![IL-6 stimulates proliferation of naive and memory CD8 T cells in synergy with IL-7 or IL-15. A, Peripheral lymph node cells from naive Rag1 / H-Y TCR tg mice, and Rag1 / females harboring memory H-Y TCR tg T cells (see Materials and Methods) were stained with Abs against CD8, the tg TCR (T3.70), and one of the indicated memory cell markers. Representative data on the expression of individual memory markers in gated naive (shaded histograms ) and memory (line histogram) CD8 T3.70 H-Y TCR tg T cells are shown. B, Purified naive and memory CD8 T3.70 H-Y TCR tg T cells were stimulated with indicated cytokines at the same concentrations as in Fig. 1, and cell proliferation was measured by [ 3 H]thymidine incorporation. Representative data from two independent experiments are shown. Statistical comparison was made using Student's t test, and p values are indicated. C, MACS purification of naive (CD44 low ) and memory (CD44 high ) phenotype cells from negatively selected CD8 T cells of C57BL/6 mice. D, Expression of memory cell markers in CD44 low and CD44 high CD8 T cell subsets. E, Purified CD44 low and CD44 high CD8 T cell subsets were stimulated with indicated cytokines, and proliferation was measured by [ 3 H]thymidine incorporation. Representative results from two separate experiments are shown.](https://www.researchgate.net/profile/Alexandre-Cloutier-2/publication/51399614/figure/fig1/AS:335927600795648@1457102789127/IL-6-stimulates-proliferation-of-naive-and-memory-CD8-T-cells-in-synergy-with-IL-7-or_Q320.jpg)
![Concomitant stimulation of CD8 T lymphocytes by IL-6 or IL-21 augments STAT5 phosphorylation and its DNA-binding activity induced by IL-7 or IL-15. A, Purified P14 TCR tg CD8 T cells were stimulated with the indicated combinations of cytokines at the same concentrations used in Fig. 1. Twenty minutes after stimulation, the cells were lysed and phosphorylation of the indicated STAT molecules was evaluated by Western blot. The blots were reprobed to determine the amount of total STAT proteins. This experiment has been repeated more than three times with similar results. B, The ratio between the signal intensity of phospho-STAT and total STAT proteins shown in A was calculated from densitometric units of the protein bands corresponding to each stimulus. C, EMSA. Purified P14 TCR tg CD8 T cells were stimulated with the indicated combinations of cytokines at the same concentrations used in A. Twenty minutes after stimulation, nuclear extracts were prepared. Equivalent amount of nuclear proteins was incubated with 32 P-labeled GRR probe, and the DNA protein complexes were separated by electrophoresis and detected by autoradiography. D, Supershift assay: to determine the specificity of STAT binding, anti-STAT3 or anti-STAT5 Ab were incubated with the nuclear extracts before the addition of the labeled probe. Representative data from three independent experiments are shown. E, Synergistic stimulation by IL-21 or IL-6 does not augment the duration of IL-7-induced STAT5 activation. CD8 T cells purified from P14 TCR tg mice were stimulated with IL-7 either alone or in combination with IL-6 or IL-21 for 15 min. Stimulated cells were either lysed immediately, or washed and incubated in cytokine-free medium and lysed after indicated duration (chase). Whole cell lysates were evaluated for phospho-STAT5 and total STAT5 protein levels. Data from one of the two experiments with comparable results are shown. F, Inhibitors of Src family PTKs, PI3K, and MAPK pathway block cytokine-induced proliferation of CD8 T cells. Total lymph node cells (5 10 4 cells/well) of C57BL/6 mice were stimulated with indicated combinations of cytokines in 96-well microtiter plates. Pharmacological inhibitors of Src family protein tyrosine kinases (PP2,1.25 M), PI3K (LY 294002, 1.25 M), p38 MAPK (SB 202190, 750 nM), MEK (PD 98059, 10 M), JNK (JNK inhibitor II, 5 M), or the JAK-STAT3 pathway (JSI-124, 1.25 M) were added at the initiation of culture. [ 3 H]Thymidine was added during the last 8 –10 h of 3-day culture period. Proliferation data shown are representative of three independent experiments. Percentage of inhibition was calculated based on values obtained without the addition of any inhibitor.](https://www.researchgate.net/profile/Alexandre-Cloutier-2/publication/51399614/figure/fig2/AS:335927600795650@1457102789162/Concomitant-stimulation-of-CD8-T-lymphocytes-by-IL-6-or-IL-21-augments-STAT5_Q320.jpg)
