Interactions between B cells and T follicular regulatory cells enhance susceptibility to Brucella infection independent of the anti- Brucella humoral response

Abstract

Brucellosis, caused by facultative, intracellular Brucella spp., often results in chronic and/or lifelong infection. Therefore, Brucella must employ mechanisms to subvert adaptive immunity to cause chronic infection. B lymphocytes enhance susceptibility to infection with Brucella spp. though the mechanisms remain unclear. Here we investigated the role of antibody secretion, B cell receptor (BCR) specificity, and B cell antigen presentation on susceptibility to B. melitensis. We report that mice unable to secrete antibody do not display altered resistance to Brucella. However, animals with B cells that are unable to recognize Brucella through their BCR are resistant to infection. In addition, B cell MHCII expression enhances susceptibility to infection in a CD4+ T cell-dependent manner, and we found that follicular B cells are sufficient to inhibit CD4+ T cell-mediated immunity against Brucella. B cells promote development of T follicular helper (TFH) and T follicular regulatory (TFR) cells during Brucella infection. Inhibition of B cell and CD4+ T cell interaction via CD40L blockade enhances resistance to Brucella in a B cell dependent manner concomitant with suppression of TFH and TFR differentiation. Conversely, PD-1 blockade increases Brucella burdens in a B and CD4+ T cell dependent manner while augmenting T regulatory (TReg) and TFR responses. Intriguingly, TFR deficiency enhances resistance to Brucella via a B cell dependent, but antibody independent mechanism. Collectively, these results demonstrate B cells support TFR responses that promote susceptibility to Brucella infection independent of the antibody response.

Introduction

Consistently ranked by the World Health Organization as one of the world’s most common neglected zoonoses, brucellosis remains a major impediment to human health and economic stability [1,2]. Due to non-descript clinical signs and inadequate testing procedures, it is likely that global brucellosis incidence is grossly underestimated, and the chronicity of infection further complicates measures to mitigate disease [3,4]. Infection occurs frequently following consumption of unpasteurized dairy products or contact with tissues from livestock infected with the gram negative, facultative intracellular bacteria, Brucella [5–7]. Chronic disease is a frequent outcome of infection, even when aggressive antibiotic therapy is employed, and is typified by onset of various sequelae including relapsing undulant fever, arthritis, and neurobrucellosis [2,5]. Currently, our understanding of mechanisms that allow Brucella spp. to circumvent protective host responses are lacking and represent a major limitation for rational vaccine and therapeutic development.

IFN-γ is crucial to effective control of infection [8,9], and endogenous Th1, Th17 and CD8+ T cell responses, in the right context, can confer some level of protection [2,10–15]. However, Brucella can cause a lifelong infection in humans, livestock, and mice [10,16,17], and robust protection (that which results in one log or greater reduction in bacterial burden) typically fails to arise prior to one-month post infection, underscoring the inefficiency of this response.

B cell deficient mice display enhanced resistance to Brucella that is not altered by passive transfer of antibody [10,18]. We previously reported B cells require CD4+ T cells to promote susceptibility to infection, indicating that CD4+ T cell and B cell interactions are detrimental to control of Brucella [19]. However, the nature of this interaction remains undefined. TFH comprise a subset of CD4+ T cells specialized in providing essential help to follicular B cells (Fo B) during the germinal center (GC) response via CD40L, IL-21 and IL-4 [20]. Efficient class switching, affinity maturation, and generation of memory and plasma B cells during infection depend upon this crucial interaction; however, tight regulation of this process is vital for prevention of self-reactivity while also facilitating control of infection [20]. The role of TFR in fine-tuning this response has recently come to light. TFR share many characteristics in common with TFH populations including expression of Bcl6, CXCR5, ICOS, and PD-1 [21]. Additionally, TFR share various characteristics with TReg including expression of FoxP3, GITR, and CTLA-4 [22–25]. Compelling evidence points to a regulatory role for TFR throughout the GC response, which includes conditioning both the magnitude and quality of the response via suppression of TFH and germinal center B cells (GC B) [22–24,26–33]. While this function is likely dependent on the enhanced ability of TFR to suppress B cell responses specifically [22,24,26,34], the mechanisms involved in this process are largely undefined.

Here we show both B cell receptor (BCR) specificity and B cell antigen presentation function to enhance susceptibility to Brucella. Fo B are sufficient for inhibition of protective CD4+ T cell responses, and B cells promote TReg and TFR populations which were associated with enhanced susceptibility to Brucella. Using TFR deficient mice, we demonstrate TFR enhance susceptibility to Brucella in a B cell-dependent, but antibody independent, manner.

Results

Role of BCR specificity and secreted antibody in uptake of Brucella by B cells and host susceptibility to infection

BCR-mediated antigen uptake is 100- to 1000-fold more efficient for cognate T cell activation via MHCII than BCR-independent routes of B cell antigen presentation [35]. To investigate whether BCR specificity for Brucella alters susceptibility to infection, we challenged WT and MD4 mice, in which ~90% of B cells express a BCR specific for the irrelevant antigen hen egg lysozyme (HEL) [36] with B. melitensis. Significantly fewer Brucella were recovered from spleens of MD4 mice by four weeks post challenge (Fig 1A). Levels of total IgG and IgM were reduced ~10-fold in MD4 mice (S1A Fig), similar to what has been observed in MD4 mice in other infection models [37]. However, MD4 animals generated ~1000-fold less anti-Brucella IgG compared to WT controls at both two- and four-weeks post infection (S1B and S1C Fig), confirming a reduced affinity for Brucella antigen by MD4 B cells. Taken together, these results suggest that BCR specificity for Brucella promotes susceptibility to infection.

[Figure omitted. See PDF.]

Fig 1. Role of BCR specificity and secreted antibody in susceptibility to infection and B cell uptake of Brucella.

MD4 mice or WT littermates (n = 7-10/group/timepoint) were challenged i.p. with 1x105 CFUs of B. melitensis 16M. (A) Splenic bacterial burdens measured at one-, two-, and four-weeks post infection. (B) Viable intracellular Brucella recovered from total sorted CD19+ splenic B cells at one, two- and four-weeks post infection. (C) Viable intracellular Brucella burden per sorted B cell recovered at one, two- and four weeks post challenge. (D) Splenic bacterial loads of WT and sIgM-/-/AID-/- mice (n = 10-12/group/timepoint) at one- and four-weeks post challenge with B. melitensis. (E) Total viable intracellular Brucella recovered from sorted CD19+ splenic B cells at one- and four-weeks post infection in WT and sIgM-/-/AID-/- animals. (F) Viable intracellular Brucella burden per sorted B cell recovered at one-, and four-weeks post challenge in WT and sIgM-/-/AID-/- mice. (A-F) Data are combined from 2 independent experiments per time point.

https://doi.org/10.1371/journal.ppat.1011672.g001

Because B cell uptake of antigen is more efficient via the BCR than alternate mechanisms [35], we investigated the impact of BCR specificity on Brucella uptake by B lymphocytes. Compared to WT controls, B cell lysates collected from MD4 mice consistently harbored fewer Brucella with ~10-fold reduction at one-, two-, and four-weeks post challenge (Fig 1B). MD4 and WT mice harbor similar levels of splenic B. melitensis through the first two weeks of infection (Fig 1A) indicating that reduced B cell uptake of Brucella is a B cell specific effect rather than a result of diminished total bacterial burden in MD4 animals. Because we recovered fewer B cells from MD4 animals relative to WT mice (S1D Fig), we calculated the number of B. melitensis CFUs recovered per B cell and found this was also significantly reduced at two- and four-weeks post infection in MD4 animals (Fig 1C). Collectively, these data indicate that a diminished capacity for BCR-mediated recognition of Brucella impairs uptake of Brucella by B cells, which in turn may alter host susceptibility to infection.

Opsonization of Brucella with IgM from previously infected mice enhances uptake by B cells in vitro [4]. To investigate the effect of Brucella-specific antibody on uptake in vivo we employed sIgM-/-/AID-/- mice which express a polyclonal BCR but do not secrete IgM nor generate class-switched antibodies [38]. Total splenic Brucella burdens were similar among WT and sIgM-/-/AID-/- animals at both one- and four-weeks post-infection (Fig 1D). In contrast to MD4 mice (Fig 1B), sIgM-/-/AID-/- mice displayed similar intracellular Brucella B cell burdens one week post infection and increased B cell burdens at four weeks post infection relative to WT mice (Fig 1E and 1F). These data indicate that secreted antibody does not alter susceptibility to infection, nor is it absolutely required for Brucella entry into B cells.

B cell antigen presentation promotes deleterious CD4+ T cell responses

BCR-mediated antigen uptake, trafficking and presentation are regulated by Bruton’s tyrosine kinase (Btk) [39]. Therefore, we infected mice with a mutational defect in Btk (XID) and compared control of infection to CBA/J control mice. Bacterial burdens were similar two weeks post infection, but XID mice displayed enhanced resistance by four weeks post challenge (Fig 2A) suggesting Btk-dependent B cell antigen presentation enhances susceptibility to brucellosis. In addition to disrupting BCR-mediated antigen presentation, Btk dysfunction suppresses B-1a cell development in XID mice [40] which can impact control of infection [41]. However adoptive transfer of B-1a cells from naive CBA/J donors to XID mice prior to challenge did not alter control of Brucella (S1E Fig), indicating resistance in XID animals is independent of B-1a cell deficiency.

[Figure omitted. See PDF.]

Fig 2. B cell antigen presentation promotes deleterious CD4+ T cell responses during Brucella infection.

(A) Splenic Brucella loads of XID and CBA/J animals (n = 4-6/group/timepoint) following challenge with B. melitensis at two- and four-weeks post infection. (B) Splenic Brucella burden of CD19CreiABfl/fl or iABfl/fl littermates (n = 3-7/group/timepoint) at one-, two-, and four-weeks post infection. (C) CD19CreiABfl/fl and iABfl/fl mice were treated with CD4+ T cell-depleting antibody, or IgG isotype, and challenged with B. melitensis. Four weeks post infection splenic Brucella burdens were compared. Data are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.g002

To further examine the role of B cell antigen presentation on CD4+ T cell responses to Brucella, we compared control of infection in B cell specific MHCII deficient mice (CD19CreiABfl/fl) and iABfl/fl control animals and found B cell specific MHCII deficiency enhanced resistance to Brucella four weeks post infection (Fig 2B). Additionally, while depletion of CD4+ T cells from iABfl/fl mice enhanced resistance to infection, CD4+ T cell depletion had no effect in CD19CreiABfl/fl mice at four weeks post infection (Fig 2C). This indicated that B cell MHCII expression promotes CD4+ T cell responses that enhance susceptibility to Brucella.

