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Thursday, October 3, 2013

Why do activated T cells cluster?


Cytotoxic CD8 T cells are highly motile lymphocytes that seek out and destroy specific targets in the immune response.  Upon activation by antigen-binding, T cells aggregate together, forming clusters, but the functional significance of this clustering remains unclear.

Why do T cells aggregate?  CD8 T cell activation involves recognition of an antigen displayed on MHC class I molecules by professional APCs.  Interaction of CD8 T cell with Ag-bound APC is mediated by LFA-1, a molecule expressed on T cells that binds to ICAM-1 on APCs.  The LFA-1/ICAM-1 adhesion facilitates T cell activation by promoting the joining of T cells and APCs, as well as initiating intracellular signaling that enhances TCR-mediated signals to promote T cell proliferation and differentiation.  Because T cells express both LFA-1 and ICAM-1, the interaction between these two receptors also mediates aggregation of activated T cells!  Studies have shown that this aggregation may regulate T cell proliferation and differentiation, but the functional significance of Ag-dependent clustering of T cells remains unknown.

A recent study conducted by Zumwalde et al. (2013) addressed activated T cell aggregation, and was described in Edition 191 of The Journal of Immunology.  In this study, authors used an in vitro APC-independent CD8 T cell stimulation system to investigate the functional significance of aggregate formation during T cell activation.  A line of ICAM-1-deficient mice were bred, and conjugated fluorescent Abs were used for labeling.  Naïve eild-type and ICAM-deficient CD8 T cells were purified by negative selection.  T cells were blocked in vitro from clustering using endotoxin-free anti-LFA-1 Abs.  Flow cytometry and intracellular staining techniques as well as microscopy techniques were used to examine T cells.


Important experimental results of this study are as follows:

  •      The stimulation of purified naïve CD8 T cells resulted in enhanced expression of ICAM-1 on T cells, which was sustained by the inflammatory cytokine, IL-12. 

  •      ICAM-1 deficient T cells proliferated normally but failed to aggregate following stimulation with Ag, B7-1, and IL-12.  Compared to wild type T cells, ICAM-1-deficient cells displayed enhanced expression of both IFN-y and granzyme B, and enhanced cytotoxicity. Furthermore, inhibition of clustering of wild type T cells during activation with inhibitory anti-LFA-1 resulted in increased expression of IFN-y and granzyme B. 

  •      Disruptions of T cell clustering was shown to enhance CD8 T cell effector functions by enhancing T cell ability to kill target cells compared with control wild type T cells.  These results suggest that ICAM-1 mediated clustering negatively regulates CD8 T cell effector molecule production.  Clustering of ICAM deficient CD8 T cells with wild type T cells reduces IFN-y expression

  •      A fluorescent reporter that marked TCR strength indicated that T cell clustering limits T cell exposure to Ag during activation.  Furthermore, T cell clustering upregulates CTLA-4 inhibitory receptor and downregulates eomesodermin, which controls effector molecule expression.

  •      Activation of ICAM-1-deficient T cells enhanced the amount of KLRG-1 T cells, which are indicative of short-lived effecter molecules


These results show that T cell clustering represents a mechanism that allows continued proliferation while regulating T cell effector function and differentiation.

This study is interesting in that it suggests a functional reasoning for why T cells may aggregate after activation, an event that seems as though it would be either hindering to immune response or neutral to the immune response.  When Ag-activated T cells begin to cluster, they have the potential of blocking capillaries and must be cleared, so it seems as though aggregation would hamper responses to pathogens. 

One question that I still have after reading the article is, “How is negative regulation of CD8 T cell effector molecules advantageous to the immune response?”.  A future study that could be conducted by the authors to follow up on this question would be to compare the downstream effects that take place in wild type CD8 T cells (reduction of IFN-y expression and reduced effector molecule production and cytotoxicity) in comparison to ICAM-deficient CD8 T cells (elevated expression of IFN-y and enhanced effector molecule production and cytotoxicity).

Primary Sources:
Zumwalde NA, E Domae, MF Mescher, and Y Shimizu. 2013. ICAM-1-Dependent Homotypic Aggregates Regulate CD8 T Cell Effector Function and Differentiation during T Cell Activation. Journal of Immunolgy, 191: 3681-3693

von Andrian, U. H., and T. R. Mempel. 2003. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3: 867–878
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