategy). Similar levels of IL-17A were observed after immunization of mice for EAE (MOG35–55, CFA, and pertussis toxin), suggesting that IL-17A production is a generalized response by a portion of TIM-4+ B cells to immunizing stimuli (Supplementary Fig. 1B, C). To determine whether IL-17A production could be enhanced in vitro, sort-purified CD19+ B cells from naïve IL-17-EGFP reporter mice were stimulated with anti-IgM for 24 hours with or without the addition of various cytokines. After culture, bright EGFP-fluorescence was observed on a subset of B cells marked by the activation antigen Sca-1, and EGFP expression was increased 8–12-fold by the addition of IL-23 (Supplementary Fig. 2A, B). IL-1β increased IL-17A production to a lesser degree but had a synergistic effect when combined with IL-23. IL-6 had a small additive effect. To corroborate IL-17A production by intracellular staining and reporter expression, we examined cytokine secretion. TIM-4+ B cells stimulated for 48 hours with anti-IgM secrete IL-17A, but only in the presence of IL-23 (Fig. 1F). In contrast, TIM-4− B cells did not secrete detectable IL-17A. Finally, TIM-4+ but not TIM-4− B cells express IL-17A mRNA after stimulation with anti-IgM plus IL-23 (Supplementary Fig. 2C). Thus, TIM-4+ B cells express IL-17A and this was enhanced in vitro by IL-23.

Fig 1. TIM-4+ B cells are distinct from those expressing TIM-1+ and preferentially express IL-17A.

Fig 1.

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Splenic B cells from C57BL/6 mice 14 days after alloimmunization. (A, B) Representative flow cytometry plots: (A) TIM-1 and TIM-4 expression on CD19+ B cells and B) IL-10 and IL-17A expression on total (CD19+) B cells, TIM-1−TIM-4− (DN), TIM-1+, and TIM-4+ B cell subpopulations. IL-17A isotype (control) shown in bottom row. (GFP-negative control for IL-10-GFP reporter not shown). (C) Bar graph showing mean frequency (+ SD) of IL-10 and IL-17A expression on TIM-1+ vs. TIM-4+ B cells based on flow cytometry, as in (B) (n=9 mice per group in 3 individual experiments). (D) Bar graph showing the ratio of IL-17A+/IL-10+ cells within TIM-1+ and TIM-4+ B cell subsets (mean + SD) based on data in (C). (E) Representative FACS plots showing TIM-4 and IL-17A-EGFP expression on unstimulated vs. PIB stimulated CD19+ B cells from alloimmunized IL-17A-EGFP reporter mice. (n=3 mice per group). (F) IL-17 secretion by TIM-4+ and TIM-4− CD19+ B cells stimulated with anti-IgM +/− IL-23 for 48 hours (PMA/Ionomycin added for the final 5 hours). IL-17A concentration in the supernatants was measured by Cytometric Bead Array. (n=6 mice per group in 2 independent experiments). Statistics: * p< 0.05 vs. other groups; ** p<0.01 vs. other groups.

B cell IL-17A is an important driver of allo- and auto- immune responses

To determine the role of IL-17A produced by B cells on the immune response, we examined EAE in μMT mice receiving adoptive transfer of WT versus Il17a−/− B cells. In agreement with previous studies, μMT mice exhibit severe and unremitting EAE induced by MOG35–55 peptide, and this is reduced by the transfer of WT B cells which have been shown to contain Bregs (Fig. 2A)16. We reasoned that since WT B cells may not only include Bregs, but also IL-17A-producing Beffs, transfer of Il17a−/− B cells might further reduce EAE severity. Indeed, transfer of Il17a−/− B cells into μMT mice significantly reduced the severity of EAE well below that seen after transfer of WT B cells.

Fig 2. IL-17-deficient B cells are regulatory.

Fig 2.

Fig 2.

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(A) EAE was induced in μMT mice that received no B cells, or adoptive transfer of 10 X106 B cells from naive WT or IL-17A−/− mice. Fig. shows mean (+ SEM) EAE scores over time (n≥6 mice per group; representative of 2 independent experiments). ***p < 0.001 by 2-way ANOVA. (B) Kaplan–Meier plots showing survival of BALB/c islet allografts in diabetic μMT (B6) recipients that were untreated (control) or received 5×106 syngeneic TIM-4+CD19+ B cells from (day 14) alloimmunized WT or IL-17−/− donor mice. n=4–5 mice/group. *p <0.05; **p <0.01 versus control. (C) Representative flow cytometry plots of IL-10 expression by CD19+ and TIM-4+ B cells from alloimmunized IL-17A−/− versus WT B6 mice. (D) Bar graph showing mean frequency (+SD) of IL-10 expression on total (CD19+) and TIM-4+ B cells represented in panel (C). n = 3–5 mice/group. **p < 0.01. (E) μMT BM chimeric mice were reconstituted with 100% μMT BM or with 80% μMT BM plus 20% Il17−/−, Rorc−/−, or WT BM. Left panel: Kaplan-Meier plots showing BALB/c islet allograft survival in diabetic BM chimeric recipients (n=5–10 mice/group) *p < 0.05 vs. Controls. Right panel: Quantitation of IL-17A expression (intracellular staining) in alloimmunized WT vs. Rorc−/− BM chimeras (n=4 mice/group) *p < 0.05. (F) WT and Il17a BM chimeric mice were alloimmunized and cytokine (IL-10, IL-17, and IFN-γ) and Foxp3 expression on endogenous CD4+ T cells and CD19+ B cells was determined by intracellular flow cytometry. *p<0.05; **p<0.01. (G) Quantitation of frequency of IL-17+ B cells in RORγt-iBKO vs. Rorcfl/fl (Flox Control) mice 14 days after alloimmunization. ** p<0.01. (H) KaplanMeier plots of BALB/c islet allograft survival in Tamoxifen-treated diabetic RORγt-iBKO versus Flox Control and hCD20.ERT2.Cre+/− (Cre control) recipients. n=5–8 mice/group *p<0.05. (I) Bar Graph showing mean frequency (mean + SD) of IL-10 expression on total B cells and B cell subpopulations (n=3 mice per group). *p<0.05, **p < 0.01 RORγt-iBKO vs. Flox Control mice.

