Stimulatory and Inhibitory Killer Ig-Like Molecules Are Expressed and Functional on Lupus T Cells

This information is current as Dhiman Basu, Ying Liu, Ailing Wu, Sushma Yarlagadda, of October 1, 2021. Gabriela J. Gorelik, Mariana J. Kaplan, Anura Hewagama, Robert C. Hinderer, Faith M. Strickland and Bruce C. Richardson J Immunol 2009; 183:3481-3487; Prepublished online 12 August 2009; doi: 10.4049/jimmunol.0900034 Downloaded from http://www.jimmunol.org/content/183/5/3481

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Stimulatory and Inhibitory Killer Ig-Like Receptor Molecules Are Expressed and Functional on Lupus T Cells1

Dhiman Basu,* Ying Liu,* Ailing Wu,* Sushma Yarlagadda,* Gabriela J. Gorelik,* Mariana J. Kaplan,* Anura Hewagama,* Robert C. Hinderer,* Faith M. Strickland,* and Bruce C. Richardson2*†

T cells from lupus patients have hypomethylated DNA and overexpress normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell Ig-like receptor (KIR) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Abs to the stimulatory molecule KIR2DL4 triggered IFN-␥ release by lupus T cells, and production was pro-

portional to disease activity. Similarly, cross-linking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage Downloaded from killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN over- production and macrophage killing in human lupus, and they suggest that Abs to inhibitory KIR may be a treatment for this disease. The Journal of Immunology, 2009, 183: 3481–3487.

mpaired T cell DNA methylation contributes to the develop- active lupus, and the degree of demethylation and overex-

ment of lupus-like diseases. Injecting experimentally demethyl- pression are directly related to disease activity. ITGAL demethyl- http://www.jimmunol.org/ ated T cells into syngeneic mice causes lupus-like autoimmunity ation results in LFA-1 overexpression on autoreactive T cells (10), I ϩ (1, 2), as does decreasing T cell DNA methyltransferase 1 expression PRF1 demethylation in CD4 T cells results in aberrant perforin in transgenic mice (3). Similarly, reports that the DNA methylation expression contributing to autologous M␾ killing (7), and CD40LG inhibitors procainamide and hydralazine cause anti-nuclear Abs in demethylation contributes to B cell overstimulation in women with most patients receiving these drugs, and a lupus-like disease in a ge- active lupus (9). netically predisposed subset (4), also support a causative role for The evidence indicating a role for T cells with hypomethylated DNA demethylation in lupus-like autoimmunity. DNA in lupus pathogenesis suggests that Abs or other recombinant Methylation is a repressive DNA modification, suppressing molecules designed to deplete or inactivate this subset may be genes unnecessary or potentially detrimental to normal cellular by guest on October 1, 2021 therapeutic in human lupus, and be more selective and safer than function, but for which the cell expresses the requisite transcription ϩ current modalities such as corticosteroids or cyclophosphamide. factors. Inhibiting DNA methylation in CD4 T cells causes ab- The ideal therapeutic target would be a gene expressed on demeth- errant overexpression of genes that contribute to the development ylated but not normal T cells, and which inhibits autoreactive re- of autoimmunity in adoptive transfer models. Demethylation of ITGAL (CD11a) causes LFA-1 overexpression, resulting in MHC- sponses when ligated. specific autoreactivity (5, 6), and demethylation of PRF1 results in We recently identified the killer cell Ig-like receptor (KIR) gene perforin overexpression that contributes to autoreactive macro- family as methylation sensitive in human T cells (11). The KIR phage (M␾)3 killing and release of antigenic nucleosomes (7, 8). genes constitute a polymorphic family normally expressed on NK Similarly, demethylation of TNFSF7 and CD40LG results in over- cells but rarely on normal T cells (12). KIR molecules on NK cells expression of the B cell costimulatory molecules CD40L and recognize class I MHC molecules and possibly other self ligands, CD70, contributing to Ab overproduction (9, 10). and they either stimulate or inhibit killing and secretion of inflam- Identical changes in DNA methylation, , and matory cytokines depending on the cytoplasmic domain (13). cellular function characterize a CD4ϩ T cell subset in patients with Therefore, in the present study we investigated whether experi- mentally demethylated T cells aberrantly express KIR genes, both stimulatory and inhibitory, and whether KIR expression has func- *Department of Medicine, University of Michigan, Ann Arbor, MI 48109; and †Ann Arbor Veterans Affairs Medical Center, Ann Arbor, MI 48105 tional implications by cross-linking stimulatory KIR to induce ␥ ␾ Received for publication January 7, 2009. Accepted for publication June 22, 2009. IFN- secretion, and inhibitory KIR to prevent autoreactive M The costs of publication of this article were defrayed in part by the payment of page killing. Furthermore, since lupus T cells have hypomethylated charges. This article must therefore be hereby marked advertisement in accordance DNA and aberrantly overexpress other methylation-sensitive genes, with 18 U.S.C. Section 1734 solely to indicate this fact. we hypothesized that the KIR genes would be similarly over- 1 This work was supported by Public Health Service Grants AR42525, AR56370, expressed in T cells from patients with active lupus. We also AG25877, and ES15214, National Institute on Aging Training Grant AG000114, and a Merit Grant from the Veterans Administration. hypothesized that Abs to inhibitory KIR molecules might in- 2 Address correspondence and reprint requests to Dr. Bruce Richardson, Department hibit the autoreactive, cytotoxic responses that characterize this of Medicine, University of Michigan, 3007 Biomedical Science Research Building subset in lupus patients. We therefore compared KIR expression (BSRB), Ann Arbor, MI 48109. E-mail address: [email protected] and function on experimentally demethylated T cells and on T 3 Abbreviations used in this paper: M␾, macrophage; KIR, killer cell Ig-like receptor; SLE, systemic lupus erythematosus; SLEDAI, systemic lupus erythematosus disease cells from patients with active and inactive systemic lupus er- activity index; MS-PCR, methylation-sensitive PCR; 5-azaC, 5-azacytidine. ythematosus (SLE). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900034 3482 KIR AND LUPUS

