Murine isolated lymphoid follicles contain follicular B lymphocytes with a mucosal phenotype

Abstract

Isolated lymphoid follicles (ILFs) are organized intestinal lymphoid structures whose formation can be induced by luminal stimuli. ILFs have been demonstrated to act as inductive sites for the generation of immune responses directed toward luminal stimuli; however, the phenotype of the immune response initiated within ILFs has largely been uninvestigated. To gain a better understanding of the immune responses initiated within ILFs, we examined phenotypic and functional aspects of the largest cellular component of the murine ILF lymphocyte population, B lymphocytes. We observed that murine ILF B lymphocytes are composed of a relatively homogenous population of follicular B-2 B lymphocytes. Consistent with their proximity to multiple stimuli, ILF B lymphocytes displayed a more activated phenotype compared with their counterparts in the spleen and Peyer's patch (PP). ILF B lymphocytes also expressed higher levels of immunomodulatory B7 and CD28 family members B7X and programmed death-1 compared with their counterparts in the spleen and PP. ILF B lymphocytes preferentially differentiate into IgA-producing plasma cells and produce more IL-4 and IL-10 and less interferon-gamma compared with their counterparts in the spleen. Immunoglobulin repertoire analysis from individual ILFs demonstrated that ILFs contain a polyclonal population of B lymphocytes. These findings indicate that murine ILFs contain a polyclonal population of follicular B-2 B lymphocytes with a phenotype similar to PP B lymphocytes and that, in unchallenged animals, ILFs promote immune responses with a homeostatic phenotype.

Fig. 1

Isolated lymphoid follicules (ILFs) contain mature follicular B-2 B lymphocytes. Flow cytometry analysis was performed on cellular populations from the spleen (SP), Peyer’s patch (PP), and mature ILF as outlined in MATERIALS AND METHODS . A low-power photomicrograph of an intestine from a Balb/c mouse stained with lectin from Ulex Europaeus demonstrated the appearance of mature ILFs (arrowheads in A). Mature ILFs contained a population of B lymphocytes that was sIgD^hi sIgM^lo (B), CD5⁻ (C), and CD23⁺ (D). Mature ILF B lymphocytes have a cell surface phenotype that is most similar to that of PP B lymphocytes as opposed to splenic B lymphocytes. Flow cytometry analysis of ILFs cellular populations was performed on a pooled population from 3 mice. The percentages of positive cells in each quadrant are shown, and the plots are representative of 1 of 3 experiments. Original magnification of A: ×20.

Fig. 2

Mature ILF B lymphocytes express higher levels of CD69 and CD80 and higher levels of B7 family members. Three-color flow cytometry analysis was performed on cellular populations from the SP, PP, and mILF as outlined in MATERIALS AND METHODS. Analysis was performed by gating on sIgD^hi sIgM^lo follicular B-2 B lymphocytes from each tissue, as shown in Fig. 1B. The majority of ILF B lymphocytes (solid trace) expressed CD44, CD62L, CD86, major histocompatibility complex II (MHC II), and B7H1. Among all the markers, the ILF expressed much higher CD69, CD80, B7X, and PD-1 compared with the SP (shaded trace) and PP (dotted trace). Isotype control staining is designated by the filled shaded trace. Flow cytometry analysis of ILF cellular populations was performed on a pooled population from 3 mice. Data shown are representative 1 of 2 experiments.

Fig. 3

ILF B lymphocytes preferentially differentiate into IgA-producing plasma cells. The level of secreted Ig was quantified by ELISA (A), and the number of antibody-forming cells was quantified by ELISPOTs (B) as described in MATERIALS AND METHODS . ILF cellular populations preferentially produced IgA as opposed to other immunoglobulin isotypes; this profile was similar to that produced by PPs and intestinal lamina propia (LP) cells and dissimilar to that produced by the sP or that seen in the serum (A). ELISPOT analysis confirmed that ILF B lymphocytes preferentially differentiate into IgA- and IgM-producing plasma cells; this profile was most similar to that seen in the PP and LP and least similar to that seen in the SP (B). ILF cell populations contained higher number of IgA-producing cells on a per cell basis compared with the SP using a standard Student’s t-test (P < 0.05); no differences were noted when the IgA-producing cell populations from the PP and ILF were compared. Pooled cellular populations from 3 or 4 mice were used to measure immunoglobulin production by ELISA or ELISPOT assay. Data are represented as means ± SE from 3 independent experiments.

Fig. 4

ILF B lymphocytes produce IL-4 and IL-10 and little interferon (IFN)-γ. IL-4, IL-10, and IFN-γ secretion by ILF B lymphocytes was measured by ELISPOT as described in MATERIALS AND METHODS. Compared with splenic and PP sIgD^hi sIgM^lo B lymphocytes, ILF B lymphocytes produced more IL-4 and IL-10. Compared with splenic B lymphocytes, IFN-γ-secreting cells were not detected among the ILF and PP sIgD^hi sIgM^lo B lymphocytes. Pooled cellular populations from 3 or 4 mice were used for the measurement of each cytokine. Data are represented as means ± SD from 2 or more independent experiments. ND, none detected. *P < 0.05 compared with cytokine production by ILFs with that of the PP or spleen using one-way ANOVA.

Fig. 5

ILF B lymphocytes have a diverse immunoglobulin repertoire similar to the repertoire seen in the SP and PP. Immunoglobulin heavy-chain usage in the SP and PP and individual ILFs was analyzed using a PCR-based approach as described in MATERIALS AND METHODS. PCR was performed with primers specific for each of the three V_H families and analyzed by combining the products for each family from an individual ILF, PP, or SP (A). Examination of the usage of three V_H families revealed that the SP, PP, and individual ILF displayed a diverse immunoglobulin repertoire with multiple bands corresponding to unique rearrangements (A). When we examined the usage of individual V_H families by the individual ILFs, SP, and PP, we observed that each tissue preferentially used J558 V_H family members (B). J558 is the largest V_H family in the mouse and is used by ~50% of immunoglobulin gene rearrangements. Preferential usage of this family by all of the individual ILFs examined is most consistent with ILFs containing a population of B lymphocytes reflective of the systemic pool as opposed to ILFs arising from a small number of B lymphocytes that have expanded in situ. The above results are representative of findings from the examination of 20 individual ILFs from multiple mice.