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

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][6][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][11][12][13][14][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.T FH 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 T FR in fine-tuning this response has recently come to light.T FR share many characteristics in common with T FH populations including expression of Bcl6, CXCR5, ICOS, and PD-1 [21].Additionally, T FR share various characteristics with T Reg including expression of FoxP3, GITR, and
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 T Reg and T FR populations which were associated with enhanced susceptibility to Brucella.Using T FR deficient mice, we demonstrate T FR enhance susceptibility to Brucella in a B cell-dependent, but antibody independent, manner.

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 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

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T follicular regulatory cell mediated susceptibility to Brucella 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.
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 (CD19 Cre iAB fl/ fl ) and iAB fl/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 iAB fl/fl mice enhanced resistance to infection, CD4 + T cell depletion had no effect in CD19 Cre iAB fl/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 oneand two weeks post infection (Figs 3C and S1G), suggesting B cells may drive T Reg 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).
CXCR5 expression facilitates CD4 + T cell trafficking to B cell follicles and subsequent interaction with cognate Fo B [43][44][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 T FH , 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 T Reg , T FH , and T FR 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, T Reg , T FH , and/or T FR populations may improve control of infection.

Bcl6 expression in CD4 T cells protects the host against Brucella
Expression of Bcl6 in CD4 + T cells is essential to differentiation of both T FH and T FR [22,48,49].To ascertain whether Brucella infection is exacerbated by B cell interaction with Bcl6 expressing CD4 + T cells, we infected CD4 Cre Bcl6 fl/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 CD4 Cre Bcl6 fl/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, T FH , and T FR , though T-bet expression remained similar between groups (Figs 5B-5F and S2G).While CD4 Cre Bcl6 fl/fl mice had markedly reduced levels of GC B cells (Fig 5B and 5C), B cell depleted CD4 Cre Bcl6 fl/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 T Reg following Brucella infection in CD4 Cre Bcl6 fl/fl animals (Figs 5G, 5H and S2H).This was of interest given that CD40L blockade, which results in protection associated with suppression of T FH , T FR and GC B, significantly reduced T Reg 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 T Reg in CD4 Cre Bcl6 fl/fl mice drives enhanced susceptibility to challenge.

PD-1 blockade enhances susceptibility to Brucella
CD40L blockade and Bcl6 + CD4 + T cell deficiency both dampen T FH 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 T Reg and T FR frequencies would alter control of Brucella.PD-1 regulates both T Reg and T FR 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 T FH [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 T FH , T Reg and T FR 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.As T FR regulate antibody responses [28,62,63], we questioned whether T FR 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 Bcl6 fl/fl and FoxP3-