Follicular B cells Promote Susceptibility to Brucella

We next compared CD4+ T cell phenotypes in B cell deficient (μMT) and WT mice infected with B. melitensis. At one week post infection, we observed a significant increase in the percentage of activated (CD44+) CD4+ T cells in μMT mice (Fig 3A). μMT mice also displayed ~30% increase in T-bet expression on CD44+CD4+ T cells one week post infection, though this difference dissipated two weeks post challenge (Figs 3B and S1F). FoxP3 expression on CD44+CD4+ T cells was significantly decreased among μMT animals compared to WT at one- and two weeks post infection (Figs 3C and S1G), suggesting B cells may drive TReg differentiation in response to Brucella. Because μMT mice can exhibit altered T cell development [42], we confirmed our findings with adoptive transfer experiments. Rag1-/- animals received CD4+ T cells alone, or both CD4+ T and B cells from WT donors prior to challenge with B. melitensis. Similar to our findings in μMT mice, by two weeks post infection co-transfer of B cells enhanced FoxP3 expression and diminished T-bet expression on CD44+CD4+ T cells (S1H and S1I Fig).

[Figure omitted. See PDF.]

Fig 3. Fo B inhibit CD4+ T cell immunity to Brucella.

Splenic CD4+ T cell responses assessed via flow cytometry in naïve (n = 2) or B. melitensis infected WT and μMT mice (n = 3-6/group/time point) at one-, or four-weeks post infection. Quantification of CD44 expression on CD4+ T cells (A) along with T-bet (B), and FoxP3 (C) expression on CD44 expressing CD4+ T cells. (D) Proportion of FoxP3+ cells among CXCR5+ and CXCR5- CD44+CD4+ T cells at one-, two-, and four-weeks post infection in WT animals. (E) Proportion of FoxP3+ cells amongst CXCR5+CD44+CD4+ T cells in WT and μMT mice one- and four-weeks post infection. (F) Rag1-/- mice (n = 3-7/treatment) adoptively transferred either CD4+ T cells alone, or CD4+ T cells in combination with purified Fo B cells one day prior to B. melitensis challenge. Data are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.g003

Further analysis of CD44+CD4+ T cells in WT animals revealed FoxP3 expression was preferentially enhanced among CXCR5+ populations vs CXCR5- populations (15.40% ±1.93% vs 3.6 ±1.22) at two- and four-weeks post infection (Fig 3D). WT mice also display increased FoxP3+ cell frequencies amongst CXCR5+CD44+CD4+ T cell populations one week post infection compared to μMT animals (Fig 3E), suggesting B cells promote TReg development amongst CXCR5+CD44+CD4+ T cells during Brucella infection.

CXCR5 expression facilitates CD4+ T cell trafficking to B cell follicles and subsequent interaction with cognate Fo B [43–45]. To test the role of Fo B during Brucella infection, we adoptively transferred CD4+ T cells alone, or both CD4+ T cells and Fo B into Rag1-/- mice prior to challenge. At four weeks post infection, co-transfer of Fo B with CD4+ T cells resulted in ~10-fold increase in splenic Brucella loads compared to animals transferred CD4+ T cells alone (Fig 3F), demonstrating Fo B inhibit CD4+ T cell responses following B. melitensis infection. Notably, we observed no marked effect in adoptive transfer experiments in which B-1a cells were co-transferred with CD4+ T cells (S1J Fig).

CD40L blockade enhances resistance to Brucella

CD40:CD40L interactions between TFH, and Fo B following priming are requisite for generation of GC responses [46,47]. Thus, we hypothesized inhibition of CD40:CD40L interactions could suppress the deleterious effects resulting from interaction of CD4+ T cells and Fo B during Brucella infection. Interestingly, CD40L blockade in WT, but not uMT animals, enhanced control of splenic Brucella burdens four weeks post infection (Fig 4A and 4B), indicating the deleterious effect of CD40:CD40L interactions is B cell dependent. CD40L blockade also suppressed GC B cell responses (Figs 4C, 4D, S2A and S2B) and the proportion of TReg, TFH, and TFR four-weeks post challenge (Figs 4E–4H and S2C–S2E). Collectively these data establish that interruption of CD40:CD40L interactions enhances resistance to Brucella, and suggest disrupting GC B, TReg, TFH, and/or TFR populations may improve control of infection.

[Figure omitted. See PDF.]

Fig 4. CD40L blockade enhances resistance to Brucella.

(A-H) Mice (n = 3-5/group) were treated with CD40L blocking antibody or IgG. (A-B) Splenic Brucella burdens were measured in WT and μMT mice one (A) and four (B) weeks post infection. Representative plots (C) and quantification (D) of the percentage of GC B cells (CD19+CD43-GL7+Fas+) among CD19+ B cells in isotype or anti-CD40L treated WT animals. (E-G) Representative plots showing CXCR5 and FoxP3 expression (E) and quantification (F-H) of the percentage of TReg (FoxP3+CXCR5-) (F), TFH (FoxP3-ICOS+CXCR5+) (G), and TFR (FoxP3+ICOS+CXCR5+) (H) amongst CD44+CD4+ T cells in IgG and anti-CD40L-treated WT and μMT mice four weeks post infection. Data are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.g004

Bcl6 expression in CD4 T cells protects the host against Brucella

Expression of Bcl6 in CD4+ T cells is essential to differentiation of both TFH and TFR [22,48,49]. To ascertain whether Brucella infection is exacerbated by B cell interaction with Bcl6 expressing CD4+ T cells, we infected CD4CreBcl6fl/fl mice with B. melitensis. While depletion of total CD4+ T cells enhances resistance (Fig 2C), deletion of Bcl6+CD4+ T cells increased susceptibility to Brucella four weeks post challenge (Fig 5A). Thymocytes express both CD8 and CD4 during early development, and Bcl6 is expressed by some splenic CD8+ T cells [50,51]. However, we found enhanced susceptibility in CD4CreBcl6fl/fl mice did not require CD8+ T cells (S2F Fig). Similar to previous reports [25,52], deletion of Bcl6 in CD4+ T cells suppressed differentiation of GC B cells, TFH, and TFR, though T-bet expression remained similar between groups (Figs 5B–5F and S2G). While CD4CreBcl6fl/fl mice had markedly reduced levels of GC B cells (Fig 5B and 5C), B cell depleted CD4CreBcl6fl/fl animals displayed ~100-fold decrease in splenic Brucella loads compared to controls (Fig 5I) suggesting GC B cells are not essential for B cell mediated susceptibility to Brucella. We also observed a marked increase in both the frequency and absolute number of TReg following Brucella infection in CD4CreBcl6fl/fl animals (Figs 5G, 5H and S2H). This was of interest given that CD40L blockade, which results in protection associated with suppression of TFH, TFR and GC B, significantly reduced TReg frequencies (Fig 4F–4H). Taken together, these findings suggest one or more Bcl6 expressing CD4+ T cell populations may be necessary for efficient control of infection, or that outgrowth of TReg in CD4CreBcl6fl/fl mice drives enhanced susceptibility to challenge.

[Figure omitted. See PDF.]

Fig 5. Lack of Bcl6 expressing CD4+ T cells impairs protection against Brucella.

Bcl6fl/fl or CD4CreBcl6fl/fl mice (n = 4-5/group) were challenged with B. melitensis. (A) Splenic bacterial burdens measured four weeks post infection. (B-C) Representative plots (B) and quantification (C) of the percentage of GC B cells amongst CD19+ cells four weeks post infection. (D-F) Representative plots showing CXCR5 and PD-1 expression (D) and quantification of the percent of TFH (FoxP3-PD-1+CXCR5+) (E) and TFR (FoxP3+PD-1+CXCR5+) (F) amongst CD44 expressing CD4+ T cells four weeks post infection. (G-H) Representative plots showing FoxP3 and PD-1 expression (G) and quantification (H) of the percentage of TReg (FoxP3+CXCR5-) among CD44 expressing CD4+ T cells four weeks post infection. (I) Splenic bacterial burdens of CD4CreBcl6fl/fl animals (n = 5/treatment) treated with anti-CD20 or IgG four weeks post Brucella challenge. Data are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.g005

PD-1 blockade enhances susceptibility to Brucella

CD40L blockade and Bcl6+CD4+ T cell deficiency both dampen TFH responses yet have opposing effects on control of infection (Figs 4 and 5). Combined with our finding that B cells promote FoxP3 expression amongst CXCR5+CD44+CD4+ T cells, we questioned whether altering TReg and TFR frequencies would alter control of Brucella. PD-1 regulates both TReg and TFR differentiation and function [27,53,54]. Intriguingly, anti-PD-1 treated WT animals exhibited significantly increased bacterial loads compared to controls four weeks post Brucella challenge (Fig 6A). While PD-1 expression is a key trait of TFH [27,55,56], various cell populations signal through PD-1 [57]. However, neither PD-1 blocked CD4+ T cell-depleted mice nor μMT animals had exacerbated infection (Fig 6B and 6C), demonstrating this effect is both CD4+T and B cell dependent. PD-1 blockade enhanced TFH, TReg and TFR proportions during infection in WT animals, while these populations remained unchanged in anti-PD-1 treated μMT animals (Fig 6D–6G). Therefore, one or more of these populations may drive B cell-dependent susceptibility to Brucella infection.

[Figure omitted. See PDF.]

Fig 6. PD-1 blockade enhances susceptibility to Brucella.

