NKG2D signaling on CD8⁺ T cells represses T-bet and rescues CD4-unhelped CD8⁺ T cell memory recall but not effector responses

Abstract

CD4-unhelped CD8(+) T cells are functionally defective T cells primed in the absence of CD4(+) T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8(+) T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8(+) T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-γ production and cytolytic responses. Rescue is abrogated in CD8(+) T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4(+) T cells in a CD4-dependent influenza model and rescues HIV-specific CD8(+) T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8(+) T cells from their pathophysiological fate and may provide therapeutic benefits.

Figure 1

NKG2D engagement by Rae-1ε rescues CD4-unhelped CD8⁺ T cell memory recall expansion. (a) Experimental design for vaccination and CD4 depletion. (b) Expansion kinetics of OVA-tetramer⁺ CD8⁺ T cells (±SEM) calculated per spleen. (c) Mean number of OVA-tetramer⁺ CD8⁺ T cells (+SEM) per spleen on days 38 and 43. (d) Data from (c) shown as a fold change. Data are representative of 3–5 mice analyzed individually per group per experiment from three experiments conducted with similar results. *P < 0.05.

Figure 2

NKG2D-mediated rescue of CD4-unhelped CD8⁺ T cell memory recall responses involves cytokine and lytic molecule production and CTL lysis. (a) Splenic OVA-tetramer⁺ CD8⁺ T cell production of granzyme B, IL-2 and IFN-γ on day 43 as described in Fig. 1a. (b) Experimental design and in vivo specific lysis (%) of OVA_257–264-loaded (CFSE^lo) and irrelevant peptide-loaded (hgp100_25–33, CFSE^hi) targets from a control (Empty/Empty) mouse. (c,d) Specific lysis (%) from mice vaccinated with OVA/Empty, OVA/Rae-1ε, and OVA/H60 as described in (b) without (c) and with (d) CD4 depletion. Data are representative of 3–5 mice analyzed individually per group per experiment from at least three experiments conducted with similar results. *P < 0.05.

Figure 3

Rescue of CD4-unhelped CD8⁺ T cell memory recall responses is dependent on NKG2D expression on CD8⁺ T cells. (a) Memory recall (day 43) specific lysis (% ⁺ SEM) of C57BL/6 mice vaccinated as described in Fig. 1a and treated with NKG2D blocking antibody on days 0, 5, and 10. (b,c) NK1.1 expression in the spleen (b) and specific lysis (% ⁺ SEM) (c) of C57BL/6 mice depleted of NK1.1 cells on days −5 and −3, adoptively transferred Thy1.1-marked naïve OT-I (OVA_257–264-specific) CD8⁺ T cells (2*10⁶ per mouse) on day −1, and vaccinated on day 0 ± CD4 depletion on days −2 and 0. (d) CD45.1 expression on transferred (CD45.1⁻) and endogenous (CD45.1⁺) OVA-tetramer⁺ CD8⁺ T cells. ACT = adoptive cell transfer; WT = wild type C57BL/6. (e) Mean number of OVA-tetramer⁺ CD8⁺ T cells (+SEM) from (d) per spleen on day 43. Data are representative of 3–5 mice analyzed individually per group per experiment from three experiments conducted with similar results. *P < 0.05; NS: P > 0.05.

Figure 4

NKG2D engagement reverses the elevated T-bet expression associated with CD4-unhelped CD8⁺ T cells. (a) T-bet expression MFI (±SEM) in OVA-tetramer⁺ CD8⁺ T cells from C57BL/6 mice vaccinated as described in Fig. 1a. (b) T-bet staining on days 15 and 43. MFI = mean fluorescent intensity; gray curve = isotype. (c) T-bet expression from (a) on days 15 and 43. (d) Memory recall (day 43) T-bet expression on OVA-specific CD8⁺ T cells transferred from CD45.1⁻ NKG2D-deficient (Klrk1^−/−) or wild type C57BL/6 (WT) mice into WT CD45.1⁺ mice. (e) Memory recall (day 43) T-bet expression on OVA-specific CD8⁺ T cells in IL-15-deficient (Il15^−/−) or WT mice vaccinated as described in Fig. 1a. Data are representative of 3–5 mice analyzed individually per group per experiment from three experiments conducted with similar results. Dashed lines represent MFI of background flow cytometric staining for T-bet. *P < 0.05; NS: P > 0.05.

Figure 5

Suppression of T-bet by NKG2D/Rae-1ε engagement is mediated through JNK2. (a) CD8⁺ T cell T-bet MFI (+SEM) from NKG2D-deficient (Klrk1^−/−) and wild type C57BL/6 (WT) spleens stimulated in vitro with anti-CD3/CD28 antibodies (1 µg ml⁻¹ each) for 72 h or left untreated. (b) pJNK2 MFI (+SEM) from CD8⁺ T cells stimulated as in (a) ± NKG2D blocking antibody (αNKG2D). (c) T-bet MFI (+SEM) of OT-I CD8⁺ T cells co-cultured with OVA_257–264-loaded EL4 cells transfected with either Rae-1ε or empty vector, and treated with a JNK2 inhibitor (JNK inhibitor IX; 25 nM,). Data are representative of 3–5 mice analyzed individually per group per experiment from at least three experiments conducted with similar results. Dashed lines represent MFI of background flow cytometric staining for the respective markers. *P < 0.05; NS: P > 0.05.

Figure 6

NKG2D co-stimulation confers protection in CD4-dependent infectious disease models. (a) Experimental design for C57BL/6 mice vaccinated as described in Fig. 1a and continuously CD4 depleted. (b,c) Mean number (c) and T-bet MFI (+SEM) (d) of OVA-tetramer⁺ CD8⁺ T cells (+SEM) per spleen on days 38 and 43 from mice in (a). (d) Experimental design of Influenza-PR/8 intranasal infection. (e) Number of OVA-tetramer⁺ CD8⁺ T cells from experiment in which WT or NKG2D-deficient (Klrk1^−/−) CD8⁺ T cells were adoptively transferred into WT hosts and treated as described in (d). (f) Survival curve for experiment described in (d) and repeated twice with similar results. (g) Experimental design for stimulation of HIV-positive chronic (Chronic) and long-term non-progressor (LTNP) donor cells with pooled HIV peptides. (h–j) HIV-tetramer⁺ CD8⁺ T cell MFI (+SEM) of T-bet (h), mean % producing granzyme B, IL-2, and IFN-γ (+SEM) (i), and mean % incorporating BrdU (+SEM) (j), from at least three donors per group. Dashed lines represent MFI of background flow cytometric staining for the respective markers. *P < 0.05; NS: P > 0.05.