Differential Regulation of Two Arms of mTORC1 Pathway Fine-Tunes Global Protein Synthesis in Resting B Lymphocytes

Protein synthesis is tightly regulated by both gene-specific and global mechanisms to match the metabolic and proliferative demands of the cell. While the regulation of global protein synthesis in response to mitogen or stress signals is relatively well understood in multiple experimental systems, how different cell types fine-tune their basal protein synthesis rate is not known. In a previous study, we showed that resting B and T lymphocytes exhibit dramatic differences in their metabolic profile, with implications for their post-activation function. Here, we show that resting B cells, despite being quiescent, exhibit increased protein synthesis in vivo as well as ex vivo. The increased protein synthesis in B cells is driven by mTORC1, which exhibits an intermediate level of activation in these cells when compared with resting T cells and activated B cells. A comparative analysis of the transcriptome and translatome of these cells indicates that the genes encoding the MHC Class II molecules and their chaperone CD74 are highly translated in B cells. These data suggest that the translatome of B cells shows enrichment for genes associated with antigen processing and presentation. Even though the B cells exhibit higher mTORC1 levels, they prevent the translational activation of TOP mRNAs, which are mostly constituted by ribosomal proteins and other translation factors, by upregulating 4EBP1 levels. This mechanism may keep the protein synthesis machinery under check while enabling higher levels of translation in B cells

Rewiring of the TCR signalosome in natural intestinal Intraepithelial T lymphocytes drives non-deletional tolerance

Intraepithelial T lymphocytes (T-IEL) are a large population of cytotoxic T cells that protect the small intestinal epithelium against pathogens. Based on ontogeny, T-IEL can be categorized into two major subsets: induced and natural. Natural T-IEL are agonistically selected in the thymus on self-antigens before migrating directly to the small intestine. Despite having self-reactive T cell antigen receptors (TCR), natural T-IEL are maintained in a tolerized state in the gut by unknown mechanisms. We therefore investigated TCR signaling in T-IEL using multiplexed fluorescent cell barcoding, phosphoproteomics and TCR signaling reporter mouse models, which revealed that TCR signaling is intrinsically suppressed in natural, but not induced, T-IEL. Unexpectedly, we discover that this cell intrinsic suppression was mediated through altered TCR signalosome components. Specifically, downregulation of the key signaling adaptor, Linker for activation of T cells (LAT) during thymic selection is a vital checkpoint for the development and tolerization of natural IELs. Thus, TCR signaling is rewired in self-reactive natural T-IEL to promote tolerance and prevent inappropriate inflammation in the gut.One sentence summary Self-reactive natural intestinal intraepithelial T lymphocytes are developmentally tolerized by rewiring the T cell antigen receptor signaling pathway through the downregulation of the adaptor protein, LAT

Rewiring of the TCR signalosome in natural intestinal Intraepithelial T lymphocytes drives non-deletional tolerance

Intraepithelial T lymphocytes (T-IEL) are a large population of cytotoxic T cells that protect the small intestinal epithelium against pathogens. Based on ontogeny, T-IEL can be categorized into two major subsets: induced and natural. Natural T-IEL are agonistically selected in the thymus on self-antigens before migrating directly to the small intestine. Despite having self-reactive T cell antigen receptors (TCR), natural T-IEL are maintained in a tolerized state in the gut by unknown mechanisms. We therefore investigated TCR signaling in T-IEL using multiplexed fluorescent cell barcoding, phosphoproteomics and TCR signaling reporter mouse models, which revealed that TCR signaling is intrinsically suppressed in natural, but not induced, T-IEL. Unexpectedly, we discover that this cell intrinsic suppression was mediated through altered TCR signalosome components. Specifically, downregulation of the key signaling adaptor, Linker for activation of T cells (LAT) during thymic selection is a vital checkpoint for the development and tolerization of natural IELs. Thus, TCR signaling is rewired in self-reactive natural T-IEL to promote tolerance and prevent inappropriate inflammation in the gut.One sentence summary Self-reactive natural intestinal intraepithelial T lymphocytes are developmentally tolerized by rewiring the T cell antigen receptor signaling pathway through the downregulation of the adaptor protein, LAT

CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function

<div><p>Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β–T cells (TCRβ–null) are highly susceptible and die over 10–18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.</p></div

JEV-specific IgM, IgG and neutralizing antibody levels.

