The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-LCK interactions.

CD4+ T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.

Enhancing and inhibitory motifs regulate CD4 activity.

CD4+ T cells use T cell receptor (TCR)-CD3 complexes, and CD4, to respond to peptide antigens within MHCII molecules (pMHCII). We report here that, through ~435 million years of evolution in jawed vertebrates, purifying selection has shaped motifs in the extracellular, transmembrane, and intracellular domains of eutherian CD4 that enhance pMHCII responses, and covary with residues in an intracellular motif that inhibits responses. Importantly, while CD4 interactions with the Src kinase, Lck, are viewed as key to pMHCII responses, our data indicate that CD4-Lck interactions derive their importance from the counterbalancing activity of the inhibitory motif, as well as motifs that direct CD4-Lck pairs to specific membrane compartments. These results have implications for the evolution and function of complex transmembrane receptors and for biomimetic engineering.

Domain-swapped T cell receptors improve the safety of TCR gene therapy.

T cells engineered to express a tumor-specific αß T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αß chains with endogenous αß 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 ß 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 αß 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 CD4 and CD3δε Cytosolic Juxtamembrane Regions Are Proximal within a Compact TCR-CD3-pMHC-CD4 Macrocomplex.

TCRs relay information about peptides embedded within MHC molecules (pMHC) to the ITAMs of the associated CD3γε, CD3δε, and CD3ζζ signaling modules. CD4 then recruits the Src kinase p56(Lck) (Lck) to the TCR-CD3 complex to phosphorylate the ITAMs, initiate intracellular signaling, and drive CD4(+) T cell fate decisions. Whereas the six ITAMs of CD3ζζ are key determinants of T cell development, activation, and the execution of effector functions, multiple models predict that CD4 recruits Lck proximal to the four ITAMs of the CD3 heterodimers. We tested these models by placing FRET probes at the cytosolic juxtamembrane regions of CD4 and the CD3 subunits to evaluate their relationship upon pMHC engagement in mouse cell lines. The data are consistent with a compact assembly in which CD4 is proximal to CD3δε, CD3ζζ resides behind the TCR, and CD3γε is offset from CD3δε. These results advance our understanding of the architecture of the TCR-CD3-pMHC-CD4 macrocomplex and point to regions of high CD4-Lck + ITAM concentrations therein. The findings thus have implications for TCR signaling, as phosphorylation of the CD3 ITAMs by CD4-associated Lck is important for CD4(+) T cell fate decisions.

Functional evidence for TCR-intrinsic specificity for MHCII.

How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR-pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4(+) or CD8(+) T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR-MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or "shaved" peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR-MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.

A Transmembrane Domain GGxxG Motif in CD4 Contributes to Its Lck-Independent Function but Does Not Mediate CD4 Dimerization.

CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions. While its association with p56lck (Lck), a Src kinase, is important for these functions CD4 also has an Lck-independent role in TCR signaling that is incompletely understood. Here, we identify a conserved GGxxG motif in the CD4 transmembrane domain that is related to the previously described GxxxG motifs of other proteins and predicted to form a flat glycine patch in a transmembrane helix. In other proteins, these patches have been reported to mediate dimerization of transmembrane domains. Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation. However, using Forster's Resonance Energy Transfer (FRET), we saw no evidence that these mutations decreased CD4 dimerization either in the unliganded state or upon engagement of pMHC concomitantly with the TCR. This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation.

Piecing together the family portrait of TCR-CD3 complexes.

The pre-T-cell receptor (TCR)-, αßTCR-, and γδTCR-CD3 complexes are members of a family of modular biosensors that are responsible for driving T-cell development, activation, and effector functions. They inform essential checkpoint decisions by relaying key information from their ligand-binding modules (TCRs) to their signaling modules (CD3γε + CD3δε and CD3ζζ) and on to the intracellular signaling apparatus. Their actions shape the T-cell repertoire, as well as T-cell-mediated immunity; yet, the mechanisms that underlie their activity remain an enigma. As with any molecular machine, understanding how they function depends upon understanding how their parts fit and work together. In the 30 years since the initial biochemical and genetic characterizations of the αßTCR, the structure and function of the individual components of these family members have been extensively characterized. Cumulatively, this information has allowed us to piece together a portrait of the αßTCR-CD3 complex and outline the form of the remaining family members. Here we review the known structural and functional characteristics of the components of these TCR-CD3 complex family members. We then discuss how these data have informed our understanding of the architecture of the αßTCR-CD3 complex as well as their implications for the other family members. The intent is to provide a framework for considering: (i) how these thematically similar complexes diverge to execute their specific functions and (ii) how our knowledge of the form and function of these distinct family members can cross-inform our understanding of the other family members.

Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist.

The precise timing of signals downstream of the T cell receptor (TCR) is poorly understood. To address this problem, we prepared major histocompatibility complexes containing an antigenic peptide that is biologically inert until exposed to ultraviolet (UV) light. UV irradiation of these complexes in contact with cognate T cells enabled the high-resolution temporal analysis of signaling. Phosphorylation of the LAT adaptor molecule was observed in 4 s, and diacylglycerol production and calcium flux was observed in 6-7 s. TCR activation also induced cytoskeletal polarization within 2 min. Antibody blockade of CD4 reduced the intensity of LAT phosphorylation and the speed of calcium flux. Furthermore, strong desensitization of diacylglycerol production, but not LAT phosphorylation, occurred shortly after TCR activation, suggesting that different molecular events play distinct signal-processing roles. These results establish the speed and localization of early signaling steps, and have important implications regarding the overall structure of the network.

Disruption of extracellular interactions impairs T cell receptor-CD3 complex stability and signaling.

The alphabeta T cell antigen receptor (TCR), in complex with the CD3deltavarepsilon, gammavarepsilon, and zetazeta signaling subunits, is the chief determinant for specific CD4(+) and CD8(+) T cell responses to self and foreign antigens. Although transmembrane domain charge interactions are critical for the assembly of the complex, the location of extracellular contacts between the TCR and CD3 subunits and their contributions to stability and signal transduction have not been defined. Here we used mutagenesis to demonstrate that the CD3deltavarepsilon and CD3gammavarepsilon subunits interact with the TCR via adjacent Calpha DE and Cbeta CC' loops, respectively. The TCR-CD3deltavarepsilon interactions helped stabilize CD3gammavarepsilon within the complex and were important for normal T cell and thymocyte responses to TCR engagement. These data demonstrate that extracellular TCR-CD3 subunit interactions contribute to the structural integrity and function of this multisubunit receptor.

T cells use two directionally distinct pathways for cytokine secretion.

Activated T helper cells produce many cytokines, some of which are secreted through the immunological synapse toward the antigen-presenting cell. Here we have used immunocytochemistry, live-cell imaging and a surface-mediated secretion assay to show that there are two cytokine export pathways in T helper cells. Some cytokines, including interleukin 2 and interferon-gamma, were secreted into the synapse, whereas others, including tumor necrosis factor and the chemokine CCL3 (MIP-1alpha), were released multidirectionally. Each secretion pathway was associated with different trafficking proteins, indicating that they are molecularly distinct processes. These data suggest that T helper cells release some cytokines into the immunological synapse to impart specific communication and others multidirectionally to promote inflammation and to establish chemokine gradients.

CTLA-4: new insights into its biological function and use in tumor immunotherapy.

The discovery of multiple costimulatory cell surface molecules that influence the course of T cell activation has increased our appreciation of the complexity of the T cell response. It remains clear, however, that CD28 and cytotoxic T lymphocyte antigen 4 (CTLA-4) are the critical costimulatory receptors that determine the early outcome of stimulation through the T cell antigen receptor (TCR). Details of how the T cell integrates TCR stimulation with the costimulatory signals of CD28 and the inhibitory signals of CTLA-4 remain to be established, but unique features of the cell biology of CTLA-4 provide important insights into its function. We summarize here recent findings that suggest a previously unrecognized role for CTLA-4 in the regulation of T cell responses. We also describe preclinical and clinical results that indicate manipulation of CTLA-4 has considerable promise as a strategy for the immunotherapy of cancer.