Kinase suppressor of Ras 1 is not required for the generation of regulatory and memory T cells

The mammalian target of rapamycin (mTOR) kinase is a critical regulator of the differentiation of helper and regulatory CD4+ T cells, as well as memory CD8+ T cells. In this study, we investigated the role of the ERK signaling pathway in regulating mTOR activation in T cells. We showed that activation of ERK following TCR engagement is required for sustained mTOR complex 1 (mTORC1) activation. Absence of kinase suppressor of Ras 1 (KSR1), a scaffold protein of the ERK signaling pathway, or inhibition of ERK resulted in decreased mTORC1 activity following T cell activation. However, KSR1-deficient mice displayed normal regulatory CD4+ T cell development, as well as normal memory CD8+ T cell responses to LCMV and Listeria monocytogenes infection. These data indicate that despite its role in mTORC1 activation, KSR1 is not required in vivo for mTOR-dependent T cell differentiation

Normal effector and memory CD8+ T cell response to Listeria in KSR1-deficient mice.

<p>WT and KSR1-/- mice were infected with 2×10³ cfu of Lm-OVA, and presence of OVA epitope specific CD8+ T cells in the spleen was analyzed by flow cytometry 7 (A, B) and 30 (C, D, G, H) days after infection. 30 days after Lm-OVA infection, mice were challenged with 10⁵ cfu of Lm-OVA and presence of OVA epitope specific CD8+ T cells in the spleen was assessed 5 days later (E, F). A, C, E: Percentage of Db-GP33 Tetramer positive cells in CD8+ T cells. Plots are representative of two experiments with 3–8 mice per group. B, D, F: Number of Db-GP33 Tetramer+ CD8+ T cells per spleen. Dashed line represents average numbers in naive mice and mice infected with Lm. G, H: CD62L and CD127 expression in Db-GP33 Tetramer+ CD8+ T splenocytes 30 days after Lm-OVA infection. Data is presented as mean + SEM and is representative of two experiments with 3–8 mice per group. There were no significant differences between WT and KO mice (t-test).</p

Normal effector and memory CD8+ T cell response to LCMV in KSR1-deficient mice.

<p>WT, KSR1-/-, and WT mice treated with rapamycin were infected with 10⁵ pfu of LCMV-Armstrong, and presence of GP33 epitope specific CD8+ T cells was analyzed by flow cytometry 7 (A, B) and 30 (C–H) days after infection in the blood (A–D) and spleen (E–H). A, C, E: Percentage of Db-GP33 Tetramer positive cells in CD8+ T cells. Plots are representative of two experiments with 5 mice per group. B, D: Percentage of Db-GP33 Tetramer+ CD8+ T cells in peripheral blood mononuclear cells. F: Number of Db-GP33 Tetramer+ CD8+ T cells per spleen 30 days after infection. G, H: CD62L and CD127 expression in Db-GP33 Tetramer+ CD8+ T splenocytes 30 days after infection. Data is presented as mean + SEM and is representative of two experiments with 5 mice per group. *: p<0.05, t-test.</p

MEK inhibition decreases mTOR activity in T cells.

<p>Splenocytes from B6 mice were pretreated or not for one hour with 10 µM UO126 and cultured for 2 to 48 hrs with or without anti-CD3 + anti-CD28 stimulation (5 µg/mL each). Phosphorylation of ERK (A, B), T421/S424-S6K (C, D) and T389-S6K (E, F) in CD8+ T cells was measured by flow cytometry. A, C, E: Representative histograms of unstimulated cells (filled histogram), cells stimulated with CD3+CD28 and treated (red line) or not (black line) with UO126, gated on CD8+ T cells. Graphs are representative of 3 to 5 experiments. C, D, F: Fold increase phosphorylation of ERK, T421/S424-S6K and T389-S6K in anti-CD3 + anti-CD28 stimulated CD8+ T cells treated (red) or not (black) with UO126, compared to unstimulated cells. Each dot represents an independent experiment. p-ERK, p-T421/S424-S6K and p-T389-S6K levels were normalized within each experiment to unstimulated control T cells. *: p<0.05, paired t-test.</p

Regulatory CD4+ T cell development is normal in KSR1-deficient T cells.