<p>[A] JEV-specific IgM levels in WT B6 and TCRβ-null mice prior to (day 0) and post-infection on indicated days. n = 6 mice. [B] JEV-specific IgG levels in WT B6 and TCRβ-null mice prior to (day 0) and post-infection on indicated days. n = 6 mice. [C] Virus neutralization titers (PRNT50) in infected WT B6, TCRβ-null and TCRδ-null shown as reciprocal log 2 values. n = 5 mice per group.</p

Cytokine mRNA levels in the brains of infected B6 and TCRβ-/- mice.

<p>Relative mRNA levels of IL-1β [A], IL-6 [B], TNFα [C] and IFNγ [D] normalized to GAPDH in the brain homogenates of B6 and TCRβ-null mice infected with JEV on day 7 and 12 post-infection (mean ± SE, n > 7). $ = p<0.05, ψ = p<0.01.</p

T cells play a significant role in protection from JEV infection.

<p>[A] Survival kinetics of JEV infected WT B6 and Rag1-null mice over time. n = 10–13 mice. [B] Survival kinetics of JEV infected WT B6 and TCRδ-null mice over time. n = 15–20 mice. [C] Survival kinetics of JEV infected WT B6 and TCRβ-null mice over time. n = 12–18 mice. [D] Relative weight loss of JEV infected TCRβ-null mice as compared to uninfected TCRβ-null (mock) and infected WT B6 mice (mean ± SE, n > 10). [E] Clinical score for JEV infected WT B6 and TCRβ-null mice along with uninfected TCRβ-null (mock) mice (mean ± SE, n > 10). [F] Distribution of leukocyte subsets per brain in uninfected WT B6, infected WT B6 and infected TCRβ-null mice (mean ± SE, n as shown). [G] Frequencies of CD44highCD69+ activated cells in brains of infected WT B6 mice (mean ± SE, n as shown). [H] Frequencies of CD44highCD69+ JEV-specific cells in spleens of infected WT B6 mice (mean ± SE, n as shown). $ = p<0.05, ψ = p<0.01, # = p<0.005, φ = p<0.001.</p

Presence of T cells is essential for protection from primary JEV infection, but CD4 T cells appear less critical.

<p>[A] Survival kinetics of mock and JEV infected TCRβ-null with or without transfer of naïve T cells from WT B6 mice as indicated (n > 8). [B] Survival kinetics of mock and JEV infected TCRβ-null mice with purified naïve T cell or total spleen cell transfers from WT B6 mice (n = 7 for sorted naïve T cell transfer, for other groups n > 8). [C] Survival kinetics following JEV infection in WT B6 and MHCII-null mice over time (n = 14). [D] Relative weight loss of JEV infected MHCII-null mice as compared to infected WT B6 mice (mean ± SE, n = 14). [E] Clinical score for JEV infected WT B6 and MHCII-null mice (mean ± SE, n = 14). [F] Survival kinetics of mock and JEV infected TCRβ-null mice with or without transfer of naïve T cells from MHCII-null or WT B6 mice (n > 8). $ = p<0.05, ψ = p<0.01, # = p<0.005, n.s. = not significant.</p

Beige defect makes mice susceptible to JEV infection.

<p>[A] Survival kinetics following JEV infection in WT B6 and beige mice over time (n > 8). [B] Relative weight loss of JEV infected beige mice as compared to infected WT B6 mice (mean ± SE, n = 14). [C] Clinical score for JEV infected WT B6 and beige mice (mean ± SE, n = 14). [D] Survival kinetics of mock or JEV infected TCRβ-null mice with or without transfer of naïve T cells from beige or WT B6 mice (n > 8). $ = p<0.05, ψ = p<0.01, φ = p<0.001, ns = not significant.</p