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Recognition of Stress-Induced MHC Molecules by Intestinal Epithelial γδ T Cells

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Science  13 Mar 1998:
Vol. 279, Issue 5357, pp. 1737-1740
DOI: 10.1126/science.279.5357.1737

Abstract

T cells with variable region Vδ1 γδ T cell receptors (TCRs) are distributed throughout the human intestinal epithelium and may function as sentinels that respond to self antigens. The expression of a major histocompatibility complex (MHC) class I–related molecule, MICA, matches this localization. MICA and the closely related MICB were recognized by intestinal epithelial T cells expressing diverse Vδ1 γδ TCRs. These interactions involved the α1α2 domains of MICA and MICB but were independent of antigen processing. With intestinal epithelial cell lines, the expression and recognition of MICA and MICB could be stress-induced. Thus, these molecules may broadly regulate protective responses by the Vδ1 γδ T cells in the epithelium of the intestinal tract.

T cells expressing γδ TCRs recognize antigens without restriction by polymorphic MHC class I or class II molecules and their associated peptide ligands (1-3). Of two main subsets in humans, Vγ2/Vδ2 T cells predominate in the circulation and respond to bacterial infections by recognizing soluble nonpeptide antigens (4). The other subset defined by expression of Vδ1, however, is of unknown function and no antigens have been identified. These T cells represent 70 to 90% of the γδ T cells in the intestinal epithelium (5). Because they are oligoclonal and uniformly distributed, they are believed to recognize self antigens that may be stress-induced (6, 7).

The localization of the intestinal intraepithelial Vδ1 γδ T cells is matched by the restricted expression of MICA, a divergent MHC class I–related molecule of unknown function (8). Its characteristics include the lack of association with β2-microglobulin (β2M), stable expression without conventional class I peptide ligands, and the absence of a CD8 binding site (8, 9). Notably, the 5′-end flanking regions of the genes for MICA and a closely related molecule, MICB, include putative heat shock elements similar to those ofhsp70 genes, and the encoded mRNAs are increased in heat shock–stressed epithelial cells (8).

To explore a functional relation, we established T cell lines from lymphocytes extracted from intestinal epithelial tumors (10). Other adequate sources of human intestinal epithelium are generally unavailable. Freshly isolated tumor cells gave positive stainings with monoclonal antibodies (mAbs) 2C10 and 6D4, which are specific for MICA and for MICA and MICB, respectively (8, 11,12). Vδ1 γδ T cells isolated by cell sorting were grown as two lines, δ1A and δ1B, which were cultured in the presence of cytokines and irradiated C1R cells transfected with cDNA for MICA or MICB, respectively (10, 13). After expansion, the T cell lines were tested for phenotype and function. They were homogeneously positive for Vδ1 γδ TCRs, CD4, and CD8 (Fig.1A). As is characteristic of intestinal intraepithelial T cells, they expressed the αEβ7 integrin (12, 14). In chromium release assays with C1R- MICA or C1R-MICB cells as targets, the δ1A and δ1B T cells were cytotoxic against both of these transfectants, but not against untransfected C1R cells (Fig. 1B) (15). Two CD8+ αβ T cell lines grown and tested under identical conditions gave negative results. The same observations were made with T cell lines from a second intestinal epithelial tumor and when T cells were expanded in the absence of MICA and MICB (12).

Figure 1

Vδ1 γδ T cell lines from intestinal epithelium recognize C1R transfectants expressing MICA or MICB. (A) Surface phenotype of the δ1B T cells by immunofluorescence stainings and flow cytometry with the following mAbs: αβ TCR (anti-TCR- α/β-1) (shaded profile), γδ TCR (anti-TCR-γ/δ-1), Vδ1 (mAb δTCS1), CD4 (mAb Leu-3a), and CD8 (mAb Leu-2a) (10). Note that mAb anti-TCR- α/β-1 weakly stains γδ TCR+ T cells. The open profile is an isotype-matched control staining. Similar profiles were obtained with the δ1A T cells. (B) In chromium release assays, the δ1A and δ1B T cells lysed C1R-MICA and C1R-MICB transfectants but not untransfected C1R cells. Data are means of triplicate experiments with less than ±5% deviation. E:T, effector-to-target cell ratio.

