Differential Expression of Natural Killer Receptors on V[delta]1 [gamma][delta] T Cells in HIV-1-Infected Individuals

γδ T cells expressing the Vδ1 T-cell receptor are increased in HIV-1-infected individuals. Since T-cell activation is modulated by inhibitory and activating natural killer receptors (NKRs), we investigated the expression of NKRs on Vδ1 T cells in 22 HIV-1 infected patients by 2-color flow cytometry. We observed a strongly increased expression of several NKRs on ex vivo analyzed Vδ1 T cells from HIV⁺ patients. Furthermore, we show that the in vitro stimulation of CD94^- Vδ1 T cells induced strong expression of inhibitory NKR CD94/CD159A (NKG2A). Our results suggest that the increased expression of NKRs might result from chronic activation of Vδ1 T cells in HIV-1-infected persons.

One percent to 10% of the peripheral blood T lymphocytes express a γδ T-cell receptor (TCR) instead of the conventional αβ TCR. ¹ Significant alterations of γδ T-cell subset distribution are observed in HIV-1-infected individuals already at an early stage of the infection. While Vδ2/Vγ9 is preferentially used by peripheral blood γδ T cells in healthy adults, Vδ1 T cells dominate among γδ T cells in HIV⁺ patients. ^2-5 Increased numbers of Vδ1 T cells are present in HIV and cytomegalovirus infections but not in other chronic virus infections such as Epstein-Barr or hepatitis B. ^2,3,6,7 These observations suggest that Vδ1 T cells play a significant role during HIV infection.

Human natural killer (NK) cells and subsets of T cells including Vδ2/Vγ9 γδ T cells express members of a multigenic and mutiallelic family of natural killer receptors (NKRs) composed of Ig-like receptors or lectin-like dimers. ⁸ NKRs bind to classic or nonclassic MHC class I molecules and can deliver inhibitory or stimulatory signals. Among the Ig-like receptors, CD158a (p58.1) and CD158b (p58.2) carry immune tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tail and prevent killer-cell-mediated cytotoxicity, while p50.1 and p50.2, with a shorter cytoplasmic tail, transmit activating signals to cytotoxic cells. ⁹ Furthermore, the killer-inhibitory p70 molecule detected by monoclonal antibody (mAb) NKB1 inhibits cytotoxic cells from killing target cells that express HLA-Bw4. ¹⁰ Among the lectin-like dimers, the invariant CD94 can be associated with a variety of CD159 (NKG2) molecules that recognize nonclassic MHC molecules (e.g., HLA-E, MICA) on target cells. The CD94/CD159A (CD94/NKG2-A) heterodimer is an inhibitory receptor because the CD159A chain contains an ITIM motif. ¹¹

In the present study we analyzed the expression of NKRs on the Vδ1 T-cell subset in HIV-infected patients. Our results demonstrate a strikingly increased expression of NKRs on Vδ1 T cells in HIV-1⁺ subjects compared with healthy donors.

MATERIALS AND METHODS

Patients

Peripheral blood was obtained from 22 HIV-1 infected individuals (CDC stages A1-A3, 9 patients; B1-B3, 7 patients; C3, 6 patients) and from 15 healthy age-matched donors. None of the patients was on treatment with IL-2, while 17 of the patients received protease inhibitors or various combinations of reverse transcription inhibitors (Table 1). Informed consent was obtained from all donors.

Table 1

Isolation of Lymphocytes and Separation of CD94^- Vδ1 T Cells

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Biochrom, Berlin, Germany) density gradient centrifugation. Vδ1 T cells were separated by positive selection using the Dynabeads system (Dynal, Hamburg, Germany). PBMCs were incubated with unconjugated anti-Vδ1 mAb followed by magnetic particles coated with antimouse Ig (Dynal). Positively selected Vδ1 T cells were propagated with irradiated feeder cells (Epstein-Barr virus-transformed lymphoblastoid cell lines [LCLs] and allogeneic PBMCs), phytohemaglutinin (PHA), and IL-2 for 1 week because of the low yields of Vδ1 T cells within PBMCs. Thereafter, CD94/CD159A^- Vδ1 T cells were purified by negative depletion procedure using magnetic cell sorting with MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). Vδ1 T cells were stained with unconjugated anti-CD94/anti-CD159A mAb followed by magnetic microparticles coated with antimouse Ig (Miltenyi Biotech). The negatively selected Vδ1 T cells were routinely 99.9% pure and were restimulated again as described above. The induction of CD94/CD159A was analyzed by flow cytometry. The magnetic cell separation followed by stimulation with feeder cells was repeated 3 times.

