UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene

Apoptosis is an important defensemechanismmounted by the immune systemto control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generatedMCMVUL36 inhibited apoptosis inmacrophage lines RAW264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo

NFAT signaling is indispensable for persistent memory responses of MCMV-specific CD8+ T cells.

Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells

MCMV specific CD8⁺ T cell responses in mixed bone marrow chimeric mice.

Lethally irradiated mice were reconstituted with BM (1:1) of NFATc1 KO, NFATc2 KO, NFATc1c2 DKO or wildtype BM (CD45.2+/+) with control wildtype BM (CD45.1+/-CD45.2+/-). CD8+ T cell response kinetics were monitored for 90 days following intraperitoneal MCMV infection with 106 PFU. (A) Relative frequency of tetramer+ cells among CD45.2+/+ CD8+ T cells. (B) Absolute size of SLEC (KLRG1+ CD27-) and tetramer specific responses from control CD45.1+/-CD45.2+/- population. Data are pooled from two experiments and for each group n≥6. Statistically significant differences are highlighted; *, p (TIF)</p

Accumulation of NFATc1c2 DKO CD8⁺ T cells in Lymph nodes.

(A-C) Mixed bone marrow chimeric animals were infected with 106 PFU of MCMV and sacrificed at 7 dpi. Absolute count of different tetramer specific CD45.2+/+ CD8+ T cells in blood (A), spleen, lungs and mesenteric LN (B). (C) Relative and absolute count of CM CD45.2+/+ CD8+ T cells in spleen, lungs and mesenteric LN. (D-F) 104 naïve OTI T cells were transferred to congenic animals and activated by MCMV infection. (D) Absolute count of CM OTI T cells in different organs at 7 dpi is shown. (E) Selected genes involved in T cell migration are shown in heatmap. Data are pooled from at least two experiments and each dot represents one mouse; mean ± SEM values are plotted. (TIF)</p

Representative gating strategy.

Following singlet gating, lymphocytes were selected with forward/side scatter parameters. Live CD3+ cells were identified by excluding 7AAD stained cells, and CD8+ T cells were selected by CD8a expression. Next, CD8+ T cells (blue gate and arrow) were gated into CD45.2+ single and CD45.1+CD45.2+ double positive populations in mixed bone chimeric animals. Population arising from each bone marrow was progressively gated to define naive and primed CD8+ T cells based on CD44 and CD11a expression. Central memory cells were distinguished from effector cells by CD62L and CD27 expression within primed population. Similarly, short lived effector and memory cell populations (SLEC and Mem) were gated according to KLRG1 and CD27 expression within primed CD8+ T cells (green gate and arrow). Tetramer+ (M38+ and m139+) and CX3CR1+ populations were defined by gating directly on CD45.2+ CD8+ T cells (red gate and arrow) or control CD45.1+CD45.2+ CD8+ T cell populations. (TIF)</p

CD8⁺ T cell differentiation status during chronic infection.

Mixed bone marrow chimeric animals were infected with 106 PFU of MCMV and sacrificed at 90 dpi. (A) Absolute count of KLRG1+CD27- CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. (B) Frequency of CM and KLRG1-CD27+ T cells among primed CD45.2+/+ CD8+ T cells (CD44+CD11a+) from mesenteric LN of chronically infected mice. (C) Quantification of CM CD8+ T cells from CD45.2+/+ compartment in blood, spleen, lungs and mesenteric LN. (D) Absolute count of CX3CR1+ CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. Data are pooled from two independent experiments and each dot represent one mouse. Statistically significant differences are highlighted; *, p (TIF)</p

NFAT KO mice fail to mount inflationary CD8⁺ T cell response following chronic MCMV infection.

Animals lacking either NFATc1, NFATc2 or both were infected with 106 PFU of MCMV intraperitoneally. (A) Total CD8+ T cells and tetramer specific response kinetics in peripheral blood were tracked. Mean ± SEM are plotted for data pooled from 3 independent experiments (n≥12). Total CD4+ T cells, CD8+ T cells (B) and M38 tetramer+ T cell responses (C) in spleen at 90 dpi. (D) mice were sacrificed at 5 dpi to titrate the virus replication in spleen and lungs. (E) Infected mice were sacrificed 6 months post infection to quantify MCMV latent virus load, which is presented as virus copies per 105 host cells in spleen and salivary glands. Data in panel D and E are pooled from two experiments and each dot represents one mouse. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; (Mann-Whitney U Test).</p

Defective NFAT signaling in CD8⁺ T cells leads to accumulation of memory CD8⁺ T cells in LNs.