Materials and Methods Table I. KIR genes in lupus patients and controls Subjects KIR SLE (%) (N ϭ 35) Control (%) (N ϭ 35) Healthy subjects were recruited by advertising. Lupus patients met criteria for lupus (14) and were recruited from the Michigan Lupus Cohort and the 2DL4 35 (100) 35 (100) inpatient services at the University of Michigan Hospitals. Disease activity 2DL2 35 (100) 23 (66) was quantitated using the SLE disease activity index (SLEDAI) (15). The 3DL2 33 (94) 35 (100) protocols were reviewed and approved by the University of Michigan In- 3DL3 30 (86) 34 (97) stitutional Review Board. 2DL3 30 (86) 32 (91) 2DL1 30 (86) 29 (83) T cell isolation PBMC were isolated from peripheral blood by density gradient centrifu- gation, and T cells were purified using the MACS Pan-T cell isolation Statistical analyses (Miltenyi Biotec) and instructions provided by the manufacturer. The Pan-T cell isolation kit removes non-T cells, that is, B cells, NK cells, The significance of differences between means was determined using Student’s dendritic cells, monocytes, granulocytes, and erythroid cells, using a cock- t test, and the relationship between disease activity and measured parameters tail of biotin-conjugated Abs against CD14, CD16, CD19, CD36, CD56, were determined by linear regression and ANOVA using Systat software. CD123, and CD235a (). The non-T cells were labeled with anti-biotin microbeads and magnetically depleted, following the manufac- Results ϩ turer’s instructions. Following collection of the unlabeled T cells, CD4 5-Azacytidine (5-azaC) induces KIR expression on T cells and CD8ϩ cells were further isolated by negative selection using CD4ϩ and CD8ϩ isolation kits (Miltenyi Biotec). The resultant CD4ϩ and CD8ϩ The KIR gene family is highly polymorphic, containing up to 14 ϩ T cells were free of NK and NKT (CD56 ) cells, as determined by staining genes and pseudogenes, and multiple alleles exist, resulting in ex- Downloaded from with PE-anti-CD56 Ab (BD Pharmingen) and by flow cytometry. tensive variability between individuals (18). We therefore geno- KIR genotyping typed a panel of 35 lupus patients and 35 healthy controls. Both the lupus and control subjects were primarily (Ͼ85%) Caucasian. Ta- KIR genotypes were determined using a KIR typing kit (Miltenyi Biotec), ble I shows the most common alleles observed in the lupus patients testing the presence of 15 KIR genes and pseudogenes, using protocols provided by the manufacturer. compared with controls. 2DL4 was present in everyone, as re- ported (18), and the frequencies of the other alleles shown resem- http://www.jimmunol.org/ Antibodies ble those reported by others for Caucasians (19). Other alleles were Unconjugated and PE-conjugated anti-CD158d (KIR2DL4) were pur- variably present at a lower frequency in the lupus patients and chased from R&D Systems, and unconjugated and PE-conjugated anti- controls, but given the limited number of subjects studied the sig- NKB1 (KIR3DL1), anti-perforin Abs, and isotype-matched control Abs nificance of the variability is uncertain. were obtained from BD Pharmingen. Anti-CD4-Cy5, anti-CD8-FITC, and We then confirmed that CD4ϩ and CD8ϩ T cells demethylate PE-conjugated Abs to and KIR2DS2 (anti-CD158b) were obtained from and express KIR genes following treatment with the DNA meth- BD Pharmingen, KIR2DL1/2DS2 (CD158a/h) and KIR3DL1/3DS1 (CD158e1/e2) were from Beckman Coulter, and 2DL1/2DL3/2DS2 (anti- ylation inhibitor 5-azaC. PBMC from 11 healthy subjects were CD158 b1/b2/j (GL183)) was purchased from R&D Systems. Ten micro- stimulated with PHA, treated with 5-azaC, and then KIR expres- liters of each PE-conjugated anti-KIR Ab was mixed to form a “cocktail” sion was measured on untreated and treated CD4ϩ and CD8ϩ cells by guest on October 1, 2021 used to stain the cells. All labeling procedures were performed on ice in using a “cocktail” of anti-KIR Abs and flow cytometry as de- PBS containing 10% horse serum and normal human AB serum (Invitro- gen) and sodium azide. Intracellular staining for perforin was performed on scribed in Materials and Methods. Fig. 1 confirms that 5-azaC cells permeabilized for 20 min on ice with Cytofix/Cytoperm solution (BD Pharmingen). Following staining, the cells were fixed in 1% paraformal- dehyde and kept at 4°C until analyzed.

