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Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles

The killer cell inhibitory receptors (KIRs) are surface glycoproteins expressed by natural killer (NK) and T cells that specifically recognize defined groups of polymorphic human histocompatibility leukocyte antigen (HLA) class I molecules. Interactions between KIRs on NK or T cells and major histocompatibility complex (MHC) class I molecules on potential target cells inhibit cell-mediated cytotoxicity, presumably by delivering a negative signal preventing lymphocyte activation. In this study we examined whether KIRs also regulate cytokine production induced in response to T cell receptor-dependent T cell activation. CD4+ and CD8+ T cell clones were stimulated by bacterial superantigens in the presence or absence of monoclonal antibodies (mAbs) against the KIR NKB1 or MHC class I molecules, and production of tumor necrosis factor alpha and interferon gamma was evaluated. When bacterial superantigen was presented by an autologous antigen-presenting cell (APC) to a KIR+ T cell clone, cytokine production was always enhanced in the presence of anti-MHC class I mAb. Similarly, anti-KIR mAb also augmented cytokine production, provided that the APC expressed a HLA class I allele recognized by the KIR. These results suggest that recognition of autologous MHC class I molecules by KIR+ T cells provides a regulatory mechanism acting to modulate the potency of their responses to antigenic challenge.

T he killer cell inhibitory receptors (KilLs) are a family of structurally related cell surface molecules that are expressed on subsets of human NK and T cells (1)(2)(3). These receptors bind to polymorphic HLA class I glycoproteins (4), and this interaction prevents NK cells from killing potential target cells expressing certain HLA-B or HLA-C alleles (5)(6)(7). lLecent studies suggest that the Src homology region 2-domain phosphatase (SHP) tyrosine phosphatase may be responsible for the KilL-mediated inhibition of cytotoxicity (8). Although structurally unrelated, the Ly49 receptors expressed on mouse NK cells also recognize H-2 class I molecules and inhibit NK cell-mediated cytotoxicity (9).
While initially discovered on NK cells, KI1Ks are also present on subsets ofT lymphocytes, including both CD4 + and CD8 + T cells (10)(11)(12). The expression of KIlL by T cells is intriguing because this suggests that T cell responses are regulated by two distinct, opposing receptors for MHC/ peptide ligands. Whereas the TClL transmits positive signals after engaging MHC/peptide complexes, the KIlL may serve to dampen the extent of cellular activation, thereby regulating the signaling threshold required for effector cell function. Previously, we demonstrated that KI1Ks can inhibit the lytic function of CTLs activated by bacterial superantigens (11). Here we examine the ability of the KIlL to regulate other T cell responses and demonstrate that disruption of KI1K recognition of self-class I molecules leads to significant increases in T cell cytokine production in response to superantigen stimulation.

Materials and Methods
T Cell Clones and B Cell Lines. T cell clones were generated and maintained as described previously (13,14). The HLA class I-negative B lymphoblastoid cell line (B-LCL) 721.221 was generously provided by Dr. Robert DeMars (University of Wisconsin, Madison, WI) (15). 721.221 transfectants expressing HLA-B'5801 were generated as described previously (14).
Antibodies and Flow Cytometry. mAbs were generously provided by Becton Dickinson Immunocytometry Systems (San Jose, CA). The anti-KItL-NKB1 mAb DX9 and anti-HLA-A,-B,-C mAb DX17 were produced and characterized in our laboratory (6,11). The anti-KIN mAbs GL183 and EB6 mAbs were kindly provided by Dr. Lorenzo Moretta (University of Genoa, Genoa, Italy), and HP-3E4 by Dr. Miguel Lopez-Botet (University of Madrid, Madrid, Spain). Methods of immunofluorescence staining and flow cytometry are described elsewhere (16).
Cytokine Assays. T cell clones were cultured in 96-well flatbottom microtiter plates (Becton Dickinson Labware, Lincoln Park, NJ) with or without Staphylococcus enterotoxin B (SEB, Toxin Technology Inc., Sarasota, FL). In some assays, 721.221 cells or 721.221 cells transfected with HLA-B*5801 were precoated with SEB, washed to remove residual superantigen, and used as APCs at a stimulator to responder ratio of 1:1. After overnight incubation, triplicate 50-lxl samples of cell-free supernatant were collected from each well and IFN-~/and TNF-cl were measured by ELISA, as previously described (17,18).