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-  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) Therefore, the potential/capacity for residual B cell MHCII expression and subsequent CD4 + T cell interaction following Brucella challenge in CD19 Cre iAB fl/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 T FH, T FR 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 T Reg (Fig 4F), while we observed that deficiency of Bcl6 in CD4 + T cells results in an increase in the proportion and absolute number of T Reg following Brucella infection (Figs 5H and S2H).Given that T FR derive from thymic and/or peripheral T Reg [22,24,26,67], the increase in T Reg in CD4 Cre Bcl6 fl/f mice during Brucella infection may arise from the inability of peripheral T Reg to commit to T FR differentiation in response to infection.Bcl6 restrains CD4 + T cell IL-10 production [52], and T Reg, 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 T Reg and/or enhanced CD4 + T cell IL-10 production in CD4 Cre Bcl6 fl/fl mice results in elevated susceptibility to Brucella, though this would require additional investigation to confirm.
Upon depletion of B cells from CD4 Cre Bcl6 fl/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 T FH 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 T Reg and T FR function [27,53,54] and upregulation of PD-1 by T FH and T FR contributes to follicular migration and positioning in the developing GC response [70].Here PD-1 blockade enhanced susceptibility to Brucella and augmented T Reg, and T FR populations (Fig 6F and 6G).Notably, tumor infiltrating T FR are prevalent in several types of cancer, and PD-1 blockade enhances tumor-infiltrating T FR leading to reduced tumor control in mice [54].Collectively, these findings support a link between PD-1 blockade and promotion of T Reg and T FR which may directly impact host susceptibility to infection and the efficiency of tumor control.
Alterations in T Reg and T FR frequencies induced by PD-1 blockade were B cell dependent (Fig 6F and 6G).B cell expression of PD-L1 is implicated in T FH 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 + T FH -like cells in the lung [73].Future studies should focus on how B cell PD-L1/L2 expression affects the quantity and quality of T FH, T FR and T Reg development during infection with Brucella and other pathogens.
While T Reg and T FR promote susceptibility to Brucella, only T FR absolutely require B cells for this effect (Figs 7A and 7F and S3H).To gain insight as to how T FR impact Fo B behavior to potentially alter susceptibility to infection, we performed RNA-seq on splenic Fo B isolated from Bcl6 fl/fl and FoxP3 Cre Bcl6 fl/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 Bcl6 fl/fl and FoxP3 Cre Bcl6 fl/fl mice, 401 genes were differentially regulated.Interestingly, only ~5% of the genes differentially regulated when comparing Fo B obtained from infected Bcl6 fl/fl and FoxP3 Cre Bcl6 fl/f mice were also differentially regulated when comparing Fo B from naïve Bcl6 fl/fl and FoxP3 Cre Bcl6 fl/f mice (S10B and S10C Fig) indicating the effect of T FR on Fo B transcription is context dependent.Interestingly, RNA-seq analysis revealed decreased Fo B transcription of Tgfb3 in Brucellainfected T FR deficient mice (S10A Fig) .As TGF-β3 production by B cells has been shown to drive expansion of T Reg [74], which are deleterious to control of Brucella (S3 Fig), regulation of B cell TGF-β3 expression by T FR could alter susceptibility to Brucella.We also observed decreased transcription of dual specificity phosphatase 4 (Dusp4) in T FR 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 T FR may warrant further study.T FR deficiency also altered transcription of genes involved in splenic compartmentalization/follicular positioning (Klf2, Bcl6, Ccr6, Cxcr4, Pdlim1) [70,71,[76][77][78], and antibody production (Tgfβ3) [79] which aligns with the established role of T FR 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 T FR alter the positioning of Fo B and the frequency and/or magnitude of interactions of Fo B with CD4 + T cells.
Alternatively, T FR may negatively impact the ability of non-B cell populations to effectively control infection.Due to the reciprocal regulation inherent in generation of T FH , T FR and GC B responses [20,32,52], B cell deficiency lowers T FR proportions (Fig 4E and 4H).Therefore, B cell dependent effects of T FR may arise from their dependence on B cells for generation or maintenance.In this case, T FR could either act directly on B cells, or could function to alter other T-and/or non-B cell populations.Interestingly, T FR deficiency in mice has been linked to upregulation of granzyme B and other cytotoxicity associated genes in T FH [33], indicating that T FR can modulate the function of other CD4 + T cell populations.
In sum, our findings indicate B cells promote T FR responses that are regulated by CD40L and PD-1 dependent mechanisms.T FR in turn promote susceptibility to infection in a manner independent of the humoral response (S11 Fig) .T FR deficiency can enhance protection against influenza [62], but to our knowledge, this is the first report of an antibody independent effect of T FR altering resistance to infection.Future studies will need to determine whether T FR 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 T FR induction synergize to hamper control of infection independent of the antibody response could have broad implications for rational vaccine designs which seek to optimize T FH and GC B responses.

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% CO 2 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 1x10 5 CFUs of B. melitensis 16M in 200 μl of DPBS.The delivered dose was confirmed via plating of inoculum onto Brucella agar.

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

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T follicular regulatory cell mediated susceptibility to Brucella 37˚C/5%CO 2 , 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).

Passive antibody transfer
Bcl6 fl/fl and FoxP3 Cre Bcl6 fl/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 Bcl6 fl/fl or FoxP3 Cre Bcl6 fl/fl sample were sterilized using a 0.22 μm filter and pooled by genotype.200 μL from either Bcl6 fl/fl or FoxP3-Cre Bcl6 fl/fl pooled stock were administered i.p. to naïve FoxP3 Cre Bcl6 fl/fl mice twenty-four hours prior to infection.

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T follicular regulatory cell mediated susceptibility to Brucella 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 10 8 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.

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.T Reg 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).

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 FoxP3 Cre Bcl6 fl/fl averaged 93.5% and 97.4% respectively.Fo B cell (CD23 + CD21 lo B220 + ) purity averaged ~85% of live B220 + cells as determined by flow cytometry in both strains.Approximately 4x10 6 -1x10 7 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.

Fig 1 .
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 1x10 5 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

Fig 2 .
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 CD19 Cre iAB fl/fl or iAB fl/fl littermates (n = 3-7/group/timepoint) at one-, two-, and fourweeks post infection.(C) CD19 Cre iAB fl/fl and iAB fl/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

Fig 4 .Fig 5 .
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 T Reg (FoxP3 + CXCR5 -) (F), T FH (FoxP3 -ICOS + CXCR5 + ) (G), and T FR (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 As PD-1 blockade enhances susceptibility to infection as well as T Reg and T FR outgrowth (Fig 6A, 6F and 6G), we investigated the contribution of T Reg 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 days14 and 15 post challenge and confirmed FoxP3 expressing CD4 + T cells were diminished (S3A Fig).In line with previous reports suggesting T Reg cells inhibit control of brucellosis [60,61], T Reg 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 T FH 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 T Reg during Brucella infection (Figs 3C, 3E, S1G and S1H), depletion of T Reg in both control and B cell depleted mice enhanced resistance to infection (S3B and S3H Fig) indicating the deleterious effect of T Reg is not entirely B cell dependent.

T
FR enhance susceptibility to Brucella infection in a B cell dependent, but antibody independent, manner DTX treatment leads to T FR deficiency in DEREG mice (S3J Fig) making it unclear whether T Reg , T FR or both contribute to enhanced susceptibility to Brucella.Therefore, we employed FoxP3 Cre Bcl6 fl/fl mice, in which T FR are deficient while T Reg, T FH, and GC B cells remain intact[29] and found T FR deficiency enhanced resistance to Brucella four weeks post infection (Fig 7Aand 7F).FoxP3 Cre Bcl6 fl/fl mice displayed elevated total T Reg frequencies compared to Bcl6 fl/fl mice (Fig 7B), though this did not adversely affect control of infection.To determine whether T FR require B cells to enhance susceptibility to infection, we depleted B cells from FoxP3 Cre Bcl6 fl/fl and Bcl6 fl/fl mice.While isotype treated FoxP3 Cre Bcl6 fl/fl mice were more resistant to infection than isotype treated Bcl6 fl/fl animals, Brucella burdens four weeks post infection were similar in FoxP3 Cre Bcl6 fl/fl and Bcl6 fl/fl mice depleted of B cells (Fig7F).Thus, our results demonstrate that, in the absence of B cells, T FR are no longer deleterious to control of infection.

Cre
Bcl6 fl/fl mice four weeks post infection, FoxP3 Cre Bcl6 fl/fl animals did display reduced anti-Brucella IgG levels (S4A Fig).However, passive transfer of sera from either Bcl6 fl/fl or FoxP3-Cre Bcl6 fl/fl mice previously infected with B. melitensis conferred similar levels of protection to FoxP3 Cre Bcl6 fl/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 T FR is independent of antibody regulatory function.

Fig 7 .
Fig 7. T FR enhance susceptibility to Brucella.(A) Splenic Brucella burdens of Bcl6 fl/fl and FoxP3 Cre Bcl6 fl/fl mice (n = 5-8/group) challenged with B. melitensis four weeks post challenge.(B-D) Quantification of the mean percentage of T Reg (FoxP3 + CXCR5 -) (B), T FR (FoxP3 + PD-1 + CXCR5 + ) (C), and T FH (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 Bcl6 fl/fl or FoxP3 Cre Bcl6 fl/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 While CD19 Cre deletes the majority of MHCII expression on B cells in CD19 Cre iAB fl/fl mice, a fraction of B cells retains B cell MHCII expression, and the proportion of MHCII + B cells in CD19 Cre iAB fl/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 CD19 Cre iAB fl/fl mice treated with IgG, but not in CD19 Cre iAB fl/fl mice depleted of CD4 + T cells (S8B and S8C Fig).