Flow cytometric analysis Multicolor flow cytometry was performed using previously published pro- tocols (16).

IFN-␥ stimulation Anti-KIR2DL4 or isotype-matched control Abs were diluted in PBS, then allowed to bind to flat-bottom 96-well microtiter plates (Costar; Corning) for3hat37°C. The plates were then washed and 2 ϫ 105 T cells were added per well in RPMI 1640 supplemented with 10% FBS (Invitrogen) then cultured at 37°C in room air supplemented with 5% CO2. Twenty-four hours later the supernatants were recovered and IFN-␥ was measured using an OptEIA Duo ELISA kit (BD Biosciences) and recombinant IFN-␥ stan- dard, according to the manufacturer’s instructions.

KIR 2DL4 methylation-specific PCR (MS-PCR) The methylation status of the KIR2DL4 was determined using MS-PCR as previously described (11).

M␾ killing assays FIGURE 1. KIR expression on 5-azaC-treated CD4ϩ and CD8ϩ T cells. Monocytes were purified from PBMC by adherence to round-bottom mi- PBMC from 11 healthy subjects were stimulated with PHA, treated with crotiter wells and labeled with 51Cr as previously described (17). Purified 5-azaC, then 72 h later stained with anti-CD4-CyChrome, anti-CD8-FITC, anti-KIR3DL1 or isotype-matched control Abs were added where indi- cated. Purified T cells were then cultured with the M␾ for 18 h at 37°C in and a cocktail of PE-conjugated Abs to KIR 3DL1, 2DS2, 2DL1, and 51 2DL1/2DL3/2DS2 and then analyzed by flow cytometry. Dark hatched room air supplemented with 5% CO2, and Cr release was measured as described (17). Results are presented as the means Ϯ SEM of three to four bars represent PHA-stimulated, untreated T cells, and the hatched bars determinations per data point. represent PHA-stimulated, 5-azaC-treated T cells. The Journal of Immunology 3483

FIGURE 2. 5-azaC-induced KIR molecules are functional. T cells were stimulated with PHA and treated with 5-azaC as in Fig. 1. A, The 5-azaC-treated T cells were fractionated into CD4ϩ and CD8ϩ cells using magnetic beads, then cultured with immobilized anti-KIR2DL4 or an isotype-matched IgG and IFN-␥ release was measured by ELISA. The light hatched bars represent 5-azaC-treated cells and the dark hatched bars untreated cells. B, the treated T Downloaded from cells were cultured with 51Cr-labeled autologous monocytes/M␾ and the indicated concentrations of anti-KIR-3DL1 or isotype-matched control IgG, and 51Cr release was measured 18 h later. Results are presented as the means Ϯ SEM of three determinations. significantly increases KIR expression on both CD4ϩ and CD8ϩ a somewhat greater percentage of CD8ϩ T cells expressing KIR