(A-E) Mixed BMC animals were infected with 106 PFU of MCMV and CD8+ T cell responses were analyzed by tetramer staining and flow cytometry at 7 dpi. (A) Total number of indicated tetramer+ CD8+ T cells in spleen, lungs and mesenteric LN. (B-D) Relative size of CM, effector (B), KLRG1+CD27-, KLRG1+CD27+, KLRG1-CD27+ (C) and CX3CR1+ (D) CD8+ T cell populations from spleen are plotted. (E) Percentages of total CD45.1-/-CD45.2+/+ NFATc1c2 DKO and CD45.1+/-CD45.2+/- WT cells among CD8+ T cells in different organs. (F-I) 104 naïve OTI T cells were transferred to congenic animals and activated by MCMV infection. (F) Absolute count of total OTI T cells in spleen, lungs and different LNs at 7 dpi. (G) WT and NFATc1c2 DKO OTI T cells were isolated from spleen or LNs and transcriptional analysis was performed. Transcriptional profile of genes involved in T cell activation, migration and cell cycle regulation are shown as a heatmap. (H) Flow-cytometric plots showing Ki-67 expression on total CD8+ T cell population (left). Frequency of Ki-67+ cells among total OTI and CM OTI are shown. Panel A-E show data pooled from two experiments (n = 5–6) and panel F has data pooled from 2–3 experiments (n = 5–10). Plots show means ± SEM values, each dot represents one mouse. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; (Mann-Whitney U Test).</p

NFATc1 is crucial for persistent CD8⁺ T cell response during chronic infection.

(A) Overview of mixed bone marrow chimera (BMC) generation, MCMV infection and CD8+ T cell response monitoring. Lethally irradiated C57BL/6J mice were reconstituted with bone marrow mix (1:1) of the control WT BM (CD45.1+/-CD45.2+/-) and either NFATc1 KO, NFATc2 KO, NFATc1c2 DKO or WT BM (CD45.1-/-CD45.2+/+). CD8+ T cell response kinetics were monitored for 90 days following intraperitoneal MCMV infection with 106 PFU. Mice were sacrificed 90 dpi and CD8+ T cell responses in blood and organs were analyzed. (B) Representative flow-cytometric plots showing total CD8+ T cells during acute and chronic infection. (C) Percentage of the CD45.2+/+ subset in the CD8+ T cell population was tracked by labelling peripheral blood. (D) CD8+ T cell response was tracked by labelling peripheral blood with MCMV epitope-specific tetramers. The total number of epitope-specific CD8+ T cells among CD45.2+ population is plotted. (E) Bar plots represent the mean ± SEM of tetramer+ CD8+ T cells for each epitope in spleen, lungs, and mesenteric LN at 90 dpi. Data are pooled from two experiments and each dot represents one mouse, n = 6–10. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; (Mann-Whitney U Test); mean ± SEM values are plotted.</p

CD8⁺ T cells lacking NFATc1 and NFATc2 showed distinct transcriptional profile.

Naïve OTI T cells (104) were transferred to congenic animals and activated by acute MCMV infection. Animals were sacrificed at 7 dpi and transcriptional analysis was performed. (A) Volcano plot shows genes that are differentially regulated in NFATc1c2 DKO OTI cells as compared to WT cells. (B) Principal component analysis of RNA sequencing samples from WT and NFATc1c2 DKO CD8+ T cells from spleen and lymph nodes (LN). Replicates of the same genotype and tissue are indicated by similar color and shape, respectively. (C) Heatmap shows expression of selected cytokine and cytokine receptor genes in OTI cells that lack NFATc1, NFATc2 or both. (D) Heatmap shows expression of selected transcription factors, surface receptors and cell cycle regulation genes from CD8+ T cells. Color shows Z-score differences. (TIF)</p