Regulation of activated CD4+ T cells by NK cells via the Qa-1-NKG2A inhibitory pathway

Abstract

The ability of natural-killer cells to regulate adaptive immunity is not well understood. Here we define an interaction between the class Ib major histocompatibility complex (MHC) molecule Qa-1-Qdm on activated T cells responsible for adaptive immunity and CD94-NKG2A inhibitory receptors expressed by natural-killer cells by using Qa-1-deficient and Qa-1 knockin mice containing a point mutation that selectively abolishes Qa-1-Qdm binding to CD94-NKG2A receptors. The Qa-1-NKG2A interaction protected activated CD4+ T cells from lysis by a subset of NKG2A+ NK cells and was essential for T cell expansion and development of immunologic memory. Antibody-dependent blockade of this Qa-1-NKG2A interaction resulted in potent NK-dependent elimination of activated autoreactive T cells and amelioration of experimental autoimmune encephalomyelitis. These findings extend the functional reach of the NK system to include regulation of adaptive T cell responses and suggest a new clinical strategy for elimination of antigen-activated T cells in the context of autoimmune disease and transplantation.

Figure 1. Proliferation of Qa-1 WT and Qa-1-Deficient CD4⁺ T Cells

(A) CD4⁺ T cells isolated from either C57BL/6 (B6) Qa-1WTor B6 Qa-1-deficient (N11) mice were transferred into syngeneic Rag2^−/− or Rag2^−/−Prf1^−/− hosts. 14 days later, CD4⁺ T cells from spleen and LN were enumerated. Data are from one representative experiment of three in which each group consisted of 3–4 mice. Graphs show mean ± SD (n = 3). (B) Purified Qa-1 WT or Qa-1-deficient OTII CD4⁺ T cells were transferred (10⁶/mouse) into Rag2^−/− or Rag2^−/−Prf1^−/− hosts along with immunization of 50 µg OVA peptide emulsified in CFA. Proliferation of OTII cells was monitored by enumeration of CD4⁺V^β5⁺ cells in LN and spleen 14 days later. NK cells (0.5 × 10⁶) purified from splenocytes of Rag2^−/− mice were cotransferred with Qa-1-deficient OTII cells into Rag2^−/− Prf1^−/− hosts, as indicated. Data are from one representative experiment of three in which each group consisted of 3–4 mice. Graphs show mean ± SD (n = 3). (C) Peripheral T cell numbers in B6 Qa-1 WT and Qa-1-deficient mice: Total numbers of CD4⁺ and CD8⁺ T cells from pooled spleen and lymph node of 5- to 6-week-old Qa-1-deficient (KO) mice or its wild-type (WT) littermates (4–6 mice/group). Graphs show mean ± SD (n = 4). (D) Qa-1-deficient or WT mice were thymectomized (TX) or sham thymectomized at around 10 weeks age. The numbers of CD4⁺ and CD8⁺ cells in peripheral lymphoid tissues 6 weeks after either thymectomy or sham thymectomy of Qa-1 WT or Qa-1-deficient mice at 10 weeks of age (3 mice/group) were measured. Data shown represent the average of 3 pooled mice.

Figure 2. Cytokine Recall Response upon OTII Peptide Immunization

Qa-1 WT or Qa-1-deficient mice bearing the OTII TCR transgene were immunized with 25 µg OVA peptide emulsified in CFA. 14 days later, OTII CD4⁺ T cells were purified from draining LN. To deplete NK cells, one group of mice was injected i.v. with NK1.1 antibody (200 µg/ mouse) at day −1 and day 6 after peptide immunization. Control mice were injected with PBS (3–4 mice/group). (A) Increasing numbers of purified CD4⁺ T cells were stimulated in vitro with OVA peptide (10 µg/ml) and irradiated splenocytes (4 × 10⁵). Supernatants were collected 48 hr later and tested for IFN-γ by ELISA. Data shown represent mean ± SD (n = 3). (B) Purified OTII CD4⁺ T cells (1 × 10⁵) were stimulated with the indicated concentrations of OVA peptide before collection of culture supernatant 48 hr later and testing for IFN-γ by ELISA. Data shown represent mean ± SD (n = 3).

Figure 3. Development and Function of Qa-1 WT and Qa-1-Deficient T Cells in Mixed Bone-Marrow Chimeras

(A) Hematopoietic stem cell (HSC)-enriched bone-marrow cells (10⁶/mouse) from Qa-1 WT (Thy1.1) and Qa-1-deficient (KO; Thy1.2) mice was cotransferred into Rag2^−/− or Rag2^−/−Prf1^−/− hosts and sacrificed at 4 or 12 weeks after transfer (4 mice/group). The total number of peripheral CD4⁺ and CD8⁺ T cells from spleen and LN were enumerated. Data shown represent the average of 3 pooled mice. (B) BM chimeric hosts were immunized subcutaneously with PLP peptide 12 weeks after adoptive transfer of BM cells. 14 days after immunization, total CD4⁺ and CD8⁺ T cell numbers in draining LN were enumerated. Data shown represent the average of 3 pooled mice. (C) Pooled (n = 3) Qa-1 WT (Thy1.1) and Qa-1-deficient (Thy1.2) CD4⁺ T cells were purified from draining LN and stimulated with PLP peptide together with irradiated splenocytes. IFN-γ secretion was measured after 48 hr. (D) The indicated subset of CD4⁺ T cells from the draining LN were analyzed for CD44 and CD5 expression by FACS. Gated on CD4 cells: the solid line represents Thy1.1 (WT); the dotted line represents Thy1.2 (KO).