T cells. relative to CD4. http://www.jimmunol.org/ Previous studies demonstrated that CD11a, perforin, and KIR is functional in 5-azaC-treated T cells CD40L overexpression is directly proportional to disease ac- Stimulatory and inhibitory KIR functions were tested in tivity. We therefore compared KIR expression with disease ac- 5-azaC-treated CD4ϩ and CD8ϩ T cells. mAbs are available for tivity in the 16 lupus patients studied in Fig. 3. Fig. 4A shows some but not all KIR gene products, and some of the Abs are cross-reactive with multiple KIR genes. However, Abs reactive with CD158d (KIR2DL4), a stimulatory molecule present in all donors, and CD158e1/2 (anti-NKB1, KIR3DL1), an inhibitory by guest on October 1, 2021 molecule present in most but not all individuals (19), are available. PBMC from three healthy individuals were stimulated with PHA and treated with 5-azaC as before. CD4ϩ and CD8ϩ T cells were isolated using magnetic beads and then stimulated with immobi- lized anti-KIR2DL4 or an equal concentration of isotype-matched control Ig. IFN-␥ release was measured by ELISA. Fig. 2A con- firms that anti-KIR2DL4, but not the control Ab, stimulates IFN-␥ synthesis by 5-azaC-treated CD4ϩ and CD8ϩ T cells. Inhibitory KIR function was tested by culturing 5-azaC- treated, magnetic bead-purified T cells from a KIR3DL1-posi- tive subject with 51Cr-labeled autologous M␾, with or without graded amounts of anti-KIR3DL1 or isotype-matched control Ig. Fig. 2B shows that demethylated T cells spontaneously kill autologous M␾, as we previously reported (17), and that the killing is inhibited with anti-KIR3DL1 but not control Ig. Sim- ilar results were seen in a confirming experiment (37 Ϯ 8% vs 66 Ϯ 6% cytotoxicity (mean Ϯ SEM), KIR3DL1 vs control, p ϭ 0.016). Taken together, these results indicate that stimu- latory and inhibitory KIR function is intact in KIR-expressing, demethylated T cells. FIGURE 3. Lupus T cells express KIR molecules. A, PBMC from a Lupus T cells express KIR representative lupus patient were stained with anti-CD4-CyChrome and the cocktail of PE-conjugated anti-KIR Abs and then analyzed by flow We then asked if KIR is expressed on lupus T cells. Fig. 3A shows ϩ ϩ T cells from a lupus patient stained with anti-CD4 and the cocktail cytometry. The percentage CD4 KIR is shown in the upper right quadrant. B, PBMC from the same subject were similarly stained and of anti-KIR Abs, and Fig. 3B shows cells similarly stained with ϩ ϩ analyzed for CD8 and KIR. The percentage CD8 KIR is again shown anti-CD8 and the anti-KIR mixture. KIR is expressed on a subset in the upper right quadrant. C, PBMC from 16 lupus patients (light of T cells. Fig. 3C compares the percentage of KIRϩCD4ϩ and ϩ ϩ hatched bars) and 16 age- and sex-matched controls (dark bars) were KIR CD8 T cells in PBMC from 16 lupus patients and 16 age- stained for CD4, CD8, and KIR as in A and B. Results are represent the and sex-matched controls. There is a significant increase in KIR percentage KIRϩ CD4 or CD8 cells and are presented as the means Ϯ expression on both subsets in T cells from the lupus patients, with SEM of the 16 determinations. 3484 KIR AND LUPUS

FIGURE 4. T cell KIR expression is proportional to disease activity. A, The percentage CD4ϩKIRϩ T cells is plotted against the SLEDAI for each FIGURE 5. KIR expression on CD4ϩCD28ϩ and CD4ϩCD28Ϫ T cells Downloaded from of the 16 lupus patients reported from Fig. 3. B, The percentage CD8ϩ from patients with active lupus. T cells from six lupus patients (light KIRϩ T cells is similarly plotted against the SLEDAI for each of the 16 hatched bars) or age- and sex-matched healthy controls (dark hatched bars) lupus patients reported from Fig. 3. were stained with the KIR cocktail, anti-CD4, and anti-CD28 and then analyzed by flow cytometry. Results are presented as the means Ϯ SEM of the percentage KIRϩ cells for the six determinations. the relationship between the percentage of CD4ϩKIRϩ T cells and lupus disease activity as measured by the SLEDAI, and Fig. http://www.jimmunol.org/ ϩ ϩ 4B similarly compares the percentage of CD8 KIR T cells Stimulatory and inhibitory KIR are functional on lupus T cells with the SLEDAI. The number of KIRϩ T cells is proportional Stimulatory KIR function was tested by culturing purified T cells to disease activity, similar to other methylation-sensitive genes ϩ ϩ ϩ from nine lupus patients and nine healthy age- and sex-matched ( p ϭ 0.059 for CD4 KIR cells, and p ϭ 0.016 for CD8 ϩ controls with immobilized anti-KIR2DL4 or control Ig and mea- KIR cells). ϩ Ϫ suring IFN-␥ release as described in Fig. 2A. Fig. 7A shows that KIR genes are also expressed on “senescent” CD4 CD28 T control T cells secrete minimal amounts of IFN-␥ when cultured cells (20). Cells in this subset have shortened telomeres, decreased with either anti-KIR2DL4 or control Ig. In contrast, lupus T cells replicative potential, express large amounts of proinflammatory secreted significantly ( p ϭ 0.011) more IFN-␥ than did controls by guest on October 1, 2021 molecules such as IFN-␥ and perforin (21), and are found in the when cultured with the control Ig, and they demonstrated a further elderly (22) as well as in patients with chronic inflammatory dis- eases such as rheumatoid arthritis (23). Interestingly, these cells are cytotoxic for endothelial cells, and they have been cloned from ruptured atherosclerotic plaques in patients dying from myocardial infarctions (21). We therefore addressed the possibility that KIR was primarily expressed on CD4ϩCD28Ϫ T cells in lupus, sim- ilar to rheumatoid arthritis. Fig. 5 compares KIR expression, again measured with the anti-KIR cocktail, on CD4ϩCD28ϩ and CD4ϩCD28Ϫ T cells from six lupus patients and six healthy age- and sex-matched controls. The healthy controls had small numbers (about Յ1%) of KIRϩ T cells in both sub- sets. In contrast, KIR was expressed on significantly ( p Ͻ 0.05) greater numbers of CD4ϩCD28ϩ as well as CD4ϩCD28Ϫ T cells from lupus patients relative to the controls. There was a trend for greater KIR expression on CD4ϩCD28Ϫ T cells rel- ative to the CD4ϩCD28ϩ subset in the lupus patients, but was of borderline significance ( p ϭ 0.078).