Results and Discussion
Superantigens are viral and bacterial proteins that activate T lymphocytes by binding to MHC class II molecules on Unst im ulat ed APCs and to the V region of TCR [3 chains on the responding T cells (21). We previously demonstrated that KIP. + CTL kill superandgen-coated class I-negative B-LCL, but are inhibited when the potential target cells express a HLA class I allele reactive with the KIP`. Whereas KIP`s have been implicated in regulation of cytotoxicity in both NK and T cells, less is known about the role of these inhibitory receptors in the regulation of other antigen-induced T cell functions, in particular, cytokine synthesis. To this end, we generated KIR +, TCR.
-(x/[3 + T cell clones (both CD4 + and CD8 +) and selected them for further study based on their ability to produce cytokines in response to stimulation with SEB presented either by the HLA class I-deficient cell clone (Tc867.9) expressing the K I R NKB1 which recognizes HLA-Bw4 ligands (7). In the absence of SEB, no cytokines were produced in response to coculture with 721.221 or a 721.221 transfectant expressing HLA-B*5801 (a HLA-Bw4 allele). Higher levels of TNF-ot and I N F -y were secreted by the T cell clone when SEB was presented by the class I-negative APC, compared with the HLA-B'5801 + transfectant (Fig. 1, A and B). Suppression of cytokine production by the HLA-B*5801 + APC apparently is regulated by the K I R present on the T cell clone because anti-NKB1 mAb or anti-MHC class I mAb enhanced cytokine production to a level equal to or higher than when the SEB is presented by the HLA class I-negative APC. In parallel with experiments measuring cytokine secretion, this T cell clone was assayed for its ability to kill SEBcoated 721.221 or HLA-B*5801 + 721.221 cells, and consistent with prior findings, the NKB1 K I R also inhibited superantigen-induced cytotoxicity against targets expressing HLA-B*5801 (not shown). These results demonstrate that binding of the K I R to its class I ligand is not only capable of modulating T cell-mediated cytotoxicity but also has inhibitory effects on T cell activation pathways leading to lymphokine production. These results are consistent with a prior study demonstrating effects ofanti-KIR mAb on cytokine production (10).
Whereas studies with MHC-deficient EBV-transformed B-LCL cell lines transfected with a single class I allele are useful to analyze the specificity of KIRs, they do not address the role of these inhibitory receptors in immune responses initiated by normal, autologous APCs. Because activated human T cells express M H C class II, they are able to serve as APCs for superantigens. Therefore, this provides the opportunity to study KIRs regulation ofa T cell response in a completely autologous system with SEB presented on autologous class II molecules and KIRs interacting with self-class I ligands. Preliminary studies demonstrated that H L A -D R + human T cell clones are capable of autopresenration of SEB and that the response is blocked by anti-H L A -D R mAbs (data not shown). To examine the role of KIRs in the regulation of cytokine production induced by autologous APCs, we selected a NKB1 + T cell clone from a HLA-Bw4 + individual so that the K I R is capable of interacting with a self-MHC class I ligand. These K I R + T cells were cultured with SEB in the presence or absence of anti-NKBl' or anti-HLA class I mAbs, and cytokines were measured (Fig. 1, C and D), W h e n the T cell clone autopresented SEB, lymphokine production (TNF-~x and INF-',/) was substantially increased if self-class I recognition by the K I R was disrupted by either anti-NKB1 or anti-class I mAb. These results demonstrate that interactions between KIRs and self-HLA class I molecules may regulate T C R -i n d u c e d immune responses initiated by autologous APCs. It should be noted that we used the same NKB1 + T cell clone for the experiments shown in Fig. 1, A -D to directly compare SEB presentation by the 721.221 APC (Fig. 1, A and B) and the autologous T cell as APC (Fig. 1, C and D). Similar results were obtained in both situations.
