Diversification, expression, and gamma delta T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Abstract

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial gamma delta T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative alpha-helical regions of the alpha1 alpha2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their alpha1 alpha2 domains, they were recognized by diverse human intestinal epithelial gamma delta T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of gamma delta T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

Figure 1

Expression of MICA- and MICB-related mRNAs and proteins by nonhuman primate epithelial cell lines. (A) Hybridization of total RNA samples detected one or two MIC mRNAs in various cell lines derived from chimpanzee (WES), African green monkey (COS and CV-1), rhesus monkey (FRhK-4), owl monkey (OMK), and silvery marmoset (NZP-60). HCT116 is a human colon carcinoma cell line; the lower and upper bands correspond to MICA and MICB mRNA, respectively. Numbers on the right indicate sizes in kilobases. (B) Flow cytometry with mAb 6G6 detected MIC-related molecules on the surfaces of several primate cell lines (filled profiles). Open profiles are IgG1 isotype control stainings. Shaded profiles are stainings with mAb W6/32 (anti-MHC class I HLA-A, -B, and -C) (30).

Figure 2

Diversity of primate MIC molecules. Amino acid sequences deduced from cDNAs are compared with MICA*01 and MICB*01 (12, 23). The α₁, α₂, α₃, and transmembrane (TM) and cytoplasmic tail (CYT) sequences are shown separately. Numbering refers to the MICB*01 sequence. Dashes and dots indicate identical residues or gaps and unaligned sequences, respectively. The positions of the highly conserved cysteines are shown in black. Shaded sequences are potential NXS/T glycosylation sites. Asterisks below sequences identify amino acid residues that are conserved among all vertebrate MHC class I sequences and open triangles indicate the positions of the conserved Tyr-7 and Tyr-171 (4, 20). Bars below the α₁ and α₂ sequence alignments indicate tentative extensions of α-helical sequences inferred from the previous comparison of MICA with HLA-A2 (11).

Figure 3

Recognition of diverse primate MIC molecules by human V_δ1 γδ T cells specific for MICA and MICB. (A–D) C1R cells transfected with the cDNAs for chimpanzee Patr-MIC1, rhesus monkey Mamu-MIC1, African green monkey Ceae-MIC2, or owl monkey Aotr-MIC1 expressed the encoded MIC proteins on the cell surface (filled profiles), as shown by immunofluorescence stainings and flow cytometry using mAb 6G6 (A–C) and an anti-MICA polyclonal antiserum (D). Open profiles are control stainings of C1R cells mock-transfected with the neomycin selectable marker gene. (E and F) In standard chromium release assays, the various primate MIC transfectants, including C1R-MICB transfectants, were lysed by the human V_δ1 γδ T cell clones δ1B-S1 and δ1B-S4 (10). C1R cells gave negative results. Similar data were obtained in at least one repeat experiment, as well as with C1R transfectants expressing Mamu-MIC2 or Ceae-MIC1. E:T, effector-to-target cell ratio.

Figure 4

Hypothetical placement of amino acid residues that are conserved among human MICA*01 and MICB*01, chimpanzee Patr-MIC1, rhesus monkey Mamu-MIC1 and -MIC2, African green monkey Ceae-MIC1 and -MIC2, and owl monkey Aotr-MIC1 on the ribbon diagram of HLA-A2 (adapted from ref. 31); conserved positions are shaded. These molecules were all recognized by the various V_δ1 γδ T cell clones tested. This figure is intended to visualize sequence diversity associated with probable similar secondary structures and folds of MIC molecules, without implying any close similarity to HLA-A2. Numbers are for orientation in the sequence alignment (Fig. 2). Areas marked by broken lines indicate tentative locations of small deletions occurring in the Mamu-MIC2, Ceae-MIC2 and Aotr-MIC1 sequences.