ICAM-1–Dependent Homotypic Aggregates Regulate CD8 T Cell Effector Function and Differentiation during T Cell Activation

Abstract

A hallmark of T cell activation in vitro and in vivo is the clustering of T cells with each other via interaction of the LFA-1 integrin with ICAM-1. The functional significance of these homotypic aggregates in regulating T cell function remains unknown. We used an APC-free in vitro activation system to demonstrate that stimulation of purified naïve CD8 T cells results in enhanced expression of ICAM-1 on T cells that is sustained by the inflammatory cytokine IL-12 and associated with robust T cell aggregates. ICAM-1 deficient CD8 T cells proliferate normally but demonstrate a striking failure to aggregate. Interestingly, loss of ICAM-1 expression results in elevated levels of interferon-γ (IFN-γ) and Granzyme B, as well as enhanced cytotoxicity. Similar results were obtained when anti-LFA-1 antibody was used to block the clustering of wild-type T cells. ICAM-1 ligation is not required for IFN-γ regulation, as clustering of ICAM-1 deficient CD8 T cells with wild-type T cells reduces IFN-γ expression. Analysis using a fluorescent reporter that monitors TCR signal strength indicates that T cell clustering limits T cell exposure to antigen during activation. Furthermore, T cell clustering promotes the upregulation of the CTLA-4 inhibitory receptor and the downregulation of eomesodermin, which controls effector molecule expression. Activation of ICAM-1 deficient CD8 T cells in vivo results in an enhanced percentage of KLRG-1+ T cells indicative of short-lived effectors. These results suggest that T cell clustering represents a mechanism that allows continued proliferation but regulates T cell effector function and differentiation.

Substrate-bound CCL21 and ICAM1 combined with soluble IL-6 collectively augment the expansion of antigen-specific murine CD4+ T cells

“…For example, clustering was shown to limit cytotoxic T-cell effector function and differentiation by limiting the T cells' exposure to antigen during activation, upregulating inhibitory receptors (CTLA-4) and downregulating the expression of effector molecules. 45 In vivo work further supports this duality, and some studies hint at the importance of clusters in T-cell activation and differentiation processes. 3,40 However, ICAM1-mediated interactions, per se, are not obligatory for cell expansion to occur, as ICAM1-deficient T cells, which are unable to aggregate, can still proliferate.…”

“…3,40 However, ICAM1-mediated interactions, per se, are not obligatory for cell expansion to occur, as ICAM1-deficient T cells, which are unable to aggregate, can still proliferate. 45 As CCL21 coating induced the formation of very large, yet shortlived, 3D clusters, we assumed that the conditions within these clusters might be less favorable. Hence, we examined the possibility that coating of the culture substrate with the adhesion molecule ICAM1 might attract LFA1-expressing cells, thereby reducing cell-cell interactions and, as a consequence, massive clustering.…”

Negative Regulation of Memory Phenotype CD8 T Cell Conversion by Adhesion and Degranulation–Promoting Adapter Protein

“…Lymph nodes were harvested from CD45.2 (wild-type) and CD45.1/2 (ADAP −/− ) mice and CD8 mature naïve T cells were isolated by negative magnetic bead enrichment similar to previously described methods (20). Briefly, single-cell suspensions were incubated with the following FITC-conjugated antibodies: CD4, B220, F4/80, CD16/32, I-A b , Ter119, and CD44.…”

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

“…Fluorescence microscopy shows cells, initially seeded at the center of the well, migrate away from the center over time. The formation of alloCTL aggregates after 4 hr (Figure 3, white arrows) is likely reflective of autocrine growth patterns exhibited by activated, IL-2-producing T cell populations 13 .…”