Figure 4. Reduced Ability of Qa-1-Deficient 2D2 CD4⁺ T Cells to Transfer EAE into Rag2^−/− Mice

(A) 2D2 CD4⁺ T cells (1 × 10⁶) from either Qa-1 WT or Qa-1-deficient mice were transferred into syngeneic Rag2^−/− or Rag2^−/−Prf1^−/− mice (5 mice/group) immunized with MOG 35–55 and CFA and pertussis toxin. Development of EAE was scored as described in Experimental Procedures. The bar graph represents the disease incidence of the different groups. (B) 2D2 CD4⁺ T cells (1 × 10⁶) from either Qa-1 WT or Qa-1-deficient mice were transferred into C57BL/6 hosts (5 mice/group) followed by immunization with (150 µg) MOG 35–55 peptide with pertussis toxin on the same day and day 2 to induce EAE. The development of EAE is shown, as in (A), and disease incidence of the different groups is shown in the bar graph. (C) Antigen-driven proliferation of OTII cells in Rag2^−/− and Rag2^−/−Prf1^−/− hosts. OTII CD4⁺ T cells (1 × 10⁶) were transferred into Rag2^−/− or Rag2^−/− Prf1^−/− mice (4 mice/group) before immunization with OVA peptide (75 µg peptide–CFA) immediately after transfer. Mice were taken down 72 hr later, and total OTII cells in the draining LN and spleen were enumerated. On the right panel, OTII CD4⁺ T cells infected with lentivirus expressing either GFP control or Qdm-β₂m-Qa-1 fusion protein were also transferred into Rag2^−/− or Rag2^−/−Prf1^−/− hosts (4 mice/group) before immunization with OVA peptide (75 µg peptide–CFA). 72 hr later, expansion of OTII cells was analyzed as described above. Data shown represent mean ± SD of the recovery OTII CD4⁺ T cells from draining lymph nodes.

Figure 5. In Vitro Lysis of Qa-1-Deficient CD4⁺ T Cells by IL-2-Activated NKG2A⁺ NK Cells

(A) NKG2A⁺ and NKG2A⁻ NK cells isolated from B6 mice and activated individually by IL-2 were used at the indicated E:T ratios as effector killer cells to kill ConA-activated CD4⁺ T cells from Qa-1 wild-type and Qa-1-deficient mice in a standard killing assay. This experiment is representative of a total of four experiments. Data shown represent mean ± SD (n = 3). (B) Protection of Qa-1-deficient CD4 cells from NK lysis by lentiviral-mediated surface expression of a covalent Qdm-β₂m-Qa-1 complex. Qa-1-deficient (Qa-1 KO) OTII cells were infected with lentivirus expressing either a Qdm-β₂m-Qa-1 fusion protein (QbQ) or a HSP60 peptide-β₂m-Qa-1 fusion protein (HbQ) and used as target cell in the killing assay by NKG2A⁺ NK cells. Qa-1 WT and Qa-1-deficient OTII cells were used as control. Percentage of killing is shown at the indicated E:T ratios. (C) CD4 cells were purified from Qa-1 WT and Qa-1-deficient OTII TCR transgenic mice, and splenic DCs were purified from Qa-1 WT and Qa-1-deficient mice and activated by anti-CD40. NKG2A⁺ and NKG2A⁻ NK cells were purified from B6 mice and stimulated with 1000 U/ml IL-2 for 5 days. OTII CD4⁺ T cells (5 × 10⁴) were stimulated with 1 µg/ml OVA peptide and 2.5 × 10⁴ activated DC. The indicated numbers of IL-2-activated NK cells were added to cultures 48 hr before proliferation of CD4⁺ T cells was measured. Data shown represent mean ± SD (n = 3). (D) Adoptive transfer and homeostatic expansion of NK cells. NKG2A⁻ and NKG2A⁺ NK cells were purified from the spleens of B6 mice by FACS sorting and injected i.v. (5 × 10⁵) into Rag2^−/− γc-deficient mice. 10 days later, spleens of host mice were analyzed for NK cells by staining with antibodies against NK1.1 and NKG2A. This result represents three different experiments with 3–4 mice/group.

Figure 6. NK Killing Susceptibility of Qa-1-Deficient Cells Can Be Mimicked by a Point Mutation at R72 that Disrupts Its Binding to NKG2A

(A) NK cell susceptibility of R72A mutant CD4 cells. CD4⁺ T cells from Qa-1 WT, Qa-1-deficient, and Qa-1-R72A mutant mice were activated by ConA for 48 hr, labeled with ⁵¹Cr, and used as targets for IL-2-activated NKG2A⁺ and NKG2A⁻ NK cells in a standard 4 hr killing assay. Percentage of killing at E:T ratio of 20:1 is shown. Data shown represent mean ± SD (n = 3). (B) Homeostatic expansion of R72A mutant CD4 cells. CD4⁺ T cells (1 × 10⁶) were isolated from Qa-1 WT, Qa-1-deficient, and Qa-1-R72A mutant mice and i.v. transferred into Rag2^−/− and Rag2^−/−Prf1^−/− host (3 mice/group). 14 days later, host mice were killed and total CD4⁺ T cells were enumerated from spleen and LN. Data shown represent mean ± SD (n = 3).

Figure 7. Antibody Blockade of Qa-1–NK Cell Interaction Inhibits the Development of EAE

EAE were induced in C57BL/6 mice as described in Experimental Procedures via MOG peptide. Qa-1 antibody was injected i.v. (200 µg/injection) at days 5, 9, and 12 after peptide immunization. In a separate group, anti-NK1.1 was administered (150 µg, i.v.) at days 3, 6, and 10 to deplete NK cells with or without injection of Qa-1 antibody on days 5, 9, and 12, as above. Control groups were injected with either PBS or mouse IgG1 isotype control antibody (5 mice/group). The development of disease was monitored as described in Experimental Procedures. Data shown represent two independent experiments.