The NF-κB RelA transcription factor is not required for CD8+ T-cell function in acute viral infection and cancer

CD8+ T cells are critical mediators of pathogen clearance and anti-tumor immunity. Although signaling pathways leading to the activation of NF-κB transcription factors have crucial functions in the regulation of immune responses, the CD8+ T cell-autonomous roles of the different NF-κB subunits, are still unresolved. Here, we investigated the function of the ubiquitously expressed transcription factor RelA in CD8+ T-cell biology using a novel mouse model and gene-edited human cells. We found that CD8+ T cell-specific ablation of RelA markedly altered the transcriptome of ex vivo stimulated cells, but maintained the proliferative capacity of both mouse and human cells. In contrast, in vivo experiments showed that RelA deficiency did not affect the CD8+ T-cell response to acute viral infection or transplanted tumors. Our data suggest that in CD8+ T cells, RelA is dispensable for their protective activity in pathological contexts.


Introduction
NF-kappaB (NF-kB) is a family of transcription factors with pleiotropic functions in inflammation and immunity.The family comprises 5 subunits that share a Rel-homology domain.RelA (also known as p65, encoded by Rela), c-Rel and NF-kB1 are activated upon stimulation of the canonical signal transduction pathway, whereas RelB and NF-kB2 are the terminal effectors of the noncanonical (or alternative) pathway (1).
The canonical NF-kB pathway can be activated in T cells following engagement of T-Cell Receptor (TCR)/CD28, different members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and CD28 families, as well as several cytokine receptors, that all lead to the formation of the activating Inhibitor of KappaB Kinase (IKK)a/b/g complex, allowing degradation of the inhibitor IkBa/b and subsequent nuclear translocation of NF-kB subunits (2).NF-kB dimers then bind to DNA to repress or activate transcription.The canonical pathway has been implicated in various aspects of T-cell biology, ranging from thymic development to effector functions (3).
Among T-cell subsets, cytotoxic CD8 + T cells which are critical mediators of anti-tumor and anti-pathogen immunity, have been proposed to rely on NF-kB activity at different levels, including activation, survival, proliferation, or cytokine expression (3)(4)(5)(6)(7).Nevertheless, the CD8 + T-cell-autonomous roles of NF-kB subunits, are incompletely understood.Indeed, whereas RelA is ubiquitously expressed and known as a quick and critical mediator of gene expression following stimulation, knowledge on its functions in CD8 + T cells is scarce.This is largely due to the embryonic lethality of mice with germline ablation of Rela (8).In mice, overexpression of a dominant-negative mutant form of Rela impaired CD8 + T-cell survival, as well as EOMES expression in memory T-cells (9,10).Elegant studies have also demonstrated a direct function of RelA in IFNg production and proliferation, in mouse and human CD8 + T-cells, respectively (11,12).In patients with heterozygous, dominant negative loss-of-function (LOF) mutations in RELA, decreases in circulating central memory CD8 + T cells were reported (13,14), although this was not confirmed in another cohort of patients (15).However, whether RelA is intrinsically required to orchestrate CD8 + T-cell gene expression and function in vivo, is unknown.Herein, we selectively ablated Rela in CD8 + T cells using a novel mouse model and engineered human cells, in order to investigate its putative roles ex vivo and in disease contexts.

Mice
Rela-Floxed (B6.129S1-Rela tm1Ukl/J ) mice were previously described (16).CD8 cre (C57BL/6-Tg(Cd8a-cre) 1Itan/J ) were a gift from Ichiro Taniuchi (RIKEN Center for Integrative Medical Sciences, Japan) (17).CD4 cre (Tg(CD4-cre) 1Cw1 ) on a C57Bl/6 J background were purchased from the Jackson Laboratory.Rag2 -/- and C57Bl/6 CD45.1 (Ptprc a Pepc b /BoyJ) mice were purchased from Charles River Laboratories France.Mice were bred and used in specific-pathogen-free (SPF) conditions at the CRCL animal facility (P-PAC) or the Ecole Normale Supeŕieure de Lyon BSL-2 facility (PBES, for LCVM experiments only).Animals were housed in individually ventilated cages with temperature-controlled conditions under a 12-h-light/dark cycle with free access to drinking water and food.Adult (6-to 30-week-old) male or female mice were used for all experiments.Studies were conducted in accordance with the animal care guidelines of the European Union and French laws.Protocols were validated by the local Animal Ethics Evaluation Committee (C2A15).Project references were #16772, 30346 and 43605.