We next wished to confirm the activity of Il17a−/− B cells in an allograft setting which results in a robust innate and polyclonal T cell response 17,18. To remove the potentially confounding influence of Bregs from the findings, we transferred sort-purified TIM-4+ Beffs from alloimmunized syngeneic (B6) mice into chemically diabetic μMT (B6) islet allograft recipients. As we previously demonstrated, transfer of WT TIM-4+ B cells accelerated islet allograft rejection compared to control μMT recipients without B cell transfer (Fig. 2B). Surprisingly, Il17a−/− TIM-4+ B cells from alloimmunized mice not only failed to accelerate graft rejection, but markedly prolonged graft survival. In attempts to explain the suppressive activity of TIM-4+ B cells from Il17a−/− mice, we examined their cytokine expression. Compared to WT B cells, those from Il17a−/− mice produced IL-10 with twice the normal frequency (Fig. 2C, D). In particular, IL-10 frequency amongst Il17a−/− TIM-4+ B cells increased 3-fold, now similar to that of WT TIM-1+ B cells (Fig. 1).

To ensure that the effect on B cell IL-10 observed above was not due to dysregulation of B cells arising in globally IL-17A-deficient mice, we generated mixed bone marrow (BM) chimeras where B cells specifically lacked IL-17 (lethally irradiated μMT mice reconstituted with a 1:5 mixture of Il17a−/− plus μMT bone marrow). B cell Il17a−/− BM chimeras exhibited prolonged islet allograft survival with 33% of recipients never rejecting their allografts (Fig. 2E). In contrast, control uMT BM chimeras with WT B cells (reconstituted with a 1:5 mixture of WT plus μMT marrow) or uMT BM allograft recipients (reconstituted with 100% μMT BM), both acutely rejected islet allografts (Fig 2E) with a tempo similar to unmodified naive μMT allograft recipients (Fig 2B). We also generated mixed bone marrow (BM) chimeras where B cells specifically lacked lacked Rorc (encoding RORγt) by reconstituting lethally irradiated μMT hosts with a 1:5 mixture of Rorc−/− plus μMT BM. Compared to control μMT BM chimeras with WT B cells, B cells from Rorc−/− BM chimeras produced markedly reduced IL-17A (Fig. 2E). This demonstrates that like T cells, B cell IL-17 is RORγt dependent. Similar to B cell Il17a−/− BM chimeras, these B cell Rorc−/− BM chimeric mice also exhibited prolonged islet allograft survival with 30% of recipients never rejecting their allografts (Fig. 2E).

Endogenous CD4+ T cells in B cell IL-17A-deficient chimeras expressed significantly less IL-17A and IFNγ, and although IL-10 was not increased, there was a 2-fold increase in FOXP3+ Tregs (Fig. 2F). To determine how IL-17A-deficient B cells alter an antigen-specific T cell response within the context of an alloimmune response, B cell Il17a−/− BM chimeras vs. WT B cell control BM chimeras were immunized with Act-mOva.BALB/c.B6 F1 splenocytes, followed by adoptive transfer of CFSE-labelled OTII CD4+ T cells19,20. IL-17A-deficiency in B cells was associated with decreased proliferation (day 4) and altered frequency of cytokine production (day 7) by OTII CD4+ T cells. This included a 2-fold decrease in IFNγ, and ≥1.7-fold increase in IL-10 and IL-4 (Supplementary Fig. 3A). Similar changes were observed in transferred CD8+ OT-1 cells (Supplementary Fig. 3B). Thus, in the absence of Beff-derived IL-17A, T cell proliferation was decreased, and the T cell response was skewed towards less inflammatory and more regulatory activity. Examination of endogenous B cells in alloimmunized Il17a−/− BM chimeras versus WT B cell control BM chimeras demonstrated that, as expected, B cell IL-17A was negligible, but IFNγ was also reduced by ~30%, and once again B cell-derived IL-10 was increased more than 2-fold (Fig. 2F).

In a further attempt to separate IL-17/RORγt deficiency from increased IL-10, we generated mice with an inducible knockout of Rorc in B cells by crossing hCd20-ERT2.Cre and Rorcfl/fl mice (RORγt-iBKO). These mice are entirely normal until being placed on Tamoxifen (TAM) chow to acutely induce B cell-specific deletion of RORγt. In comparison to TAM-treated Rorcfl/fl (Flox control) mice, B cells from RORγt-iBKO mice exhibited an 80% reduction in B cell-derived IL-17A (Fig. 2G). RORγt-iBKO mice exhibited prolongation of islet allograft survival compared to either Flox control or hCd20-ERT2.Cre (Cre control) mice, with ~35% surviving long-term (Fig. 2H), very similar to that seen in the B cell Il17a−/− or Rorc−/− BM chimeras above. Furthermore, RORγt-iBKO mice also exhibited significantly decreased EAE severity (Supplementary Fig. 3C). Analysis of B cells in alloimmunized RORγt-iBKO mice again demonstrated increased frequency of IL-10 production, ~2-fold by total B cells and 5-fold by TIM-4+ B cells (Fig. 2I and Supplementary Fig. 3D). Thus, even the acute loss of RORγt and IL-17A in developmentally normal mice results in dysregulated IL-10 production in the TIM-4+ Beff subset. Furthermore, the loss of proinflammatory IL-17A and increased IL-10 production by B cells is associated with increased allograft survival and decreased EAE severity.

