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T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.

The T cell receptor (TCR) and associated CD3[gamma][epsilon], [delta][epsilon], and [zeta][zeta] signaling dimers allow T cells to discriminate between different antigens and respond accordingly, but our knowledge of how these parts fit and work together is incomplete. In this study, we provide additional evidence that the CD3 heterodimers congregate on one side of the TCR in both the [alpha][beta] and [gamma][delta]TCR-CD3 complexes. We also report that the other side of the [alpha][beta]TCR mediates homotypic [alpha][beta]TCR interactions and signaling. Specifically, an erythropoietin receptor-based dimerization assay was used to show that, upon complex assembly, the CD3[epsilon] chains of two CD3 heterodimers are arranged side-by-side in both the [alpha][beta] and [gamma][delta]TCR-CD3 complexes. This system was also used to show that [alpha][beta]TCRs can dimerize in the cell membrane and that mutating the unusual outer strands of the C[alpha] domain impairs this dimerization. Finally, we present data showing that, for CD4 T cells, the mutations that impair [alpha][beta]TCR dimerization also alter ligand-induced calcium mobilization, TCR accumulation at the site of pMHC contact, and polarization toward the site of antigen contact. These data reveal a 'functional-sidedness' to the [alpha][beta]TCR constant region, with dimerization occurring on the side of the TCR opposite from where the CD3 heterodimers are located. T cell | T cell receptor | CD3 | organization | dimerization | signaling doi/ 10.1073/pnas.1000925107

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.10.033 Byline: Michael S. Kuhns (1), Mark M. Davis (1)(2) Abstract: In this issue, provide evidence for a new mechanism of T cell receptor regulation. Prior to activation, basic residues in the cytoplasmic domain of the signaling subunits of the T cell receptor associate with the plasma membrane such that the key signaling tyrosines are sequestered in the bilayer. Author Affiliation: (1) Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA (2) The Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA

T cells express clonotypic T cell receptors (TCRs) that recognize peptide antigens in the context of class I or II MHC molecules (pMHCI/II). These receptor modules associate with three signaling modules (CD3γe, δe, and ζζ) and work in concert with a coreceptor module (either CD8 or CD4) to drive T cell activation in response to pMHCI/II. Here, we describe a first-generation biomimetic fivemodule chimeric antigen receptor (5MCAR). We show that 1) chimeric receptor modules built with the ectodomains of pMHCII assemble with CD3 signaling modules into complexes that redirect cytotoxic T lymphocyte (CTL) specificity and function in response to the clonotypic TCRs of pMHCII-specific CD4+ T cells, and 2) surrogate coreceptor modules enhance the function of these complexes. Furthermore, we demonstrate that adoptively transferred 5MCAR-CTLs can mitigate type I diabetes by targeting autoimmune CD4+ T cells in NOD mice. This work provides a framework for the construction of biomimetic 5MCARs that can be used as tools to study the impact of particular antigen-specific T cells in immune responses, and may hold potential for ameliorating diseases mediated by pathogenic T cells. [ABSTRACT FROM AUTHOR]

We examined how cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates heterogeneous [CD4.sup.+] T cell responses by using experimental autoimmune encephalomyelitis (EAE), a [CD4.sup.+] T cell-mediated disease that is subject to regulation by CTLA-4. Disease incidence and severity were used as measures of in vivo [CD4.sup.+] T cell responses. The frequency, cytokine production, and reactivity of primed T cells were determined from animals immunized with proteolipid protein (PLP)-139-151 (disease agonist), PLP-Q (disease antagonist), or both peptides, and treated with control or anti-CTLA-4 antibody to analyze the responding population. CTLA-4 blockade exacerbated disease in PLP-139-151-primed animals and overcame disease antagonism in coimmunized animals, but did not permit disease induction in PLP-Q-primed animals. Experimental autoimmune encephalomyelitis enhancement was associated with increased frequencies of cytokine-producing cells and increased ratios of IFN-[Gamma] to IL-4 secretors responsive to PLP-139-151. Priming with PLP-Q elicited IL-4 and IL-2, but not IFN-[Gamma] secretors cross-reactive with PLP-139-151. Strikingly, CTLA-4 blockade was found to decrease rather than increase the frequencies of cross-reactive IL-4 and IL-2 secretors. Thus, CTLA-4 engagement limits the size, but increases the breadth, of reactivity of a primed pool of [CD4.sup.+] T cells, consequently regulating its function.

T cell recognition of peptides presented within self-major histocompatibility complex (pMHC) molecules is essential for long-lived protective immunity. As mice age the number of naïve CD4+ and CD8+ T cells declines. However, unlike for CD8+ T cells, there are more naïve and memory phenotype CD4+ T cells that bind foreign pMHCII in old mice (18–22 months) than adults (12–15 weeks), suggesting increased promiscuity of pMHCII recognition with aging. Here we asked if CD4+ T cell responses to immunization or infection increase with aging since the magnitude of a CD4+ T cell response to a foreign pMHCII is proportional to the size of the precursor population in adult mice. We observed no difference in the number of pMHCII-specific CD4+ T cells in adult versus old mice for pooled secondary lymphoid organs after immunization, bacterial infection, or viral infection, but we did observe diminished numbers of pMHCII-specific CD4+ T cells in both the draining lymph node and brain of old mice after West Nile virus infection. These data indicate that an increased precursor frequency does not translate into more robust responses upon immunization or infection in old mice. [ABSTRACT FROM AUTHOR]

The article presents the research that investigates the correlation between the rise of CD4+ T cells and the decline of immunity functions of aging mice and the onset of autoimmune age-related diseases in the U.S.

T cells specific for the cytochrome c Ag are widely used to investigate many aspects of TCR specificity and interactions with peptide-MHC, but structural information has long been elusive. In this study, we present structures for the well-studied 2B4 TCR, as well as a naturally occurring variant of the 5c.c7 TCR, 226, which is cross-reactive with more than half of possible substitutions at all three TCR-sensitive residues on the peptide Ag. These structures alone and in complex with peptide-MHC ligands allow us to reassess many prior mutagenesis results. In addition, the structure of 226 bound to one peptide variant, p5E, shows major changes in the CDR3 contacts compared with wild-type, yet the TCR V-region contacts with MHC are conserved. These and other data illustrate the ability of TCRs to accommodate large variations in CDR3 structure and peptide contacts within the constraints of highly conserved TCR-MHC interactions. [ABSTRACT FROM AUTHOR]