Co-engagement of alpha(4)beta(1) integrin (VLA-4) and CD4 or CD8 is necessary to induce maximal Erk1/2 phosphorylation and cytokine production in human T cells

Abstract

The alpha(4)beta(1) integrin VLA-4 (very-late activation antigen-4) and the lineage-specific CD4 and CD8 receptors have been proposed as putative co-stimulatory receptors on T cells. To assess the relative contribution of signaling through the TCR, CD28 and these accessory molecules, we activated human T cells using soluble antibodies recognizing all four of these T-cell receptor classes (CD3, CD28, CD4/CD8, and VLA-4), and we assessed the degree of activation using higher-order flow cytometry detecting intracellular Erk1/2 phosphorylation and production of IL-2 and IFN-gamma. We found that: (1) co-stimulation via CD4/CD8, in addition to CD28, is required for optimal T-cell activation; (2) VLA-4 binding consistently potentiates CD4(+) and CD8(+) T-cell activation; (3) augmentation of T-cell activation through VLA-4 binding is most pronounced following engagement of CD4/CD8. These results confirm that multiple signals, including VLA-4 engagement, are necessary for maximal T-cell activation beyond that induced via the TCR and CD28.

Figure 1. Co-engagement of α₄β₁ integrin and CD4/CD8 results in maximal Erk1/2 phosphorylation

(a) Representative Erk1/2 phosphorylation in CD4+ and CD8+ T cells from a single subject, demonstrating augmented activation following CD3/D28 stimulation following co-engagement of VLA-4 and CD4 or CD8. (b) Scatter plots demonstrating the synergistic effect of α₄β₁ integrin and CD4/CD8 co-engagement on intracellular Erk1/2 phosphorylation in CD4+ and CD8+ T cells from six healthy donors. Intergroup comparisons were performed using the Wilcoxon signed-rank test. All P values were two-tailed and considered significant if less than 0.05 (filled black box, lower panel). Positive trends (P<0.1) are also indicated (filled gray box, lower panel).

Figure 2. Co-engagement of α₄β₁ integrin and CD4/CD8 results in maximal CD4+ and CD8+ T cell cytokine production

(a) Representative IL-2/IFNγ production in CD4+ and CD8+ T cells from a single subject, demonstrating augmented activation following CD3/CD28 stimulation following co-engagement of VLA-4 and CD4 or CD8. (b) Scatter plots demonstrating the synergistic effect of α₄β₁ integrin and CD4/CD8 co-engagement on IL-2 and IFNγ production in CD4+ and CD8+ T cells from six healthy donors. Intergroup comparisons were performed using the Wilcoxon signed-rank test. All P values were two-tailed and considered significant if less than 0.05 (filled black box, lower panel). Positive trends (P<0.1) are also indicated (filled gray box, lower panel).

Figure 3. Co-engagement of α₄β₁ integrin augments Erk1/2 phosphorylation and the production of IL-2 in memory T cells

(a) Representative CD45RA/CD27 and α₄β₁ integrin VLA-4 staining in CD4 T cells. CD45RA/CD27 delineates CD4 T cell maturation subsets (naïve (N): CD45RA+CD27+, early memory (M1): CD45RA−CD27+, intermediate memory (M2): CD45RA−CD27−) (left). The expression of α₄β₁ integrin in memory CD4 T cell subsets (M1, M2) is higher than naïve subset (I: isotype control (black dot), N: naïve (thin black), M1: early memory (thick gray), M2: intermediate memory (thick black)) (right). (b) Representative Erk1/2 phosphorylation (left) and IL-2/IFNγ production (right) in CD4+ T cell maturation subsets from a single subject, demonstrating augmented activation in memory CD4+ T cell subsets (M1, M2) following CD3/CD28/CD4 stimulation following co-engagement of VLA-4. For the most analyses, at least 300,000 total events were acquired, with sequential gating of PBMC in a lymphocyte region by scatter, on CD4+ T cells and assessment of intracellular p-Erk1/2 and IL-2 & IFNγ within three naïve and memory T cell maturation subsets demarcated by CD45RA and CD27 expression. 2-D dot plots reflect p-Erk1/2 on side scatter and IL-2 vs. IFNγ staining. (c) Scatter plots demonstrating the synergistic effect of α₄β₁ integrin on intracellular Erk1/2 phosphorylation and IL-2/IFNγ production in CD4+ T cells from six healthy donors. Intergroup comparisons were performed using the Wilcoxon signed-rank test. N.S.: not significant, * P<0.05.