<p>Thymocytes (A, B) and splenocytes (C, D) of WT (white bars) and KSR1-/- (black bars) mice were analyzed for the presence of regulatory CD4+ T cells by flow cytometry. A, C: Percentage of FoxP3+ CD25+ cells in CD4+ CD8- single positive (SP) thymocytes (A) and CD4+ CD3+ T cells (B). B, D: Numbers of FoxP3+ CD25+ CD4 SP by thymuses (B) and CD4+ T cells by spleens (D). Data is presented as mean + SEM and represent 10-12 mice per group. There were no significant differences between WT and KO mice (t-test).</p

mTOR activity is decreased in KSR1-deficient T cells.

<p>Splenocytes from WT or KSR1-/- mice were cultured with or without anti-CD3 + anti-CD28 (5 µg/mL each) stimulation for 2 to 48 hrs. Phosphorylation of ERK (A, B), T421/S424-S6K (C, D) and T389-S6K (E, F), and expression of CD127 (G,H) in CD8+ T cells was measured by flow cytometry. A, C, E: Representative histograms of unstimulated WT cells (filled grey histogram), unstimulated KO cells (blue line), and WT (black line) or KO (red line) cells stimulated with CD3+CD28, gated on CD8+ T cells. Graphs are representative of 3 to 4 experiments. C, D, F: Fold increase phosphorylation of ERK, T421/S424-S6K and T389-S6K in stimulated WT (black) and KO (red) CD8+ T cells compared to non-stimulated cells. Each dot represents an independent experiment. p-ERK, p-T421/S424-S6K and p-T389-S6K levels were normalized within each experiment to unstimulated control T cells. *: p<0.05, paired t-test. G-H. Representative histograms (G) and fold increase in comparison to non-stimulated control T cells (H) of CD127 expression in WT (black) and KO (red) cells stimulated with CD3+CD28, gated on CD8+ T cells. Grey line: isotype control. H. Data is presented as mean + SEM and is representative of two independent experiments. *: p<0.05, unpaired t-test.</p

Growth hormone enhances thymic function in HIV-1–infected adults

Growth hormone (GH) is an underappreciated but important regulator of T cell development that can reverse age-related declines in thymopoiesis in rodents. Here, we report findings of a prospective randomized study examining the effects of GH on the immune system of HIV-1–infected adults. GH treatment was associated with increased thymic mass. In addition, GH treatment enhanced thymic output, as measured by both the frequency of T cell receptor rearrangement excision circles in circulating T cells and the numbers of circulating naive and total CD4+ T cells. These findings provide compelling evidence that GH induces de novo T cell production and may, accordingly, facilitate CD4+ T cell recovery in HIV-1–infected adults. Further, these randomized, prospective data have shown that thymic involution can be pharmacologically reversed in humans, suggesting that immune-based therapies could be used to enhance thymopoiesis in immunodeficient individuals

Neoadjuvant CD40 agonism remodels the tumor immune microenvironment in locally advanced esophageal/gastroesophageal junction cancer

Sotigalimab is an agonistic anti-CD40 monoclonal antibody that can modulate anti-tumor immune responses. In a phase II clinical trial of sotigalimab combined with neoadjuvant chemoradiation (CRT) in locally advanced esophageal/gastroesophageal junction (E/GEJ) cancer with the primary outcome of efficacy as measured by pathologic complete response (pCR) rate, the combination induced pCR in 38% of treated patients. We investigated the mechanism of action of sotigalimab in samples obtained from this clinical trial. Tumor biopsies and peripheral blood samples were collected at baseline, following an initial dose of sotigalimab, and at the time of surgery after CRT completion from six patients. High dimensional single cell techniques were used, including combined single cell RNA sequencing and proteomics (CITEseq) and multiplexed ion beam imaging (MIBI), to analyze immune responses. Sotigalimab dramatically re-modeled the immune compartment in the periphery and within the tumor microenvironment (TME), increasing expression of molecules related to antigen processing and presentation and altering metabolic pathways in myeloid cells. Concomitant with these changes in myeloid cells, sotigalimab treatment primed new T cell clonotypes and increased the density and activation of T cells with enhanced cytotoxic function. Sotigalimab treatment also induced a decrease in the frequency of Tregs in the TME. These findings indicate that a single dose of sotigalimab leads to enhanced antigen presentation that can activate T cells and induce new T cell clones. This restructuring of the TME provides elements which are critical to the development of effective antitumor immune responses and improved clinical outcomes