Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells

Abstract

The T helper (Th) cell pool is composed of specialized cells with heterogeneous effector functions. Apart from Th1 and 2 cells, CXCR5+ T cells have been suggested to be another type of effector T cell specialized for B cell help. We show here that CXCR5+ T cells are heterogeneous, and we identify subsets of CXCR5+ CD4 T cells that differ in function and microenvironmental localization in secondary lymphoid tissues. CD57+CXCR5 T cells, hereafter termed germinal center Th (GC-Th) cells, are localized only in GCs, lack CCR7, and are highly responsive to the follicular chemokine B lymphocyte chemoattractant but not to the T cell zone EBI1-ligand chemokine. Importantly, GC-Th cells are much more efficient than CD57-CXCR5+ T cells or CXCR5- T cells in inducing antibody production from B cells. Consistent with their function, GC-Th cells produce elevated levels of interleukin 10 upon stimulation which, with other cytokines and costimulatory molecules, may help confer their B cell helper activity. Our results demonstrate that CXCR5+ T cells are functionally heterogeneous and that the GC-Th cells, a small subset of CXCR5+ T cells, are the key helpers for B cell differentiation and antibody production in lymphoid tissues.

Figure 1

Identification of CD57⁺ CXCR5⁺ T cells and their localization in GCs. (A) Flow analyses of CD57 and CXCR5 expression on tonsil CD4 T cells. (B) Specific localization of CD57⁺ CD4 T cells in GCs. (C) Localization of CD57⁺ CXCR5⁺ CD4 T cells in GCs. Antibodies to CD57 (green), CD4 (blue), and IgD (red; B) or CXCR5 (red; C) were used for in situ immunohistochemistry.

Figure 1

Identification of CD57⁺ CXCR5⁺ T cells and their localization in GCs. (A) Flow analyses of CD57 and CXCR5 expression on tonsil CD4 T cells. (B) Specific localization of CD57⁺ CD4 T cells in GCs. (C) Localization of CD57⁺ CXCR5⁺ CD4 T cells in GCs. Antibodies to CD57 (green), CD4 (blue), and IgD (red; B) or CXCR5 (red; C) were used for in situ immunohistochemistry.

Figure 2

Chemotactic responses (A) and chemokine receptor expression (B) by CD57^+/2CXCR5^+/− CD4 T cells. Optimal concentrations of 5 μg/ml BLC, 1 μg/ml ELC, and 100 ng/ml SDF-1 were used for chemotaxis experiments. Freshly isolated tonsil cells were used for chemotaxis and flow analyses of tonsil CD4 T cell subsets. Representatives of three independent experiments are shown.

Figure 2

Chemotactic responses (A) and chemokine receptor expression (B) by CD57^+/2CXCR5^+/− CD4 T cells. Optimal concentrations of 5 μg/ml BLC, 1 μg/ml ELC, and 100 ng/ml SDF-1 were used for chemotaxis experiments. Freshly isolated tonsil cells were used for chemotaxis and flow analyses of tonsil CD4 T cell subsets. Representatives of three independent experiments are shown.

Figure 3

Effector machinery of CD57^+/−CD45RA^+/− or CD57^+/− CXCR5^+/− CD4 T cells. (A) Intracellular cytokine analyses of TNF-α, IL-2, IL-10, IFN-γ, and IL-4. (B) ELISA of IL-10 and IL-4. Isolated T cells were activated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h for intracellular cytokine analyses, or 24 h for ELISA of IL-10 and IL-4. Representatives of at least four independent experiments are shown.

Figure 3

Effector machinery of CD57^+/−CD45RA^+/− or CD57^+/− CXCR5^+/− CD4 T cells. (A) Intracellular cytokine analyses of TNF-α, IL-2, IL-10, IFN-γ, and IL-4. (B) ELISA of IL-10 and IL-4. Isolated T cells were activated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h for intracellular cytokine analyses, or 24 h for ELISA of IL-10 and IL-4. Representatives of at least four independent experiments are shown.

Figure 4

Spontaneous B cell helper activity of CD57^+/− CXCR5^+/− CD4 T cells. Sorted CD57⁺CXCR5⁺, CD57⁻ CXCR5⁺, and CXCR5⁻ CD4 T cells were cocultured with B cells from the same tonsil for 11–13 d in the absence of any stimulatory agents followed by analyses of secreted IgG, IgA, and IgM. Representatives of at least three independent experiments are shown.

Figure 5

Phenotype and effector function of circulating CXCR5⁺ T cells. (A) Surface phenotype of circulating naive or CXCR5^+/− memory T cells. **Significant differences between CXCR5⁺ and CXCR5⁻ memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5^+/− T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5⁺ T cells after T cell receptor activation. Naive (IgD⁺) or memory (IgD⁻) B cells (2 × 10⁴) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10³, 5 × 10³, 10⁴, 2.5 × 10⁴, 5 × 10⁴, and 10⁵) of autologous T cells (CXCR5⁻CD45RA⁺, CXCR5⁺CD45RA⁻, or CXCR5⁻ CD45RA⁻). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Figure 5

Phenotype and effector function of circulating CXCR5⁺ T cells. (A) Surface phenotype of circulating naive or CXCR5^+/− memory T cells. **Significant differences between CXCR5⁺ and CXCR5⁻ memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5^+/− T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5⁺ T cells after T cell receptor activation. Naive (IgD⁺) or memory (IgD⁻) B cells (2 × 10⁴) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10³, 5 × 10³, 10⁴, 2.5 × 10⁴, 5 × 10⁴, and 10⁵) of autologous T cells (CXCR5⁻CD45RA⁺, CXCR5⁺CD45RA⁻, or CXCR5⁻ CD45RA⁻). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Figure 5

Phenotype and effector function of circulating CXCR5⁺ T cells. (A) Surface phenotype of circulating naive or CXCR5^+/− memory T cells. **Significant differences between CXCR5⁺ and CXCR5⁻ memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5^+/− T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5⁺ T cells after T cell receptor activation. Naive (IgD⁺) or memory (IgD⁻) B cells (2 × 10⁴) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10³, 5 × 10³, 10⁴, 2.5 × 10⁴, 5 × 10⁴, and 10⁵) of autologous T cells (CXCR5⁻CD45RA⁺, CXCR5⁺CD45RA⁻, or CXCR5⁻ CD45RA⁻). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Figure 5

Phenotype and effector function of circulating CXCR5⁺ T cells. (A) Surface phenotype of circulating naive or CXCR5^+/− memory T cells. **Significant differences between CXCR5⁺ and CXCR5⁻ memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5^+/− T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5⁺ T cells after T cell receptor activation. Naive (IgD⁺) or memory (IgD⁻) B cells (2 × 10⁴) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10³, 5 × 10³, 10⁴, 2.5 × 10⁴, 5 × 10⁴, and 10⁵) of autologous T cells (CXCR5⁻CD45RA⁺, CXCR5⁺CD45RA⁻, or CXCR5⁻ CD45RA⁻). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.