Patients with Human Hyper IgM syndrome (HIGM), usually develop symptoms in infancy and are prone to pulmonary and gastrointestinal infections and complications (Moazzami et al., 2019). HIGM syndrome was first described back in 1961 and was first molecularly characterised as a mutation in the CD40 ligand (CD40L) gene in 1992 (Cabral-Marques et al., 2014). Patients are characterised by presenting with higher or similar concentrations of serum IgM but lower IgG, IgA, or absent concentrations of IgE compared to healthy subjects, accordingly, serum IgG immunoglobulin replacement is an effective host treatment for decreasing chronic infections among these patients (Davies and Thrasher, 2010; Yazdani et al., 2019). CD40L deficiency ranks as the 28th most frequent type of primary immunodeficiency disorder plus defects to the CD40L (48-70%) molecule are the most common cause of HIGM syndromes worldwide (Yazdani et al., 2019).

The CD40L also known as CD154, is a type II transmembrane protein expressed mainly by activated T cells and platelets and its expression is also induced under inflammatory conditions on basophils, monocytes, natural killers, and mast cells (Meng et al., 2018). In contrast, CD40 is a constitutively expressed receptor that initially was characterised on B-cells and also expressed on dendritic cells (DCs), monocytes, platelets, macrophages, and many non-hematopoietic cells (myofibroblasts, fibroblasts, epithelial, and endothelial cells) (Elgueta et al., 2009). The wide expression of this costimulatory pair of molecules indicates the pivotal roles they play in different cellular immune processes, e.g., CD40 signalling of B-cells by CD40L on activated T cells is essential for the formation of Germinal Centres (GCs) inside Secondary Lymphoid Organs (SLOs). GCs are highly specialized microstructures comprised of B-cells undergoing clonal expansion and affinity maturation (Klein and Dalla-Favera, 2008; Victora and Nussenzweig, 2022). Within GCs, B-cells undergo the Darwinian process of somatic diversification, and affinity-driven selection of immunoglobulins producing high-affinity (IgG, IgA, and IgE) antibodies, long-lived antibody-secreting plasma cells, and memory B-cells, that together are essential for effective humoral immunity, including the elimination of pathogens and protecting the host against reinfections (Klein and Dalla-Favera, 2008; Victora and Nussenzweig, 2022).

Focusing on intestinal antibody production during homeostasis, it is well known that it is dominated by IgA production, albeit intestinal B-cell IgG class switching also occurs although at a low level (Fleming et al., 2022). Furthermore, it has been demonstrated that within mice MLN and PP commensal-reactive IgG2b and IgG3 responses. These responses are largely generated independently of T cells (TI), requiring B-cell Toll-like receptor (TLR) signalling, therefore, gut microbiota-induced IgG antibodies predominantly IgG2b and IgG3, it is well known that TI IgG3 responses in mice predominantly target microorganisms’ carbohydrate antigens (Pone et al., 2010; Rawlings et al., 2012; Koch et al., 2016; Fleming et al., 2022).

Additionally, to the conventional B-2 cell population, there is another population of B-cells known as B-1 cells that produce antibodies that do not require the signalling between CD40 and CD40L on B-cells and on activated T cells, respectively. B-1 cells are more abundant in the pleural and peritoneal cavities, anatomical regions continuously exposed to microorganisms (Kroese et al., 1989; Gao et al., 2012). Therefore, B-1 cells migrate to both SLOs and intestinal lamina propria spontaneously secreting natural IgM, IgA, and IgG3 antibodies, in the absence of exogenous immunization, mainly against non-protein antigens including those present on pathogenic microbiota microorganisms (Meyer-Bahlburg, 2015; Prieto and Felippe, 2017; Castro-Dopico and Clatworthy, 2019). B-1 cells switch to IgA expression more readily than other B-cell populations (B-2) when exposed to certain soluble cytokines e.g., transforming growth factor beta (TGFβ), a proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF) or bacterial molecules like lipopolysaccharides (LPS) that signal through TLR4, resulting in IgA production in a T cell-independent manner, that is, in the absence of CD40L-CD40 interaction (Lebman and Edmiston, 1999; Cerutti, 2008).

