NFAT single-deficient murine T cells reduce the risk of aGvHD while controlling cytomegalovirus infection

Abstract

NFAT is a family of transcription factors whose activation is inhibited by calcineurin inhibitors (CNIs). In allogeneic hematopoietic stem cell transplantation (allo-HCT), CNIs are employed to prevent and treat graft-versus-host disease (GvHD). Unfortunately, control of cytomegalovirus (CMV), which exacerbates clinical outcomes, is simultaneously lost. Since single NFAT deficiency in T cells ameliorates GvHD in our major mismatch model, we investigated whether protection is maintained during CMV infection. Reassuringly, NFAT-deficient T cells still improved GvHD upon acute CMV infection and after allo-HCT in latently CMV-infected mice, showing reduced proinflammatory and cytotoxic potential. In sharp contrast, CMV-specific NFAT-deficient CD8⁺ inflated memory T cells expanded more and with higher levels of interferon gamma (IFN-γ) and GzmB expression, effectively controlling CMV. Notably, NFAT-deficient inflated memory T cells could migrate to non-lymphoid tissues and fight CMV. Therefore, CMV infection does not interfere with the protective effect of NFAT inhibition to attenuate GvHD while allowing an anti-CMV response.

Keywords: Biological sciences; Immunology; Microbiology; Natural sciences; Virology.

Graphical abstract

Figure 1

Co-transfer of single-deficient NFATc1 and NFATc2 or double-deficient NFATc1c2 T cells in a haploidentical major mismatch model limits signs of aGvHD even after an acute MCMV infection (A) Experimental in vivo set up of the induction of aGvHD in a haploidentical mouse model due to an H-2^b → H-2^b+d transfer with WT or NFAT-deficient T cells along with BM, preceded by lethal irradiation (9 Gy) of CB6F1 recipients. Two days post-transplantation, mice were acutely infected with 1 × 10⁶ PFU of WT-MCMV Smith strain. Six or twelve days post-infection, ex vivo analyses were conducted on spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), salivary gland (SG), lungs, and liver. (B) Weight changes were determined over 14 days post-irradiation (dpi) in mice without (aGvHD) or with an acute MCMV infection (aGvHD + acute MCMV). Mice were evaluated every day and weight loss was calculated considering day 0 weight as 100%; n ≥ 5. (C) Clinical scores were determined over 14 days. (B and C) Two-way ANOVA and Tukey’s multiple comparisons test (∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n ≥ 5, two independent experiments. (D) Virus DNA concentration of acutely infected mice 14 dpi in indicated organs was determined by RT-PCR. (E) Survival over 14 days.

Figure 2

NFAT-deficient T cells can still provide protection when transferred into latently infected recipients (A) Experimental in vivo set up. Latent infected CB6F1 recipient (90 days prior transplantation) with WT-MCMV Smith strain. Acute GvHD was initiated by transferring H-2^b donor T cells together with BM cells into H-2^b+d recipients, preceded by lethal irradiation (9 Gy) of CB6F1 recipients. Six or thirty days post-transplantation, ex vivo analyses were conducted on various tissues. Symbols representing studied genotypes in only aGVHD (D−/R−) and latently infected CB6F1 recipients (D−/R+). (B) Weight loss and (C) clinical scores of GvHD-induced mice in latently infected recipients were monitored daily for short (6 dpi) or long term (30 dpi). Two-way ANOVA and Tukey’s multiple comparisons test (∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n ≥ 5, two independent experiments. (D) Virus DNA concentration 30 dpi, determined by RT-PCR, mean ± SD, n = 3, unpaired Student’s t test (∗p < 0.05, ∗∗p < 0.005). (E) Survival was monitored daily for 30 days.

Figure 3

Independent from MCMV infection, NFAT-deficient Tregs are significantly enriched during GvHD (A) Frequency and absolute cell numbers—calculated back to all cells of the particular organ—of donor CD90.1⁺CD4⁺CD25⁺Foxp3⁺ T cells 8 days post-transplantation in non-infected or acutely infected (MCMV+) recipients (spleen, pLN, mLN, salivary gland [SG], lungs, and liver). Student’s two-tailed t test (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n ≥ 4, three independent experiments. (B) Flow cytometric analyses of indicated organs harvested 6 days post-transplantation from mice without or with latent MCMV infection (R+). Percentage of CD25⁺Foxp3⁺ Tregs within donor CD90.1⁺CD4⁺ T cells and absolute cell numbers. (A and B) Student’s two-tailed t test (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n ≥ 3, two independent experiments.

Figure 4

The reduction of proinflammatory cytokines and cytotoxic molecules in NFAT-deficient CD8⁺ T cells during aGvHD remains significant regardless of a concurrent MCMV infection (A and B) Flow cytometric analyses of recipient mice without (R−) and with latent MCMV infection (R+) were conducted 6 days post-transplantation. (A) Frequency of donor CD90.1⁺CD8⁺IFN-γ⁺ T cells in the depicted organs, n ≥ 3. (B) Frequency of donor CD90.1⁺CD8⁺GzmB⁺ T cells. (A and B) Student’s two-tailed t test (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n ≥ 3, two independent experiments.

Figure 5

NFAT deficiency reduces CD8⁺ T cell exhaustion while promoting terminal differentiation of MCMV-specific CD8⁺ T cells after allo-HCT (A) Percentage of PD-1^hiTOX^hi in CD8⁺ T cells in only aGVHD and latently infected CB6F1 recipients (+MCMV) 6 days post-transplantation, n ≥ 3. (B) Percentages of the chemokine receptor CX3CR1⁺ and (C) type II transmembrane protein receptor KLRG1⁺ within MCMV-specific donor CD90.1⁺CD8⁺M38⁺ T cells was determined through tetramer staining and subsequent flow cytometry analysis. Fold change was calculated relative to WT. (A, B and C) Student’s two-tailed t test (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n ≥ 3, two independent experiments.

Figure 6

NFAT deficiency in CD8⁺ T cells is sufficient to increase the frequency of IFN-γ⁺ MI T cells (A–C) Flow cytometric analyses of allotransplanted WT CD4⁺ T cells and WT or NFAT-deficient CD8⁺ T cells (1:1 ratio) 6 days post-transplantation. (A) Absolute numbers and (B) fold change of the percentage of KLRG1⁺ within MCMV-specific donor CD90.1⁺CD8⁺CD44⁺CD27^–M38⁺ T cells was determined through tetramer staining and subsequent flow cytometry analysis. Fold change was calculated specifically on WT. (C) Frequency of IFN-γ⁺ within MCMV-specific donor CD90.1⁺CD8⁺CD44⁺CD27^–M38⁺ T cells (in vitro restimulated). (A, B and C) Student’s two-tailed t test (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n = 5.

Figure 7

After allo-HCT in latently MCMV-infected mice, NFAT-deficient MI T cells present with a higher frequency of IFN-γ⁺ and GzmB⁺ cells Percentage of (A) IFN-γ⁺ and (B) GzmB⁺ cells of either M38^– or MCMV-specific M38⁺, M38⁺KLRG1⁺, and M38⁺KLRG1⁺CX3CR1⁺ CD8⁺ T cells after 6 days post-transplantation and in vitro restimulation. (A and B) Student’s two-tailed t test (∗p < 0.05, ∗∗p < 0.0051, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), mean ± SEM, n = 3.