<i>Cd2ap</i>-deficiency enhances T_FH differentiation in a cell-intrinsic manner.

<p>(A) Schematic of experimental strategy for the generation of mixed BM chimeras and subsequent analysis of anti-LCMV responses. (B) Contribution of CD45.2 <i>Cd2ap</i>^F/F or <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F cells to B cells, activated CD4 T cells and T_FH cells in the mixed BM chimeras 22 days after LCMV-c13 infection. Representative Data from 2 independent experiments (n = 4 of each genotype) are shown with means and standard deviations. Statistical difference was tested by Student’s <i>t</i>-test. (C) A volcano plot showing differentially expressed genes between Cd2ap-deficient (KO, n = 2) and -sufficient (WT, n = 2) T_FH cells harvested from mixed BM chimeras 22 days after infection. Genes that were differentially expressed by >2-fold are shown.</p

<i>Cin85</i>-Deficiency results in defective clearance of LCMV-c13 and is correlated with decreased neutralizing antibody titer.

<p>(A, B) Analysis of LCMV abundance in plasma in <i>Cin85</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cin85</i>^F/F mice as determined by LCMV <i>gp</i> transcript levels (A) or focus forming assay (B) at day 80. Horizontal lines indicate median. The limit of detection is shown by dashed lines. Statistical significance was tested by Mann Whitney U-test. (C) Frequencies of Fas⁺ GL7⁺ B220⁺ GC B cells at day 35 after LCMV-c13 infection. (D) anti-LCMV IgG antibody titers of plasma from <i>Cin85</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cin85</i>^F/F mice 30 days after LCMV-c13 infection. (E) Neutralizing activity of plasma from <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F mice 80 days after LCMV-c13 infection. Reduction in focus forming units (FFU) in the presence of 1:5 dilution of plasma compared to untreated control (% Neutralization) is shown with mean and standard deviation. Student <i>t</i>-test. Data combined from 3 independent experiments are shown with n = 3–4 mice per genotype.</p

The adaptor molecule CD2AP in CD4 T cells modulates differentiation of follicular helper T cells during chronic LCMV infection

<div><p>CD4 T cell-mediated help to CD8 T cells and B cells is a critical arm of the adaptive immune system required for control of pathogen infection. CD4 T cells express cytokines and co-stimulatory molecules that support a sustained CD8 T cell response and also enhance generation of protective antibody by germinal center B cells. However, the molecular components that modulate CD4 T cell functions in response to viral infection or vaccine are incompletely understood. Here we demonstrate that inactivation of the signaling adaptor CD2-associated protein (CD2AP) promotes CD4 T cell differentiation towards the follicular helper lineage, leading to enhanced control of viral infection by augmented germinal center response in chronic lymphocytic choriomeningitis virus (LCMV) infection. The enhanced follicular helper differentiation is associated with extended duration of TCR signaling and enhanced cytokine production of CD2AP-deficient CD4 T cells specifically under T_H1 conditions, while neither prolonged TCR signaling nor enhanced follicular helper differentiation was observed under conditions that induce other helper effector subsets. Despite the structural similarity between CD2AP and the closely related adaptor protein CIN85, we observed defective antibody-mediated control of chronic LCMV infection in mice lacking CIN85 in T cells, suggesting non-overlapping and potentially antagonistic roles for CD2AP and CIN85. These results suggest that tuning of TCR signaling by targeting CD2AP improves protective antibody responses in viral infection.</p></div

<i>Cd2ap</i> deficiency has a minimal impact on T_FH differentiation and GC B cell responses following immunization with SRBCs.

<p>(A, B) Flow cytometric analysis of expression of PD-1 and CXCR5 on pre-gated CD4⁺ B220⁻ T cells (A) and GL7 and Fas expression on CD19⁺ B220⁺ B Cells (B) 12 days following SRBC immunization. (C-E) Numbers and frequencies of total CD4 T cells and CXCR5⁺ PD-1⁺ T_FH in the spleen of <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F and control <i>Cd2ap</i>^F/F mice 12 days after immunization with SRBCs. (F-H) Numbers and frequencies of total B cells and Fas⁺ GL7⁺ GC B cells in the spleen of <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F and control <i>Cd2ap</i>^F/F mice 12 days after immunization with SRBCs. Data are pooled from 2 independent experiments shown as means and standard deviation.</p

Enhanced control of LCMV-c13 infection in <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F is associated with elevated T_FH response.