Human subjects
Blood samples from healthy volunteers were obtained through the Etablissement Francais du Sang (EFS) (French Blood Transfusion Society).

Mixed bone marrow chimeras
Bone marrow cells were retrieved from tibia and femur of donor mice.Red blood cells were lysed with Ammonium-Chloride-Potassium lysis Buffer and enumerated.1/3 WT CD45.1 bone marrow and 2/3 CD45.2 bone marrow of interest (CD4 cre or CD4 cre Rela flox/flox ) were mixed to prepare bone marrow solutions.After sub-lethal irradiation (7 Gy), recipient mice were transplanted intravenously (retro orbital sinus) with 10×10 6 bone marrow cells.Mice were given Neomycin (200 ng/mL, Sigma catalog #N6386) in drinking water for ten days.8 weeks after reconstitution tissues were harvested for subsequent analyses.
Electroporation was performed using the Neon transfection system with the following parameters: 1600V, 10 ms, 3 pulses.Subsequently, cells were transferred into 1.9 mL complete RPMI with IL-2 (20 U/mL) and Dynabeads CD3/CD28 (1 bead for 4 T cells).Cells were cultured for 3 days, washed, and stained with DAPI (Cell Signaling Technologies, catalog #4083S) in FACS buffer.DAPI -ATTO550 + cells were sorted using a FACS ARIA II cytometer and used for subsequent assays.

In vitro culture assays Human T cells
Following sorting, human CD8 + T cells were left to rest for 2 days in complete RPMI with IL-2 (Proleukin, Novartis Pharma, 50 U/mL).Cells were then labeled using the CellTrace Violet Cell Proliferation Kit (CTV; Thermo Fisher Scientific, catalog #C34557A) and 2x10 4 cells were stimulated with Dynabeads anti-CD3/CD28 (1 bead:4 T cells, Thermo Fisher Scientific) in complete RPMI with IL-2 (25 U/mL).After 4 days of culture, supernatants were harvested and stored at -80°C until further use and proliferation and cytokine expression were assessed by FACS.

Preparation of cell suspensions
Single cell suspensions from LN, thymus and spleens were obtained by mechanical dilaceration in FACS Buffer (PBS 1X + 2% FBS, 2 mM EDTA) with glass slides, strained and washed in complete RPMI.
After being sliced in small pieces tumors were digested in RPMI 1640 (Gibco) supplemented with 1 mg/mL collagenase type IV (Sigma-Aldrich, catalog #C2674) and 250 µg/mL DNase I (Sigma-Aldrich catalog #DN25) for 25 min at 37°C followed by mechanical dissociation.Reaction was stopped by the addition of 15 mL PBS1X containing 5 mM EDTA.The solution was filtered through a 70 mm cell strainer, and any remaining solid pieces were mechanically disrupted.After centrifugation, cell pellets were resuspended in 8 mL of Percoll 40% (Sigma-Aldrich, catalog #17-08-91-01) and then carefully layered onto 4 mL of Percoll 80% in a 15 mL polypropylene tube.Tubes were centrifuged at 2,500 rpm for 20 min at RT. Mononuclear cells were retrieved from the interface of the 40:80% Percoll gradient and washed in complete RPMI.