Antibodies

The following monoclonal antibodies were used as FITC- or phycoerythrin (PE)-conjugates or unconjugated (unconj.): Vδ1-FITC from Endogen (Biozol, Eching, Germany); unconj. Vδ1, unconj. CD94, CD94-PE (HP-3B1), unconj. CD159A (NKG2A), CD159A-PE (Z199), CD158a-PE (p58.1 and p50.1; clone EB6), and CD158b-PE (p58.2 and p50.2; clone GL183) from Immunotech/ Beckman Coulter (Krefeld, Germany); NKB1-PE (clone DX9), CD28-PE, CD45RO-PE, CD3-PE, and TCR γδ-1-PE from Becton Dickinson (Heidelberg, Germany). As a second step antibody FITC-conjugated goat F(ab)₂ antimouse IgG&M (Medac, Hamburg, Germany) was used. After staining, cells were fixed in 1% paraformaldehyde and analyzed on a FACScan flow cytometer (BD) using the Cell Quest software.

Statistical Analysis

Student t test was used to analyze the statistical significance.

RESULTS AND DISCUSSION

Increased NKR Expression on Vδ1 T Cells From HIV⁺ Donors

Increased expression of NKR on T lymphocytes is frequently observed in HIV-1 infected individuals. ^12,13 Moreover, HIV infection is also characterized by a striking increase in Vδ1-expressing γδ T cells. ^2-6,14 NKR expression on Vδ1 γδ T cells from HIV⁺ individuals, however, has not yet been investigated in detail. Results of a representative experiment with an HIV-1-seronegative and a seropositive donor are shown in Figure 1. As can be seen, there was a strongly enhanced expression of all analyzed NKRs (CD94, CD159A/NKG2A, CD158a, CD158b, NKB1) on Vδ1 cells from the HIV-1⁺ as compared with the HIV-1^- donor. Two populations of CD94^bright and CD94^dim Vδ1 cells were clearly discernible (Fig. 1B). Others have reported that CD94^bright staining as detected with mAb HP-3B1 correlates with the expression of the inhibitory CD94/CD159A (NKG2-A) heterodimer. ¹⁵ Therefore, we used HP-3B1 (CD94) and Z199 (CD159A) mAb in our analysis. The results obtained with 15 HIV-1^- and 22 HIV-1⁺ donors are summarized in Figure 2. Vδ1 T cells of healthy donors expressed lower levels of NKR (CD158a: 4.2 ± 3.1%, CD158b: 22.3 ± 16.1%, NKB-1: 4.2 ± 3.3%, Fig. 2A) than HIV-1⁺ donors. CD158a as well as CD158b mAb do not allow to distinguish between killer inhibitory receptor and killer activatory receptor by flow cytometry. CD94^dim expression was determined on 25.4 ± 21.6% of Vδ1 T, while a CD94^bright phenotype was observed on 12.6 ± 23.8% of Vδ1 T cells from 12 of 14 tested healthy donors (Fig. 2A). In fact, however, only in 4 of the 14 healthy donors did >10% of Vδ1 T cells express CD94^bright. Conversely, the expression of NKRs was significantly increased on Vδ1 T cells from HIV-1 infected individuals (CD158a: 19.5 ± 13.9%; CD158b: 48.2 ± 19.4%, NKB-1: 14.6 ± 11.8%, Fig. 2B). Interestingly, higher levels of CD94^bright (39.3 ± 17.8%) and of CD94^dim phenotype (44.2 ± 21.1%, Fig. 2B) were present on Vδ1 T cells from 22 HIV⁺ donors. The increase in the NKR expression on Vδ1 T cells occurred independently of the stage of HIV infection, the CD4 cell count, secondary diseases, or the treatment with nucleoside reverse transcription inhibitors or protease inhibitors as demonstrated in Table 1. All differences between HIV^- and HIV⁺ donors were statistically significant at P < 0.01. Further analysis of the Vδ1 T cells from healthy donors and HIV⁺ patients revealed that increased NKR expression was associated with an absence of CD28 (Fig. 2) and an increased expression of CD45RO (Fig. 1G). These data are consistent with a memory phenotype of inhibitory NKR-expressing T cells ¹³ and additionally indicate that Vδ1 T cells are in an activated state in vivo.