WT and/or μMT mice (n = 4-5/treatment) were treated with PD-1 blocking antibody, CD4+ T cell depleting antibody, PD-1 blocking and CD4-depleting antibody, or isotype control. (A & C) Splenic bacterial loads in IgG and PD-1 blocked WT (A) or μMT (C) mice. (B) Splenic bacterial burden of CD4-depleted and CD4-depleted/PD-1 blocked WT mice. (D) Representative flow plots showing CXCR5 and FoxP3 expression and the percentage of TFH (FoxP3-ICOS+CXCR5+) (E), TFR (FoxP3+ICOS+CXCR5+) (F), and TReg (FoxP3+CXCR5-) (G) present in IgG and anti-PD-1 treated WT and μMT mice spleens four weeks post challenge. (E-G) Quantification of the percentage of each indicated population of CD44 expressing CD4+ T cells four weeks post challenge. Data are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.g006

B cells are not absolutely required for TReg- mediated susceptibility to Brucella

As PD-1 blockade enhances susceptibility to infection as well as TReg and TFR outgrowth (Fig 6A, 6F and 6G), we investigated the contribution of TReg to control of infection. DEREG mice bear a diphtheria toxin receptor transgene under the control of the Foxp3 promoter, allowing for depletion of FoxP3 expressing cells by administration of diphtheria toxin (DTX). Because efficient FoxP3+ cell depletion is short-lived [58,59], we treated DEREG and WT groups with (DTX) on days 14 and 15 post challenge and confirmed FoxP3 expressing CD4+ T cells were diminished (S3A Fig). In line with previous reports suggesting TReg cells inhibit control of brucellosis [60,61], TReg depletion significantly enhanced resistance to Brucella at four weeks post-infection (S3B Fig). DTX-treated DEREG mice also presented with a decreased frequency of B cells compared to DTX-treated WT animals (S3C and S3D Fig); however, GC B and TFH proportions were increased in DTX treated DEREG animals compared to WT controls (S3E, S3F,S3I and S3K Fig). T-bet expressing CD4+ T effector levels were similar among FoxP3-depleted and WT groups (S3G Fig). While B cells enhance the proportion of TReg during Brucella infection (Figs 3C, 3E, S1G and S1H), depletion of TReg in both control and B cell depleted mice enhanced resistance to infection (S3B and S3H Fig) indicating the deleterious effect of TReg is not entirely B cell dependent.

TFR enhance susceptibility to Brucella infection in a B cell dependent, but antibody independent, manner

DTX treatment leads to TFR deficiency in DEREG mice (S3J Fig) making it unclear whether TReg, TFR or both contribute to enhanced susceptibility to Brucella. Therefore, we employed FoxP3CreBcl6fl/fl mice, in which TFR are deficient while TReg, TFH, and GC B cells remain intact [29] and found TFR deficiency enhanced resistance to Brucella four weeks post infection (Fig 7A and 7F). FoxP3CreBcl6fl/fl mice displayed elevated total TReg frequencies compared to Bcl6fl/fl mice (Fig 7B), though this did not adversely affect control of infection. To determine whether TFR require B cells to enhance susceptibility to infection, we depleted B cells from FoxP3CreBcl6fl/fl and Bcl6fl/fl mice. While isotype treated FoxP3CreBcl6fl/fl mice were more resistant to infection than isotype treated Bcl6fl/fl animals, Brucella burdens four weeks post infection were similar in FoxP3CreBcl6fl/fl and Bcl6fl/fl mice depleted of B cells (Fig 7F). Thus, our results demonstrate that, in the absence of B cells, TFR are no longer deleterious to control of infection.

[Figure omitted. See PDF.]

Fig 7. TFR enhance susceptibility to Brucella.

(A) Splenic Brucella burdens of Bcl6fl/fl and FoxP3CreBcl6fl/fl mice (n = 5-8/group) challenged with B. melitensis four weeks post challenge. (B-D) Quantification of the mean percentage of TReg (FoxP3+CXCR5-) (B), TFR (FoxP3+PD-1+CXCR5+) (C), and TFH (FoxP3-PD-1+CXCR5+) (D) of CD44 expressing CD4+ T cells in the spleen of infected animals four weeks post challenge. (E) Percentage of splenic GC B cells (Fas+GL7+ of CD19+) amongst CD19+ B cells four weeks post infection. (F) Splenic bacterial burdens of IgG or anti-CD20-treated Bcl6fl/fl or FoxP3CreBcl6fl/fl mice (n = 4-7/treatment) four weeks after challenge with B. melitensis. Data are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.g007

As TFR regulate antibody responses [28,62,63], we questioned whether TFR deficiency altered antibody responses which could in turn impact control of Brucella. While we did not find a difference in the quantity of Brucella-specific IgM generated by Bcl6fl/fl and FoxP3CreBcl6fl/fl mice four weeks post infection, FoxP3CreBcl6fl/fl animals did display reduced anti-Brucella IgG levels (S4A Fig). However, passive transfer of sera from either Bcl6fl/fl or FoxP3CreBcl6fl/fl mice previously infected with B. melitensis conferred similar levels of protection to FoxP3CreBcl6fl/fl mice (S4B Fig). Coupled with our finding that control of infection is not altered in mice lacking the ability to secrete antibody (Fig 1D), these data indicate the deleterious effect of TFR is independent of antibody regulatory function.

Discussion

We previously reported B cell mediated susceptibility to Brucella is CD4+ T cell dependent [19]. Here we report that an inability of B cells to recognize Brucella via BCR specificity results in host resistance to infection and reduced B cell uptake of Brucella in vivo. While B cells enhance expression of FoxP3 and decrease expression of CD44 and T-bet by CD4+ T cells in the first two weeks after infection (Figs 3A–3C and S1G), we found expression of CD44, T-bet, and FoxP3 was similar in CD4+ T cells from WT and MD4 mice at one- and two-weeks post-infection (S5A–S5F Fig). Total B cell deficiency results in a ~100-fold decrease in CFUs at 4 weeks post-infection (Figs 4B and 7F), while Brucella counts were reduced less than 10-fold in MD4 mice at the same timepoint (Fig 1A). The difference in phenotypes observed in MD4 mice versus mice with a total B cell deficiency could be explained by several factors. While ~90% of B cells in MD4 mice express a BCR specific for HEL [36], we did detect some Brucella-specific Ig MD4 mice (S1B and S1C Fig). Therefore, this residual population of non-HEL specific B cells in MD4 animals could potentially recognize Brucella through their BCR resulting in the dampened phenotype observed. Alternatively, while less efficient than antigen specific mechanisms, Brucella uptake and antigen presentation by B cells could also occur via BCR-independent processes [35].

Others have reported opsonization of B. abortus with IgM improves B cell uptake of bacteria in vitro [4]. However, we found the presence of Brucella specific antibody in vivo did not alter resistance to infection or reduce B cell Brucella burden (Fig 1D–1F). Interestingly, at later timepoints we found greater numbers of Brucella in B cells from sIgM-/-/AID-/- mice (Fig 1E and 1F). B cells from mice that lack the ability to secrete IgM can have increased BCR signaling including elevated Btk activation [64]. This was of interest, as uptake of Brucella by B cells appears to be BCR specific (Fig 1), Btk is required for BCR-mediated antigen internalization [39], and because we found Btk deficiency enhances resistance to Brucella (Fig 2). Therefore, it is possible that enhanced BCR signaling in sIgM-/-/AID-/- mice leads to increased uptake of Brucella by B cells. Alternatively, we found both the proportion and absolute number of B-1 cells in sIgM-/-/AID-/- mice was increased >2.5 fold (S6A–S6H Fig), which is similar to what others have reported in sIgM-/-/AID-/- mice [38]. As some B-1 cells can have phagocytic activity [65], the increased number of B-1 cells in sIgM-/-/AID-/- mice could potentially lead to higher levels of Brucella within the B cell compartment.

Brucella burdens in the spleens of mice lacking B cell MHCII expression are ~10 fold lower than in control animals (Fig 2B), which is a lesser phenotype than the ~100-fold decrease in Brucella in mice depleted of B cells (Fig 7F) or in mice with a genetic B cell deficiency (Fig 4B). In the first week after infection, FoxP3 expression is lower while T-bet expression is higher on activated CD4+ T cells in μMT mice (Fig 3B and 3C); however, we found expression of FoxP3 and T-bet was similar in activated CD4+ T cells from B cell MHCII deficient mice and control animals one week post-infection (S7A–S7C Fig). In a previous study, we found increased levels of IFN-γ, TNF-α, IL-17, and IL-1β in the spleens of μMT mice at two weeks post-infection [19]. However, the levels of IFN-γ, TNF-α, IL-17, and IL-1β were similar in spleens of control and B cell MHCII deficient mice two weeks post-infection (S7D–S7I Fig). While CD19Cre deletes the majority of MHCII expression on B cells in CD19CreiABfl/fl mice, a fraction of B cells retains B cell MHCII expression, and the proportion of MHCII+ B cells in CD19CreiABfl/fl mice increases over the course of infection from ~7% to ~25% (S8A Fig). Moreover, we observed a correlation between B cell MHCII expression and susceptibility to infection in CD19CreiABfl/fl mice treated with IgG, but not in CD19CreiABfl/fl mice depleted of CD4+ T cells (S8B and S8C Fig). Therefore, the potential/capacity for residual B cell MHCII expression and subsequent CD4+ T cell interaction following Brucella challenge in CD19CreiABfl/fl mice could contribute to the diminished magnitude of the phenotype observed in these animals relative to total B cell deficiency.

Cotransfer of Fo B with CD4+ T cells inhibited the ability of CD4+ T cells to protect against infection (Fig 3F). Following initial antigen encounter, Fo B and T lymphocytes that have migrated to the B:T border reciprocally regulate the activation and differentiation of one another. This process relies on both B cell mediated antigen presentation and CD40:CD40L engagement [55]. Strikingly, CD40L blockade conferred protection against Brucella infection in WT, but not B cell deficient animals (Fig 4B). Similar to other reports [66], CD40L blockade suppressed TFH, TFR and GC B cell responses, signaling the GC response was ablated in treated animals (Figs 4C–4H and S2B–S2E). This suggests CD40L blockade hinders CD4+ T and Fo B CD40L:CD40 engagement during Brucella infection. Deletion of Bcl6 in CD4+ T cells and CD40L blockade both thwart development of GC responses in Brucella infected mice yet have opposing effects on control of infection (Figs 4 and 5). CD40L blockade results in a decrease in the proportion of TReg (Fig 4F), while we observed that deficiency of Bcl6 in CD4+ T cells results in an increase in the proportion and absolute number of TReg following Brucella infection (Figs 5H and S2H). Given that TFR derive from thymic and/or peripheral TReg [22,24,26,67], the increase in TReg in CD4CreBcl6fl/f mice during Brucella infection may arise from the inability of peripheral TReg to commit to TFR differentiation in response to infection. Bcl6 restrains CD4+ T cell IL-10 production [52], and TReg, along with other CD4+ T cell subsets, can produce IL-10 [68] which in turn promotes susceptibility of Brucella [69]. In particular, CD4+ T cell derived IL-10 diminishes TNF-α levels during Brucella infection [69], and we found Bcl6 deficiency in CD4+ T cells markedly reduced TNF-α levels in the spleens of Brucella-infected mice (S9 Fig). Therefore, it is possible that an outgrowth of TReg and/or enhanced CD4+ T cell IL-10 production in CD4CreBcl6fl/fl mice results in elevated susceptibility to Brucella, though this would require additional investigation to confirm.