Flow cytometry
Cells were washed in PBS1X and incubated with purified anti-CD16/CD32 (Biolegend, catalog #101302) and a viability marker for 10 min at RT in the dark.After a wash in PBS1X cells were incubated with the surface marker antibody mix in FACS Buffer (PBS1X, 2% FBS, 2 mM EDTA) for 20 min at 4°C in the dark.Cells were then washed in FACS buffer and fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, catalog #00-5523-00) according to the manufacturer's instructions.Cells were washed in permeabilization buffer and incubated with the intracellular marker antibody mix for 20 min at 4°C in the dark.Cells were then washed in permeabilization buffer and resuspended in FACS buffer.At times, biotin-coupled antibodies were employed.In such instances, an additional stage of staining with fluorochromecoupled streptavidin was required (in FACS buffer for cell surface labeling or wash buffer for intracellular labeling).
NP396-404 PE and GP33-41 APC class I tetramers were obtained through the NIH tetramer facility.The complete list of antibodies can be found in Supplementary Table 1.Acquisition was performed on a LSR Fortessa (BD Biosciences) or an Aurora spectral cytometer (Cytek Bioscience).Data were analyzed with FlowJo software v10.9.0.

RNA-sequencing and analyses
RNA from 0.25 to 1x10 6 CD8 + T cells was isolated with Nucleospin RNA extraction kits (Macherey Nagel, catalog #740955.250);libraries were prepared using an Illumina TruSeq Library Kit and sequenced with an Illumina NovaSeq instrument.Reads were aligned on reference genomes (mm10 for mouse data, GRCh38 for human data) using the STAR universal RNA-seq aligner; DEGs were calculated with DESeq2.Heatmaps were created with Morpheus (Morpheus (broadinstitute.org)).For functional enrichment analyses, we used the enricher function (default parameters) from the clusterProfiler package v4.2.2 to perform hypergeometric tests for functional enrichment analysis.Only down-regulated genes were used as the input, and the universe/background was defined as all detected genes in our RNA-Seq.The human hallmark, C2 and GOBP (C5:BP) gene sets were retrieved from the Molecular Signatures Database [MSigDB (20)] using the msigdbr function and package v7.4.1 Finally, we applied the Benjamini-Hochberg method to control false discoveries in multiple hypothesis testing.

Statistics
Statistical analyses were performed using GraphPad Prism Software v9 (https://www.graphpad.com/scientific-software/prism/).For FACS data and tumor weights, two-tailed Mann-Whitney tests or paired T-tests (when 2 groups) and Kruskall-Wallis followed by Dunn's post-test (when more than 2 groups) were used to calculate statistical significance.For tumor volume 2-way ANOVA followed by Bonferroni-Dunn's post-test (when more than 2 groups), and twotailed Mann-Whitney test (when only 2 groups) were used.