Figure 1

Figure 2

Stimulation of CD94^- Vδ1⁺ T Cell-Induced Upregulation of CD94/CD159A

Upregulation of CD94^bright expression Vδ1 T cells in HIV⁺ donors could be due to the chronic activation status occurring during HIV infection. Therfore, we analyzed whether in vitro stimulation induced CD94 expression on CD94^- Vδ1 T cells. As shown in Figure 3, negatively isolated CD94^- Vδ1 T cells from 1 healthy donor and 2 HIV⁺ individuals (1 representative is shown) were stimulated with irradiated feeder cells, PHA, and IL-2. All γδ T cells expressed Vδ1 (Fig. 3B, I) and were CD94^- and CD159A (NKG2-A)^- after magnetic sorting (Fig. 3C, D, J, K). Interestingly, expression of CD94 was induced on CD94^- Vδ1 T cells within 7 to 8 days of in vitro culture. The induction of CD94 closely correlated with the induction of CD159A in all 3 tested donors (Fig. 3G, N). All Vδ1 T-cell populations lacked CD8 and CD28 whereas they all expressed CD45RO, before and after restimulation (not shown). Furthermore, we depleted the CD94⁺ Vδ1 T cells each time before restimulation (total of 3 times) and observed a reinduction of CD94/CD159A expression every time in all tested Vδ1 T cell lines. Additionally, we observed a strong upregulation of CD94/CD159A Vδ1 T cells in freshly isolated PBMCs 6 days after stimulation with LCLs (not shown). In contrast to CD94, no significant modulation of Ig superfamily type NKR expression on Vδ1 T cells was observed under the different culture conditions (not shown).

Figure 3

In addition to their role in regulating NK cell cytotoxicity, NKRs also modulate the reactivity pattern of T cells. In this respect, it has been shown that NKRs expressed on Vδ2Vγ9 T cells regulate the activation and cytolytic effector function, thereby preventing damage to normal cells by otherwise self-reactive γδ T cells. ^12,16-19 Moreover, inhibitory NKRs can also downregulate TCR-mediated T-cell activation. Thus, triggering of CD94/CD159A downmodulates γδ TCR expression upon stimulation with phosphoantigens and inhibits the cytokine production and proliferation of γδ T cells. ^18,20,21 In agreement with results of Poccia et al., ²⁰ we observed that Vδ1 T cells of healthy donors expressed low levels of NKRs. Surprisingly, however, NKRs and especially inhibitory receptors such as CD94/CD159A and NKB1 (p70) were much more strongly expressed on the Vδ1 T-cell subset of HIV-infected individuals. It has been shown that Vδ1 T cells recognize unidentified ligands on LCLs, on some Burkitt lymphoma cells, and on activated B lymphocytes from HIV⁺ individuals. ^22-24 We observed an upregulation of CD94/CD159A on freshly isolated Vδ1 T cells from HIV⁺ donors after culture with LCL (not shown) and an induction of CD94/CD159A when CD94^- Vδ1 T cells were stimulated with feeder cells plus PHA (Fig. 3). In conclusion, we have shown strongly increased expression of NKRs on Vδ1 T cells in HIV-1⁺ patients. Our in vitro analysis using CD94^- Vδ1 T cells suggests a functional significance of the induced expression of the inhibitory receptor CD94/CD159A that requires further investigation.

Acknowledgments:

The authors thank Dr. D. Schuster for kindly providing blood samples of HIV-1 infected patients. The technical assistance of Hoa Ly and Claudia Wild is gratefully acknowledged.