![Prestimulation of CD8 T cells with IL-6 and IL-7 lowers the TCR signaling threshold. A, Purified P14 TCR tg CD8 T cells were stimulated with indicated combinations of plate-bound anti-CD3 mAb (left panel) or the gp33– 41 peptide presented by irradiated splenocytes as APC (right panel), in the absence or presence of IL-7, IL-6, or IL-21. Cell proliferation was measured by [ 3 H]thymidine incorporation. Representative data from three separate experiments are shown. B, Purified P14 TCR tg CD8 T cells, either freshly isolated (none) or cultured with indicated cytokines for 3 days and washed, were stimulated with anti- CD3 mAb (left panel) or the gp33– 41 peptide and APC (right panel) without any cytokine supplement. Cell proliferation was measured by [ 3 H]thymidine incorporation. Results from one of the two independent experiments are shown. C, Purified P14 cells were stimulated as described in B, except that IL-15 was used instead of IL-7 either alone or in combination with IL-6 or IL-21 for priming. Unlike the IL-7 stimulation, cell recovery following IL-15 stimulation alone was meager, and these cells responded poorly to subsequent stimulation via the TCR.](https://www.researchgate.net/profile/Alexandre-Cloutier-2/publication/51399614/figure/fig3/AS:335927600795651@1457102789200/Prestimulation-of-CD8-T-cells-with-IL-6-and-IL-7-lowers-the-TCR-signaling-threshold-A_Q320.jpg)
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IL-6, in Synergy with IL-7 or IL-15, Stimulates TCR-Independent Proliferation and Functional Differentiation of CD8+ T Lymphocytes
Abstract and Figures
Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8(+) T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8(+) T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8(+) T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8(+) T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8(+) T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8(+) T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.
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... Among the downregulated genes were TCF1 and LEF1, two critical regulators of stemness and memory formation [24]. Likewise, the two chemokine receptors IL6R and IL7R were downregulated, which are involved in stimulation of naïve precursor T cells [25]. Furthermore, they are crucial mediators of anti-tumor activity as well as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), the most frequently observed immune-mediated adverse events following CAR T cell administration [26]. ...
Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.
... IFN-α, IFN-β and IL-6, known to induce CD8 + T cell effector phenotype [34] [35], were increased in the supernatants of LCMV-infected BMMCs compared with uninfected control cells ( Figure 5). Therefore, LCMV-infected BMMCs not only upregulated important costimulatory molecules but also produced cytokines that promote CD8 + T cell differentiation. ...
Efficient anti‐viral responses of CD8 ⁺ T cells require signals that promote their effector cell differentiation, that are mainly provided by dendritic cells (DCs). Mast cells (MCs) are key drivers of DC maturation, but also influence their migration and antigen presenting properties and therefore indirectly mediate CD8 ⁺ T cell activation. MCs initiate innate immune responses at pathogen entry sites, promote the development of adaptive immune responses after infection, and release mediators including chemokines that recruit and activate immune cells including T cells during viral infections. However, whether MCs can directly activate virus‐specific CD8 ⁺ T cells remains largely unknown. Here, we used an in vitro viral infection model with lymphocytic choriomeningitis virus (LCMV)‐infected MCs or DCs co‐cultured with either LCMV‐specific CD8 ⁺ T cells or with WT (unspecific) CD8 ⁺ T cells. Similar to LCMV‐infected DCs, LCMV‐infected MCs clustered with virus‐specific CD8 ⁺ T cells and induced their activation and production of antiviral cytokines. In addition, the co‐stimulatory molecules CD86 and OX40L, but not CD80, were upregulated on MCs and an increased production of IL‐6 and type I interferons after LCMV infection was shown. Our findings suggest that MCs can promote CD8 ⁺ T cell activation during viral infections. MC‐mediated CD8 ⁺ T cell activation might be especially important within infected tissues where direct cellular interaction can take place. A better understanding of anti‐viral functions of MCs may help developing new strategies to better treat viral infections.