Regulation of IL-17A and IL-10 expression in B cells by IL-23 and IL-17

The data above (Figs. 1 and S2) suggested that IL-17A production by TIM-4+ B cells is IL-23-dependent while Il17 or Rorc deletion in B cells, augments IL-10 production. To gain further insight into the regulation of these cytokines, we examined Il17a and Il10 expression at the transcriptional level by performing quantitative PCR (qPCR) on sort-purified TIM-4+(TIM-1−) and TIM-1+(TIM-4−) B cells from WT or Il23r−/− mice after anti-IgM stimulation with or without additional cytokines for 24 hours. Stimulation of TIM-4+ B cells with anti-IgM induced detectable Il17a mRNA, and this was increased ~3-fold with the addition of IL-17A and ~10-fold with IL-23 (Fig. 3A). Consistent with reporter expression (Supplementary Fig. 2B), IL-1b synergized with IL-23 in augmenting Il17a mRNA (Fig. 3A). Neutralizing IL-17 in the culture by adding anti-IL-17A mAb decreased Il17a mRNA expression induced by either IL-23 alone or IL-23 combined with IL-1β, suggesting that part of the effect of IL-23 on increased IL-17A transcription is mediated by IL-17A secreted into the medium in a positive feedback loop. TIM-4+ B cells from Il23r−/− mice exhibited markedly reduced Il17a expression, confirming the requirement for IL-23 signaling to induce Il17a expression by TIM-4+ B cells (Fig. 3A). In contrast, neither WT nor Il23r−/− TIM-1+ B cells expressed detectable Il17a mRNA. The mRNA expression data was confirmed at the protein level by comparing IL-17A secretion by sort-purified TIM-1+ versus TIM-4+ B cells in vitro after 48-hour culture (Fig. 3B). IL-23 markedly enhances IL-17A secreted by anti-IgM-treated TIM-4+ B cells. As expected, anti-IL-17A completely neutralized IL-17A detected in the supernatants. TIM-1+ B cells secrete minimal IL-17A even after IL-23 treatment.

Fig 3. Regulation of IL-17A and IL-10 expression by IL-23 and IL-17.

Fig 3.

Fig 3.

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(A) TIM-1+TIM-4 and TIM-4+TIM-1− CD19+ B cells from alloimmunized Il23r−/− and WT B6 mice were stimulated for 24 hours with anti-IgM and cytokines or neutralizing anti-IL-17A as indicated. IL-17A expression was compared to that of GAPDH by qPCR. The bar graph shows the relative expression of IL-17A mRNA as 2-ΔΔCT performed in triplicate wells on cells pooled from 3 mice per group. **p<0.01; ***p<0.001. Data representative of three independent experiments. (B-C) TIM-1+TIM-4− and TIM-4+TIM-1− B cells from alloimmunized WT B6 mice were stimulated with anti-IgM alone or anti-IgM plus IL-23, IL-17A or anti-IL-17A neutralizing antibody as indicated. Bar graphs display the concentration of IL-17A (B) and IL-10 (C) in the supernatants (performed in triplicate wells on cells pooled from 3 mice per group). Data representative of three independent experiments. **p<0.01 and ***p<0.001.

(D) TIM-1+TIM-4− and TIM-4+TIM-1−B cells from Il23r−/− and WT B6 mice, were stimulated and IL-10 mRNA expression assessed by qPCR as in (A) above. The bar graph shows the relative expression of IL-10 as 2-ΔΔCT in triplicate wells on cells pooled from 3 mice per group. **p<0.01; ***p<0.001. Data representative of three independent experiments. (E) Violin plot depicting mean frequency (+SD) of IL-10 expression (intracellular staining) on splenic CD19+ B cells from IL-17RCfl/flCD19-Cre+/− (IL-17RC-BKO) and CD19-Cre (Cre Control) mice. n=5 mice. *p<0.05.

Our data (Fig. 2 and Supplementary Fig. 3) indicate that deletion of either Rorc or Il17a in B cells increases IL-10 production by TIM-4+ B cells. This suggests that B cell-derived IL-17A directly or indirectly inhibits IL-10 and maintains Beff function. To address this, we examined IL-10 secretion by sort-purified TIM-4+ versus TIM-1+ B cells in vitro. Anti-IgM-stimulated TIM-4+ B cells secrete only 17% as much IL-10 as TIM-1+ B cells (Fig. 3C), in agreement with the frequency of IL-10 expression determined by intracellular staining (Fig. 1). The addition of IL-23 to TIM-4+ B cells, which promotes IL-17A secretion, inhibits their secretion of IL-10 ~3-fold. However, when IL-17A in the supernatant is neutralized with anti-IL-17A, anti-IgM plus IL-23-stimulated TIM-4+ B cells increase their IL-10 secretion almost 8-fold (~2.5-fold above the baseline with anti-IgM alone; Fig. 3C). In contrast, IL-10 secretion after anti-IgM plus IL-23 treatment is further reduced by the addition of exogenous IL-17A. These data suggest that IL-17A directly inhibits IL-10 secretion by TIM-4+ B cells and contributes to the inhibitory effect of IL-23.

TIM-1+ B cells secrete higher levels of IL-10 in response to anti-IgM than TIM-4+ B cells and IL-10 production is markedly inhibited by IL-23 (Fig. 3C). While IL-23 induces detectable IL-17A secretion by TIM-1+ B cells (Fig. 3B), this is unlikely to be the main mechanism by which IL-23 inhibits IL-10 production in TIM-1+ B cells since IL-17A neutralization only slightly restores IL-10, and addition of exogenous IL-17A has no significant effect on IL-10 secretion compared to IL-23 alone (Fig. 3C). Thus, IL-23 does signal and inhibit IL-10 secretion in both TIM-1+ and TIM-4+ B cells. However, in TIM-4+ B cells the inhibition of IL-10 by IL-23 signaling can be overcome by IL-17A neutralization, whereas in TIM-1+ B cells IL-23-mediated suppression of IL-10 appears largely independent of IL-17A.

