Fig 1: Loss of intestinal goblet cells or inhibition of GAPs results in expansion of plasma cells in small intestinal lamina propria.(A-B) Bulk RNAseq was performed on RNA isolated from small intestinal tissue from mAChr4f/f and mAChr4f/f Math1Cre*PR mice treated with RU486 to activate Cre for seven days. (A) Graph shows cumulative gene enrichment score upregulated in mAChr4f/f Math1Cre*PR mice (positive enrichment score) compared with mAChr4f/f mice (negative enrichment score). (B) Volcano plot depicting DEGs. Yellow dots represent significantly upregulated genes in mAChr4f/f Math1Cre*PR mice and blue dots represent significantly upregulated genes in mAChr4f/f mice. (C) Graph depicts small intestinal IgA-secreting cells measured by ELISpot assay in mAChr4f/f and mAChr4f/f Math1Cre*PR mice. (D) Small intestinal IgA-secreting cells measured by ELISpot assay in Math1f/f and Math1f/f Vil-Cre-ERT2 mice treated with tamoxifen to induce goblet cell deletion. (E) Small intestinal IgA-secreting cells measured by ELISpot assay in C57BL/6 mice gavaged with PBS or recombinant EGF for seven days. (F) ELISA analysis of IgA concentration in small intestinal lamina propria culture for 3 days. (G) Graph depicts relative expression of PIGR in small intestinal tissue. (H) Concentration of BAFF was determined in tissue homogenate. (C-G) Data pooled from 2–3 independent experiments. Statistics were calculated by unpaired t-test. *p< 0.05, **p<0.01.
Fig 2: Bone marrow B cell mobilization following alteration of intestinal GAPs.(A-D) Bone marrow (B220+), (B220+ IgM+ IgD−, immature B cells) and (B220+ IgM+ IgD+, mature B cells) subsets were quantified following inhibition of GAPs in mAChR4fl/fl Math1Cre*PR mice and littermate controls. (E) CXCL12 was measured in the bone marrow extracellular fluid from single femur bone per mouse. (F) Expression level of CXCR4 on bone marrow B220+ cells was determined by flow cytometry. (G) Frequency and absolute numbers of mature neutrophils (Gr1+ CD11bhi) were measured in bone marrow of mAChR4fl/fl Math1Cre*PR mice and littermate controls. Data pooled from 2–3 independent experiments. Statistics were calculated by unpaired t-test. *p<0.05.
Fig 3: Enteric infection promotes intestinal IgA+ plasma cell accumulation and bone marrow B cell mobilization.(A) Graph depicts small intestinal IgA-secreting cells measured by ELISpot assay in C57BL/6 mice infected orally with Salmonella for 4 days or in uninfected mice treated with PBS. (B) IgA concentration were measured by ELISA in the small intestinal luminal content and in the serum (C) of these mice. (D) ELISA data from Fecal content of Salmonella infected mice. (E) J-chain levels were measured using ELISA in the cultures of small intestinal immune cells from infected and uninfected mice. (F) Relative expression of PIGR and TSLP was determined by RT-PCR on small intestinal tissue. (G) Flow cytometry analysis of ICOS staining among non-Tfh CD4+ CD45+ cells in Salmonella infection and uninfected mice. (H) Bone marrow B220+ cells were assessed following 4 days of Salmonella infection. Bone marrow subsets pre-B cell (B220+ IgM− IgD−), immature B cells (B220+ IgM+ IgD−) and mature B cells (B220+ IgM+ IgD+) were quantified in Salmonella infected and uninfected mice. Expression level of CXCR4 on bone marrow B220+ cells was determined by flow cytometry measurement. (I) Frequency and absolute numbers of mature neutrophils (Gr1+ CD11bhi) were measured in bone marrow of Frequency and absolute numbers of mature neutrophils (Gr1+ CD11bhi) were measured in bone marrow of Salmonella infected and uninfected mice (J). Data pooled from 2–3 independent experiments. Statistics were calculated by unpaired t-test. *p<0.05, **p<0.01.
Fig 4: PD-1CAR Tregs are activated by PD-1+ target cells and secrete IL-10 and TGF-β1, but not IFN-γ and IL-2(A) Bar graph summarizing Ki67 expression in 2D2 PD-1KO cells and 2D2 cell line-derived PD-1CAR Tregs with and without the stimulation of PD-1+ cells at a 1:1 ratio for three days.(B) Bar graph summarizing Ki67 expression in primary parent cells and PD-1CAR Tregs with and without stimulation PD-1+ cells at a 1:1 ratio for three days.(C–E and G) Bar graph summarizes cytokine secretion by primary parent cells and PD-1CAR Tregs with and without PD-1+ cell stimulation (1:1) for three days. (C) IL-10, (D) TGF-β1, (E) IFN-γ, and (G) IL-2.(F) Flow cytometry dot plots show IFN-γ expression in primary PD-1CAR Tregs activated by PD-1+ cells at a 1:1 ratio for three days, compared to parent cells under Th1 polarization conditions as a control.Data of (A–E and G) are mean ± SEM, n = 4; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, as determined by Student’s two-tailed t test.
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