RelA orchestrates mouse CD8 + T cell activation and gene expression at steady-state
To investigate the T-cell autonomous functions of RelA, we used mice carrying floxed alleles of Rela, which we crossed with CD4 cre mice, resulting in the deletion of the gene across all T-cell subsets and the concomitant expression of green fluorescent protein (GFP) (16).
In order to avoid indirect perturbations in CD8+ T cells that may rely on a role of RelA in other T-cell subsets, and thus specifically study the intrinsic functions of Rela within CD8 + T cells, we conducted mixed bone marrow (BM) transfer experiments of WT CD45.1 + cells and CD4 cre (control) or CD4 cre Rela F/F (Rela-cKO T ) CD45.2 + cells (Figure 1A).Flow cytometry analyses performed 8 weeks after BM transfer, showed that the distribution of thymocyte subsets was similar regardless of the genotype (Figure 1B).Interestingly, analysis of secondary lymphoid organs unveiled a slight competitive disadvantage for Rela-cKO CD8 + T cells (Figure 1C).Among CD8 + T cells, a decline in CD44 high activated cells was detected in the absence of RelA compared to controls; however, proliferation, illustrated by Ki67 expression, remained unchanged (Figure 1D).As NF-kB signaling has been linked to the production of inflammatory cytokines by immune cells, we explored cytokine expression upon PMA-ionomycin restimulation in vitro.We observed a dramatic decrease in the proportion of IFNg, TNFa and IL-2-expressing Rela-deficient CD8 + T cells, in the spleen and, to a lesser extent, in LN compared to controls.In contrast, Granzyme B expression was similar between groups (Figure 1E).These data were corroborated by measuring cytokine levels in culture supernatants of sorted CD8 + T cells showing decreased concentrations of IFNg, TNFa and IL-2 (Figure 1F).
Seeking deeper insights into the role of RelA in regulating global gene expression, we conducted RNA-sequencing analyses on both control and mutant CD8 + T cells that were stimulated with anti-CD3/ CD28 and IL-2 for 4 h.Differential gene expression analyses highlighted substantial changes in the transcriptome of Rela-cKO T CD8 + T cells, with 293 genes significantly up-or down-regulated when applying a fold change cut-off of 2. This number increased to 493 genes when the cutoff was reduced to 1.5 (Figure 1G, Supplementary Table 2).Of note, most differentially expressed genes (DEGs) were underrepresented in Rela-deficient conditions, supporting a transcription-promoting function of this NF-kB subunit.Functional enrichment analysis of down-regulated genes with Gene Ontology Biological Processes (GOBP), Hallmarks and canonical pathways revealed a number of differentially enriched pathways, including NF-kB-related pathways as well as T-cell activation or response to virus (Figure 1H).When looking at specific gene expression, we found massive down-regulation in NF-kB pathway-related transcripts, including negative regulators (Nfkbia, Tnfaip3), but also NF-kB subunits themselves (with the exception of Rel), establishing the apex function of RelA in the regulation of NF-kB-driven genes.Furthermore, genes involved in T-cell function (Ifng, Il2) or maturation (Tbx21, Eomes) were down-regulated in Rela-deficient cells.Surprisingly, the top enriched pathways were related to IFN signaling, as the expression of many Interferon-Stimulated Genes (ISGs) was dampened in the absence of Rela (Figure 1I).Although it cannot be excluded that this phenotype stems from the reduced expression of Ifng itself, this confirms that, as proposed in other cell types, the NF-kB and IFN pathways are strongly interconnected.Taken together, these observations establish RelA as a critical regulator of the CD8 + T-cell phenotype and transcriptome following polyclonal stimulation.

T-cell distribution is unaffected in mice with CD8 + T-cell-restricted ablation of Rela
Next, to explore with greater specificity the CD8 + T-cellautonomous functions of RelA, we crossed mice carrying Rela-foxed alleles with mice expressing Cre recombinase driven by a combination of the core E8I enhancer and the Cd8a promoter (called CD8 cre in the manuscript) (17).This allowed the conditional ablation of Rela and concomitant expression of GFP in peripheral CD8 + T cells (hereafter named Rela-cKO CD8 for conditional knock-out mice) (Figure 2A).Flow cytometric analyses performed on adult control and cKO animals revealed that the distribution of CD8 + T cells in the thymus, spleen and peripheral lymph nodes was similar between genotypes (Figure 2B).Moreover, levels of in vivo activation and proliferation, assessed by the expression of Ki67, CD44 and CD62L, were unaltered upon Rela ablation (Figures 2C, D).Following polyclonal stimulation with PMA and ionomycin, the percentage of IFNg-producing CD8 + T-cells (but not TNFa, GzmB or IL-2) was reduced in Rela-cKO CD8 mice (Figure 2E).Hence, CD8 + T-cellrestricted Rela ablation did not strongly impact steady-state homeostasis and function of T cells, suggesting that the strong impairment in T-cell homeostasis detected in Figure 1 likely relied on the competitive environment of mixed BM chimeras.

Cytokine expression following in vitro culture of CD8 + T cells is altered in the absence of RelA
Next, we assessed the impact of Rela ablation on CD8 + T cell responses in vitro.Naïve CD8 + T cells isolated from the spleen and LN of control and Rela-cKO CD8 mice, displayed similar levels of proliferation following 4 days of CD3/CD28 stimulation, in the presence or absence of IL-2 (Figures 3A, B).In contrast, we observed a dramatic impact of Rela ablation on the ability of CD8 + T cells to produce inflammatory cytokines, as illustrated by the reduction in the percentage of IFNg-and TNFa-expressing cells as well as the quantity of secreted IFNg, TNFa and GzmB in Rela-deficient cells compared to control (Figures 3C, D).RelA thus appears to exert critical functions in cytokine expression upon long-term TCR/CD28 engagement.