KIR2DL4 is demethylated in lupus T cells Our earlier report on T cell DNA methylation and KIR expression demonstrated demethylation of 7 CG pairs, located between bp Ϫ158 and Ϫ126 relative to the start site, in the 2DL4 promoter of FIGURE 6. The KIR2DL4 promoter is demethylated in lupus T cells. 5-azaC-treated CD4ϩ and CD8ϩ T cells. MS-PCR was used to MS-PCR was used to compare methylation of the KIR2DL4 promoter in T cells from five lupus patients and five age- and sex-matched controls. Prim- compare methylation of CG pairs located Ϫ158 and Ϫ26 in T cells ers designed to hybridize with bisulfite-treated methylated (M) and or un- from 5 lupus patients and age- and sex-matched controls (Fig. 6). methylated (U) CG pairs were used to amplify bisulfite-treated DNA from ϭ There was significantly ( p 0.006) less methylation of the each subject and quantitated relative to a control amplification of an adja- KIR2DL4 promoter in the lupus T cells relative to controls, thus cent sequence lacking CG pairs. The methylation index was then calculated resembling T methylation patterns observed in normal T cells de- as M/(U ϩ M). Results are presented as the means Ϯ SEM of the five methylated with 5-azaC (24). determinations per group. The Journal of Immunology 3485

FIGURE 7. KIR2DL4 on lupus T cells is functional. A, T cells from nine lupus patients and nine age- and sex- matched controls were stimulated with immobilized anti-KIR2DL4 (light hatched bars) or isotype matched con- trol IgG and IFN-␥ release measured as in Fig. 2A. Results represent the means Ϯ SEM of the nine determina- tions. B, The amount of IFN-␥ pro- duced is plotted against the SLEDAI for each of the nine lupus patients. Re- gression analysis was performed as in Fig. 4. increase when stimulated with similar amounts of anti-KIR2DL4 both CD28ϩ and CD28Ϫ T cells from patients with active lu- ( p ϭ 0.001 relative to IgG control). Fig. 7B shows that the amount pus. The mechanism appears to involve demethylation of reg- of IFN-␥ secreted is proportional to disease activity as measured ulatory elements, and it confers INF-␥ secretion and cytotoxic ϭ by the SLEDAI ( p 0.002). This indicates that KIR2DL4 is function on the cells affected. Downloaded from functional. The distribution of KIR genes was similar in lupus patients and We also reported that T cells from patients with active lupus kill controls, although the number of subjects studied was relatively autologous M␾ without added Ag (7, 17). Inhibition of autoreac- small. However, a larger study from Germany also failed to find an tive autologous M␾ killing was therefore used to test inhibitory association of specific KIR genes with SLE (25). We found a KIR function. T cells from 6 KIR3DL1-positive patients with somewhat different distribution of KIR genes in lupus patients and mildly active lupus (SLEDAI 2–4) were cultured with 51Cr-la-

controls relative to this report (25), likely reflecting a different http://www.jimmunol.org/ beled autologous M␾ alone, with anti-KIR3DL1, or with isotype- ethnic makeup of the two populations (19) and/or the smaller num- matched IgG. Fig. 8 shows that anti-KIR3DL1 completely inhibits ber of subjects studied in the present report. the M␾ killing while the control IgG does not, similar to effects ob- KIR molecules on NK cells have stimulatory or inhibitory ϩ served with 5-azaC-treated, KIR T cells (Fig. 2). These results functions, depending on the length of the cytoplasmic domain. indicate that the KIR3DL1 molecules expressed on lupus T cells KIR molecules with short cytoplasmic tails, designated by the are also functional. “S” in the name, are stimulatory, while molecules with long (“L”) cytoplasmic domains are inhibitory, with the exception of Discussion 2DL4, which stimulates IFN-␥ secretion (13). KIR molecules ϩ ϩ KIR genes are expressed on a small subset of CD4 and CD8 on T cells serve similar functions. Others have confirmed that by guest on October 1, 2021 T cells in healthy individuals, and on a somewhat larger senes- KIR molecules on the senescent CD4ϩCD28Ϫ subset have sim- Ϫ cent CD28 subset found in patients with acute coronary syn- ilar stimulatory function, as measured by IFN-␥ secretion (26), dromes (unstable angina and acute myocardial infarctions), or as measured by redirected killing assays (20). KIR function chronic inflammatory diseases such as rheumatoid arthritis, and on 5-azaC-treated T cells appears to serve similar functions. We in the elderly (20–23). This report describes KIR expression on find that cross-linking KIR2DL4 stimulates IFN-␥ secretion, and that cross-linking KIR3DL1 inhibits autoreactive cytotoxic responses to autologous M␾. Thus, the necessary signaling pathways are present in the demethylated T cells to permit KIR function analogous to those found in NK cells. However, since the M␾ killing we used in this report is an autologous system, one would expect that inhibitory KIR molecules recognizing self ligands on the M␾ would prevent the killing. It is possible that a stronger interaction between anti-KIR Abs and the KIR molecules than between KIR and its ligands would result in a stronger inhibitory signal. T cell DNA demethylates in lupus in proportion to disease ac- tivity, and we have found that genes activated by 5-azaC in T cells, including ITGAL (CD11a), PRF1 (perforin), TNFSF7 (CD70), and CD40LG (CD40L) are similarly demethylated and expressed in CD4ϩ T cells from lupus patients with active disease (5, 8–10). The KIR gene family appears to be similarly affected in lupus, with expression directly proportional to disease activity. To the best of our knowledge, this is the first report of T cell KIR expression in lupus. The observation that expression is increased on both CD28ϩ FIGURE 8. KIR3DL1 inhibits autoreactive monocyte/M␾ killing by lu- Ϫ 51 and CD28 T cells in lupus is potentially interesting. KIR expres- pus T cells. T cells from six lupus patients were cultured with Cr-labeled Ϫ autologous monocytes/M␾ and the indicated concentrations of anti- sion on senescent CD28 cells is well described in the literature KIR3DL1 or isotype-matched control IgG as in Fig. 2B. Results are pre- (20, 21, 23). However, the KIR genes were also overexpressed on ϩ sented as the means Ϯ SEM of the results from the six subjects, each the CD28 subset in lupus relative to age- and sex-matched performed in replicates of four. controls. We have reported that inhibiting DNA methylation in 3486 KIR AND LUPUS