Upon depletion of B cells from CD4CreBcl6fl/f mice we observed a ~100-fold decrease in splenic bacterial burdens despite the absence of GC B cells in these animals (Fig 5B, 5C and 5I). This is of interest as it suggests GC B cells are not absolutely required for B cells to mediate enhanced susceptibility and implicates early CD4+ T and Fo B interactions as determinants of enhanced susceptibility to infection rather than interactions between fully committed TFH and GC B cells in the GC.

PD-1 has been studied extensively in the context of chronic infection, particularly regarding its role in T cell exhaustion [57]. T cell responses to Brucella are inefficient at controlling infection, and CD8+ T cell exhaustion is associated with chronic disease in a murine model of brucellosis [11]. Intriguingly, we found PD-1 blockade enhanced susceptibility to infection in a CD4+ T and B cell-dependent manner (Fig 6A–6C). PD-1 restrains TReg and TFR function [27,53,54] and upregulation of PD-1 by TFH and TFR contributes to follicular migration and positioning in the developing GC response [70]. Here PD-1 blockade enhanced susceptibility to Brucella and augmented TReg, and TFR populations (Fig 6F and 6G). Notably, tumor infiltrating TFR are prevalent in several types of cancer, and PD-1 blockade enhances tumor-infiltrating TFR leading to reduced tumor control in mice [54]. Collectively, these findings support a link between PD-1 blockade and promotion of TReg and TFR which may directly impact host susceptibility to infection and the efficiency of tumor control.

Alterations in TReg and TFR frequencies induced by PD-1 blockade were B cell dependent (Fig 6F and 6G). B cell expression of PD-L1 is implicated in TFH differentiation and migration [71], and PD-L1 expression by B cells has been linked to B cell regulatory function which can inhibit CD4+ T cell responses [72]. Interestingly, antigen-specific B cells protect against Mycobacterium tuberculosis infection, specifically through PD-L1 expressing B cells engaging PD-1+ TFH-like cells in the lung [73]. Future studies should focus on how B cell PD-L1/L2 expression affects the quantity and quality of TFH, TFR and TReg development during infection with Brucella and other pathogens.

While TReg and TFR promote susceptibility to Brucella, only TFR absolutely require B cells for this effect (Figs 7A and 7F and S3H). To gain insight as to how TFR impact Fo B behavior to potentially alter susceptibility to infection, we performed RNA-seq on splenic Fo B isolated from Bcl6fl/fl and FoxP3CreBcl6fl/fl mice at two weeks post challenge, when Brucella burdens are similar in these strains (S9A Fig), and on Fo B from naïve animals (S10 Fig and S1 Table). When comparing the transcriptional profile of Fo B from infected Bcl6fl/fl and FoxP3CreBcl6fl/fl mice, 401 genes were differentially regulated. Interestingly, only ~5% of the genes differentially regulated when comparing Fo B obtained from infected Bcl6fl/fl and FoxP3CreBcl6fl/f mice were also differentially regulated when comparing Fo B from naïve Bcl6fl/fl and FoxP3CreBcl6fl/f mice (S10B and S10C Fig) indicating the effect of TFR on Fo B transcription is context dependent. Interestingly, RNA-seq analysis revealed decreased Fo B transcription of Tgfb3 in Brucella-infected TFR deficient mice (S10A Fig). As TGF-β3 production by B cells has been shown to drive expansion of TReg [74], which are deleterious to control of Brucella (S3 Fig), regulation of B cell TGF-β3 expression by TFR could alter susceptibility to Brucella. We also observed decreased transcription of dual specificity phosphatase 4 (Dusp4) in TFR deficient mice (S10A Fig). Dusp4 is induced upon B cell activation involving both CD40 engagement and BCR signaling and promotes apoptosis via negative regulation of JNK (1/2) [75]. As CD40:CD40L interactions and BCR signaling both play a part in B cell mediated susceptibility to Brucella (Figs 1 and 4), the role of alteration of Dusp4 expression in B cells by TFR may warrant further study. TFR deficiency also altered transcription of genes involved in splenic compartmentalization/follicular positioning (Klf2, Bcl6, Ccr6, Cxcr4, Pdlim1) [70,71, 76–78], and antibody production (Tgfβ3) [79] which aligns with the established role of TFR in conditioning B cell reactions during GC responses. Changes in the positioning of Fo B could alter interactions with CD4+ T cells. Thus, as B cell mediated susceptibility to Brucella is CD4+ T cell dependent ([19] and Fig 2), in the future we will investigate whether TFR alter the positioning of Fo B and the frequency and/or magnitude of interactions of Fo B with CD4+ T cells.

Alternatively, TFR may negatively impact the ability of non-B cell populations to effectively control infection. Due to the reciprocal regulation inherent in generation of TFH, TFR and GC B responses [20,32,52], B cell deficiency lowers TFR proportions (Fig 4E and 4H). Therefore, B cell dependent effects of TFR may arise from their dependence on B cells for generation or maintenance. In this case, TFR could either act directly on B cells, or could function to alter other T- and/or non-B cell populations. Interestingly, TFR deficiency in mice has been linked to upregulation of granzyme B and other cytotoxicity associated genes in TFH [33], indicating that TFR can modulate the function of other CD4+ T cell populations.

In sum, our findings indicate B cells promote TFR responses that are regulated by CD40L and PD-1 dependent mechanisms. TFR in turn promote susceptibility to infection in a manner independent of the humoral response (S11 Fig). TFR deficiency can enhance protection against influenza [62], but to our knowledge, this is the first report of an antibody independent effect of TFR altering resistance to infection. Future studies will need to determine whether TFR mediate B cell responses which in turn promote infection, or whether their deleterious effect is mediated by altering responses of non-B cell populations. Finally, investigating how B cell antigen specificity, presentation and TFR induction synergize to hamper control of infection independent of the antibody response could have broad implications for rational vaccine designs which seek to optimize TFH and GC B responses.

Materials and methods

Ethics statement

All mouse experiments were approved by the University of Missouri Animal Care and Use Committee (ACUC protocol 27761).

Growth conditions and bacterial strains

All experiments were performed using Brucella melitensis 16M obtained from Montana State University (Bozeman, MT) in biosafety level 3 (BSL-3) facilities. Bacteria were grown on Brucella agar (Becton Dickinson) at 37°C/5% CO2 before colonies were picked and cultured in Brucella broth overnight at 37°C in an orbital shaker. Challenge doses were approximated by measurement of optical density at 600 nm and diluted using sterile Dulbecco’s Phosphate Buffered Saline (DPBS) (Thermofisher). All in vivo studies employed an intraperitoneal injection of 1x105 CFUs of B. melitensis 16M in 200 μl of DPBS. The delivered dose was confirmed via plating of inoculum onto Brucella agar.

Mice

Animals challenged with B. melitensis were of mixed sex and were age (6–12 weeks) and sex matched for all experiments. Mice were maintained in individually ventilated caging under high efficiency particulate air-filtered barrier conditions with 12 hr light and dark cycles within ABSL-3 facilities at the University of Missouri. Food and water were provided to animals ad libitum. B6.129S2-Ighmtm1Cgn/J (μMT), B6.129S7-Rag1tm1Mom/J (Rag1-/-), C57BL/6-Tg(IghelMD4)4Ccg/J (MD4), B6.129X1-H2-Ab1tm1Koni/J (iABfl/fl), C.B6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax (DEREG), B6.129S(FVB)-Bcl6tm1.1Dent/J (Bcl6fl/fl), CBA/CaJ, and CBA/CaHN-BtkXID/J (XID) and C57BL/6J (WT) mice were obtained from the Jackson Laboratory. With the exception of XID mice and their controls (CBA/CaJ mice), all animal strains were on a C57BL/6 background. sIgM-/-/AID-/- mice were a gift from Dr. Nicole Baumgarth at the University of California, Davis. AID-/- mice were originally generated at Kyoto University [80] and were bred to sIgM-/- mice at the Trudeau Institute [81]. B6.129P2(C)-Cd19tm1(cre)Cgn/J (CD19Cre) and B6.Cg-Tg(Cd4-cre)1Cwi/BfluJmice (CD4Cre) were a gift from Dr. Mark Daniels (University of Missouri). CD19Cre animals were intercrossed with iABfl/fl animals to generate CD19CreiABfl/fl mice. FoxP3Cre+/+Bcl6fl/fl (Bcl6FC) animals were gifted from Dr. Alexander Dent (University of Indiana). CD4Cre and FoxP3Cre+/+ animals were intercrossed with Bcl6fl/fl mice to generate Cd4CreBcl6fl/fl and FoxP3Cre+/+Bcl6fl/fl respectively. Experiments involving challenge of MD4, or CD19CreiABfl/fl mice utilized HEL-negative or iABfl/fl litter mates as control animals respectively. In all experiments using CD4CreBcl6fl/fl or FoxP3CreBcl6fl/fl animals, Bcl6fl/fl mice were used as controls. A description of the phenotypes of mice employed in this study are shown in Table 1.

[Figure omitted. See PDF.]

Table 1. Mouse Strains used in this Study.

https://doi.org/10.1371/journal.ppat.1011672.t001

Quantification of bacterial burden

Spleens were mechanically homogenized, serially diluted, and aliquots plated in triplicate onto Brucella agar as previously described [82]. Plated samples were incubated for 3–4 days at 37°C/5%CO2, and colonies counted to quantify the total CFUs/tissue. For enumeration of viable intracellular B cell burdens, B cells were isolated from spleens of infected animals using positive selection with anti-CD19 magnetic bead isolation (Miltenyi Biotec). For each sample, an aliquot of spleen homogenate was used for B cell purification, and total splenic cells, total B cells harvested, and total B cells/spleen were calculated. To kill extracellular bacteria, spleen homogenates were incubated in complete medium (CM; RPMI 1640, 0.1 HEPES, 1 mM sodium pyruvate, 1 mM nonessential amino acids, and 10% fetal bovine serum [FBS]) containing 50 μg/ml gentamicin for 30 minutes. Homogenates were then washed, and the remaining isolation protocol carried out using MACs isolation buffer (PBS, pH 7.2, 0.5% BSA, and 2 mM EDTA) supplemented with 5 μg/mL gentamicin. CD19+ and CD19- fractions were washed three times with DPBS and lysed in icy cold molecular grade water. Cell lysates were plated in triplicate to determine the Brucella burden for each fraction. Flow cytometric analysis of an aliquot of each sample was performed to confirm CD19+ B cell fractions were >90% pure. For measurement of cytokines, homogenized tissues were centrifuged at 2000 X G for 5 minutes, and supernatants were filter sterilized (0.22 μm) and stored at −70°C prior to analysis. Cytokines were measured with a Luminex (Austin, TX) MagPix instrument using Milliplex magnetic reagents according to manufacturer’s instructions (MilliporeSigma, Burlington, MA). Luminex data were analyzed with Milliplex Analyst Software (MilliporeSigma).