... Finally, analysis of CD8 + TCR, agnostic to gene expression profiles, corroborates the mechanistic consequences of rs16906115 allelic variation in melanoma, with patients who carry the risk allele displaying distinct clonal responses to treatment, consisting of clonal skewing characterized by fewer small clones but increased repertoire occupancy by expanded large clones. Although IL-7 has been described as an important mediator of naive T cell homeostasis in the resting state, it also has a vital role in T cell activation and memory formation 10,25,26,[37][38][39] . Consequently, these observations suggest the importance of the latter in the context of malignancy-related chronic antigen stimulation and ICB therapy. ...
Treatment with immune checkpoint blockade (ICB) frequently triggers immune-related adverse events (irAEs), causing considerable morbidity. In 214 patients receiving ICB for melanoma, we observed increased severe irAE risk in minor allele carriers of rs16906115, intronic to IL7 . We found that rs16906115 forms a B cell-specific expression quantitative trait locus (eQTL) to IL7 in patients. Patients carrying the risk allele demonstrate increased pre-treatment B cell IL7 expression, which independently associates with irAE risk, divergent immunoglobulin expression and more B cell receptor mutations. Consistent with the role of IL-7 in T cell development, risk allele carriers have distinct ICB-induced CD8 ⁺ T cell subset responses, skewing of T cell clonality and greater proportional repertoire occupancy by large clones. Finally, analysis of TCGA data suggests that risk allele carriers independently have improved melanoma survival. These observations highlight key roles for B cells and IL-7 in both ICB response and toxicity and clinical outcomes in melanoma.
... Overall, the inflammatory profiles of various analytes were largely similar between NK cell-depleted and control groups, suggesting the most robust factor was infection itself with likely only minor changes due to depletion. However, some analytes important for T cell activation, recruitment, and development (IL-6, IL-7, CCL3, IL-12p70) were higher in the NK cell-depleted animals (Fig. 7A) (31)(32)(33)(34)(35). A deeper evaluation with term enrichment analysis using log 2 transformed fold changes between control and NK cell-depleted groups also revealed several network enrichments (Fig. 7B). ...
Natural killer (NK) cells as major effector cells of the innate immune system can contribute significantly to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) control. However, a specific role for NK cells in blocking lentivirus transmission remains incompletely clear.
Suppressor of cytokine signaling 1 (SOCS1) is a potent regulator immune cell responses and a proven tumor suppressor. Inhibition of SOCS1 in T cells can boost antitumor immunity, whereas its loss in tumor cells increases tumor aggressivity. Investigations into the tumor suppression mechanisms so far focused on tumor cell-intrinsic functions of SOCS1. However, it is possible that SOCS1 expression in tumor cells also regulate antitumor immune responses in a cell-extrinsic manner via direct and indirect mechanisms. Here, we discuss the evidence supporting the latter, and its implications for antitumor immunity.
BACKGROUND
CD8 ⁺ T cells (CD8Ts) have been implicated in hypertension. However, the specific mechanisms are not fully understood. In this study, we explore the contribution of the P2X7 (purinergic receptor P2X7) receptor to CD8T activation and subsequent promotion of sodium retention in the kidney.
METHODS
We used mouse models of hypertension. Wild type were used as genetic controls, OT1 and Rag2/OT1 mice were utilized to determine antigen dependency, and P2X7-knockout mice were studied to define the role of P2X7 in activating CD8Ts and promoting hypertension. Blood pressure was monitored continuously and kidneys were obtained at different experimental end points. Freshly isolated CD8Ts from mice for activation assays and ATP stimulation. CD8T activation-induced promotion of sodium retention was explored in cocultures of CD8Ts and mouse DCTs.
RESULTS
We found that OT1 and Rag2/OT1 mice, which are nonresponsive to common antigens, still developed hypertension and CD8T-activation in response to deoxycorticosterone acetate/salt treatment, similar to wild-type mice. Further studies identified the P2X7 receptor on CD8Ts as a possible mediator of this antigen-independent activation of CD8Ts in hypertension. Knockout of the P2X7 receptor prevented calcium influx and cytokine production in CD8Ts. This finding was associated with reduced CD8T-DCT stimulation, reversal of excessive salt retention in DCTs, and attenuated development of salt-sensitive hypertension.
CONCLUSIONS
Our findings suggest a novel mechanism by which CD8Ts are activated in hypertension to exacerbate salt retention and infer that the P2X7 receptor on CD8Ts may represent a new therapeutic target to attenuate T-cell-mediated immunopathology in hypertension.