Examination of Il10 mRNA by qPCR generally corroborates cytokine secretion data (Fig. 3D). Neutralization of IL-17 in IL-23 treated B cells augmented Il10 expression, particularly in the TIM-4+ subset which expresses more IL-17. Il23r−/− TIM-4+ and TIM-1+ B cells both expressed more Il10 than their WT counterparts but did not respond to IL-17 neutralization, consistent with their lack of IL-17 expression (Fig. 3A, D). Yet, addition of exogenous IL-17A reduced IL-10 expression in TIM-4+ and TIM-1+ B cells, even in the absence of IL-23 signaling. Taken together, our data indicate that IL-17A has direct and independent effects to suppress Il10 expression in both TIM-4+ and TIM-1+ B cells. Since B cells from BM chimeras specifically lacking B cell-derived IL-17 exhibit increased IL-10 production (Fig. 2D), our findings suggest that B cell-derived IL-17A acts as an autocrine factor to suppress B cell IL-10. If IL-17A has a direct effect on B cell Il10 expression, IL-17R-deficient B cells should express more Il10. To test this, we generated Il17RCfl/fl X CD19-Cre (IL-17RC BKO) mice and compared these to CD19-Cre controls. Consistent with our hypothesis, B cells from immunized IL-17RC BKO mice produced over 40% more IL-10 than Cre control B cells (Fig. 3E).

IL-17A production in TIM-4+ vs. TIM-1+ B cells is due to selective expression of RORγt.

Both TIM-1+ and TIM-4+ B cells respond to IL-17 and IL-23 with reduced IL-10 expression, however, only TIM-4+ B cells respond with increased IL-17 expression. Thus, these two distinct subsets of B cells exhibit major differences in response to cytokines regulating their effector function. In this regard, we demonstrated above that B cell IL-17 expression is dependent on RORγt. Therefore, we asked whether differences in RORγt levels might underlie the major difference in regulation of cytokine expression by TIM-4+ vs. TIM-1+ B cells. RORγt is almost exclusively expressed by TIM-4+ B cells and this correlates directly with IL-17A expression (Fig 4 A,B). Only a very small fraction of DN or TIM-1+ B cells express RORγt or IL-17, and this is at low MFI. RORγt is expressed in ~3% of TIM-4+ B cells from naïve mice and increased in frequency to 5% after alloimmunization (Supplementary Fig 4A). In vitro stimulation (PIB) further increases RORγt expression on TIM-4+ B cells (compare Supplementary Fig 4B and Fig 4B).

Figure 4. RORγt is selectively expressed by TIM-4+ B cells and this correlates directly with IL-17A expression.

Figure 4

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Splenic B cells from C57BL/6 mice 14 days after alloimmunization. (A) Representative flow cytometry plots show RORγt and IL-17A expression on total DN (TIM-1−TIM4−), TIM-1+, and TIM-4+ B cell subpopulations. isotype (control) shown in bottom row. (B) Bar graph showing mean frequency (+ SD) of RORγt and IL-17A expression on TIM-4+, TIM-1+ and DN B cells based on flow cytometry, as in (A). * p < 0.05; *** p < 0.001. (n=6 mice per group in 2 independent experiments). (C, D) Purified CD19+B cells from spleens of TAM-treated RORγt iBKO and Cre control mice were stimulated in vitro with anti-IgM alone plus IL-23, or IL-17A for 48 hours. Bar graphs display the concentration IL-17A (C) , and IL-10 (D) in the supernatants, as measured by Cytometric Bead Array. * p <0.03 vs. other Control groups; ** p<0.001 vs. corresponding Control group; ***p<0.001 vs. all other groups. (n=6 mice per group, in 2 independent experiments).

Consistent with intracellular staining (Fig. 2G) B cells from TAM-treated RORγt iBKO mice exhibit significantly reduced IL-17A secretion in response to anti-IgM. Moreover, RORγt deletion essentially abrogated the increase in IL-17A secretion induced by IL-23 compared to Cre-control mice (Fig 4C). Low level expression of IL-17A secretion is consistent with incomplete deletion of RORγt in these iBKO mice. Also consistent with intracellular staining (Supplementary Fig. 3D), deletion of RORγt from RORγt iBKO mice, resulted in increased IL-10 secretion by TIM-4+ B cells (Fig 4D). Notably, exogenous IL-23 reduced IL-10 secretion by TIM-4+ B cells lacking RORγt, suggesting that IL-23 does exert an IL-17-independent effect. Although exogenous IL-17 inhibits IL-10 secretion by RORγt-deficient TIM-4+ B cells, IL-10 remained far higher than by Cre-control TIM-4+ B cells. This suggests that RORγt inhibits IL-10 secretion through other means. In this regard, we previously showed that RORγt can bind to the IL-10 promotor and suppress IL-10 expression in Th17 cells 21.

Profiling TIM-4+B cells reveals expression of multiple pro-inflammatory cytokines in a pattern resembling pathogenic Th17 cells

Based on their expression of RORγt, we asked whether TIM-4+ B cells expressed additional pro-inflammatory cytokines, in comparison to the anti-inflammatory profile of TIM-1+ B cells. To address this, we performed population level transcriptomic sequencing (RNA-seq) on RNA isolated from highly sort-purified TIM-4+(TIM-1−), TIM-1+(TIM-4−), and TIM-4-TIM-1- (DN) B cells from spleens of alloimmunized mice stimulated for 24 hours with anti-IgM plus IL-23. This allowed us to compare TIM-4+ Beffs and TIM-1+ Bregs, separately from the majority DN B cell population which expresses little IL-17, IFNγ, or IL-10 (Fig. 1 and 9).