C57BL6-CD40L deficient mice (C57BL6-cd40l ^−/−) provide a model that has helped to unveil the physiopathology of human HIGM syndrome (Bernal-Reynaga et al., 2013; Lopez-Saucedo et al., 2015). These mice, in the absence of immunisation, have been shown to express elevated or similar serum concentrations of IgM, accompanied by reduced levels of IgG and IgA, and no IgE as observed in humans presenting HIGM syndrome (Renshaw et al., 1994; Bernal-Reynaga et al., 2013). This animal model has been an excellent tool for studying the interactions between the host and human and mouse gastrointestinal pathogens, such as enterotoxigenic Escherichia coli and Citrobacter rodentium (C. rodentium), respectively (Bernal-Reynaga et al., 2013; Lopez-Saucedo et al., 2015). C57BL6-cd40l ^−/− C. rodentium model revealed that after immunization against this bacterial pathogen specific serum IgG concentrations were lower than in WT mice, nevertheless, they were functional as they exhibited a bactericidal effect against C. rodentium (Lopez-Saucedo et al., 2015). Compared to WT mice intestinal IgA concentrations in C57BL6-cd40l ^−/− mice were significantly higher, IgG1 and IgG2 were significantly lower, and IgG3 levels were similar between mouse strains. For these reasons, the present study seeks to compare and evaluate the presence and localization of GCs, IgA-producing cells, and B-cell populations in both gut-associated (PP and MLN) and non-gut-associated SLOs (spleen and ILN) between unimmunised C57BL6-cd40l ^−/− and WT mice.

The importance of the HIGM syndromes in six Latin-American countries (Argentina, Brazil, Chile, Costa Rica, Peru, and México) was described in 2014, revealing that most HIGM patients (94.5%) have mutations in the cd40l gene. The authors also described that the bacteria Salmonella spp, the fungus Microsporidium spp, and the protozoa Criptosporidium parvum, Giardia lamblia, Entamoeba histolytica and Isospora belli, are the most common microorganism associated with chronic diarrheal among HIGM patients in Latin-America (Cabral-Marques et al., 2014).

We described for the first time that in unimmunised C57BL6-cd40l ^−/− mice exhibit significantly higher concentrations of IgA in their faeces in comparison with WT mice (Bernal-Reynaga et al., 2013). IgA is the most abundant immunoglobulin in the intestinal tract, which derives from B-cells, receiving stimulating signals from intestinal microorganisms, in a T-dependent and T-independent fashion. Mice gut IgA responses to intestinal bacteria contribute to shaping bacterial topography, composition, growth, invasiveness, and even modulating the host immunometabolic functions, and immune responses (Chen et al., 2020). Therefore, to understand the observation of higher concentrations of gut IgA in unimmunised C57BL6-cd40l ^−/− mice, we determined the prevalence of several B-cell populations and plasma cells (total, IgA-, IgG- and IgM-positive plasma cells) in gut-associated (MLN and PP) and non-gut-associated SLOs (spleen and ILN) in WT mice and C57BL6-cd40l ^−/− mice.

In unimmunised WT mice, GCs were present in-gut associated SLOs but absent in non-gut-associated SLOs, this could be due to the fact of the continuous antigen stimulation of intestinal SLOs by microorganisms and food antigens, as it is well established (Forchielli and Walker, 2005; Van den Broeck et al., 2006; Suzuki et al., 2010a; Stebegg et al., 2018). In line, it has also been described that after WT mice were orally immunised with ovalbumin-cholera toxin, IgA-positive cells were absent or present in low numbers within the spleen and other non-gut associated lymph nodes, respectively (Weiberg et al., 2018), also indicating that antigen SLOs stimulation is influenced by immunisation site (Drayson, 1986). In contrast, in PP and MLN of WT mice a higher frequency of IgA-positive cells within the GC, known as IgA-GC, was observed, as has been shown in antigen-driven reactions (Mora and von Andrian, 2008). IgA-positive cells were observed within gut-associated SLOs of C57BL6-cd40l ^−/− mice even in the absence of GC, revealing a differential abundance and localization of IgA-positive cells, which were more abundant and located within the BZ in MLN, and less abundant and located within the TZ in PP. Overall our results suggest that in unimmunised mice GC formation and production of IgA-positive cells, are mainly limited to gut-associated SLOs that are constantly exposed to microbial and food antigens (Suzuki et al., 2010b).