ϩ otherwise normal cloned or polyclonal CD4 T cells induces au- 3. Sawalha, A. H., M. Jeffries, R. Webb, Q. Lu, G. Gorelik, D. Ray, J. Osban, toreactivity (27), and that the demethylated T cells in lupus dem- N. Knowlton, K. Johnson, and B. Richardson. 2008. Defective T-cell ERK sig- naling induces interferon-regulated gene expression and overexpression of meth- onstrate similar autoreactivity (17). It is possible that the autore- ylation-sensitive genes similar to lupus patients. Genes Immun. 9: 368–378. activity in lupus results in chronic stimulation leading to 4. Yung, R. L., and B. C. Richardson. 1994. Drug-induced lupus. Rheum. Dis. Clin. N. Am. 20: 61–86. senescence. This is consistent with in vitro models where KIR 5. Lu, Q., M. Kaplan, D. Ray, D. Ray, S. Zacharek, D. Gutsch, and B. Richardson. expression appears on chronically stimulated T cells, correlating 2002. Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus with loss of CD28 expression (28). erythematosus. Arthritis Rheum. 46: 1282–1291. 6. Yung, R., D. Powers, K. Johnson, E. Amento, D. Carr, T. Laing, J. Yang, Expression of KIR molecules on T cells may contribute to S. Chang, N. Hemati, and B. Richardson. 1996. Mechanisms of drug-induced lupus pathogenesis. IFNs contribute to inflammatory processes lupus, II: T cells overexpressing lymphocyte function-associated antigen 1 be- in SLE. While type I IFN overproduction plays an important come autoreactive and cause a lupuslike disease in syngeneic mice. J. Clin. In- ϩ vest. 97: 2866–2871. role (29), KIR T cells responding to self class I MHC or other 7. Kaplan, M. J., Q. Lu, A. Wu, J. Attwood, and B. Richardson. 2004. Demethyl- self molecules may also contribute through IFN-␥ production ation of promoter regulatory elements contributes to perforin overexpression in ϩ (30). Furthermore, autoreactive M␾ killing by demethylated T CD4 lupus T cells. J. Immunol. 172: 3652–3661. 8. Lu, Q., A. Wu, D. Ray, C. Deng, J. Attwood, S. Hanash, M. Pipkin, cells results in increased amounts of antigenic apoptotic mate- M. Lichtenheld, and B. Richardson. 2003. DNA methylation and chromatin struc- rial contributing to autoantibody responses in murine systems ture regulate T cell perforin gene expression. J. Immunol. 170: 5124–5132. 9. Lu, Q., A. Wu, L. Tesmer, D. Ray, N. Yousif, and B. Richardson. 2007. De- and likely in human lupus (31). This autoreactive response may methylation of CD40LG on the inactive X in T cells from women with lupus. be mediated at least in part by stimulatory KIR molecules. The J. Immunol. 179: 6352–6358. observation that cross-linking inhibitory KIR molecules pre- 10. Lu, Q., A. Wu, and B. C. Richardson. 2005. Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with vents this killing suggests a potential therapeutic approach to lupus-inducing drugs. J. Immunol. 174: 6212–6219. Downloaded from lupus, based on recombinant Abs to inhibitory KIR molecules. 11. Liu, Y., R. Kuick, S. Hanash, and B. Richardson. 2008. DNA methylation inhi- Prevention of M␾ killing may decrease antigenic apoptotic ma- bition increases T cell KIR expression through effects on both promoter meth- ylation and transcription factors. Clin. Immunol. 130: 213–224. terial, helping to decrease the autoantibody response. Further- 12. Vilches, C., and P. Parham. 2002. KIR: diverse, rapidly evolving receptors of more, since demethylated T cells are sufficient to cause lupus- innate and adaptive immunity. Annu. Rev. Immunol. 20: 217–251. like autoimmunity, anti-KIR Abs may deplete the demethylated 13. Natarajan, K., N. Dimasi, J. Wang, R. A. Mariuzza, and D. H. Margulies. 2002. Structure and function of natural killer cell receptors: multiple molecular solu- subset also ameliorating the disease. NK cells would also be tions to self, nonself discrimination. Annu. Rev. Immunol. 20: 853–885. depleted, but this subset may be functionally impaired in human 14. Tan, E. M., A. S. Cohen, J. F. Fries, A. T. Masi, D. J. McShane, N. F. Rothfield, http://www.jimmunol.org/ J. G. Schaller, N. Talal, and R. J. Winchester. 1982. The 1982 revised criteria for SLE (32). Furthermore, KIR genes are clonally expressed on the classification of systemic lupus erythematosus. Arthritis Rheum. 25: NK cells (33), so it is possible that only a subset would be 1271–1277. affected. Inhibiting DNA methylation also induces expression 15. Bombardier, C., D. D. Gladman, M. B. Urowitz, D. Caron, and C. H. Chang. 1992. Derivation of the SLEDAI: a disease activity index for lupus patients: The of suppressed KIR genes in NK cells (33), but whether DNA Committee on Prognosis Studies in SLE. Arthritis Rheum. 35: 630–640. methylation is impaired in lupus NK cells is unknown. 