Principal component analysis (PCA) revealed that PC1 separated the expression profiles of TIM-1+ and TIM-4+ B cells, while PC2 separated DN B cells from both TIM-4+ and TIM-1+ B cells (Supplementary Fig. 5A). Variability in TIM-1+ samples from different mice along PC1 related to total number of unique genes detected. Comparison of TIM-4+ and TIM-1+ B cells by differential gene expression analysis revealed 3425 upregulated genes (613 with fold change >1.5) and 7858 downregulated genes (7393 with fold change >1.5) in the TIM-4+ population (FDR <0.05; Fig. 5A). Genes of interest are displayed in the volcano plot (Fig. 5B). Compared to TIM-1+ B cells, TIM-4+ B cells not only expressed higher levels of Il17a, but also a pro-inflammatory module comprising Il17f, Il22, Csf2, Il6, Il1β, TNFα, and Il2 (Fig. 5B–D). These transcripts were not detected in the absence of IL-23 (not shown). As expected, TIM-1+ cells expressed more Il10, and also Ctla4, Nte5 (expressing CD73), ENTpd1 (expressing CD39), Cd274 (expressing PD-L1), and Pdcd1lg2 (expressing PD-L2)7. Of note, IFNγ expression, showed a trend, but was not statistically higher in TIM-4+ B cells (Fig. 5C), even though IFNγ protein expression is higher in freshly isolated TIM-4+ versus TIM-1+ B cells9. Surprisingly, in these stimulated cells, the TIM-4 gene (Timd4) was expressed at higher levels by TIM-1+ B cells even though TIM-4 protein was not detected by flow cytometry. Many of the same proinflammatory cytokines noted above were also increased in TIM-4+ B cells compared to DN B cells (Fig. 5C, D). The top 50 upregulated and downregulated genes in TIM-4+ vs TIM-1+ B cells are listed in Table S1. Intracellular staining confirms increased expression of IL-1β, GM-CSF, IL-22, and IL-6 at the protein level in TIM-4+ B cells (Supplementary Fig 5B). Examination of these same cytokines reveals reduced frequency of expression in TIM-4+ B cells from Rorc KO mice, indicating partial, but not complete, dependence.

Fig 5. Differential gene expression in TIM-1+ and TIM-4+ B cells from alloimmunized mice.

Fig 5.

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A-B: Amongst genes of interest, pro-inflammatory genes are depicted in red, anti-inflammatory genes are in blue, and other genes are in black text. A) Heatmap showing differentially expressed genes identified by RNA-seq (FDR<0.05). B) Volcano plot with selected genes of interest. The y-axis is capped at 25 for visual clarity. C) Heatmap comparing expression of selected genes of interest between three groups: TIM-1+, TIM-4+ and DN B cells annotated on the right. Vertical bars represent genes that are upregulated (orange), downregulated (black), or not statistically differentially expressed (gray, FDR ≥ 0.05) in a given comparison between TIM-4+ B cells (T4) and either TIM-1+ (T1) or DN B cells. D) Fold change in expression between select genes in TIM-4+ vs. TIM-1+ B cells or TIM-4+ vs. all other (TIM-4−) B cells. Data are representative of a second independent experiment comparing TIM-4+ vs TIM-4− B cells.

The pattern of cytokine genes expressed by TIM-4+ B cells closely resembles that of pathogenic Th17 cells (pTh17) 22–24. Comparing gene expression by TIM-4+ B cells to TIM-1+ B cells, demonstrates that in addition to the cytokines pathognomonic for pTh17 cells like Csf2 and Il22, TIM-4+ B cells exhibit increased expression of a number of other genes in the pTh17 signature that we previously identified by polarizing pTh17 cells with TGFβ3 and IL-6, compared to non-pTh17 cells polarized with TGFβ1 and IL-6 (Supplementary Fig. 5C) 23. These include chemokines such as Ccl3, Ccl4, and Ccl5, as well as Stat4, and Lgals3 (encoding Galectin 3). However, not all genes upregulated in that pTh17 signature were upregulated in TIM-4+ B cells. For example, Icos, Cxcl3, Gzmb, Casp1 (encoding caspase I, required for IL-1β maturation) and Tbx21, were either expressed at lower levels or were not statistically higher in TIM-4+ B cells. Amongst genes downregulated in pTh17 cells, Il10, Maf, and Ikzf3 (IKAROS family Zinc Finger 3) are also downregulated in TIM-4+ B cells, while Cd5l was upregulated and Ahr expression was variable in TIM-4+ B cells (Supplementary Fig. 5D). Of genes identified in several other pTh17 signatures24–29, only Batf, Bhle40, and Cd44 were upregulated in TIM-4+ B cells, but not Il23r, the canonical Notch signaling mediator Rbpj, IL1rn, Gpr65, Toso, Plzp, Batf3, Cd44, Prdm1, Protein receptor C (PROCR), Foxo1, or IRF4, (Supplementary Fig. 5C). These data suggest that while expressing similar cytokines, the regulation of TIM-4+ B cell induction and/or differentiation may be distinct from that of pTh17 cells.

DISCUSSION

In addition to antibody production, B cells play an important role enhancing or inhibiting immune responses through antigen presentation, the elaboration of cytokines and expression of coinhibitory and costimulatory ligands 1,2,4–6. Despite the strong influence of various B cell-derived proinflammatory cytokines on anti-microbial, tumor, allo-, and auto- immunity, no broad marker for Beffs has been previously identified. Herein, we demonstrated that TIM-4 identifies proinflammatory Beff cells that selectively express RORγt, IL-17A, and additional proinflammatory cytokines resembling those expressed by pTh17 cells. Production of IL-17A by TIM-4+ B cells is dependent on IL-23, RORγt, and IL-17 itself, which is required as a B cell autocrine factor. In the absence of these underlying pro-inflammatory signals, TIM-4+ B cells are dysregulated and now produce IL-10 and exhibit potent regulatory activity.

