Functional evidence for TCR-intrinsic specificity for MHCII

Abstract

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.TCR | MHC | restriction | CD4 | Lck P ositive and negative selection limit the αβT-cell repertoire to cells expressing clonotypic T-cell receptors (TCRs) that distinguish the antigenicity of peptides embedded within class I and class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) based on their source of origin (i.e., self or foreign) (1-4). Approximately 7.5% of CD4 + CD8+ doublepositive (DP) thymocytes express TCRs that interact with selfpMHC above an affinity threshold required for positive selection, whereas 7.5% cross a higher affinity threshold that mediates negative selection and the remaining TCRs fail to direct positive selection (5). The rules that restrict TCR recognition of antigenic peptides within MHCI or MHCII are unresolved.Two models have been proposed to explain MHC restriction. One posits that restriction is imposed by CD4 or CD8 during thymocyte development to eliminate TCRs that recognize non-MHC ligands (2, 6). Here, the CD4-and CD8-associated Src kinase, p56Lck [lymphocyte-specific protein tyrosine kinase (Lck)], is sequestered away from the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-associated CD3δe, CD3γe, and CD3ζζ signaling modules. Positively selecting signals are then generated in thymocytes expressing TCRs that bind MHCII or MHCI together with CD4 or CD8, respectively, as this localizes Lck to the ITAMs. T...

The CD4 and CD3δε Cytosolic Juxtamembrane Regions Are Proximal within a Compact TCR–CD3–pMHC–CD4 Macrocomplex

“…When a CD3T subunit was expressed as a fusion with monomeric enhanced GFP (mEGFP), we used a GGGSAAAG linker. C-terminally truncated versions of CD4 (CD4T: aa1–421), with or without the Δbind mutation (aa:68–73 KGVLIR to DGDSDS), fused to mCherry via a AAAG linker were used in this study (26, 29). Glycine-based linkers were used for our fusion proteins because flexibility is required to allow equivalent probability of dipole alignment between FRET pairs, and even a single glycine in a dipeptide linker can allow a strand to double back upon itself (30–34).…”

“…After washing, the beads were then probed with anti-TCRα (α–Vα11 RR8-1 APC eBioscience) and anti-CD3ε (145–2C11 PE BD) with fluorescently labeled mAbs. Analysis was then performed by flow cytometry to assess co-precipitation of TCRα, CD3ε, and CD3xT G (mEGFP) with TCRβ similarly to previously described protocols (29, 40, 41). …”

Revealing the TCR bias for MHC molecules

“…Such a potential study would lend insight into more conserved regions of TCRs and how different TCRs have devised unique strategies to interact with their ligands. The present study (7) provides further clues into how 500 million years of evolution has led to a recognition system that is paradoxically both specific and flexible. Although many questions linger and require additional investigation, whether the TCR has an inherent bias for MHC is becoming an answerable one.…”

“…Despite years of work exploring reasons for this predisposition, a clear consensus remains elusive. In PNAS, Parrish et al, using a sensitive in vitro system, provide new evidence to suggest that TCRs possess an inherent bias to recognize MHC molecules (7).…”

“…When Parrish et al (7) replaced the TCR-exposed residues in the peptides with alanines, both the 5C.C7 and OT-II CD4-Lck cells continued to be stimulated in a MHC-restricted manner. Interestingly, this response was not MHC-allele specific because OT-II hybridomas responded to I-E k and 5C.C7 hybridomas responded to I-A b .…”