T-cell signalling: ITAMs: quantity over quality

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A systematic analysis of the CD3 immunoreceptor tyrosine-based activation motifs (ITAMs), which transmit intracellular signals following T-cell receptor (TCR) ligation, illuminates the requirement for multiple ITAMs in T-cell development and function.

The 6 CD3 adaptor molecules of the T-cell receptor (TCR) complex (CD3 gamma , CD3 delta and two each of CD3 epsildot and CD3 zeta chains) contain a total of 10 immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains. Although it is known that ITAMs are essential for the transmission of intracellular signals following TCR ligation, the reason why the TCR complex contains so many ITAMs is not well understood. Now, Vignali and colleagues report a systematic analysis of CD3-ITAM function that expands our knowledge on how the quantity and quality of ITAM signalling regulates several aspects of T-cell development and function.

To assess the functional importance of overall ITAM numbers, as well as the contribution of individual CD3 ITAMs in vivo, the authors generated 25 groups of mice, each expressing a TCR complex with a different combination of wild-type and mutant CD3 ITAMs. To achieve this, 'retrogenic mice' were generated by reconstituting Rag1^-/- mice (which are deficient in the recombination-activating gene 1 and therefore lack lymphocytes) with bone marrow transduced with a retrovirus encoding either wild-type CD3 chains or CD3 chains containing various combinations of ITAM mutations. As the donor bone marrow was from mice that lacked expression of endogenous CD3 proteins, the functionality of retrovirally expressed CD3 chains that contained the entire range of 1–10 ITAMs could be assessed in the recipient mice.

Mice that received bone marrow transduced with Cd3 containing 2–6 functional CD3 ITAMs developed a lethal multi-organ autoimmune syndrome, whereas mice that expressed 7 or more CD3 ITAMs remained healthy. The autoimmunity in the diseased mice was mediated mainly by CD4⁺ T cells and in some mice was accompanied by the production of autoantibodies. Closer examination of the diseased mice revealed that their populations of forkhead box P3 (FOXP3)-expressing regulatory T cells were intact, whereas negative selection was impaired, meaning that large numbers of autoreactive T cells escaped from the thymus into the periphery. So, a breakdown in central rather than peripheral tolerance mechanisms led to autoimmunity in mice with less than 7 functional CD3 ITAMs, and relatively subtle changes in TCR-signalling strength resulted in defective negative selection of autoreactive T cells.

Precise numbers of CD3 ITAMs were also found to be essential for other aspects of T-cell development. A minimum of 7 ITAMs was needed for normal thymocyte numbers, whereas 4 were required for splenic T-cell populations and a minimum of 2 was needed for normal CD4⁺:CD8⁺ T-cell ratios. In contrast to the precise thresholds that were found to regulate aspects of T-cell development, the relationship between proliferative capacity and the number of CD3 ITAMs expressed was proportional. Surprisingly, the number of CD3 ITAMs had very little influence on cytokine production.

Although these results indicate that quantity is the crucial determinant for many aspects of T-cell development and function, individual CD3 ITAMs were found to be important in some cases. For example, intact CD3 epsildot chains alone were uniquely able to regulate CD4⁺:CD8⁺ T-cell ratios, whereas both CD3 and CD3 zeta chains were important for peripheral T-cell homeostasis. So, subtle alterations in the location or sequence of CD3 ITAMs can also influence the function of the TCR complex.

These findings indicate that the multiple ITAMs of the TCR complex enable a dynamic range of scalable signal strength, allowing for the generation of diverse downstream events such as negative selection, tolerance induction, proliferation and cytokine production.

Sarah Allan

References

Holst, J. et al. Scalable signaling mediated by T cell antigen receptor–CD3 ITAMs ensures effective negative selection and prevents autoimmunity. Nature Immunol. 9, 658–666 (2008) | Article | PubMed |