Adoptive transfers

Spleens were collected from naïve animals and mechanically homogenized. For CD4+ T cell and total B cell isolations, cells were magnetically purified using either CD4 or CD19 magnetic bead isolation kits (Miltenyi Biotec). Fo B cells were isolated from spleen using the MZ and FO B cell isolation kit (Miltenyi Biotec). Isolated lymphocytes were transferred via intravenous injection in 200 μL of DPBS into the tail vein of recipient mice one day prior to challenge with B. melitensis. Each animal received ~1.5 x 107 CD4+ T cells alone, ~1.5 x 107 CD4+ T cells in tandem with 5 x 107 (total B cell), or ~1.5 x 107 CD4+ T cells in tandem with 3-5x107 Fo B cells. For B-1a cell transfers, cells were harvested from the peritoneal cavity and pleural space of naïve mice [83]. Cells from the peritoneum and pleural cavity were pooled and B-1a cells purified using the mouse B-1a cell isolation kit (Miltenyi Biotec). B-1a transfer groups received ~2x105 B-1a cells in 200 μl of PBS or ~2x105 B-1a cells in 200 μl of PBS concomitant with intravenous administration of CD4+ T cells (1.5x107). The purity of sorted cell populations was confirmed to be ≥90% purity via flow cytometry. Purified CD4+ T and total B cells were assessed using anti-CD4 (GK1.5 Biolegend), anti-CD3 (145–2011 Biolegend), anti-CD8 (53–6.7 eBioscience), anti-CD19 (1D3 Leinco) and/or anti-B220 (RA3-6B2 Biolegend). B-1a cell purity was determined using anti-CD19 (1D3 Leinco) and/or anti-B220 (RA3-6B2 Biolegend), anti-CD5 (53–7.3 Biolegend), and anti-CD43 (S11 Biolegend).

Passive antibody transfer

Bcl6fl/fl and FoxP3CreBcl6fl/fl mice were challenged with B. melitensis and whole blood drawn via intracardial exsanguination four weeks post infection. Sera were collected by centrifuging blood samples at 10,000X G for 10 minutes at room temperature. Sera samples were stored at -80°C until passive transfer. Anti-Brucella IgM and IgG were quantified via ELISA as described below. For passive transfer, sera from each individual Bcl6fl/fl or FoxP3CreBcl6fl/fl sample were sterilized using a 0.22 μm filter and pooled by genotype. 200 μL from either Bcl6fl/fl or FoxP3CreBcl6fl/fl pooled stock were administered i.p. to naïve FoxP3CreBcl6fl/fl mice twenty-four hours prior to infection.

Anti-Brucella antibody ELISA

ELISA performed as previously described with minor modifications [8,84]. Briefly, 96-well high binding plates (Nunc), were coated overnight at 4°C with 108 CFU equivalents of the heat killed B. abortus S19 vaccine strain (University of Wyoming) in 0.05 M carbonate/bicarbonate coating buffer (pH 9.6). For measuring total Ig levels and for the standard curve, either unlabeled rat anti-mouse IgM (5 μg/ml) or goat anti-mouse IgG (0.5 μg/ml) (Southern Biotech) were used to coat IgM or IgG ELISA plates respectively. Plates were then washed using PBS-T buffer (0.05% Tween-20 in 1x PBS) before blocking for 1 hr at room temperature using 1% BSA in PBS. Plates were washed again before addition of serially diluted serum, or serially diluted IgM/IgG (standard curve) and allowed to incubate for 2 hrs at room temperature. ELISA plates were subsequently washed, and Goat anti-mouse IgM-HRP (1:1000) or Goat anti-mouse IgG-HRP (1:4000) antibody (Southern Biotech) added before incubation for 1 hr at room temperature. Plates were washed a final time before development with TMB substrate (Invitrogen) and addition of stop solution (2N Sulfuric acid solution). Absorbance was measured at 450 nm using a SpectraMax (Molecular Devices, San Jose, CA). Standard curves with unlabeled mouse IgM or IgG were employed to estimate Ig concentrations and data are presented in Units/ml (U/ml), where 1 U/ml roughly correlates with 1 pg/ml of antibody. The limit of detection for anti-Brucella antibody was 30.9 U/ml for IgM, and 3.43 U/ml for IgG.

Flow cytometry

Spleens were homogenized and cell suspensions filtered through sterile 40 μm mesh following red blood cell lysis. Splenocytes were Fc blocked (2.4G2 Leinco) in fluorescence-activated cell-sorting (FACS) buffer (2% heat inactivated fetal bovine serum in DPBS) before extracellular staining with fluorochrome-conjugated mAbs: anti-CD4 (GK1.5 Biolegend), anti-CXCR5 (L138D7 Biolegend), anti-CD44 (IM7 Biolegend), anti-CD23 (B3B4 Biolegend), anti-CD19 (1D3 Biolegend) or anti-CD19 (6D5 Biolegend), anti-CD43 (S11 Biolegend), anti-CD21 (7E9 Biolegend), anti-B220 (RA3-6B2 eBioscience), anti-CD8 (53–6.7 Biolegend), anti-CD3 (145-2C11 Biolegend or BD Biosciences), anti-I-A/I-E(MHCII) (M5/114.15.2 Biolegend), anti-CD279 (PD-1) (29F.1A12 Biolegend), anti-CD278 (ICOS) (7E.17G9 BD Biosciences), anti-mu/HU GL7 antigen (GL7 Biolegend), anti-CD95 (Fas) (SA367H8 Biolegend), and eBioscience Fixable Viability Dye eFluor 780 (Invitrogen). Cells were then fixed in 4% formalin at 4°C overnight before washing with and resuspension in FACS buffer. For intracellular staining, samples were fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermofisher) for 30 minutes at room temperature following extracellular staining. Samples were then stained with anti-T-bet (4B10 Biolegend) and anti-FoxP3 (FJK.16s Invitrogen) for two hours before fixation with 4% formalin at 4°C overnight, washing and resuspension in FACS buffer. Fluorescence was measured using a CyAn ADP High-Performance Flow Cytometer, BD LSR Fortessa X-20, or a Cytek Aurora spectral analyzer. Fluorescence Minus One (FMO) controls were utilized to identify rare or dim populations such as CXCR5 and PD-1 expressing cells (S12 Fig). Data were analyzed using FlowJo (Tree Star) software.

In vivo cell depletions

Animals were depleted of CD4+ or CD8+ T cells as previously described [19,85]. Briefly, animals were treated with 0.5 mg of rat anti-CD4 mAb GK1.5 (Leinco) or 0.2 mg rat anti-CD8 mAb 2.43 (Leinco) in 200 μl of DPBS i.p. one day prior to challenge. Treatment was repeated once weekly for the entirety of each study. For depletion of B cells, animals were treated with 250 μg of rat anti-CD20 mAB MB20-11 (BioXcell) in 200μl of DPBS i.p. one week prior to challenge [86]. Anti-CD20 treatment was repeated on day 14 post infection. Control animals for both T and B cell depletions received equivalent dosages of rat or mouse IgG (Leinco or Southern Biotech) respectively. Upon euthanasia, depletion of splenic CD4+ T, CD8+ T, or B cell populations were confirmed to be ≥ 90% effective via flow cytometry. TReg were depleted in DEREG animals by administering 1 μg DTX (List Biological Laboratories) resuspended in 100 μL DPBS i.p. to each animal on day 14 and 15 post challenge [59]. WT animals were administered an identical dose of DTX as a control. Blood was collected from animals to confirm systemic depletion of FoxP3+CD4+ T cells seven days post treatment (S3A Fig).

CD40L and PD-1 blockade

WT and/or μMT animals were administered 250 μg, hamster anti-CD154 (CD40L) mAB MR-1 (BioXcell), or 250 μg of rat anti-CD279 (PD-1) mAB RMP1-14 (Leinco) in 200 μL of DPBS i.p. one day prior to the start of the study. Treatments were repeated every three days thereafter for the duration of the study based on the regimens of others [87,88]. Control animals were treated with equivalent dosages of either hamster IgG (CD40L) (Southern Biotech) or rat IgG (PD-1) (Leinco).

RNA-Seq analysis

Fo B cells were sorted from naïve or infected animals as described above, and were washed, and placed in 1 ml of RNAlater (Thermo) and stored at 4°C overnight. The B cell purity of cells isolated from Bcl6 and FoxP3CreBcl6fl/fl averaged 93.5% and 97.4% respectively. Fo B cell (CD23+CD21loB220+) purity averaged ~85% of live B220+ cells as determined by flow cytometry in both strains. Approximately 4x106-1x107 Fo B were harvested from each animal. RNA was purified according to manufacturer instructions using a RNeasy Mini kit (Qiagen). Poly A enriched stranded mRNA libraries were generated, which were then sequenced on a NovaSeq 6000 (Illumina) as described elsewhere [89]. RNA-seq data were analyzed by the University of Missouri Bioinformatics Core Facility. Initial quality control of raw paired-end reads (100bp) was performed FastQC (v.0.11.8, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Subsequently, fastp [90] with default parameters was used to remove adapter sequences and quality trim reads. Trimmed reads were aligned to the mouse genome assembly (mm39, annotation V109, Ensembl: http://useast.ensembl.org/Mus_musculus/Info/Index) and gene read count was quantified using STAR [91]. The gene counts for each sample were transformed and normalized using the variance-stabilizing transformation method implemented in the Bioconductor package DESeq2 [92] in R (v4.2.1; https://www.r-project.org/). Linear regression models within DESeq2 were used to identify differentially expressed genes between case vs. control sample sets. Final values for differential expression are log2 fold change ≥ 1 or ≤ -1 with false discovery rate < 0.05 (FDR, Benjamini-Hochberg) as significant.

