Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion

Abstract

Although a fraction of human blood memory CD4(+) T cells expresses chemokine (C-X-C motif) receptor 5 (CXCR5), their relationship to T follicular helper (Tfh) cells is not well established. Here we show that human blood CXCR5(+)CD4(+) T cells share functional properties with Tfh cells and appear to represent their circulating memory compartment. Blood CXCR5(+)CD4(+) T cells comprised three subsets: T helper 1 (Th1), Th2, and Th17 cells. Th2 and Th17 cells within CXCR5(+), but not within CXCR5(-), compartment efficiently induced naive B cells to produce immunoglobulins via interleukin-21 (IL-21). In contrast, Th1 cells from both CXCR5(+) and CXCR5(-) compartments lacked the capacity to help B cells. Patients with juvenile dermatomyositis, a systemic autoimmune disease, displayed a profound skewing of blood CXCR5(+) Th cell subsets toward Th2 and Th17 cells. Importantly, the skewing of subsets correlated with disease activity and frequency of blood plasmablasts. Collectively, our study suggests that an altered balance of Tfh cell subsets contributes to human autoimmunity.

Figure 1. Blood CXCR5⁺ CD4⁺ T cells induce naïve B cells to differentiate into Ig-producing plasmablasts

A. CXCR5 expression by blood CD4⁺ T cells. PBMCs were stained with CD3, CD4, CD45RA, and CXCR5 mAbs. Gated to CD3⁺CD4⁺ T cells. B. Blood naive, CXCR5⁻, and CXCR5⁺ CD4⁺ T cells were cultured with autologous naïve B cells in the presence of SEB. Ig concentrations were measured at day 12. One way ANOVA test. Data from seven independent experiments. ** p<0.01, *** p<0.001. C-D. CXCR5⁻ or CXCR5⁺ CD4⁺ T cells were cultured with naïve (C) or memory (D) B cells in the presence of titrated doses of SEB. Ig concentrations at day 12 in (C) and day 6 in (D). n=3, Mean ± s.d. Representative data from three independent experiments. E. Ig concentrations at different time points in the cultures of CXCR5⁻ or CXCR5⁺ CD4⁺ T cells with naïve B cells. n=3, Mean ± s.d. Representative data from four independent experiments. F. Number of viable CD4⁺ T and B cells at different time points. Representative data from two independent experiments. G. Number of viable CD4⁺ T cells at day 8. Paired t-test. Data from three independent experiments. H. CD38⁺ plasmablast population in the co-culture of blood Th subsets and naive B cells at day 8. Representative data from three independent experiments. I. Number of plasmablasts in the co-cultures. One way ANOVA test. Data from three independent experiments. J. Measurement of BCL6, PRDM1, and AICDA mRNA expression by real-time RT-PCR in naïve B cells cultured with blood Th subsets. Expression of each mRNA was normalized to that of ACTB mRNA. Before and after 7 d culture with CXCR5⁻ or CXCR5⁺ CD4⁺ T cell subsets (B cells were purified after culture). Representative data from two independent experiments.

Figure 2. Blood CXCR5⁺ CD4⁺ T cells depends on IL-21, IL-10 and ICOS for B cell help

A. IL-21 secretion by blood Th subsets cultured with naïve B cells. Data from six independent experiments. One way ANOVA test. ** p<0.01 B. CXCL13 secretion by blood Th subsets cultured with naïve B cells. Mean ± s.d, n=3. Representative data from three independent experiments. C. Titrated amounts of IL-21R-Fc were added to the co-culture of CXCR5⁺ CD4⁺ T cells and naïve B cells. Ig concentrations at day 12. IgG is not shown due to the cross-reactivity to the Fc portion of IL-21R-Fc. Representative data from two independent experiments. D. Recovery of viable B cells at day 12. Normalized to the culture of CXCR5⁺ CD4⁺ T cells and naïve B cells. One way ANOVA test, n=4. *** p<0.001. E. Titrated amounts of IL-21 were added to the co-culture of naïve or CXCR5⁻ CD4⁺ T cells with naïve B cells. Ig concentrations at day 12. n=3, Mean ± s.d. Representative data from three independent experiments. F. Ig concentrations at day 12 in the co-culture of CXCR5⁺ CD4⁺ T cells and naïve B cells with an ICOS blocking reagent. Mean ± s.d, n=3. Representative data from two independent experiments. G. IL-10 secretion in supernatants of blood Th subsets cultured with naïve B cells. Data from four independent experiments. One way ANOVA test. * p<0.05, ** p<0.01. H. Ig concentrations at day 12 in the co-culture of CXCR5⁺ CD4⁺ T cells and naïve B cells with indicated amounts of anti-IL-10. Mean ± s.d., n=3. Representative data from three independent experiments. I. CFSE-labeled blood Th subsets were cultured with autologous monocytes incubated with inactivated Flu virus or CMV. Cell proliferation was analyzed at day 5. Representative data from two independent experiments. J. IL-2 and IFN-γ secretion in supernatants at day 2. Data from five independent experiments. Paired t-test. *** p<0.001, * p<0.05. K. PBMCs were stimulated with none, Flu vaccine, inactivated Flu virus, or SEB for 6 h in the presence of Brefeldin A and monensin, and the intracytoplasmic expression of CD154 in CXCR5⁺ or CXCR5⁻ CD4⁺ T cells was analyzed. Representative data from four independent experiments.