Further experiments were performed to evaluate the ki-netics and the effects ofsuperantigen concentration on K I R regulation of cytokine production. As shown in Fig. 2 A, a NKB 1 + CD4 + T cell clone (AD 130.2) was stimulated with SEB in the presence or absence ofanti-NKB1 or anti-HLA class I mAb. To examine early time points in the SEBinduced response, cytokines were detected by using immunofluorescence staining of intracytoplasmic lymphokines. Substantially more TNF-o~ and I N F -y were present in the cytoplasm of K I R + T cells stimulated in the presence of anti-NKB1 or anti-HLA class I mAb even within 4 h after SEB activation, After overnight culture, the ability of KIRs to suppress cytokine secretion by SEB-activated T cells was evident at all concentrations of superantigen tested, although at higher antigen doses the inhibition was not absolute, implying a quantitative regulation of activation. In general, K I R regulation of TNF-ix production was more profound than control of I F N -y secretion. IL-2 was not detected in these cultures and superantigen stimulation did not induce substantial proliferation under the conditions studied. Whereas KIRs are present on a subset o f T cells, many T cell clones lack expression of these molecules. For comparison with the K I R + T cell clones, we selected T cell clones that were unreactive with all existing anti-KIR rnAbs (including EB6, GL183, HP-3E4, and D x g ) and did not possess K I R transcripts as determined by Northern blot analysis (Fig. 3). These K I R -T cells were evaluated for SEB responsiveness in the presence or absence of anti-HLA class I mAb. A representative K I R CD4 + T cell clone (T81) is shown in Fig. 2 B. In this case, anti-HLA class I mAb had no effect on cytokine production induced by SEB, consistent with the lack of inhibitory receptor for self-HLA class I.
Whereas the NKB1 KIRs recognize HLA-Bw4 ligands, other members of the K I R family react with H L A -C or HLA-A molecules (5,22,23). K I R reactive with H L A -C may also regulate SEB-induced cytokine production. For example, the CD8 + T cell clone TC867.4 expresses a GL183 K I R that presumably can interact with this individual's HLA-Cw*0703 allele. Consistent with this possibility, anti-HLA class I mAb enhanced SEB-induced cytokine production by this T cell clone (Table 1). An individual N K cell clone can simultaneously express two or more KIRs on the cell surface (22,24). Moreover, often KIRs are present on N K cells of individuals who apparently lack self-class I alleles able to bind the receptor (25). Similarly, we have observed T cell clones expressing more than one K I R and  T celt clones were analyzed by flow cytometry for their KIR expression. IFN-3~ and TNF-a were analyzed by ELISA after stimulation for 20 h with 10 ng/rrd of SEB in the presence or absence ofanti-NKBl (DX9, 10 I~g/ml) or anti-MHC class-I (DXIT, 10 I~g/ml) mAbs. IgG1 control mAb had no effect on cytokine production (not shown). *SEB concentration = 5 ng/ml. receptors apparently unable to interact with self-class I ligands. T cell clone AD169.5 represents a cell expressing two KIRs, NKB1 and GL183; however, this individual does not express a HLA-B or HLA-C allele known to interact with NKB1 or GL183 (Table 1). Nonetheless, in functional assays, anti-HLA class I mAb augmented cytokine production, suggesting that yet another KIR or inhibitory receptor for a self-class I allele may be expressed by this clone. Results from these studies indicate that the distribution and function of KIRs on T cells are quite similar to NK cells and reveal the complexity of analyzing T cell clones able to simultaneously express multiple KIRs, only some of which may interact with self-class I ligands.
In summary, our findings suggest that KIRs affect not only NK cell-mediated cytotoxicity, but also cytokine production by CD4 + and CD8 + T cells, implying a more general regulatory role in the immune system. The exact mechanisms governing KIR expression on NK and T ceils are unclear at present and an understanding of how the KIR repertoire of an individual is shaped by self-HLA is limited. We have observed that KIRs are preferentially expressed on peripheral T cells with a "memory" phenotype and are rare in thymus and cord blood, suggesting that these inhibitory receptors may be expressed late during differentiation and serve to regulate responses against antigens, although this remains to be proven. It is tempting to speculate that in the T cell lineage, KIP. may provide a novel mechanism to increase the threshold of activation for effector T cells, thereby prohibiting stimulation due to encounters with low affinity, self-peptide/MHC complexes. That these receptors, upon binding to their class I ligands, are able diminish the potency of T cell responses, suggests a mechanism for limiting T cell activation possibly operative in the maintenance of tolerance or prevention of autoimmunity.