<p>(A) Analysis of LCMV abundance in plasma in mice as determined by LCMV <i>gp</i> transcript levels. Horizontal bars indicate medians. The limit of detection is shown by a dashed line. Statistical significance was tested by Mann Whitney U-test. (B-E) Expression of B220, GL7, Fas, CD4, CD44, PD-1 and CXCR5 and binding of I-A^b (gp66-77) tetramer of splenocytes from <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F mice 22 days after LCMV-c13 infection. Representative plots are shown with percentages of gated cells. Statistical analyses from at least 7–10 mice per genotype in 2 independent experiments are shown with means and standard deviation in (D) and (E). (F) (Left) Neutralizing activity and (Right) anti-LCMV IgG2c antibody titers of plasma from <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F mice 60 days after LCMV-c13 infection. Reduction in focus forming units (FFU) in the presence of 1:10 dilution of plasma compared to untreated control (%Neutralization) is shown with mean and standard deviation. <i>Cd2ap</i>^F/F: n = 7, <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F: n = 9.</p

<i>Cd2ap</i>-deficiency causes sustained TCR signaling specifically in T_H1 cells <i>in vitro</i>.

<p>(A) Schematic of experimental strategy for culture of CD4 T cells. (B) Concentration of IFN-γ or IL-4 in supernatant measured by ELISA 24 hours after re-stimulation of polarized <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T_H1 cells and T_H2 cells with plate-bound anti-CD3/anti-CD28 antibodies. (C) Immunoblotting showing phosphorylation of MEK1/2 in polarized <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T_H1 or T_H2 cells following re-stimulation with plate-bound anti-CD3/anti-CD28 for the indicated times at 37°C. Anti-CD2AP, -CIN85 and ERK were used as control. Data are representative of three experiments. (D) Downregulation of surface TCR of <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T cells that were polarized under indicated conditions. Cells were stimulated with plate-bound anti-CD3 and surface TCR levels were quantitated at the indicated time points. Data are representative of three experiments. (E, F) Intracellular staining for IFN-γ and TNF-α of polarized <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T_H1 cells after re-stimulation with PMA and Ionomycin or plate-bound anti-CD3 and anti-CD28 for 4 or 24 hours in the presence of Brefeldin A for the last 2 hours before harvest. Representative plots (E) and statistical analyses with means and standard deviations (F) from three experiments are shown.</p

Enhanced T_FH and GC B cell responses in T cell-specific <i>Cd2ap</i>-deficient mice in response to acute viral infection.

<p>(A) Flow cytometric analysis of expression of CD4, CD8 and CD44 and binding of H-2D^b(gp33-41) tetramer in splenocytes 8 days after LCMV-Armstrong infection. Total CD8 T cells and LCMV-gp33-specific CD8 T cells are shown with rectangular gates. (B) Expression of CD8 and KLRG1 in splenocytes 8 days after LCMV-Armstrong infection. (C) Expression of CD44 and binding of I-A^b(gp66-77) tetramer in CD4⁺ splenic T cells 8 days after LCMV-Armstrong infection. LCMV-gp66-specific CD4 T cells are shown with rectangular gates. (E, G) Expression of PD-1, CXCR5 and Ly6C and binding of I-A^b (gp66-77)⁺ tetramer of CD4⁺ T cells and Fas and GL7 expression in B cells in the spleens of <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F and control <i>Cd2ap</i>^F/F mice 8 (E) and 22 (G) days after infection. T_FH cells and GC B cells are shown with rectangular gates. Representative plots are shown with percentages of gated cells. Statistical analyses from 4–6 mice in 2 independent experiments are shown with means and standard deviation in (D, F, H).</p