Figure 3. Blood CXCR5⁺ CD4⁺ T cells are composed of Th1, Th2, and Th17 cells

A. CXCR3 and CCR6 expression on blood CXCR5⁻ or CXCR5⁺ CD4⁺ T cell population. Gated to CD3⁺CD4⁺CD45RA⁻ cells. B. Frequency of populations within blood CXCR5⁻ and CXCR5⁺ CD4⁺ T cells of ten healthy adults. C. The seven blood Th populations were co-cultured with naïve B cells and the secreted IL-21 was measured at 48 h. n=3, Mean ± s.d. Representative data from four independent experiments. D. Other cytokine secretion in the co-cultures of the seven blood Th populations and naïve B cells. n=3, Mean ± s.d. Representative data from four independent experiments. E. Expression of each transcriptional factor in the seven blood Th populations was assessed by RT-PCR. Representative data from two independent experiments. F. The seven Th populations were sorted from PBMCs of three donors, and expression of BCL6 and PRDM1 mRNA was analyzed by real-time RT-PCR. Normalized to ACTB mRNA expression in each Th subset.

Figure 4. CXCR5⁺ Th2 and Th17 cells efficiently help naïve B cells

A. Ig secretion by naïve B cells co-cultured with the seven blood Th populations for 12 d. n=3-4, Mean ± s.d. Representative data from three independent experiments. B. Ig secretion from naïve B cells co-cultured with blood CXCR5⁺ Th subsets. Data of three independent experiments. One way ANOVA test. * p<0.05, ** p<0.01. C. Number of viable CD4⁺ T cells at day 8. n=3-4, Mean ± s.d. Representative data from two independent experiments. D. Number of viable B cells and plasmablasts at day 8. n=3-4, Mean ± s.d. Representative data from two independent experiments. E. IgA1 and IgA2 production. Data from three independent experiments. Student’s t-test. * p<0.05. F. Addition of IL-21R-Fc chimera protein to the co-cultures of naïve B cells and CXCR5⁺ Th2 or Th17 cells. n=3, Mean ± s.d. Student’s t-test. * p<0.05, ** p<0.01, *** p<0.001. Representative data from three independent experiments. G. Addition of IL-4 blocking antibody to the culture of CXCR5⁺ Th2 cells and naïve B cells. n=3, Mean ± s.d. Student’s t-test. ** p<0.01. Representative data from two independent experiments.

Figure 5. Blood CXCR5⁺ Th subsets are altered in JDM

A. Percentage of CXCR5⁺ cells within CD4⁺ T cells in samples from JDM patients (n=52), PSOA patients (n=31), and age-matched healthy controls (n=43). B. Percentage of each Th subset within blood CXCR5⁺ CD4⁺ T cells. One way ANOVA test, ** p<0.01, *** p<0.001. C. Ratio of CXCR5⁺ (Th2 + Th17) / Th1 cells. One way ANOVA test. D. Frequency of CXCR5⁺ Th1 cells and ratio of (Th2+Th17) / Th1 in JDM patients with different disease activities. One way ANOVA test. E. Frequency of CXCR5⁺ Th1 cells and ratio of (Th2+Th17) / Th1 in active JDM patients receiving none, intravenous corticosteroids or high-dose Ig treatments. F. The absolute cell numbers in blood were calculated based on the complete blood cell count, lymphocyte frequency within white blood cells, and the frequency of CXCR5⁺ Th subsets within the lymphocyte population. Student’s t-test.

Figure 6. Skewing in blood CXCR5⁺ Th subsets correlates with B cell alteration

A. The absolute number of plasmablasts in blood of JDM patients, PSOA patients, and healthy controls (left), and in JDM patients with different disease activities (right). One way ANOVA test. B. Percentage of plasmablast within total CD19⁺ B cells. C. The absolute number of plasmablasts in blood of active JDM patients receiving none, intravenous corticosteroids or high-dose Ig treatments. D. Correlation between the percentage of plasmablasts within CD19⁺ B cells and the ratio of CXCR5⁺ (Th2+Th17) / Th1 cells (left) or the frequency of CXCR5⁺ Th1 cells (right) in JDM. Pearson correlation coefficient and two-tailed p-value are shown. E. Correlation between the percentage of plasmablasts within CD19⁺ B cells and the frequency of total CXCR5⁺ CD4⁺ T cells (left) or of ICOS⁺ CXCR5⁺ CD4⁺ T cells (right). F. Correlation between the percentage of plasmablasts and the ratio of CXCR5⁻ (Th2+Th17) / Th1 (left) or the frequency of CXCR5⁻ Th1 cells (right) in active JDM patients.