Receptors of CD4 T Cells Expressing Killer Ig-Like Phenotypic And

The Journal of Immunology Phenotypic and Functional Characterization of CD4 T Cells Expressing Killer Ig-Like Receptors1 Jeroen van Bergen,2* Allan Thompson,* Arno van der Slik,* Tom H. M. Ottenhoff,* Jacobijn Gussekloo,† and Frits Koning* Killer Ig-like receptors (KIR) are commonly found on human NK cells, ␥␦ T cells, and CD8 T cells. Although KIR؉ CD4 T cells are found in certain patients, their prevalence in healthy donors is controversial. We now provide definitive proof that such cells are present in most individuals, and report on their frequency, surface phenotype, cytokine profile, and Ag specificity. The number of KIR؉ CD4 T cells detected in peripheral blood increased with age. In contrast with regular KIR؊ CD4 T cells, the majority ,of KIR؉ CD4 T cells lacked surface expression of CD27, CD28, CCR4, and CCR7, but did express CD57 and 2B4. In addition KIR were detected on approximately one-tenth of CD28؊ and CD57؉ memory CD4 T cells. In line with the absence of the Th2 ,marker CCR4, the KIR؉ CD4 cells produced mainly IFN-␥ and little IL-4, IL-10, or IL-17 upon TCR triggering. Furthermore the KIR؉ population contained cells that responded to recall Ags in an HLA class II-restricted fashion. Together, our data indicate that KIR-expressing CD4 T cells are predominantly HLA class II-restricted effector memory Th1 cells, and that a significant, previously unrecognized fraction of effector memory Th1 cells expresses KIR. The Journal of Immunology, 2004, 173: 6719–6726. atural killer cells use a wide variety of surface receptors KIR expression by CD4 T cells is scarce. However, in patients to recognize infected and malignantly transformed cells. with certain diseases KIR are frequently detected on an expanded The killer Ig-like receptors (KIR)3 constitute the largest CD28Ϫ subset of CD4 T cells (13, 14). In rheumatoid arthritis, N Ϫ family of human NK receptors, with multiple inhibitory and acti- CD28 CD4 T cells are cytolytic and they proliferate in response vating members (1). The inhibitory receptors bind classical HLA to autologous PBMC (15, 16). Moreover, their number correlates class I molecules and mediate “missing self” recognition. The with disease severity (17). When compared with CD28Ϫ CD4 T physiological ligands for the activating KIR are unknown. In ad- cells from healthy control subjects, the cells preferentially express dition to NK cells, T cells also frequently express KIR. However, the disease-associated activating KIR2DS2 molecule (9), which the role these NK receptors play in T cell responses remains costimulates TCR-mediated IFN-␥ production and proliferation largely enigmatic (2). (13). In acute coronary syndromes, CD28Ϫ CD4 T cells can be The KIR family displays a high degree of polymorphism, which activated by KIR cross-linking directly, without the need for TCR is associated with disease. The KIR locus is polymorphic with engagement (14). The Ag specificity of the above-described KIRϩ by guest on September 30, 2021. Copyright 2004 Pageant Media Ltd. respect to both gene content and sequence (3–5). KIR haplotypes CD4 cells is unknown. contain between 5 and 12 highly homologous KIR genes (4), and There are conflicting reports on KIR expression on CD4 T cells up to 9 alleles for individual KIR genes have been described (5). in healthy individuals. According to Anfossi et al. (12), on average KIR3DS1 and its proposed ligand HLA-Bw4 confer resistance to 7 Ϯ 3% (ϮSEM) of KIRϩ T cells carry CD4, indicating that the the development of full-onset AIDS in HIV-infected individuals large majority of healthy donors has KIRϩ CD4 T cells. Indeed, a (6). Also, the KIR2DS2 gene is associated with various autoim- number of KIRϩ CD4 T cell clones have been isolated from mune conditions, such as psoriatic arthritis (7), diabetes (8), and healthy donors (18, 19). In contrast, Warrington et al. (20) detected vascular complications in rheumatoid arthritis (9). KIR expression selectively on CD28Ϫ CD4 T cells, an extremely KIR expression by TCR-␥␦ϩ T cells and a subset of CD8ϩ rare subset in healthy individuals. By consequence, they detected https://www.jimmunol.org TCR-␣␤ϩ cells is firmly established (10–12), but information on KIRϩ CD4 T cells only in a small minority of individuals, con- taining increased frequencies of CD28Ϫ CD4 cells (9, 14). We now provide definitive proof that KIRϩ CD4 T cells are present in *Department of Immunohematology and Blood Transfusion and †Section of Geron- most individuals, and report on their frequency, surface phenotype, tology and Geriatrics, Department of General Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands cytokine production, and Ag specificity. Downloaded from Received for publication June 23, 2004. Accepted for publication September 17, 2004. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page Subjects charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. After informed consent was obtained, peripheral blood cells were obtained 1 This work was supported by the Netherlands Organisation for Scientific Research from healthy adult and 90-year-old donors. Healthy adults were random (NWO) VENI Grant 916.36.025 (to J.v.B.) and the Diabetes Fonds Nederland/Juve- blood bank donors ranging in age from 18 to 70 years (n ϭ 30). Healthy nile Diabetes Research Foundation/Netherland Organisation for Health Research and 90-year-old donors (n ϭ 10) were enrolled in the cohort of the Leiden Development Grant 2001.10.004 (to A.v.d.S.). T.H.M.O. is supported by the NWO, 85-plus study (21, 22), a prospective population-based study of inhabitants the Netherlands Leprosy Foundation, and the European Commission. of Leiden, The Netherlands. Health was defined as no acute illness, no 2 Address correspondence and reprint requests to Dr. Jeroen van Bergen, Department of death within 3 mo after blood sampling, no severe cognitive impairment Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box and a plasma C-reactive protein level below 10 mg/L. Umbilical cord blood 9600, 2300 RC Leiden, The Netherlands. E-mail address: [email protected] (UCB) cells were derived from the umbilical vein after normal full-term 3 Abbreviations used in this paper: KIR, killer Ig-like receptor; ILT, Ig-like transcript; deliveries (n ϭ 7). Heparinized blood was collected and PBMC or cord LAK, lymphokine-activated killer; UCB, umbilical cord blood. blood mononuclear cells were isolated by density gradient centrifugation Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 6720 KIR EXPRESSING Th CELLS: EFFECTOR MEMORY Th1 CELLS over Ficoll-Hypaque, and subsequently stored at Ϫ80°C in 10% DMSO/ temperature of 61°C, followed by 29 cycles with an annealing temperature 20% FCS until use. of 60°C. All PCRs were conducted in a Peltier Thermal Cycler (PTC-200; MJ Research, Waltham, MA). Antibodies TCR repertoire analysis Directly conjugated Abs to CD3, CD8, CD25, CD27, CD45RA, CD45RO, CD56, CD57, CD94, CCR4, CCR7, TCR-␣␤, KIR3DL1 (CD158e1, clone TCR-␤ chain variable gene segment usage of the clones was established by DX9), and isotype controls were obtained from BD Biosciences (San Jose, FACS using the IOTest ␤ Mark TCR kit (Beckman Coulter), TCR-␣ chain CA) and to CD4 from DAKO (Cytomation, Glostrup, Denmark). The PE- variable gene segment usage was determined by RT-PCR using TCR-␣ conjugated EB6 (CD158a, CD158h, anti-KIR2DL1, KIR2DS1), GL183 chain variable gene segment family specific primers (26). The CDR3 re- (CD158b, CD158j, KIR2DL2, KIR2DL3, KIR2DS2), FES172 (CD158i, gions of the TCRs of selected clones were amplified by PCR, cloned into KIR2DS4), and CD244 (2B4) Abs were from Beckman Coulter (Mijdre- the pCR2.1 vector using TA cloning kit (Invitrogen Life Technologies), cht, The Netherlands). and then sequenced. Unconjugated Abs to NKG2D (BAT221), NKp30 (A76), NKp46 T cell functional assays (BAB281), NKp44 (Z231), and Ig-like transcript (ILT)-2 (F278) were a kind gift from Dr. D. Pende (Instituto Nazionale per la Ricerca sul Cancro, PBMC from HLA-DR3/HLA-DR4 heterozygous individuals were stained Genova, Italy), anti-KIR2D Ab (NKVSF1) from Dr. A. Poggi (Instituto with CD4-FITC and a mix of PE-conjugated Abs (EB6, GL183, DX9, Nazionale per la Ricerca sul Cancro), and anti-KIR2DL4 Ab (cl-33; Ref. FES172), and subsequently plated at 1000 cells/well by a BD Biosciences 23) from Dr. E. O. Long (National Institutes of Health, Bethesda, MD). FACSVantage flow cytometer using CellQuest software. Three wells (3000 Binding of unconjugated Abs was detected using goat anti-mouse IgG cells) of KIRϩ and three wells of KIRϪ CD4 cells were expanded twice, F(ab)2-RPE (DAKO). using mixed irradiated allogeneic PBMC supplemented with 1 ␮g/ml PHA and 20 U/ml IL-2, resulting in 50–100 million cells of each type. This in Flow cytometry vitro expansion did not affect surface KIR expression patterns. ϩ Ϫ Cells were stained with CD3-PerCP, CD4-allophycocyanin, KIR-PE (EB6, The KIR and KIR CD4 bulk cultures were stimulated with plate- ␮ GL183, DX9, FES172 mixed or separately), combined with an Ab to an bound anti-CD3 (1 g/ml in PBS; Janssen-Cilag, Tilburg, The Nether- additional surface marker conjugated to FITC. PE-conjugated Abs to a lands) in 96-well flat-bottom plates (25,000 cells/well). Culture superna- Ϫ limited number of surface markers (CD94, CD244, CCR4, CCR7, CXCR4) tants were harvested 24 h after stimulation and stored at 20°C. Cytokine were combined with the CH-L FITC Ab (directed to KIR2DL2, KIR2DL3, concentrations were determined using the Becton Dickinson Th1/Th2 Cy- KIR2DS2). Staining was performed at 4°C for 1 h, followed by three tometric Bead Array kit according to the manufacturer’s protocols.

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