Statistical analysis

All comparisons of means between two groups were assessed via Student t test with significance set at P ≤ 0.05. Comparisons of three or more groups were conducted using one-way ANOVA, followed by Tukey’s test for correction of multiple comparisons unless otherwise noted. For all experiments, error bars represent the standard deviation of the sample mean. N values and the number of experimental repeats are provided in the figure legends. All statistical analyses were performed with Prism software (version 9.2, GraphPad) and all error bars indicate standard deviation (S.D.). Statistically significant differences are indicated as *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; and NS, not significant.

Supporting information

S1 Fig. MD4 animals have reduced anti-Brucella antibody levels.

Total IgM and IgG levels were measured in the serum of MD4 and WT mice (n = 8–10 group) four weeks after infection with B. melitensis (A). Serum anti-Brucella IgM (B) and IgG (C) in MD4 and WT mice (n = 5-11/group/time point) at two- and four-weeks post challenge with B. melitensis. Total B cell (CD19+) numbers (D) were determined in the spleens of WT and MD4 mice (n = 8-10/group/time point) 1, 2, or 4 weeks after infection challenge with B. melitensis. (E) Splenic Brucella burdens four weeks post challenge of XID mice (n = 5/treatment) adoptively transferred B-1a cells or DPBS one day prior to infection. (F-I) Quantification of splenic CD4+ T cell responses assessed via flow cytometry in B. melitensis infected animals. T-bet (F) and FoxP3 (G) were measured on CD44+CD4+ T cells in WT and μMT mice (n = 3-6/group/time point) at two weeks post infection. FoxP3 (H) and T-bet (I) were assessed on CD44+CD4+ T cells in Rag1-/- mice (n = 5-6/treatment) that received either CD4+ T cells alone, or CD4+ T cells in tandem with B cells, one day prior to challenge with B. melitensis. (J) Splenic bacterial burdens in Rag1-/- mice (n = 5/treatment) administered PBS, CD4+ T cells alone, or CD4+ T cells in combination with B-1a cells one day prior to B. melitensis challenge. Data in (A-C and E-I) are from a single experiment. Data in (D) are pooled from two experiments, and data in (J) are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.s001

(TIF)

S2 Fig. Enhanced susceptibility of CD4CreBcl6fl/fl mice is CD8+ T cell independent.

The total number of CD19+ B cells (A), germinal center B cells (CD19+Fas+GL7+) (B), TReg (FoxP3+CXCR5-CD44+CD4+) (C), TFH (FoxP3-ICOS+CXCR5+CD44+CD4+) (D) and TFR (FoxP3+ICOS+CXCR5+CD44+CD4+) (E) were counted in the spleens of WT mice treated with IgG or anti-CD40L (n = 5/treatment) four weeks after infection with B. melitensis. Splenic bacterial burdens (F) in Bcl6fl/fl and CD4CreBcl6fl/fl mice (n = 4-5/treatment) treated with CD8-depleting antibody or IgG as an isotype four weeks after B. melitensis infection. (G) Quantification of the percent T-bet+ cells amongst activated (CD44+) CD4+ T cells in the spleens of Bcl6fl/fl and CD4CreBcl6fl/fl animals (n = 4-5/group) four weeks post B. melitensis infection. The number of TReg (FoxP3+CXCR5-CD44+CD4+) (H) was determined four weeks after B. melitensis infection in the spleens of Bcl6fl/fl and CD4CreBcl6fl/fl mice (n = 5-6/treatment). Data in (A-F and H) are from a single experiment, and data in (G) are representative of at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1011672.s002

(TIF)

S3 Fig. TReg driven enhanced susceptibility to Brucella infection does not absolutely require B cells.

WT or DEREG mice (n = 5/group) were challenged with B. melitensis and treated with DTX on D14 and D15 post infection. (A) Quantification of the percentage of TReg amongst CD44+CD4+ cells in the blood of WT and DEREG mice seven days post DTX treatment. (B) Splenic bacterial burdens in DTX-treated WT and DEREG animals. (C-D) Representative flow plots of the gating strategy (C) and quantification of (D) the percentage of B cells present in the spleens of DTX treated WT and DEREG animals four weeks post infection. (E-F) Representative flow plots (E) and quantification of the percentage of GC B cells amongst CD19+ B cells (F) present in the spleens of DTX-treated WT and DEREG mice. (G) Percentage of Th1 effector cells (T-bet+) amongst CD44+CD4+ T cells in WT and DEREG DTX-treated animals. (H) WT and DEREG mice (n = 6-8/treatment) treated with DTX and anti-CD20 or IgG isotype control and CFUs were measured four weeks post-infection. (I) Representative flow plots of the gating strategy to identify TFR (FoxP3+CXCR5+PD-1+) and TFH (FoxP3-CXCR5+PD-1+) within splenic tissue in infected animals. (J-K) Quantification of the indicated populations of TFR (J) and TFH (K) amongst CD44+CD4+ T cells in DTX treated WT and DEREG animals. Data in (H) are combined from two independent experiments. All other data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s003

(TIF)

S4 Fig. TFR enhance susceptibility to Brucella independent of antibody.

(A) Anti-Brucella IgM and IgG titers four weeks post B. melitensis challenge in the serum of Bcl6fl/fl and FoxP3CreBcl6fl/fl animals (n = 10/group). (B) Splenic bacterial burdens of FoxP3CreBcl6fl/fl mice (n = 4-5/transfer) administered pooled sera from previously infected Bcl6fl/fl or FoxP3CreBcl6fl/fl mice one day prior to infection with B. melitensis. Data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s004

(TIF)

S5 Fig. CD4+ T cell responses in MD4 mice.

MD4 mice or WT littermates (n = 3-5/group/timepoint) were challenged i.p. with 1x105 CFUs of B. melitensis 16M. At one (A-C) or two (D-F) weeks post-infection flow cytometry was performed to assess CD44 expression on CD4+ T cells (A,D), and the expression of T-bet (B,E) and FoxP3 (C,F) on CD44 expressing CD4+ T cells. Data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s005

(TIF)

S6 Fig. B cell populations in WT and sIgM-/-/AID-/- mice.

WT or sIgM-/-/AID-/- mice (n = 4-5/group) were challenged i.p. with 1x105 CFUs of B. melitensis 16M. At four weeks post-infection the proportion (A-D) and number (E-H) of CD19+ B cells (A,E), Fo B cells (CD23+CD21loCD43-CD19+) (B,F), MZ B cells (CD23loCD21+CD43-CD19+) (C,G), and B-1 cells (CD43+CD19+) (D,H) was determined in the spleen. Data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s006

(TIF)

S7 Fig. CD4+ T cell responses and cytokine levels in mice lacking B cell MHCII expression.

CD19CreiABfl/fl or iABfl/fl littermates (n = 5-6/group) were challenged i.p. with 1x105 CFUs of B. melitensis 16M (A-C). At one-week post-infection, flow cytometry was performed to assess CD44 expression on CD4+ T cells (A), and the expression of T-bet (B) and FoxP3 (C) on CD44 expressing CD4+ T cells. CD19CreiABfl/fl or iABfl/fl littermates (n = 3-7/group) were challenged i.p. with 1x105 CFUs of B. melitensis 16M (D-I). Two weeks after infection, colonization of the spleen was measured (D) and the splenic levels of IFN-γ (E), TNF-α (F), IL-17 (G), IL-1β (H) and IL-4 (I) were determined. Data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s007

(TIF)

S8 Fig. Residual MHCII expression on B cells from CD19CreiABfl/fl mice.

CD19CreiABfl/fl or iABfl/fl littermates (n = 5-6/group) were challenged i.p. with 1x105 CFUs of B. melitensis 16M (A). At one and four weeks after challenge flow cytometry was performed to assess MHCII expression on CD19+ B cells (A). CD19CreiABfl/fl mice (n = 5/group) were treated with IgG (B) or anti-CD4 (C) and challenged i.p. with 1x105 CFUs of B. melitensis 16M. Four weeks after infection, colonization of the spleen was plotted against B cell MHCII expression in these animals in order to perform a linear regression. Data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s008

(TIF)

S9 Fig. Cytokine levels in mice lacking TFH or TFR.

Bcl6fl/fl, CD4CreBcl6fl/fl or FoxP3CreBcl6fl/fl mice (n = 4-6/group) were challenged i.p. with 1x105 CFUs of B. melitensis 16M (A-F). Two weeks after infection, colonization of the spleen was measured (A) and the splenic levels of IFN-γ (B), TNF-α (C), IL-17 (D), IL-1β (E) and IL-4 (F) were determined. Data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s009

(TIF)

S10 Fig. TFR deficiency alters Fo B transcription during Brucella infection.

RNA-Seq of Fo B (n = 3-5/group) from TFR deficient and control animals. (A) Z-scored heat map of selected genes differentially expressed in Fo B amongst naive Bcl6fl/fl and FoxP3CreBcl6fl/fl and infected Bcl6fl/fl and FoxP3CreBcl6fl/fl mice 14 days post Brucella challenge. (B) Table depicting the total number of differentially expressed genes (DE genes) detected when comparing infected and naïve Bcl6fl/fl and FoxP3CreBcl6fl/fl animals (filtering criteria: FDR <0.05 and Log2 FC ≥ 1 or Log2 FC ≤ -1). (C) Venn diagram depicting the percent overlap of differentially expressed genes. Data are from a single experiment.

https://doi.org/10.1371/journal.ppat.1011672.s010

(TIF)

S11 Fig. Working model.

(A) Ag-specific B cell presentation to splenic CD4+ T cells results in inefficient CD4+ T cell mediated control of infection in WT mice. TFR, TFH, GC B and Brucella-specific antibody responses develop in response to infection. (B) Inhibition of Fo B and CD4+ T cell interaction in WT animals via treatment with CD40L blocking antibody results in enhanced control of splenic Brucella burdens. TFR, TFH and GC B responses are suppressed, suggesting one or more of these populations may enhance susceptibility during infection. (C) Alteration of Fo B and CD4+ T cell regulation via PD-1 blockade promotes TFR outgrowth and enhances susceptibility to Brucella. (D) Genetic TFR specific deficiency results in reduced splenic Brucella loads despite similar TFH, GC B cell and Brucella-specific antibody responses compared to control animals. This indicates TFR promote susceptibility during Brucella infection through a mechanism that is independent of their role in shaping the humoral response to infection. Image created with Biorender.

https://doi.org/10.1371/journal.ppat.1011672.s011

(PNG)

S12 Fig. FMO controls.