16. Oelke, K., Q. Lu, D. Richardson, A. Wu, C. Deng, S. Hanash, and B. Richardson. Interestingly, NK and NKT cells are stimulated by hypom- 2004. Overexpression of CD70 and overstimulation of IgG synthesis by lupus T ␥ cells and T cells treated with DNA methylation inhibitors. Arthritis Rheum. 50: ethylated DNA to secrete IFN- (34, 35), and immune com- 1850–1860. plexes containing hypomethylated DNA can be found in the 17. Richardson, B. C., J. R. Strahler, T. S. Pivirotto, J. Quddus, G. E. Bayliss, by guest on October 1, 2021 peripheral circulation of patients with active lupus (36). How- L. A. Gross, K. S. O’Rourke, D. Powers, S. M. Hanash, and M. A. Johnson. 1992. Phenotypic and functional similarities between 5-azacytidine-treated T cells and ever, this NK response is diminished in lupus patients (37), and a T cell subset in patients with active systemic lupus erythematosus. Arthritis NKT cell numbers are reduced and have impaired function in Rheum. 35: 647–662. 18. Hsu, K. C., S. Chida, D. E. Geraghty, and B. Dupont. 2002. The killer cell lupus (38), so the relevance of this response to lupus pathogen- immunoglobulin-like receptor (KIR) genomic region: gene-order, haplotypes and esis is unclear. allelic polymorphism. Immunol. Rev. 190: 40–52. In summary, KIR molecules are aberrantly overexpressed in 19. Denis, L., J. Sivula, P. A. Gourraud, N. Kerdudou, R. Chout, C. Ricard, J. P. Moisan, K. Gagne, J. Partanen, and J. D. Bignon. 2005. Genetic diversity of T cells from patients with active lupus. They may contribute to KIR natural killer cell markers in populations from France, Guadeloupe, Finland, disease pathogenesis by promoting killing of M␾ and release of Senegal and Reunion. Tissue Antigens 66: 267–276. IFN-␥. Since KIRϩCD4ϩCD28Ϫ T cells have been implicated 20. Nakajima, T., O. Goek, X. Zhang, S. L. Kopecky, R. L. Frye, J. J. Goronzy, and C. M. Weyand. 2003. De novo expression of killer immunoglobulin-like recep- in the pathogenesis of acute coronary syndromes, the KIR mol- tors and signaling proteins regulates the cytotoxic function of CD4 T cells in ecules may also play a role in the cardiovascular complications acute coronary syndromes. Circ. Res. 93: 106–113. of human SLE. Importantly, KIR expression may serve as a 21. Nakajima, T., S. Schulte, K. J. Warrington, S. L. Kopecky, R. L. Frye, J. J. Goronzy, and C. M. Weyand. 2002. T-cell-mediated lysis of endothelial cells marker for pathologic T cells in lupus, and that recombinant Ab in acute coronary syndromes. Circulation 105: 570–575. approaches designed to target and deplete this subset, and po- 22. Weyand, C. M., J. C. Brandes, D. Schmidt, J. W. Fulbright, and J. J. Goronzy. 1998. Functional properties of CD4ϩCD28Ϫ T cells in the aging . tentially inactivate it as well, may be useful therapeutically. Mech. Ageing Dev. 102: 131–147. 23. Namekawa, T., M. R. Snyder, J. H. Yen, B. E. Goehring, P. J. Leibson, C. M. Weyand, and J. J. Goronzy. 2000. Killer cell activating receptors function Acknowledgments as costimulatory molecules on CD4ϩCD28null T cells clonally expanded in rheu- The authors thank Cindy Bourke for her expert secretarial assistance. matoid arthritis. J. Immunol. 165: 1138–1145. 24. Liu, Y., R. Kuick, M. Hanash, and B. Richardson. 2009. DNA methylation in- hibition increases T cell KIR expression through effects on both promoter meth- Disclosures ylation and transcription factors. Clin. Immunl. 130: 213–224. 25. Momot, T., R. E. Schmidt, and T. Witte. 2006. Regulation of killer immuno- The authors have no financial conflicts of interest. globulin-like receptors in systemic lupus erythematosus. J. Rheumatol. 33: 1029. 26. Snyder, M. R., M. Lucas, E. Vivier, C. M. Weyand, and J. J. Goronzy. 2003. Selective activation of the c-Jun NH2-terminal kinase signaling pathway References by stimulatory KIR in the absence of KARAP/DAP12 in CD4ϩ T cells. J. Exp. 1. Quddus, J., K. J. Johnson, J. Gavalchin, E. P. Amento, C. E. Chrisp, R. L. Yung, Med. 197: 437–449. and B. C. Richardson. 1993. Treating activated CD4ϩ T cells with either of two 27. Richardson, B. 1986. Effect of an inhibitor of DNA methylation on T cells, II: distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is suf- 5-Azacytidine induces self-reactivity in antigen-specific T4ϩ cells. Hum. Immu- ficient to cause a lupus-like disease in syngeneic mice. J. Clin. Invest. 92: 38–53. nol. 17: 456–470. 2. Yung, R. L., J. Quddus, C. E. Chrisp, K. J. Johnson, and B. C. Richardson. 1995. 28. Michel, J. J., C. Turesson, B. Lemster, S. R. Atkins, C. Iclozan, T. Bongartz, Mechanism of drug-induced lupus, I: Cloned Th2 cells modified with DNA meth- M. C. Wasko, E. L. Matteson, and A. N. Vallejo. 2007. CD56-expressing T cells ylation inhibitors in vitro cause autoimmunity in vivo. J. Immunol. 154: that have features of senescence are expanded in rheumatoid arthritis. Arthritis 3025–3035. Rheum. 56: 43–57. The Journal of Immunology 3487