RELA contributes to human CD8 + T-cell identity and functions in vitro
Although a few patients with RELA loss-of-function mutations have been reported, its cell-autonomous roles within human CD8 + T cells are unknown.We established a CRISPR-Cas9 Ribonucleoprotein electroporation protocol to ablate RELA in in vitro stimulated primary human CD8 + T cells from healthy donors (Figure 4A).RELA (encoded by RELA) ablation was verified by Western blotting and exceeded 80% (Figure 4B).We first analyzed the gene expression profiles of both normal and KO cells through RNA-sequencing after 4 h of re-stimulation with anti-CD3/CD28 and IL-2.Loss of RELA resulted in significant changes in the expression of 322 genes (Fold change >1.5, q-value <0.005) (Figure 4C, Supplementary Table 2).Consistent with mouse cells, enrichment analyses on down-regulated genes showed that RELA governed, in human CD8 + T cells, the expression of genes associated with the NF-kB pathway (NF-kB subunits and negative regulators of the pathway), markers of function, and cytokines, especially the response to type I and type III IFNs (Figures 4D, E).Thus, RELA controlled different aspects of human CD8 + T cell biology, suggesting a CD8 + T-cell-intrinsic role for RELA in the immunodeficiency features detected in patients with RELA LOF (13-15).After 4 days of stimulation with anti-CD3/ 28, a similar level of proliferation was observed in control and RELA-deficient cells, in accordance with our data from mouse experiments (Figure 4F).Flow cytometry and ELISA analyses  showed a reduction in the expression of IFNg-producing cells in RELA-deficient cells compared to control, while the expressions of TNF-a-or GZMB remained unaltered (Figures 4G, H).These results reinforce the role of RELA in shaping the transcriptome and functions of CD8 + T cells both in humans and mice.

Rela is dispensable for CD8 + T-cell responses during acute LCMV infection
Our RNAseq data suggested altered expression of genes related to the response to viral infections in both mouse and human Rela-deficient CD8 + T-cells.To directly assess the contribution of RelA to CD8 + T-cell-directed antiviral responses, we infected control and Rela-cKO CD8 mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) that induces a strong, acute and well-defined T-cell response.Ten days post infection, the viral load, assessed by qPCR quantification of LCMV-glycoprotein (GP) and nucleoprotein (NP) encoding mRNAs, in the spleen and liver of infected mice was comparable between genotypes (Figure 5A).We also assessed the accumulation and phenotype of LCMV-specific CD8 + T cells in the spleen, using tetramers against both GP 33-41 (GP33) and NP 396-404 (NP396), by flow cytometry.The proportion of CD8 + T cells with either specificity, was unaltered in the absence of Rela (Figure 5B).Accordingly, the gross distribution of naïve/ TCM/TEM compartments was similar between groups, as well as their proliferation levels (Figure 5C).Rather counter-intuitively, the proportion of GzmB and PD-1-expressing GP33-specific T cells was slightly increased in Rela-cKO CD8 spleens.Nevertheless, the proportion of TNFa-and IFNg-expressing cells was not altered (Figures 5D, E).Altogether, these data suggest that Rela is dispensable for the establishment of optimal CD8 + T-cell responses during acute LCMV infection.