As previously described in C57BL6-cd40l ^−/− mice GCs were absent in non-gut-associated (Spleen and ILN) SLOs, and we described for the first time that GCs are also absent in gut-associated SLOs (PP and MLN) of C57BL6-cd40l ^−/− mice. Furthermore, CD40 receptor-deficient C57BL6 mice also lacked GCs in gut-associated (PPs and MLN) SLOs (Castigli et al., 1994; Bergqvist et al., 2006). Together these results confirm that the interaction between CD40 (on B-cells) and CD40L (on activated T-cells) is essential for the formation of GCs (Kawabe et al., 1994; Renshaw et al., 1994; Takemori et al., 2014).

The characterisation of IgA-positive cells in vivo and in situ in non-gut- and gut-associated SLOs in both unimmunised mouse strains, revealed that in non-gut-associated SLOs (spleen and ILN) IgA-positive cells were identified within the TZ, and their frequency was very low. In contrast, a high and similar number of IgA-positive cells were observed in the small intestine MV of WT and C57BL6-cd40l ^−/− mice. Confirming that most intestinal IgA production does not require CD40-CD40L interaction, GC formation, and arises from a T-independent manner (Fleming et al., 2022).

In C57BL6-cd40l ^−/− mice the total numbers of IgM-positive plasma cells were significantly higher, while IgA-positive plasma cells were significantly lower at all non-gut-associated SLOs studied, in comparison with WT mouse SLOs; corroborating that C57BL6-cd40l ^−/− mice are producing a higher or equal number of serum IgM and lower IgA antibodies (Bernal-Reynaga et al., 2013) comparable to HIGM patients (Meng et al., 2018).

Contrary to gut-associated SLOs in C57BL6-cd40l ^−/− mice IgA-positive plasma cells were significantly higher compared to gut-associated SLOs of WT mice, consistent with the previous report of higher concentrations of IgA in the faeces of C57BL6-cd40l ^−/− mice than in WT mice (Bernal-Reynaga et al., 2013). It has also been reported that IgG in faeces of C57BL6-cd40l ^−/− mice are significantly lower than in WT mice (Bernal-Reynaga et al., 2013); in line, we observed that SLOs IgG-positive cells frequency was very low or absent in C57BL6-cd40l ^−/− mice than in WT mice, confirming that most IgG responses take place in GCs (Elsner and Shlomchik, 2020). Together our results confirm the role of PP and MLN as mucosal inductive sites, whose characteristic features are to initiate an IgA preferential immune response production in these anatomical sites (Koch et al., 2016; Weiberg et al., 2018). IgA antibodies play a pivotal role in neutralizing, eliminating, and regulating potential pathogens and microorganisms in the gut (Gommerman et al., 2014; Takeuchi and Ohno, 2021).

Furthermore, we have shown that immunized C57BL6-cd40l ^−/− mice with C. rodentium, have complement-mediated bactericidal effect on C. rodentium, and indeed was observed in the sera of C57BL6-cd40l ^−/− mice contained IgM and IgG2b antibodies that are capable of fixing complement. (Lopez-Saucedo et al., 2015); hence, it is possible that intestinal IgM and IgG will also have a bactericidal effect against intestinal bacterial pathogens in C57BL6-cd40l ^−/− mice (Lopez-Saucedo et al., 2015), but the presence of IgG2b in faeces must be shown.

B-1 cells, which are an important source of secreted IgA and IgM natural antibodies, in intestinal and pulmonary mucosae, were as well characterised in peritoneal lavages and SLOs of both animal strains. (Suzuki et al., 2010a; Baumgarth, 2011; Prieto and Felippe, 2017). Notably, B-1 cells that have not been previously characterised in the peritoneal lavage of C57BL6-cd40l ^−/− mice, were significantly less abundant than in WT mice peritoneal lavages, but were more abundant within the gut and non-gut associated C57BL6-cd40l ^−/− mouse SLOs than in WT mouse SLOs. Indicated that in C57BL6-cd40l ^−/− mice, B-1 cells are migrating to immune effector sites, all around the mouse body, in order to produce natural antibodies (Hardy and Hayakawa, 2005). It has been shown that B-1 cells and their antibodies play an important role in the protection against mice pneumococci illness (caused by Streptoccocus pneumoniae), animals lacking B-1 cells were unable to survive pneumococci infections, may be due to the fact that natural IgM and IgA are not produced, especially against the pneumococcal capsular polysaccharide serotype 3 (PPS)-3, a non-protein antigen, found on the cell walls of these bacteria (Dasu et al., 2009; Rodriguez-Zhurbenko et al., 2019). Also, the human B-1 cell population generates antibodies against the capsular polysaccharides of S. pneumoniae and polysaccharide Vi of Salmonella typhi (causative agent of typhoid fever) suggesting the important role of the B-1 population in mucosal immune responses in animals and humans (Suzuki et al., 2010b; Marshall et al., 2012; Rodriguez-Zhurbenko et al., 2019).