A representative plot including a sample, and fluorescence minus one (FMO) controls to show how PD-1 and CXCR5 expressing CD4+ T cell populations were identified.

https://doi.org/10.1371/journal.ppat.1011672.s012

(TIF)

S1 Table. Table depicting expression of genes in Fo B isolated from Brucella infected and naïve Bcl6fl/fl and FoxP3CreBcl6fl/fl animals (related to S10 Fig).

https://doi.org/10.1371/journal.ppat.1011672.s013

(XLSX)

Citation: Dadelahi AS, Abushahba MFN, Ponzilacqua-Silva B, Chambers CA, Moley CR, Lacey CA, et al. (2023) Interactions between B cells and T follicular regulatory cells enhance susceptibility to Brucella infection independent of the anti-Brucella humoral response. PLoS Pathog 19(9): e1011672. https://doi.org/10.1371/journal.ppat.1011672

[/RAW_REF_TEXT]

References

1. Franc KA, Krecek RC, Häsler BN, Arenas-Gamboa AM. Brucellosis remains a neglected disease in the developing world: A call for interdisciplinary action. BMC Public Health. 2018;18: 1–9. pmid:29325516

2. Pascual DW, Goodwin ZI, Bhagyaraj E, Hoffman C, Yang X. Activation of mucosal immunity as a novel therapeutic strategy for combating brucellosis. Front Microbiol. 2022;13: 5041. pmid:36620020

3. Hull NC, Schumaker BA. Comparisons of brucellosis between human and veterinary medicine. Infect Ecol Epidemiol. 2018. pmid:30083304

4. Goenka R, Guirnalda PD, Black SJ, Baldwin CL. B Lymphocytes provide an infection niche for intracellular bacterium Brucella abortus. J Infect Dis. 2012;206: 91–8. pmid:22561364

5. Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, Walravens K, et al. From the discovery of the Malta fever’s agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Veterinary Research. 2005. pp. 313–326. pmid:15845228

6. Kaufmann AF, Fox MD, Boyce JM, Anderson DC, Potter ME, Martone WJ, et al. Airborne spread of brucellosis. Ann N Y Acad Sci. 1980;353: 105–114. pmid:6939379

7. Grillá MJ, Blasco JM, Gorvel JP, Moriyán I, Moreno E. What have we learned from brucellosis in the mouse model? Veterinary Research. 2012. pmid:22500859

8. Murphy EA, Sathiyaseelan J, Parent MA, Zou B, Baldwin CL. Interferon-γ is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice. Immunology. 2001;103: 511–518. pmid:11529943

9. Skyberg JA, Thornburg T, Kochetkova I, Layton W, Callis G, Rollins MF, et al. IFN-γ-deficient mice develop IL-1-dependent cutaneous and musculoskeletal inflammation during experimental brucellosis. J Leukoc Biol. 2012;92: 375–387. pmid:22636321

10. Goenka R, Parent MA, Elzer PH, Baldwin CL. B Cell–deficient mice display markedly enhanced resistance to the intracellular bacterium Brucella abortus. J Infect Dis. 2011;203: 1136–1146. pmid:21451002

11. Durward-Diioia M, Harms J, Khan M, Hall C, Smith JA, Splitter GA. CD8+ T cell exhaustion, suppressed gamma interferon production, and delayed memory response induced by chronic Brucella melitensis infection. Infect Immun. 2015;83: 4759–71. pmid:26416901

12. Hanot Mambres D, Machelart A, Potemberg G, De Trez C, Ryffel B, Letesson J-J, et al. Identification of immune effectors essential to the control of primary and secondary intranasal infection with Brucella melitensis in mice. J Immunol. 2016;196: 3780–3793. pmid:27036913

13. Vitry M-A, De Trez C, Goriely S, Dumoutier L, Akira S, Ryffel B, et al. Crucial role of gamma interferon-producing CD4+ Th1 cells but dispensable function of CD8+ T cell, B cell, Th2, and Th17 responses in the control of Brucella melitensis infection in mice. Infect Immun. 2012;80: 4271–80. pmid:23006848

14. Clapp B, Yang X, Thornburg T, Walters N, Pascual DW. Nasal vaccination stimulates CD8+ T cells for potent protection against mucosal Brucella melitensis challenge. Immunol Cell Biol. 2016;94: 496–508. pmid:26752510

15. Clapp B, Skyberg JA, Yang X, Thornburg T, Walters N, Pascual DW. Protective live oral brucellosis vaccines stimulate Th1 and Th17 cell responses. Infect Immun. 2011;79: 4165–4174. pmid:21768283

16. EJ Y. Human brucellosis. Rev Infect Dis. 1983;5. pmid:6356268

17. Durward M, Radhakrishnan G, Harms J, Bareiss C, Magnani D, Splitter GA. Active evasion of CTL mediated killing and low quality responding CD8+ T cells contribute to persistence of brucellosis. PLoS One. 2012;7: e34925. pmid:22558103

18. Vitry M-A, Hanot Mambres D, De Trez C, Akira S, Ryffel B, Letesson J-J, et al. Humoral immunity and CD4 + Th1 cells are both necessary for a fully protective immune response upon secondary infection with Brucella melitensis. J Immunol. 2014;192: 3740–3752. pmid:24646742

19. Dadelahi AS, Lacey CA, Chambers CA, Ponzilacqua-Silva B, Skyberg JA. B cells inhibit CD4+ T cell mediated immunity to Brucella infection in a major histocompatibility complex class ii-dependent manner. Infect Immun. 2020;88. pmid:32071068

20. Crotty S. T follicular helper cell biology: A decade of discovery and diseases. Immunity. 2019;50: 1132–1148. pmid:31117010

21. Sage PT, Sharpe AH. T follicular regulatory cells. Immunol Rev. 2016;271: 246–259. pmid:27088919

22. Chung Y, Tanaka S, Chu F, Nurieva RI, Martinez GJ, Rawal S, et al. Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat Med. 2011;17: 983–988. pmid:21785430

23. Sage PT, Paterson AM, Lovitch SB, Sharpe AH. The coinhibitory receptor CTLA-4 controls B cell responses by modulating T follicular helper, T follicular regulatory, and T regulatory cells. Immunity. 2014;41: 1026–1039. pmid:25526313

24. Linterman MA, Pierson W, Lee SK, Kallies A, Kawamoto S, Rayner TF, et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat Med. 2011;17: 975–982. pmid:21785433

25. Wing JB, Kitagawa Y, Locci M, Hume H, Tay C, Morita T, et al. A distinct subpopulation of CD25− T-follicular regulatory cells localizes in the germinal centers. Proc Natl Acad Sci U S A. 2017;114: E6400–E6409. pmid:28698369

26. Wollenberg I, Agua-Doce A, Hernández A, Almeida C, Oliveira VG, Faro J, et al. Regulation of the germinal center reaction by Foxp3 + follicular regulatory T cells. J Immunol. 2011;187: 4553–4560. pmid:21984700

27. Sage PT, Francisco LM, Carman C V., Sharpe AH. The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood. Nat Immunol. 2013;14: 152–161. pmid:23242415

28. Clement RL, Daccache J, Mohammed MT, Diallo A, Blazar BR, Kuchroo VK, et al. Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses. Nat Immunol. 2019;20: 1360. pmid:31477921

29. Wu H, Chen Y, Liu H, Xu LL, Teuscher P, Wang S, et al. Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice. Eur J Immunol. 2016;46: 1152–1161. pmid:26887860

30. Laidlaw BJ, Lu Y, Amezquita RA, Weinstein JS, Vander Heiden JA, Gupta NT, et al. Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response. Sci Immunol. 2017;2. pmid:29054998

31. Sayin I, Radtke AJ, Vella LA, Jin W, Wherry EJ, Buggert M, et al. Spatial distribution and function of T follicular regulatory cells in human lymph nodes. J Exp Med. 2018;215: 1531–1542. pmid:29769249

32. Xie MM, Dent AL. Unexpected help: follicular regulatory T cells in the germinal center. Front Immunol. 2018;9: 1536. pmid:30013575

33. Xie MM, Fang S, Chen Q, Liu H, Wan J, Dent AL. Follicular regulatory T cells inhibit the development of granzyme B-expressing follicular helper T cells. JCI insight. 2019;4. pmid:31434804

34. Sage PT, Sharpe AH. T follicular regulatory Cells in the regulation of B cell responses. Trends Immunol. 2015;36: 410. pmid:26091728

35. Barroso M, Tucker H, Drake L, Nichol K, Drake JR. Antigen-B Cell Receptor complexes associate with intracellular major histocompatibility complex (MHC) class II molecules. J Biol Chem. 2015;290: 27101. pmid:26400081

36. Goodnow CC, Crosbie J, Adelstein S, Lavoie TB, Smith-Gill SJ, Brink RA, et al. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature. 1988;334: 676–682. pmid:3261841

37. Wieland A, Shashidharamurthy R, Kamphorst AO, Han JH, Aubert RD, Choudhury BP, et al. Antibody effector functions mediated by Fcγ-receptors are compromised during persistent viral infection. Immunity. 2015;42: 367–378. pmid:25680276

38. Kumazaki K, Tirosh B, Maehr R, Boes M, Honjo T, Ploegh HL. AID−/−μs−/− Mice are agammaglobulinemic and fail to maintain B220−CD138+ plasma cells. J Immunol. 2007;178: 2192–2203. pmid:17277124

39. Sharma S, Orlowski G, Song W. Btk regulates B cell receptor-mediated antigen processing and presentation by controlling actin cytoskeleton dynamics in B cells. J Immunol. 2009;182: 329–339. pmid:19109164

40. Khan WN, Alt FW, Gerstein RM, Malynn BA, Larsson I, Rathbun G, et al. Defective B cell development and function in Btk-deficient mice. Immunity. 1995;3: 283–299. pmid:7552994

41. Smith FL, Baumgarth N. B-1 cell responses to infections. Curr Opin Immunol. 2019;57: 23. pmid:30685692

42. Serrán MG, Boari JT, Vernengo FF, Beccaría CG, Ramello MC, Bermejo DA, et al. Unconventional pro-inflammatory CD4+ T cell response in B cell-deficient mice infected with Trypanosoma cruzi. Front Immunol. 2017;8. pmid:29209313