Rela is not required for CD8 + T-cell antitumoral function and response to immune checkpoint-blockade
As CD8 + T cells are known to be critical actors of anti-tumor immunity, we wondered whether Rela was required in this context.To address this, control (CD8 cre ) and Rela-cKO CD8 mice were inoculated with B16-F10 melanoma cells.Intriguingly, Rela deletion had no impact on tumor growth (Figures 6A, B).These results were confirmed in the MC38 colon carcinoma cells (Figures 6C, D).Using spectral cytometry 19 days after MC38 transfer, we observed that the proportion of CD8 + T cells in the tumors was similar between strains (Figure 6E).Furthermore, the activation level of tumor-infiltrating CD8 + T cells was unchanged in the absence of Rela (Figure 6F), leaving the proportion of cytokineproducing cells following PMA-ionomycin restimulation unaltered, with the exception of a slight increase in GzmB + CD8 + T cells in Rela-cKO CD8 mice (Figure 6G).
Given the role of CD8 + T cells in the response to checkpointblockade cancer immunotherapies, we subsequently investigated whether Rela activity might be a requisite for optimal response.Control (CD8 cre ) and Rela-cKO CD8 mice were transplanted with Braf V600E -Pten -/-melanoma cells and injected with anti-PD-L1 or isotype control mAb at D7, 9 and 11.As with the B16-F10 and MC38 models, the ablation of Rela did not affect tumor growth compared to control littermates treated with an isotype antibody (Figures 6H, I).Furthermore, PD-L1 blockade was equally efficient at inducing tumor regression in control and Rela-cKO mice (Figures 6H,  I).Thus, our data indicate that Rela does not support antitumor immunity in CD8 + T cells or response to immunotherapy.

Discussion
RelA has long been established as a master regulator of inflammation and immunity-especially through its roles in dendritic cells or macrophages.Although evidence also suggested a prominent role in shaping the CD8 + T-cell compartment, our study is, to the best of our knowledge, the first to directly investigate the cell-autonomous functions of RelA in vivo.
We show that RelA is required for the expression of numerous genes following TCR/CD28 stimulation in vitro, including as expected many members of the NF-kB pathway and CD8 + T-cell maturation drivers such as Tbx21 or Eomes.In line with previous reports (11, 21), Ifng expression was largely down-regulated in the absence of RelA.Intriguingly, numerous IFN signaling-related genes and IFN-stimulated genes (ISGs) were also strongly impaired in mouse Rela-deficient CD8 + T cells.This critical function of RelA was further consolidated in human T cells.This link between NF-kB and IFN pathways is documented in innate immune cells (22) and our data now establish its existence in T cells.As type I and type II IFN signaling impact T-cell function (23-25), this crosstalk may have consequences on the outcome of pathological conditions.This conclusion is in stark contrast to the observations made in patients with germline RELA haploinsufficiency (loss-of-function mutations), who suffer from various autoimmune or inflammatory conditions and are characterized by enhanced IFNs and ISG expression (13-15).This suggests that these phenotypes mostly relied on CD8+T-cell extrinsic functions of RELA.
These in vitro observations led us to explore whether viral infections, whose clearance largely relies on IFNs, might be impaired in Rela-cKO CD8 animals.However, Rela ablation did not modify LCMV loads or the priming of LCMV-specific CD8 + T cells.This was in line with a report showing that the absence of Rela in all T cells (Lck cre Rela F/F mice) led to similar responses to LCMV as those observed in control animals (26).Because complete ablation of the canonical pathway in T cells in Cd4 cre Ikk2 F/F mice, was shown to impair CD8 + T-cell cytotoxic function in cancer, resulting in enhanced tumor growth (27), we also challenged our Rela-cKO CD8 mice with different types of tumors.However, we observed that RelA was completely dispensable for CD8 + T-cell priming, accumulation and function in melanoma and colon adenocarcinoma.Whereas PD-1 inhibition was shown to increase NF-kB activation (28), and CD8 + Tcells are critical mediators of the clinical response to checkpointblockade cancer immunotherapies (29, 30), we also show that tumor regression induced by PD-L1 blockade is entirely maintained in Rela-cKO CD8 animals.Thus, at odds with our in vitro observations, CD8 + T-cellrestricted ablation of Rela did not drive observable phenotypes in disease contexts.This was in accordance with data from patients with RELA haploinsufficiency, in which no increased susceptibility to infections or cancer was reported.This could rely on different mechanisms.First, while in vitro T cells were stimulated with optimal levels of TCR/CD28 and IL-2/IL-7 engagement, their in vivo function may rely on other stimulators such as TNFSF members, which may lead to the activation of other pathways or other NF-kB subunits (31,32).Second, it is possible that the absence of RelA in vivo could be compensated by other NF-kB subunits, in particular c-Rel, as shown in other contexts (33,34).In fact, the different subunits appear to share their DNA-binding sequence and might thus be, to some extent, interchangeable in vivo (35-37).In this context, it would be interesting to develop mouse models that lack other NF-kB subunits in CD8 + T cells and even mice fully devoid of canonical NF-kB subunits, such as c-Rel and RelA-double deficient mice.