Until recently, our understanding of Breg biology was hampered by the lack a unifying marker. We have since shown that TIM-1 is a broad marker for Bregs, and regulates the expression of an array of anti-inflammatory cytokines and coinhibitory molecules7,8. Its specific deletion in B cells results in spontaneous systemic autoimmunity, including an EAE-like paralytic neuroinflammatory disorder7. In contrast, B cells can also produce a variety of proinflammatory cytokines that play important roles in inflammatory immune responses. These include: B cell production of TNFα and IL-2 (required for the clearance of H. polygyrus); IL-6 (augments EAE severity); GM-CSF (pathogenic role in EAE and multiple sclerosis); and IFNγ (requisite role in proteoglycan-induced arthritis, and determines the relative size of the germinal center versus extrafollicular response during infection) 2,5,6,12,30. Yet, few studies have examined the phenotype of B cells producing any of these proinflammatory cytokines. We previously showed that TIM-4+ B cells were proinflammatory, enhancing both tumor and allograft rejection9. This was initially attributed to their increased expression of IFNγ. We now show that TIM-4 identifies B cells that also express IL-17A, IL-17F, IL-22, IL-6, IL-1β, and GM-CSF. Thus, TIM-4+ is a broad marker for B cells that express many of the proinflammatory cytokines previously attributed to B cell effector function. This allows us to begin to address how proinflammatory cytokine production is regulated in TIM4+ Beffs and differs from the signals regulating TIM-1+ Breg activity.

Bermejo et al previously reported that in the isolated setting of T. cruzii infection, a pathogen-specific transialidase alters CD45 glycosylation, and this induces B cell IL-17A expression that is RORγt-independent13. Whereas adoptive transfer of WT B cells into μMT mice enhanced clearance of T. cruzii, IL-17−/− B cells had no effect. We now demonstrate that B cell IL-17A expression is a much more generalized component of B cell function, expressed in response to various types of immunization at a frequency similar to that of B cell-derived IL-10. In these settings, IL-17A is RORγt-dependent and predominantly expressed by TIM-4+ B cells. In mice lacking B cell-derived IL-17A or RORγt, TIM-4+ B cells are dysregulated and significantly upregulate IL-10 resulting in strong regulatory activity leading to decreased CD4 and CD8 proliferation, increased Tregs, decreased Th17 and Th1 responses, reduced EAE severity, and prolonged allograft survival. Conversion between B cells with regulatory versus pro-inflammatory activity has not been previously described.

Non-pathogenic Th17 (npTh17) cells express IL-10 along with IL-17A, and while not frankly suppressive, they play an essential role maintaining the integrity of the gut mucosal barrier 31,32. In contrast, pTh17 cells express little IL-10, but express IL-17A in conjunction with a host of other pro-inflammatory cytokines including, for example, GM-CSF which plays an important inflammatory role in EAE 32,33. The equilibrium between npTh17 and pTh17 cells is carefully regulated by balancing IL-10 and IL-23R expression, respectively. For example, npTh17 cells express increased Cd5L, which interacts with fatty acid synthase to alter the lipid biosynthesis and reduce RORγt-agonistic ligands 21. This reduces RORγt-DNA binding, resulting in decreased activation of the Il23r locus and reduced repression of the Il10 locus, which together, favor the differentiation of npTh17 rather than pTh17 cells. In contrast, increased RBPJ, a mediator of NOTCH signaling, enhances Il23r expression which drives pTh17 differentiation 34.

IL-23 is also an important driver of the TIM-4+ pro-inflammatory module which shares many similarities to that of pTh17 cells including cytokines, chemokines (Ccl3, Ccl4, Ccl5), Lgals3 (encoding Galectin3), and Stat4 23. Despite this resemblance, the regulation of proinflammatory cytokine versus IL-10 expression by TIM-4+ B cells, and between TIM-4+ Beffs and the opposing TIM-1+ Breg subset, differs from that in npTh17 and pTh17 cells. For example, compared to TIM-1+ Bregs, TIM-4+ B cells express increased Cd5l, which as noted above, is more highly expressed in npTh17 cells, where it promotes IL-10 and inhibits Il23r expression, thereby inhibiting proinflammatory function. While TIM-4+ B cells express little Il10 and are proinflammatory, CD5L may act to modulate their proinflammatory activity. Moreover, neither RBPJ nor Il23r are differentially expressed, consistent with IL-23 responsiveness by both TIM-1 and TIM-4+ B cells. Indeed, IL-23R signaling reduces IL-10 expression in both TIM-1+ and TIM-4+ B cells. Yet, IL-23 only induces inflammatory cytokine expression in TIM-4+ B cells, likely due to their almost exclusive expression of RORγt. In addition to its requisite role in IL-17A expression, RORγt augments the expression of other proinflammatory cytokines by TIM-4+ Beffs, as in pTh17 cells. In this regard, TIM-4+ B cells differentially express Bhle40, a basic helix-loop-helix transcription factor that promotes Csf2 and inhibits Il10 expression in pTh17 cells, and BatF, an AP-1 transcription factor that transactivates Il17 and Il22 expression 35,36. Thus, TIM-1+ and TIM-4+ B cells with opposing functions as Beffs and Bregs, respectively, exhibit distinct signals that differentially regulate regulatory and proinflammatory cytokine loci.

We showed that exogenous IL-17A directly inhibits IL-10 production by TIM-4+ B cells in vitro. IL-23 also inhibits IL-10 production by TIM-4+ B cells, including those from RORγt iBKO mice, indicating an IL-17-independent effect. However, in WT TIM-4+ B cells, IL-10 expression in enhanced by neutralizing IL-17, despite exogenous IL-23 and intact (endogenous) RORγt, suggesting that IL-17 plays a dominant role. This is supported by BM chimeric mice with a B cell-specific deletion of Il17a which exhibit significantly increased IL-10 production by B cells - also indicating that IL-17 plays an autocrine role inhibiting B cell IL-10 production in vivo. This suggests that TIM+4+ B cells largely reside near other B cells and away from other endogenous sources of IL-17. To our knowledge, a pathway leading from IL-17A signaling to IL-10 inhibition has not been previously described and will require further study. This IL-17 pathway does not predominate in TIM-1+ B cells where IL-23 signals strongly inhibit IL-10 despite barely detectable IL-17A expression, and only modest recovery of IL-10 expression occurs upon IL-17A neutralization. Addition of exogenous IL-17 does not suppress IL-10 expression by TIM-1+ B cells beyond that seen with IL-23 alone. However, in the absence of IL-23/IL-23R signaling, exogenous IL-17 does inhibit Il10 expression by TIM-1+ B cells, indicating that IL-17 and IL-23 independently inhibit Il10 expression. Finally, we showed that RORγt plays an IL-17 and IL-23 independent role on the inhibition of IL-10 expression in TIM-4+ B cells. This is consistent with our previous findings that RORγt can bind to the IL-10 promotor and inhibit its expression 21.