We used the δ1B line to analyze the apparent recognition of MICA and MICB. MICA transfectants of Daudi cells, which lack β2M and surface MHC class I (16), were as effectively lysed as Daudi-β2M- MICA double transfectants with normal expression of class I (Fig.2A) (13). Transfectants of the lymphoblastoid cell line mutant 5.2.4, which lacks expression of most MHC class II molecules (17), were also recognized (Fig. 2B) (13), as were transfectants of mouse T cell lymphoma EL4 cells (12). No lytic activity was observed against B cell lines with diverse MHC haplotypes. Thus, the δ1B T cell responses were independent of MHC class I and class II and were not secondary to cross-reactivity with some alleles of these molecules. Cytotoxicity against the transfected target cells was inhibited when these cells were preincubated with mAbs 2C10 or 6D4 (Fig. 2, C and D). The epitopes recognized by these mAbs are within the α1α2 domains of MICA and MICB, as determined by stainings of C1R transfectants expressing mouse class I H-2Db or Kb hybrid molecules in which the α1α2 or α3 domains have been substituted with the corresponding sequences of MICA (8, 12). The δ1B T cells lysed C1R-MICAα1α2-Db cells, but not C1R- MICAα3-Kb cells (12). Thus, Vδ1 γδ T cells from intestinal epithelium were restricted by MICA and MICB and recognized an epitope or epitopes associated with the α1α2 domains of these molecules.

Figure 2

Vδ1 γδ T cell responses are restricted by MICA and MICB and are independent of β2M and conventional class I antigen processing. (A andB) The δ1B T cells lysed Daudi-MICA (β2M, class I), Daudi-β2M-MICA (class I+), and 5.2.4-MICA (DR, DQ, TAP) transfectants but showed no or minimal lytic activity against the untransfected cells. (C and D) Binding of mAbs 2C10 (anti-MICA) and 6D4 (anti-MICA and -MICB) inhibited lysis of C1R-MICA and C1R-MICB targets, respectively. Treatment with the anti–HLA-A, -B, and -C mAb W6/32 (21) or isotype control IgG had no effect. (E and F) Gel filtration analysis of acid-dissociated immunoprecipitates isolated with mAbs 2C10 or W6/32 from C1R-MICA cells after metabolic labeling with [3H]amino acids (20).

We examined whether the recognition of MICA involved antigen processing and presentation of peptide ligands. With conventional MHC class I, the peptides are generated by proteasomes in the cytosol and are translocated into the endoplasmic retic- ulum by the transporters associated with antigen processing (TAP) (18). Treatment of C1R-MICA cells with lactacystin, which blocks proteasome functions (19), had no effect on the recognition of MICA by the δ1B T cells, but this did not preclude the presence of long-lived MICA-peptide complexes. However, the transfected mutant 5.2.4 cells, which lack TAP (17), were also proficient targets (Fig. 2B); this result implies that MICA has no function in the MHC class I pathway.

We sought physical evidence for MICA- peptide complexes by gel filtration chromatography of acid-dissociated immunoprecipitates that were isolated with mAb 2C10 from lysate of C1R-MICA cells after metabolic labeling with tritiated amino acids. The eluted fractions contained a single peak of radiolabeled polypeptide that was of high molecular weight and corresponded to MICA (Fig. 2E) (20). Analysis of MHC class I complexes isolated with mAb W6/32 (anti–HLA-A, -B, and -C) yielded fractions of high and low molecular weights (Fig.2F) (21). Thus, under these experimental conditions, there was no evidence for an association of MICA with peptides. This was consistent with its recognition by the δ1B T cells independent of conventional class I antigen processing.

These results were in agreement with previous models of antigen recognition by γδ T cells (2, 3) and supported a role of MICA and MICB as self antigens. We used intestinal epithelial cell lines to investigate the expression, regulation, and T cell recognition of these molecules. Semiconfluent DLD-1, Lovo, HCT116, and HUTU-80 cells that were rapidly proliferating expressed large amounts of MICA and MICB. They were lysed by the δ1B T cells in a specific interaction that was inhibited by mAb 6D4 (12, 22).

The expression of MICA in intestinal epithelium may be stress-induced rather than constitutive (8). In proliferating cell lines, however, transcription of the hsp70 gene is activated in the absence of cellular stress (23). We used Lovo, HCT116, and HUTU-80 cells grown as nonproliferating confluent monolayers to investigate the expression of MICA and MICB before and after heat shock induction. Uninduced cells had very low steady-state levels of MICA and MICB mRNA and expressed small amounts of the encoded surface molecules. However, heat shock induction resulted in large increases in mRNA and protein expression (Fig.3, A and B) (24). Concurrently, hsp70 mRNA was potently induced, whereas class I HLA-B mRNA and surface class I HLA-A, -B, and -C detected with mAb W6/32 on HCT116 and HUTU-80 cells (Lovo lacks β2M and thus class I surface expression) were unchanged (Fig. 3A) (12). The heat shock–treated cells were sensitized to lysis by the δ1B T cells, whereas minimal lytic activity was observed with the uninduced target cells. As with the proliferating cell lines, cytotoxicity was inhibited by mAb 6D4 (Fig. 3C). Thus, the expression of MICA and MICB and their recognition by the δ1B T cells were regulated by cell stress. Because these results were obtained with cell lines derived from intestinal epithelium, which is the only peripheral site where expression of MICA has been observed (8), MICA and presumably MICB were functionally associated with Vδ1 γδ T cells in this compartment.