43. Breitfeld D, Ohl L, Kremmer E, Ellwart J, Sallusto F, Lipp M, et al. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med. 2000;192: 1545–1551. pmid:11104797

44. Kim CH, Rott LS, Clark-Lewis I, Campbell DJ, Wu L, Butcher EC. Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. J Exp Med. 2001;193: 1373–1381. pmid:11413192

45. Stebegg M, Kumar SD, Silva-Cayetano A, Fonseca VR, Linterman MA, Graca L. Regulation of the germinal center response. Front Immunol. 2018;9: 2469. pmid:30410492

46. Awe O, Hufford MM, Wu H, Pham D, Chang H-C, Jabeen R, et al. PU.1 expression in T follicular helper cells limits CD40L-dependent germinal center B cell development. J Immunol. 2015;195: 3705–3715. pmid:26363052

47. Elgueta R, Benson MJ, De Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev. 2009;229: 152–172. pmid:19426221

48. Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD, et al. Bcl6 mediates the development of T follicular helper cells. Science (1979). 2009;325: 1001–1005. pmid:19628815

49. Johnston RJ, Poholek AC, DiToro D, Yusuf I, Eto D, Barnett B, et al. Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science. 2009;325: 1006–1010. pmid:19608860

50. Ichii H, Sakamoto A, Hatano M, Okada S, Toyama H, Taki S, et al. Role for Bcl-6 in the generation and maintenance of memory CD8 + T cells. 2002. pmid:12021781

51. Carpenter AC, Bosselut R. Decision checkpoints in the thymus. Nat Immunol. 2010;11: 666. pmid:20644572

52. Hollister K, Kusam S, Wu H, Clegg N, Mondal A, Sawant D V., et al. Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model. J Immunol. 2013;191: 3705–3711. pmid:23980208

53. Tan CL, Kuchroo JR, Sage PT, Liang D, Francisco LM, Buck J, et al. PD-1 restraint of regulatory T cell suppressive activity is critical for immune tolerance. J Exp Med. 2021;218. pmid:33045061

54. Eschweiler S, Clarke J, Ramírez-Suástegui C, Panwar B, Madrigal A, Chee SJ, et al. Intratumoral follicular regulatory T cells curtail anti-PD-1 treatment efficacy. Nat Immunol. 2021 228. 2021;22: 1052–1063. pmid:34168370

55. Vinuesa CG, Linterman MA, Yu D, MacLennan ICM. Follicular helper T cells. Annu Rev Immunol. 2016;34: 335–368. pmid:26907215

56. Haynes NM, Allen CDC, Lesley R, Ansel KM, Killeen N, Cyster JG. Role of CXCR5 and CCR7 in follicular Th cell positioning and appearance of a programmed cell death gene-1 high germinal center-associated subpopulation. J Immunol. 2007;179: 5099–108. pmid:17911595

57. Jubel JM, Barbati ZR, Burger C, Wirtz DC, Schildberg FA. The role of PD-1 in acute and chronic infection. Front Immunol. 2020;11: 487. pmid:32265932

58. Wang J, Siffert M, Spiliotis M, Gottstein B. Repeated long-term DT application in the DEREG mouse induces a neutralizing anti-DT antibody response. J Immunol Res. 2016;2016. pmid:28074191

59. Lahl K, Sparwasser T. In vivo depletion of FoxP3+ Tregs using the DEREG mouse model. Methods Mol Biol. 2011;707: 157–172. pmid:21287334

60. Adetunji SA, Faustman DL, Adams LG, Garcia-Gonzalez DG, Hensel ME, Khalaf OH, et al. Brucella abortus and pregnancy in mice: Impact of chronic infection on fertility and the role of regulatory t cells in tissue colonization. Infect Immun. 2020;88. pmid:32690635

61. Pasquali P, Thornton AM, Vendetti S, Pistoia C, Petrucci P, Tarantino M, et al. CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice. Microbes Infect. 2010;12: 3–10. pmid:19772948

62. Fu W, Liu X, Lin X, Feng H, Sun L, Li S, et al. Deficiency in T follicular regulatory cells promotes autoimmunity. J Exp Med. 2018;215: 815. pmid:29378778

63. Xie MM, Chen Q, Liu H, Yang K, Koh B, Wu H, et al. T follicular regulatory cells and IL-10 promote food antigen–specific IgE. J Clin Invest. 2020;130: 3820–3832. pmid:32255767

64. Tsiantoulas D, Kiss M, Bartolini-Gritti B, Bergthaler A, Mallat Z, Jumaa H, et al. Secreted IgM deficiency leads to increased BCR signaling that results in abnormal splenic B cell development. Sci Rep. 2017;7. pmid:28615655

65. Parra D, Rieger AM, Li J, Zhang Y-A, Randall LM, Hunter CA, et al. Pivotal advance: peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities and present phagocytosed antigen to CD4+ T cells. J Leukoc Biol. 2012;91: 525–536. pmid:22058420

66. Voynova E, Mahmoud T, Woods LT, Weisman GA, Ettinger R, Braley-Mullen H. Requirement for CD40/CD40L interactions for development of autoimmunity differs depending on specific checkpoint and costimulatory pathways. ImmunoHorizons. 2018;2: 54. pmid:30607385

67. Aloulou M, Carr EJ, Gador M, Bignon A, Liblau RS, Fazilleau N, et al. Follicular regulatory T cells can be specific for the immunizing antigen and derive from naive T cells. Nat Commun. 2016 71. 2016;7: 1–10. pmid:26818004

68. Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 2010;10: 170–181. pmid:20154735

69. Xavier MN, Winter MG, Spees AM, Nguyen K, Atluri VL, Silva TMA, et al. CD4+ T cell-derived IL-10 promotes Brucella abortus persistence via modulation of macrophage function. PLoS Pathog. 2013;9: e1003454. pmid:23818855

70. Nie Y, Waite J, Brewer F, Sunshine MJ, Littman DR, Zou YR. The role of CXCR4 in maintaining peripheral B cell compartments and humoral immunity. J Exp Med. 2004;200: 1145. pmid:15520246

71. Shi J, Hou S, Fang Q, Liu X, Liu X, Qi H. PD-1 controls follicular T helper cell positioning and function. Immunity. 2018;49: 264–274.e4. pmid:30076099

72. Khan AR, Hams E, Floudas A, Sparwasser T, Weaver CT, Fallon PG. PD-L1hi B cells are critical regulators of humoral immunity. Nat Commun. 2015;6: 5997. pmid:25609381

73. Swanson R V, Gupta A, Foreman TW, Lu L, Choreno-Parra JA, Mbandi SK, et al. Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control. Nat Immunol 2023. 2023; 1–14. pmid:37012543

74. Shah S, Qiao L. Resting B cells expand a CD4+CD25+Foxp3+ Treg population via TGF-beta3. Eur J Immunol. 2008;38: 2488–2498. pmid:18792402

75. Rickert R. DUSP4 phosphatase puts the brakes on DLBCL. J Exp Med. 2015;212: 598–599. pmid:25941318

76. Hoek KL, Gordy LE, Collins PL, Parekh V V., Aune TM, Joyce S, et al. Follicular B cell trafficking within the spleen actively restricts humoral immune responses. Immunity. 2010;33: 254–265. pmid:20691614

77. Choi J, Crotty S. Bcl6-Mediated transcriptional regulation of follicular helper T cells (TFH). Trends Immunol. 2021;42: 336. pmid:33663954

78. Elgueta R, Marks E, Nowak E, Menezes S, Benson M, Raman VS, et al. CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen. J Immunol. 2015;194: 505. pmid:25505290

79. Tsuchida Y, Sumitomo S, Ishigaki K, Suzuki A, Kochi Y, Tsuchiya H, et al. TGF-β3 inhibits antibody production by human B cells. PLoS One. 2017;12. pmid:28052118

80. Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 2000;102: 553–563. pmid:11007474

81. Carragher DM, Kaminski DA, Moquin A, Hartson L, Randall TD. A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus. J Immunol. 2008;181: 4168–4176. pmid:18768874

82. Abushahba MF, Dadelahi AS, Lemoine EL, Skyberg JA, Vyas S, Dhoble S, et al. Safe subunit green vaccines confer robust immunity and protection against mucosal Brucella infection in mice. Vaccines. 2023;11:546. pmid:36992130

83. Yenson V, Baumgarth N. Purification and immune phenotyping of B-1 Cells from body cavities of mice. Humana Press, New York, NY; 2014:17–34. https://doi.org/10.1007/978-1-4939-1161-5_2 pmid:25015270

84. Ledbetter L, Cherla R, Chambers C, Zhang Y, Zhang G. Eosinophils affect antibody isotype switching and may partially contribute to early vaccine-induced immunity against Coxiella burnetii. Infect Immun. 2019;87. pmid:31427447

85. Grčevicgrčevic D, Lee S-K, Marušic AM, Lorenzo JA. Depletion of CD4 and CD8 T Lymphocytes in mice in vivo enhances 1,25-Dihydroxyvitamin D3-stimulated osteoclast-like cell formation in vitro by a mechanism that is dependent on prostaglandin Ssynthesis. J Immunol. 2000;165: 4231–4238. pmid:11035056

86. Hamaguchi Y, Uchida J, Cain DW, Venturi GM, Poe JC, Haas KM, et al. The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti-CD20 immunotherapy in mice. J Immunol. 2005;174: 4389–4399. pmid:15778404

87. Husaini Y, Tsai VWW, Manandhar R, Zhang HP, Lee-Ng KKM, Lebhar H, et al. Growth differentiation factor-15 slows the growth of murine prostate cancer by stimulating tumor immunity. PLoS One. 2020;15. pmid:32502202

88. Wiley JA, Harmsen AG. CD40 ligand is required for resolution of Pneumocystis carinii pneumonia in mice. J Immunol. 1995;155: 3525–3529.

89. Moley CR, Chambers CA, Dadelahi AS, Ponzilacqua-Silva B, Abushahba MFN, Lacey CA, et al. Innate lymphoid cells and interferons limit neurologic and articular complications of brucellosis. Am J Pathol. 2023;0. pmid:37263343

90. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34: i884–i890. pmid:30423086

91. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29: 15–21. pmid:23104886

92. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15. pmid:25516281