Derek B. Sant'Angelo

The Specificity and Orientation of a TCR to its Peptide–MHC Class II Ligands

A T cell-mediated immune response is mainly determined by the 3-5 aa residues that protrude upwards from a peptide bound to an MHC molecule. Alterations of these peptide residues can diminish, eliminate or radically alter the signal that the T cell receives through its T cell receptor (TCR). We have used peptide immunizations of normal mice and mice carrying alpha or beta chain TCR transgenes to identify three distinct peptide contact points. One, near the carboxyl terminus of the peptide, involves the beta chain CDR3 region; the second was centrally located and interacted with both the alpha and beta chain CDR3 loops; the third was near the amino terminus of the peptide, and affected V alpha gene usage, but not the structure of CDR3 of either TCR chain. Based on these results, we propose an orientation for the TCR of this cloned line and argue for its generality.

The Imprint of Intrathymic Self-Peptides on the Mature T Cell Receptor Repertoire

The analysis of T cell receptor alpha (TCR alpha) chains in mice transgenic for a TCR beta chain has allowed us to demonstrate a central role for self-peptides in the positive intrathymic selection of major histocompatibility complex (MHC) class II-restricted T cells. Analysis of specific V alpha-J alpha joins in mature CD4+ TCRhigh thymocytes and in peripheral CD4+ T cells revealed a limitation in amino-acid sequences. By analysis of immature thymocytes, we could show that this limited repertoire was selected from a more diverse repertoire. By analysis of the same beta chain-transgenic mice bred to H-2Ma-deficient mice that express one or a very limited number of peptides, we could demonstrate that the V alpha-J alpha join repertoire was now altered and much more limited. Together, these data provide molecular and genetic evidence that the intrathymic positive selection of the TCR repertoire is critically affected by self-peptides presented by MHC class II molecules, most likely on thymic cortical epithelial cells.

Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers

T cell receptor (TCR) allelic exclusion is believed to be primarily mediated by suppression of further recombination at the TCR locus after the expression of a functional TCR protein. Genetic allelic exclusion has been shown to be leaky for the β chain and, more commonly, for the α chain. Here, we demonstrate an additional mechanism by which T cells can maintain monoclonality. T cells from double TCR transgenic mice express only one or the other of the two available TCRs at the cell surface. This “functional allelic exclusion” is apparently due to control of the TCR assembly process because these T cells express RNA and protein for all four transgenic TCR proteins. Lack of cell surface expression of the second TCR may be controlled by a failure to assemble the TCR heterodimer.

Negative selection of thymocytes expressing the D10 TCR

We have analyzed the patterns of positive and negative selection of thymocytes expressing the T cell antigen receptor (TCR) from the D10.G4.1 T cell clone. This TCR confers reactivity to several non-self MHC class II alleles with a remarkably broad range of avidities. Therefore, negative selection can be studied when induced by high-, intermediate-, or low-avidity interactions with endogenous peptide–MHC complexes, all within the same TCR transgenic system. These data directly demonstrate that MHC class II–peptide ligands that fail to activate mature T cells can promote negative selection of immature thymocytes. Additionally, we show that negative selection of thymocytes can occur at two distinct “time points” during development depending on the avidity of the TCR for the MHC–peptide complex. Finally, we show that the self-peptide repertoire plays a significant role in selection because alteration of the self-peptide repertoire by disruption of the H2-Ma gene drastically alters selection of D10 TCR-expressing thymocytes.

TCR[alpha]-TCR[beta] pairing controls recognition of CD1d and directs the development of adipose NKT cells

The interaction between the T cell antigen receptor (TCR) expressed by natural killer T cells (NKT cells) and the antigen-presenting molecule CD1d is distinct from interactions between the TCR and major histocompatibility complex (MHC). Our molecular modeling suggested that a hydrophobic patch created after TCRα–TCRβ pairing has a role in maintaining the conformation of the NKT cell TCR. Disruption of this patch ablated recognition of CD1d by the NKT cell TCR but not interactions of the TCR with MHC. Partial disruption of the patch, while permissive to the recognition of CD1d, significantly altered NKT cell development, which resulted in the selective accumulation of adipose-tissue-resident NKT cells. These results indicate that a key component of the TCR is essential for the development of a distinct population of NKT cells.

A transgenic TCR directs the development of IL-4+ and PLZF+ innate CD4 T cells.

MHC class II–expressing thymocytes can efficiently mediate positive selection of CD4 T cells in mice. Thymocyte-selected CD4 (T-CD4) T cells have an innate-like phenotype similar to invariant NKT cells. To investigate the development and function of T-CD4 T cells in-depth, we cloned TCR genes from T-CD4 T cells and generated transgenic mice. Remarkably, positive selection of T-CD4 TCR transgenic (T3) thymocytes occurred more efficiently when MHC class II was expressed by thymocytes than by thymic epithelial cells. Similar to polyclonal T-CD4 T cells and also invariant NKT cells, T3 CD4 T cell development is controlled by signaling lymphocyte activation molecule/signaling lymphocyte activation molecule–associated protein signaling, and the cells expressed both IL-4 and promyelocytic leukemia zinc finger (PLZF). Surprisingly, the selected T3 CD4 T cells were heterogeneous in that only half expressed IL-4 and only half expressed PLZF. IL-4– and PLZF-expressing cells were first found at the double-positive cell stage. Thus, the expression of IL-4 and PLZF seems to be determined by an unidentified event that occurs postselection and is not solely dependent on TCR specificity or the selection process, per se. Taken together, our data show for the first time, to our knowledge, that the TCR specificity regulates but does not determine the development of innate CD4 T cells by thymocytes.

Corrigendum: TCRα-TCRβ pairing controls recognition of CD1d and directs the development of adipose NKT cells

Eliana Mariño, James L Richards, Keiran H McLeod, Dragana Stanley, Yu Anne Yap, Jacinta Knight, Craig McKenzie, Jan Kranich, Ana Carolina Oliveira, Fernando J Rossello, Balasubramanian Krishnamurthy, Christian M Nefzger, Laurence Macia, Alison Thorburn, Alan G Baxter, Grant Morahan, Lee H Wong, Jose M Polo, Robert J Moore, Trevor J Lockett, Julie M Clarke, David L Topping, Leonard C Harrison & Charles R Mackay Nat. Immunol.; doi:10.1038/ni.3713; corrected online 5 April 2017

Production of the Semi-Invariant TCR and PLZF Function in Innate Programming of iNKT Cells

Natural killer T (NKT) cells are endowed with phenotypic and functional characteristics that place them at the boundary between adaptive and innate immune responses. One subset, known as invariant NKT (iNKT) cells, expresses an invariant T cell recptor α (TCRα) chain paired with a limited number of TCRβ chains. This TCR recognizes lipids presented by the nonclassical MHC I–like molecule CD1d. Although iNKT cells are thought to arise from the same thymic precursors as conventional T cells, iNKT selection by interaction with DP thymocytes triggers a unique differentiation pathway associated with intrathymic proliferation and the acquisition of innate effector functions. Critical elements that are required for the differentiation of NKT cells but not conventional T cells have been identified. However, other unique signals that guide development of the NKT lineage remain to be elucidated. In this article we summarize our current understanding of the development of NKT cells.