Data availability statement
The datasets presented in this study can be found in online repositories.The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/,GSE254685.

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FIGURE 2 T-cell homeostasis in mice with CD8 + T-cell restricted ablation of Rela.(A) Western blot validation of RelA ablation in Rela-cKO CD8 CD8 + T cells.(B-E) Spleen and peripheral LN from control and Rela-cKO CD8 mice were analyzed by flow cytometry.(B) Proportion of live TCR-b + CD8 + cells in peripheral tissues (right) and CD4 -CD8 -(DN), CD4 + CD8 + (DP) CD4 + CD8 -(SP CD4) and CD4 -CD8 + (SP CD8) in the thymus (left) among live cells.(C) Proportion of Ki67 + in CD8 + T cells.(D) Representative dot plots in the spleen and cumulative data of CD44 and CD62L expression in spleen and LN CD8 + T cells.TCM: T central memory; TEM: T effector memory.(E) Cytokine expression by CD8 + T cells following PMA/ionomycin restimulation, measured by FACS.Data are shown as mean ± SEM of 3-5 independent experiments; each dot represents an individual mouse; Mann-Whitney tests were used.

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FIGURE 3    In vitro features of Rela-deficient CD8 + T cells.Naïve CD8 + T cells isolated from spleen and LN of control and Rela-cKO CD8 were stained with the CellTrace Violet Cell Proliferation Kit (CTV) and cultured with the indicated doses of anti-CD3/CD28 mAbs supplemented with IL-7 or IL-7 + IL-2 for 4 days and their phenotype was analyzed by FACS and ELISA.(A, B) Representative CTV profile (left) and cumulative proliferation index of live cells (right).(C) Representative CTV profile (left) and cytokine expression after PMA-ionomycin restimulation (right), measured by FACS.(D) ELISA analysis of culture supernatants.(A, B) Data are shown as mean ± SEM of 4 experiments; Mann-Whitney tests were used.(C, D) each dot represents an individual mouse from 4 independent experiments; multiple paired t-tests were used.(D) Rela-cKO samples are normalized against control samples from the same experiment.

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FIGURE 4 The roles of RELA in human CD8 + T cells.(A) Schematic representation of the experimental protocol.Created with BioRender.com(B) Western blot validation of gene editing efficacy after sorting of ATTO550 + live cells (C-E) RNA-seq analysis of 6 independent donors following 4 h stimulation with anti CD3/CD28 and IL-2.(C) Volcano Plot of 322 DEGs (log2 fold change >1.5, q < 0.005).(D) Heatmaps of selected DEGs.(E) Functional enrichment analysis on down-regulated genes.Representative signatures are shown.(F-H) RELA-edited CD8 + T cells were sorted and left to rest for two days and then labeled with CTV and stimulated 4 days with anti-CD3/CD28 and IL-2.(F) Representative CTV profile (left) and proliferation index of live cells from 11 donors/independent experiments.(G, H) Cytokines were analyzed by FACS after PMA-ionomycin restimulation (G) or in culture supernatant by ELISA (H).In F-H, each dot represents an individual donor.Multiple paired T-tests were used.Data are from 8 donors (FACS) and 6 donors (ELISA) analyzed in independent experiments.

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FIGURE 5 Unaltered CD8 + T-cell response to LCMV in the absence of RelA.CD8 cre (ctrl) and Rela-cKO CD8 mice were infected with 2x10 5 PFU LCMV Armstrong (A) qPCR quantification of LCMV in spleen and liver 10 days after infection.(B-E) FACS analysis at day 10 in spleens without (B-D) or with (E) PMA-ionomycin restimulation.Each dot represents a mouse; data are shown as mean ± SEM.Mann-Whitney tests were used.