Cross-Reactivity of Herpesvirus-Specific CD8 T Cell Lines Toward Allogeneic Class I MHC Molecules

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65495–503/A*0201, BMLF1259–267/A*0201, or BZLF154–64/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-α production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-α production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection

Antagonism of cytotoxic T-lymphocyte activation by soluble CD8.

The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8alphaalpha receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 zeta-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics

Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2.

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit

Crystal structure of the complex between CD8αα human and HLA-A2

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8αα or the heterodimer αβ stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class l/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 Å resolution of a complex between CD8αα and the human MHC molecule HLA-A2, which is associated with peptide. CD8αα binds one HLA-A2/peptide molecule, interfacing with the α2 and α3 domains of HLA-A2 and also contacting β2- microglobulin. A flexible loop of the α3 domain (residues 223-229) is damped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the α3 domain is different from that in uncomplexed HLA-A2 (refs 3, 4), being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in α3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is dearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions

T cell receptor and coreceptor CD8 alphaalpha bind peptide-MHC independently and with distinct kinetics.

The T cell surface glycoprotein CD8 enhances T cell antigen recognition by binding to MHC class I molecules. We show that human CD8 alphaalpha binds to the MHC class I molecule HLA-A2 with an extremely low affinity (Kd approximately 0.2 mM at 37 degrees C) and with kinetics that are between 2 and 3 orders of magnitude faster than reported for T cell receptor/peptide-MHC interactions. Furthermore, CD8 alphaalpha had no detectable effect on a T cell receptor (TCR) binding to the same peptide-MHC class I complex. These binding properties provide an explanation as to why the CD8/MHC class I interaction is unable to initiate cell-cell adhesion and how it can enhance TCR recognition without interfering with its specificity

Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2.

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8alpha(alpha) or the heterodimer alpha beta stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class I/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 A resolution of a complex between CD8alpha(alpha) and the human MHC molecule HLA-A2, which is associated with peptide. CD8alpha(alpha) binds one HLA-A2/peptide molecule, interfacing with the alpha2 and alpha3 domains of HLA-A2 and also contacting beta2-microglobulin. A flexible loop of the alpha3 domain (residues 223-229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the alpha3 domain is different from that in uncomplexed HLA-A2, being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in alpha3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is clearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions