Cognate T and B cell interaction and association of follicular helper T cells with B cell responses in Vibrio cholerae O1 infected Bangladeshi adults

Abstract

Vibrio cholerae O1 can cause life threatening diarrheal disease if left untreated. T cells can play critical roles in inducing B cell mediated immunity. As the mechanism of T cell dependent B cell maturation is not well established, we hypothesized that a specific population of T (follicular helper T, Tfh) cells, are involved in B cell maturation following cholera. We found flowcytometrically that V. cholerae infection induces significant increases in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to V. cholerae membrane preparation (MP) and V. cholerae cytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstrated in vitro that Tfh cells isolated after cholera can stimulate class switching of co-cultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype colorimetrically. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in gut mucosa of biopsied samples, at the single cell level if feasible.

Keywords: B cells; CD40L; CXCR5; Cholera; Follicular helper T cells.

FIG. 1.. Frequency of Tfh cells and CD40L expressing Tfh cells after onset of cholera.

(A) Frequency of Tfh cells (CD3+CD4+CXCR5+) and (B) frequency of CD40L-expressing Tfh cells (CD3+CD4+CXCR5+CD40L+) plotted as a percentage of total CD4+ cells in peripheral blood at day 2 (D2), day 7 (D7) and day 30 (D30) following onset of cholera (n=20). Data are presented as the mean percentage of Tfh cells and the CD40L+ Tfh cells, with error bars representing standard error of the mean. An asterisk represents a significant increase from day 2 or decrease from day 7 (* <0.05, **<0.01, ***<0.001) using a paired t-test.

FIG. 2.. Antigen-specific Tfh cell proliferation and B cell maturity associated protein CD40L expression after cholera.

(A) Evaluation of antigen-specific proliferation (n=18) of Tfh cells and (B) CD40L-expressing Tfh cells after cholera; cells were stimulated with G33D mutant cholera toxin (mCT), V. cholerae membrane preparation (MP), and V. cholerae cytolysin (VCC). Data are presented as mean stimulation indices (SI) of Tfh cells and CD40L expressing Tfh cells, with error bars representing standard errors of the mean. Asterisks represent a P-value less than or equal to 0.05 and represent a significant increase from day 2.

FIG. 3.. Antigen-specific Tfh cells and CD40L-expressing Tfh cells in the circulation following cholera.

Correlation of day 7 circulating antigen-specific Tfh and CD40L-expressing Tfh cells with subsequent antigen-specific memory B cells at day 30 in the same patients (n=10). The stimulation index (SI) of Tfh cells (CXCR5+) or CD40L-expressing Tfh cells were analyzed for correlation with rCTB-specific IgG memory B cells (A, B), and with rCTB-specific IgA memory B cells (C, D). Correlation was determined by the Pearson correlation coefficient.

FIG. 4.. Depletion of memory Tfh cells and co-culture of cells recovered from peripheral blood of cholera patients.

In vitro co-culture of Tfh cells or Tfh depleted T helper cells with B cells after 7 days, with antigenic stimulation (MP, mCT, or SEB) and a media only control (n=11). Data are presented as the mean concentration of IgG in ng/ml of culture supernatant, with error bars representing the standard errors of the mean. B, Tfh+ and Tfh- cells are defined as CD19+ cells, CD3+CD4+CD45R0+CXCR5+ cells and CD3+CD4+CD45R0+CXCR5- cells, respectively. An asterisk represents a significant decrease of IgG after depletion of Tfh compared to nondepleted Tfh cells (* <0.05, **<0.01).