Since TGFβ superfamily members contribute to the maturation, and differentiation of B-cells, particularly promoting IgA production by inducing the differentiation of B-cells to IgA-producing plasma cells (Li et al., 2006; Tamayo et al., 2018; Takeuchi and Ohno, 2021). After evaluating the presence of TGFβ receptor 1 (TGFβR1) on splenic B-cells of both mouse strains a similar expression of this receptor was observed. In contrast, TGFβR1 expression on B-cells of MLN, a gut-associated SLOs, was significantly higher in C57BL6-cd40l ^−/− mice compared to MLN B-cells of WT mice. These observations together with the fact that in C57BL6-cd40l ^−/− mice the number of IgA-positive cells and B-1 cells in MLN, were significantly higher than in WT mice, suggest that the microenvironment in MLN is promoting the induction of IgA plasma cells that in turn will produce gut IgA antibodies in C57BL6-cd40l ^−/− mice.

Together our results may explain our initial observation of a higher concentration of IgA antibodies in C57BL6-cd40l ^−/− mice faeces in the absence of GCs. Additionally, it was described that IgA-positive cells, IgA-positive plasma cells, B-1 cells, and B-cells TGFβR1 are more abundant in the gut-associated SLOs of C57BL6-cd40l ^−/− mice than in SLOs of WT mice. The B-cell populations identified in gut-associated SLOs and the microenvironment (due to overexpression of TGFβR1) are contributing in C57BL6-cd40l ^−/− mice to increase the production of total IgA antibodies in the gut. The high concentration of intestinal IgA constitutes a barrier for the adhesion, growth, elimination, and/or clearance of intestinal microbial pathogens in mice and in HIGM patients.

Most HIGM patients (95%) have mutations of the cd40l gene, patients are characterised by higher/similar IgM and lower IgG, and IgA serum concentrations and by the absence of GC in SLOs. C57BL6-cd40l ^−/− mouse, has serum immunoglobulin concentrations parallel levels observed in HIGM patients and lacks GC in SLOs, thus is an animal model mimicking HIGM patient characteristic. Our group has reported that C57BL6-cd40l ^−/− mice have lower IgG and higher IgA faecal antibodies than WT mice and specific serum (IgM and Ig2b) antibodies against C. rodentium that have a complement-mediated bactericidal effect on these bacteria. Using the C57BL6-cd40l ^−/− mouse model under unimmunised conditions, it was revealed that all SLOs tested (Slpleen, ILN, MLN, and PP) lacked GCs and contained higher numbers of B-1 cells, all gut-associated SLOs harboured higher numbers of IgA-positive plasma cells that WT mice, whereas both mouse strains have similar expression of IgA-positive cells in small intestine lamina propria. IgA-positive plasma cells and B-1 cells are directly involved in the production of IgA antibodies in the intestine. Our recently acquired knowledge will help to develop specific treatments for the elimination of harmful pathogens in the intestine of HIGM patients, like priming the intestine with pathogens antigens that in turn will induce the production of specific gut IgA, IgM, IgG2b, and IgG3 antibodies.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by CINVESTAV Ethics Committee, # 254994.

LF-R, TE-G, and FH-C conceived the experiments. FH-C, RM-A, and CL-S performed the experiments. FH-C worked on the quality of the images and figures.JY-P, JM-B, and SE-P provided guidance, resources, and input. FH-C and TE-G wrote the manuscript, which was reviewed and approved by all authors. All authors contributed to the article and approved the submitted version.