29. Crow, M. K. 2007. Type I interferon in systemic lupus erythematosus. Curr. Top. 34. Verthelyi, D., K. J. Ishii, M. Gursel, F. Takeshita, and D. M. Klinman. 2001. Microbiol. Immunol. 316: 359–386. Human peripheral blood cells differentially recognize and respond to two distinct 30. Seery, J. P. 2000. IFN-␥ transgenic mice: clues to the pathogenesis of systemic CPG motifs. J. Immunol. 166: 2372–2377. lupus erythematosus? Arthritis Res. 2: 437–440. 35. Paget, C., E. Bialecki, J. Fontaine, C. Vendeville, T. Mallevaey, C. Faveeuw, and F. Trottein. 2009. Role of invariant NK T lymphocytes in immune responses to 31. Denny, M. F., P. Chandaroy, P. D. Killen, R. Caricchio, E. E. Lewis, CpG oligodeoxynucleotides. J. Immunol. 182: 1846–1853. B. C. Richardson, K. D. Lee, J. Gavalchin, and M. J. Kaplan. 2006. Accelerated 36. Sato, Y., M. Miyata, T. Nishimaki, H. Kochi, and R. Kasukawa. 1999. CpG macrophage apoptosis induces autoantibody formation and organ damage in sys- motif-containing DNA fragments from sera of patients with systemic lupus ery- temic lupus erythematosus. J. Immunol. 176: 2095–2104. thematosus proliferate mononuclear cells in vitro. J. Rheumatol. 26: 294–301. 32. Ohtsuka, K., J. D. Gray, M. M. Stimmler, B. Toro, and D. A. Horwitz. 1998. 37. Zeuner, R. A., D. M. Klinman, G. Illei, C. Yarboro, K. J. Ishii, M. Gursel, and ␤ Decreased production of TGF- by lymphocytes from patients with systemic D. Verthelyi. 2003. Response of peripheral blood mononuclear cells from lupus lupus erythematosus. J. Immunol. 160: 2539–2545. patients to stimulation by CpG oligodeoxynucleotides. Rheumatology (Oxford) 33. Santourlidis, S., H. I. Trompeter, S. Weinhold, B. Eisermann, K. L. Meyer, 42: 563–569. P. Wernet, and M. Uhrberg. 2002. Crucial role of DNA methylation in determi- 38. Kojo, S., Y. Adachi, H. Keino, M. Taniguchi, and T. Sumida. 2001. Dysfunction nation of clonally distributed killer cell Ig-like receptor expression patterns in NK of T cell receptor AV24AJ18ϩ, BV11ϩ double-negative regulatory natural killer cells. J. Immunol. 169: 4253–4261. T cells in autoimmune diseases. Arthritis Rheum. 44: 1127–1138. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021