The essential role played by B cells in supporting protective immunity against Trichuris muris infection is dependent on host genetic background and is independent of antibody

This study investigates the role of B cells in immunity to Trichuris muris (T. muris) infection in two genetically distinct strains of mouse, using anti-CD20 monoclonal antibody (mAb) (Genentech-clone 5D2) to deplete B cells. Data is presented for the mouse strains: C57BL/6 and BALB/c, which mount mixed Th1/Th2, and highly polarised Th2 immune responses to T. muris, respectively. C57BL/6 mice receiving anti-CD20 treatment prior to and during, or anti-CD20 treatment that commenced two weeks post infection (p.i.), were susceptible to T. muris. Parasite-specific IgG1 antibodies were absent and Th2 type cytokines produced by mesenteric lymph nodes cells from mice receiving α-CD20 mAb treatment were significantly lower than produced by cells from isotype control treated mice. T follicular helper cells were also significantly reduced. Importantly, and in complete contrast, BALB/c mice were still able to expel T.muris in the absence of B cells, revealing that the essential role played by B cells in protective immunity was dependent on genetic background. To explore whether the important role played by the B cell in the protective immune response of C57BL/6 mice was in enabling strong Th2 responses in the presence of IFN-γ, IFN-γ was blocked using anti-IFN-γ mAb post B cell depletion. Depleting IFN-γ, in the absence of B cells restored worm expulsion in the absence of parasite-specific IgG1/IgG2c and partially rescued the T. muris specific IL-13 response. Thus, our data suggest an important, antibody independent role for B cells in supporting Th2 type immune responses in mixed IFN-γ-rich Th1/Th2 immune response settings. Author summary How B cells contribute to protective immunity against parasitic nematodes remains unclear, with their importance as accessory cells under-explored. This study reveals that, on some genetic backgrounds, B cells are important for the expulsion of T. muris by acting as accessory cells, supporting Th2 immune responses.


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Infecting over two billion people around the world, mostly in resource-limited 39 countries, the ability of parasitic helminths to maintain long standing chronic infections 40 makes them a major health care issue (1). Trichuris trichiura (T. trichiura) is one of the most 41 common gastrointestinal nematodes, infecting approximately 465 million people worldwide 42 (2), primarily children. In infected children, trichuriasis is strongly associated to malnutrition, 43 growth stunting, and intellectual retardation; whereas in pregnant adults it is related to 44 anaemia and low birth weight babies (3). 45 For decades, T. muris in the mouse has provided a useful and relevant model system 46 with which to explore immunity to T. trichiura in man. Infection of mice with intestinal 47 nematode parasite T. muris drives polarized T helper cell (Th) responses which associate with 48 resistance (Th2) or susceptibility (Th1). However, the key cellular contributions which 49 support Th2 cell polarization during T. muris infection are still not well understood. One of 50 the cells thought to be important is the B cell. It is well established that B cell function is not 51 only related to antibody production, but B cells can also act as antigen presenting cells 52 (APCs) due to the expression of MHC class II molecules and several co-stimulators, 53 including CD40, CD80, and CD86 on its surface (4-6), and as accessory cells, acting as a 54 cellular source of multiple cytokines (7).  103 As a transmembrane calcium channel, CD20 is important in B cell activation, 104 proliferation, and differentiation (25). The CD20 molecule is normally expressed on the 105 surface of B cells during the late pre-B cell. Therefore, the injection of anti-CD20 mAb will 106 deplete all B cells, except early pre-B cells and plasma cells (26). The vast majority of IgM + 107 or B220 + cells in the spleen, lymph nodes, peritoneal cavity, and blood express CD20, whilst the MLNs, spleen, and blood by day 35 p.i. (Fig. 1A&B). Therefore, a second anti-CD20 116 injection was administered at day 10 p.i. in order to maintain B cell depletion in the blood 117 and secondary lymphoid organs throughout infection (Fig. 1A&B). CD19 + cells were still 118 present in the bone marrow after anti-CD20 treatment (Fig. 1B), however these CD19 + cells 119 were pro-B cells (Fig. 1C) as has been shown previously (26).  Fig. 2A. Previous studies have shown 128 that C57BL/6 mice take up to 35 days to completely expel the parasite (31). As shown in Fig.   129 2B, C57BL/6 depleted of B cells were significantly more susceptible to infection than control 130 treated mice. 131 It has previously been shown that IgG1 antibodies are associated with resistance to T. 132 muris infection, whilst IgG2c antibodies are related to susceptibility (32). Therefore, the 133 levels of parasite specific antibodies in the sera of anti-CD20 treated mice and isotype control 134 treated mice were compared. Mice depleted of B cells using anti-CD20 mAb failed to secrete 135 IgG1 and IgG2c parasite specific antibodies, in contrast to control treated mice (Fig. 2C&D).