In summary, in TIM-4+ B cells, IL-23 drives the expression of a proinflammatory module which includes RORγt-dependent IL-17A production. IL-17A acts in an autocrine manner to enhance its own expression, and along with RORγt, suppresses IL-10 and enforces pro-inflammatory Beff activity. In contrast, IL-23 has minimal effect on proinflammatory cytokine expression by TIM-1+ B cells which lack RORγt, while IL-23 and IL-17A independently inhibit IL-10 expression. These findings suggest that cross-regulation may occur at several levels. IL-23 in the microenvironment should inhibit Il10 expression by TIM-1+ B cells and augment expression of the TIM-4+ proinflammatory module. IL-17A expressed by TIM-4+ B cells not only enforces their Beff activity but may suppress Il10 expression by TIM-1+ Bregs. Finally, impaired IL-17 production by TIM-4+ Beffs results in their increased Il10 expression and Breg activity. Thus, we have identified a reciprocal relationship between B cells with regulatory and effector activity. In this regard, we recently showed that deletion of TIM-1 expression in B cells inhibits tumor progression and results in the expansion of B cells in the draining LNs that express proinflammatory cytokines in a type I IFN-mediated manner37. Whether this is due to the acquisition of inflammatory function by B cells that were destined to become TIM-1+ Bregs, or due to increased TIM-4+ Beffs in the absence of TIM-1+ Bregs, is not yet clear. In conclusion, TIM-4 is a broad marker for RORγt-expressing B cells that exhibit Beff function. IL-23 and IL-17A selectively enhance proinflammatory cytokine expression by TIM-4+ B cells, while preventing their conversion into IL-10-expressing B cells with regulatory activity. Further elucidation of these signals may allow therapeutic manipulation of TIM-4+ B cells to appropriately inhibit or enhance the immune response in settings of autoimmunity, transplantation, and cancer.

METHODS:

Study Design:

To further study the role of TIM-4+ B cells, we examined their cytokine production by flow cytometry and confirmed results using IL-17-reporter mice, and in vitro cytokine secretion. The role of B cell IL-17 was studied in EAE and islet allograft models by B cell adoptive transfer, generation of BM chimeras, and inducible B cell specific RORγt knockouts. These studies revealed an inverse relationship between IL-17 and IL-10 expression. The role of various cytokines in regulating IL-17 vs. IL-10 expression was directly tested in Tim-4+ as well as Tim-1+ Bregs in vitro by RT-PCR and cytokine secretion assays. Finally, Bulk RNAseq was performed to reveal that Tim-4+ Beffs express a module of proinflammatory cytokines, allowing a comparison to the genes signature of pTh17 cells. Previously published methods and those not used in the main Figs. are described in Supplementary Methods.

Mice:

C57BL/6 (B6; H-2b), BALB/c (H-2d), Rorc−/−, Rorcfl/fll, Il17a–/–, B6 IL-17A–EGFP reporter, OT-I transgenic, OT-II transgenic and μMT (B6) mice were from The Jackson Laboratory. hCd20-ERT2.Cre (from Mark Shlomchik, University of Pittsburgh38 were crossed with Rorcfl/fl mice to generate Rorcfl/fl.hCd20Cre+/− (RORγt iBKO) mice. Act-mOVA F1 mice (from Fadi Lakkis, University of Pittsburgh) were generated by breeding BALB/c with B6.Act-OVA mice19. Mice were used at 6–10 weeks of age and were housed with food and water ad libitum.

Islet transplantation and alloantigen immunization:

400 allogeneic islets from BALB/c donors were transplanted under the left renal capsule of sex-matched B6 recipients with streptozotocin-induced diabetes, as previously described 8,9. In some experiments, mice were alloimmunized by i.p. injection of 2 × 107 mitomycin C–treated BALB/c splenocytes8,9.

Flow cytometry:

Fluorochrome-conjugated mAbs were purchased from BD Biosciences, eBioscience, and BioLegend. Staining was performed in the presence of Fc block(anti-CD16/CD32) and LIVE/DEAD Fixable Aqua viability indicator as described 8,9. Flow acquisition was performed on LSRII analyzers (BD Biosciences), and data were analyzed using FlowJo software (BD). Background staining was determined with isotype-matched controls or GFP− littermates. Detection of intracellular cytokine detection by flow cytometry was previously described 8,9. Briefly, T cells were cultured 4 h with PMA (50 ng/ml), ionomycin (500 ng/ml), and GolgiPlug (1 μl/ml). For IL-10, B cells were cultured 5 hours with LPIM (LPS (10 μg/ml), PMA, ionomycin, and monensin (1 μl/ml)). For IL-17, and other proinflammtory cytokines, B cells were cultured 5 hours with PIB (PMA, ionomycin, and Brefeldin A (1 μl/ml)). Cells were permeabilized using intracellular staining kits from BD Biosciences or eBioscience.

Cell preparation and adoptive transfer:

B cells were isolated from spleens of naïve or alloimmunized (d14) WT or IL17−/− mice by negative selection (EasySep; StemCell Technologies; purity >98%). CD19+TIM-4+ and CD19+ TIM-4– B cells were subsequently isolated by FACS using BD FACSAria (>95% purity). 5–7 ×106 purified B cell subsets from naïve or alloimmunized syngeneic mice were adoptively transferred (i.v.) into otherwise untreated μMT recipients followed by islet transplants or EAE induction.