Figure 3

Stress-induced expression and T cell recognition of MICA and MICB on quiescent intestinal epithelial cell lines. Lovo, HCT116, and HUTU-80 cells cultured for 8 days as confluent monolayers had very low steady-state levels of MICA and MICB mRNAs by blot hybridization of total cellular RNAs (A) (24). They expressed small amounts of the encoded cell surface proteins by indirect immunofluorescence staining with mAb 6D4 and flow cytometry (shaded profiles in B) and were poorly lysed by the δ1B T cells (C). Heat shock treatment strongly increased MICA and MICB mRNA (A) and protein expression (filled profiles in B), and also sensitized target cells to lysis, which was inhibited by mAb 6D4 (C); hsp70 mRNA was potently induced and control HLA-B mRNA was unaltered (A). The hsp70 blot was exposed to film for a much shorter time. Open profiles in (B) are isotype IgG1 control stainings. HS, heat shock.

We investigated whether MICA and MICB were recognized by T cells expressing diverse γδ TCRs and sought evidence for TCR engagement. A total of 16 T cell clones derived from the δ1A and δ1B lines showed functional activity against C1R-MICA and C1R-MICB targets. Analysis of cDNA sequences identified five distinct γ and δ chain pairs (Fig. 4, A and B) (25). The γ chains included Vγ1.3, 1.4, 1.5, or 1.8, and Jγ2.1 or 2.3. All of the δ chains expressed Vδ1 and Jδ1 with diverse junctions encoded by one or two D segments and nontemplated N region nucleotides (Fig. 4A) (1, 26). Because prolonged culture frequently resulted in loss of functional activity of T cell clones, these sequences were a minimal representation of different Vδ1 γδ T cells capable of recognizing MICA and MICB. We tested the ability of T cell clones 1, 3, and 5 to recognize C1R-MICA targets in the presence of the Vδ1 mAb δTCS1 (10, 15). With all three clones, inhibitory effects were observed (Fig. 4C). These data showed that MICA and MICB were recognized by Vδ1 γδ T cells expressing diverse TCRs and supported an engagement of these molecules by these TCRs. The diversity of TCRs implied that most, if not all, intestinal epithelial Vδ1 γδ T cells may be capable of interacting with MICA and MICB.

Figure 4

MICA is recognized by T cell clones expressing diverse Vδ1 γδ TCRs. (A) Five different γ and δ chain sequence pairs were identified by reverse transcription– PCR and direct sequencing among the 16 T cell clones derived from the δ1A and δ1B lines. Of the V and J region sequences flanking the variable N or N(D)N regions, only a few amino acids are shown (25). Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; G, Gly; H, His; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (B) T cell clones expressing the different γδ heterodimers were cytotoxic against C1R-MICA targets. (C) Inhibition of cytotoxicity of T cell clones expressing TCR sequences 1, 3, and 5 by Vδ1 mAb δTCS1 (10). Control IgG1 antibody was used under the same conditions. Data shown are representative of several independent assays and were obtained at a constant effector-to-target ratio of 5 to 1.

Our results define a T cell subset–MHC ligand interaction. Intestinal epithelial Vδ1 γδ T cells recognize epithelial cell lines without restriction by polymorphic MHC class I or class II molecules (27). We have now shown that stress-induced MHC class I–related molecules, MICA and MICB, function as target antigens recognized by these T cells. A number of allelic variants of MICA of uncertain significance have been identified (28). We observed no differences in the recognition by the δ1B T cells of C1R transfectants expressing three alleles representing most of the sequence variation in the α1α2 domains of MICA (29). Thus, although MICA and MICB are encoded in the MHC, their recognition was “MHC-unrestricted.” This was in accord with the recognition of all of the intestinal epithelial cell lines tested. Because MICA and MICB were recognized on diverse target cells without an apparent requirement for antigen processing, and there was no evidence for associated peptide ligands, it seems probable that these molecules alone conferred specificity in the recognition by the Vδ1 γδ T cells. This inference would be consistent with current models of γδ T cell recognition of antigen but remains tentative until the absence of peptide or nonpeptide ligands is conclusively demonstrated.

The stress-induced expression of MICA and MICB and their recognition by diverse Vδ1 γδ T cells may serve as an immune surveillance mechanism for the detection of damaged, infected, or transformed intestinal epithelial cells, or may stimulate T cell secretion of growth factors for the maintenance of epithelial homeostasis, as originally proposed for murine intraepithelial T cells expressing invariant γδ TCRs (7). The irregular distribution of MICA in variable areas of intestinal epithelium may reflect such an induction (8).

  • * To whom correspondence should be addressed. E-mail: vgroh{at}fred.fhcrc.org, tspies{at}fred.fhcrc.org

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