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Further, the levels of T. muris specific IgM antibodies in the sera of anti-CD20 treated mice 137 were significantly lower than in the sera of the isotype control-treated mice (Fig. 2E). These  In common with other nematode parasites, hosts resistant to T. muris mount Th-2 141 immune responses characterised by the production of IL-4, IL-5, IL-9 and IL-13; in contrast, 142 mice susceptible to T. muris mount a Th-1 type response, dominated by the release of 34,35). B cell depleted mice on a C57BL/6 background produced significantly lower 144 Th2 cytokines, including IL-5, IL-9, and IL-13 compared to mice treated with the isotype 145 control ( Fig. 2F-H). Interestingly, the production of IFN-γ and IL-17 was similar in both 146 groups ( Fig. 2I&J)

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The importance of B cells for the formation of T FH has been shown previously in both 153 mouse (15) and man (36). In keeping with these studies, the MLN T FH population of anti-154 CD20 treated mice in C57BL/6 genetic background was significantly reduced compared to 155 isotype control treated mice ( Fig. 3A-C). Gating strategy was shown in Supplementary Fig. 1.

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In contrast, T FH development in the spleen, distal to the site of infection, was not significantly 157 affected by B cell depletion ( Supplementary Fig.2), although the lack of statistical 158 significance may be due to the high variability in the control mice.   Fig. 4A. Surprisingly, the depletion of B cells from day 14 p.i. also impaired worm expulsion 175 with significantly more parasites present at day 35 p.i. than seen in control-treated mice (Fig.  4B). T FH cells and Th2 type cytokines, including IL-5, IL-9, and IL-13 were also significantly 177 reduced in the absence of B cells (Fig, 4C-H). Interestingly, TNF-α was significantly 178 increased in B cell depleted mice (Fig. 4I), whilst IFN-γ and IL-17A remained the same 179 between groups (Fig. 4J&K). These data suggest that the B cell plays an important role in 180 maintaining Th2 responses.   (Fig. 5I&J). T FH cells have been associated with Th2 immunity due to their ability to secrete 208 IL-4, a Th2 signature cytokine (33,40,41). Given that IL-13 production persisted in B cell 209 depleted mice after anti-IFN-γ injection, we wondered whether the T FH population was also 210 altered. Anti-IFN-γ treatment did not restore the T FH population in MLNs of anti-CD20 211 treated mice at day 21 p.i. (Fig. 5I&J), however it remains a possibility that the few TfH 212 remaining were sufficient to induce the partial Th2 response.  In the current study we found that when C57BL/6 mice were depleted of IFN-γ, the 217 immune response became more highly polarised towards Th2 mice, evidenced by parasite-218 specific IgG1 antibodies, in the absence of IgG2c. In this more polarised Th2 environment 219 the depletion of B cells did not prevent worm expulsion. These data suggest that the    Overall, we present a study showing that, irrespective of host genetic background, 338 antibody is not essential in the expulsion of T. muris. However, our data do identify an  were re-injected with anti-CD20 mab or isotype control at day 10 p.i. 1 mg of α-IFN-γ 366 antibody or rat IgG1 (as a control) antibody was given at day 0, day 7, and day 14 p.i. Mice 367 were euthanised by CO2 followed by autopsy at day 21 or day 35 p.i..  During autopsy, the caecum and proximal colon were collected and stored at -20°C 403 before analysis. Before worm count, the intestine was thawed at room temperature and cut 404 longitudinally using blunt ended scissors and the epithelium was scraped using curved 405 forceps in a petri dish. Worms were counted blindly under a dissecting microscope (Leica).  Approximately 50μl of blood were placed into 1.5 ml eppendorf tube containing 50μl 417 0.5M EDTA and stored on ice before analysis. 500 μl of Red Blood Cell Lysing Buffer Hybri-Max™ (Sigma-Aldrich) were added and samples were incubated for 5 minutes at room 419 temperature. 1 ml of 1xPBS were added and cells were pelleted by centrifugation at 1500 rpm 420 for 5 minutes. Red blood cell lysis process was repeated twice before cells were resuspended 421 in 1 ml of complete RPMI 1640 medium.

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Femurs and tibias were collected at autopsy and after removing any remaining tissue 423 without damaging the bone integrity, the bone was placed on ice until ready to process. Bone   478 muris E/S at 100μg/ml (to give a final concentration of E/S in the cell culture of 50µg/ml).

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Cells were incubated for 48 hours at 37°C and 5% CO2. To collect the supernatant, plates 480 were centrifuged at 1400g for 5 minutes and the supernatant was stored at -20°C.