EAE:

Mice were immunized s.c. with MOG 35–55 (200μg) and CFA (200 μl; Difco Laboratories) and received pertussis toxin (200 ng/mouse; ip) on days 0 and 2 7. Mice were scored daily for severity of EAE, as described 7.

Bone marrow (BM) chimeric mice:

μMT (H-2b) mice were lethally irradiated (1000 rad) and reconstituted with 80:20 mixtures of syngeneic BM cells from: μMT plus WT, μMT plus Il17a−/−, or μMT plus Rorc−/− donors 9. BM chimeras were used after 8 weeks to allow immune reconstitution. B cells were fully reconstituted (from non-μMT BM).

Cytokine Secretion:

IL-17A and IL-10 concentration in supernatants of B cells/subsets after 48 hours of stimulation with anti-IgM plus various cytokine. PMA and ionomyicn were added for the final 5h. was determined by Mouse Cytokine Cytometric Bead Array (CBA) and associated software per manufacturer’s instructions (BD Biosciences). 3000 events were acquired per sample on an LSR II (BD) analyzer.

RNA isolation, real-time PCR:

Splenic CD19+ B cells, isolated from alloimmunized B6 mice using the EasySep™ Mouse Pan-B Cell Isolation Kit (STEMCELL; purity >98%), were subsequently sorted based on staining criteria as CD19+ TIM-1+TIM-4-, TIM-4+TIM-1-, and DN (DUMP-negative for CD3, CD64, Gr-1, Ter119) populations. These sorted B cells were then stimulated with anti-IgM along with various cytokines for 24 hours, with the addition of PMA and ionomycin during the final 3 hours of stimulation. Total RNA was extracted using RNeasy Micro Kit (Qiagen). 100ng of total RNA was used for cDNA synthesis by RNA to cDNA EcoDry™ Premix (Double Primed) kit (Takara), and qPCR was performed using Taqman primers with TaqMan™ Fast Advanced Master Mix on StepOnePlus Real-Time PCR Systems (Applied Biosystems). Relative expression was calculated using the ΔΔCt method and level was normalized to GAPDH. Taqman primers and probes were from Applied Biosystems: Mm00439618_m1 (IL-17A), Mm01288386_m1(IL-10), Hs02786624_g1(GAPDH).

RNA-seq:

B cells were purified and stimulated as above except sorting was repeated 2–3X until the purity was ~100%. Total RNA quality was checked using a Bioanalyzer-2100 (Agilent Technologies) and submitted to the University of Pittsburgh Health Sciences Sequencing Core. Libraries were prepared (Ultra-low input RNA-seq Preparation Kit; Takara) and subjected to paired end sequencing (2 × 75 bp) using Illumina/NextSeq. Data was returned as NextSeq Fastq files.

Analysis of bulk RNA-seq data:

Bulk RNA-seq data were preprocessed on the Google Cloud Platform via the Terra scientific computing platform (Terra.Bio). Briefly, raw reads were aligned to the mouse genome mm10 using the STAR (v2.7.5a) alignment tool. Duplicate reads were identified using Picard MarkDuplicates. RNA-SeQC 2 was used for assessing sequencing depth and mapping quality. Raw counts and transcript per million (TPM) estimates were quantified using RSEM. All samples passed quality control and were included in subsequent analyses.

Downstream analyses were carried out in R (v4.1.3). Principal Component Analysis (PCA) was conducted using the R function prcomp. The gene with the highest PC2 loading, AY036118, was removed since it represents ribosomal RNA contamination39. Differential gene expression analysis was performed using DESeq2 (v1.32.0). Lowly expressed genes with a total count lower than 10 were prefiltered. P-values were adjusted by Benjamini-Hochberg (BH) method40, and log2 fold changes were shrunk using the ‘apeglm’ algorithm41. Shrunken fold changes are depicted in all Figs. Heatmaps were created using R package ComplexHeatmap (v2.13.1) on normalized counts (normalized using the counts function from the BiocGenerics package (v0.40.0)42 and were scaled by row. Volcano plots were generated using ggplot2 (v3.4.2) and ggrepel (v0.9.3).

Statistics.

Statistical analyses of allograft survival used log-rank (Mantel-Cox) test. Differences were considered to be significant at P values <0.05. The clinical score and incidence of EAE were analyzed by 2-way Anova, and comparisons for results (mean ± SEM) for cytometric bead array. FACS, and real-time PCR were analyzed by Student’s 2-tailed t test with p <0.05 considered significant. Paired 2-tailed t-test was used when cells from the same mouse were compared. Significantly differentially expressed genes for RNA-seq analysis were identified using DESeq2 with Benjamini-Hochberg adjusted p-value <0.05. No statistical methods were used to predetermine sample sizes, but our sample sizes are standard for the field7,8. No data were excluded unless stated otherwise. Data distribution was assumed to be normal, but this was not formally tested.

Reporting summary:

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Study approval:

Animal studies were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh (Pittsburgh, PA, USA).

Supplementary Material

Supplement 1

Acknowledgements:

We would like to thank Dr. Mark Shlomchik for supplying the hCD20.ERT2.Cre mice.

Funding:

This research was supported by PO1 AI129880.

Data availability:

All bulk RNA-seq data have been deposited in the NCBI’s Gene Expression Omnibus (GEO) database and are publicly available under accession number: GSE253925. All raw data and materials will be made available to investigators upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1
NIHPP2023.09.22.558524v3-supplement-1.pdf (2MB, pdf)

Data Availability Statement

All bulk RNA-seq data have been deposited in the NCBI’s Gene Expression Omnibus (GEO) database and are publicly available under accession number: GSE253925. All raw data and materials will be made available to investigators upon request.

Articles from bioRxiv are provided here courtesy of Cold Spring Harbor Laboratory Preprints

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