We studied the relationship between the level of cytokines in the lymph of the thoracic duct and the morphometric parameters of the mesenteric lymph nodes after surgical treatment of breast cancer, chemotherapy, and administration of fragmented (double-stranded, dsDNA) human DNA. In comparison with surgical treatment and with chemotherapy alone, administration of a human dsDNA has a stimulating effect on the T-cell link of the immune response. In the paracortical zone, the relationship between the chemokine MCP-1 and increased content of small lymphocytes in this zone was revealed. Interrelations of IL-2 cytokines with small lymphocytes and of IL-4 with medium lymphocytes were revealed in germinal centers. We also observed interrelations of IL-7 with small lymphocytes and IL-4 with macrophages in the medullary cords, chemokine MIP-1α with immature and mature plasma cells (the number of these cells is reduced), and of MCP-1 with immunoblasts (the number of which is also reduced) in the medullary sinuses.
Triggering of the TCR/CD3 complex with specific antigen or anti-CD3 monoclonal antibody initiates activation-induced cell death (AICD) in mature T cells, an effect also mediated by the Fas/FasL system. We have previously shown that CD2 stimulation rescues T cells from TCR/CD3-induced apoptosis by decreasing the expression of Fas and FasL. In the present study, we examined whether the endogenous production of IL-2 plays a role in the effects mediated by CD2 triggering. The results indicated that transcription of Fas/FasL is controlled by interleukin-2 (IL-2) production and that CD2 triggering rescues a T-cell hybridoma from AICD via decreased production of IL-2. To ascertain whether modulation of IL-2 may be a general mechanism of AICD control, we examined other stimuli, capable of modulating the expression of the Fas/FasL system and the ensuing AICD, for ability to affect production of IL-2. We found that IL-6 reduced the level of TCR/CD3-induced apoptosis and the expression of Fas/FasL, yet failed to inhibit IL-2 production. Because IL-2 is involved in both apoptosis and activation events, these results indicate that, in contrast to CD2, which inhibits apoptosis and T cell activation, IL-6 inhibits apoptosis but not IL-2–induced activation. These observations may provide the basis for differential control of T-cell activation and apoptosis.
Triggering of the TCR/CD3 complex with specific antigen or anti-CD3 monoclonal antibody initiates activation-induced cell death (AICD) in mature T cells, an effect also mediated by the Fas/FasL system. We have previously shown that CD2 stimulation rescues T cells from TCR/CD3-induced apoptosis by decreasing the expression of Fas and FasL. In the present study, we examined whether the endogenous production of IL-2 plays a role in the effects mediated by CD2 triggering. The results indicated that transcription of Fas/FasL is controlled by interleukin-2 (IL-2) production and that CD2 triggering rescues a T-cell hybridoma from AICD via decreased production of IL-2. To ascertain whether modulation of IL-2 may be a general mechanism of AICD control, we examined other stimuli, capable of modulating the expression of the Fas/FasL system and the ensuing AICD, for ability to affect production of IL-2. We found that IL-6 reduced the level of TCR/CD3-induced apoptosis and the expression of Fas/FasL, yet failed to inhibit IL-2 production. Because IL-2 is involved in both apoptosis and activation events, these results indicate that, in contrast to CD2, which inhibits apoptosis and T cell activation, IL-6 inhibits apoptosis but not IL-2–induced activation. These observations may provide the basis for differential control of T-cell activation and apoptosis.
Stat3, a member of STAT, is activated by a variety of cytokines such as IL-6 family of cytokines, granulocyte CSF, epidermal growth factor, and leptin. A recent study with mice genetically deficient in the Stat3 gene has revealed its important role in the early embryogenesis. To assess the function of Stat3 in adult tissues, we disrupted the Stat3 gene specifically in T cells by conditional gene targeting using Cre-loxP system. In Stat3-deficient T cells, IL-6-induced proliferation was severely impaired. IL-6 did not enhance cell cycle progression, but prevented apoptosis of normal T cells. In contrast, IL-6 did not prevent apoptosis of Stat3-deficient T cells. Antiapoptotic protein, Bcl-2, was normally up-regulated in response to IL-6 even in Stat3-deficient T cells. These results demonstrate that Stat3 activation is involved in IL-6-dependent T cell proliferation through prevention of apoptosis independently of Bcl-2.
rIL-6/B cell stimulatory factor 2 was found to augment CTL generation from mature as well as immature human T cells stimulated with UV-treated allogeneic cells. rIL-6 also acted on peanut agglutinin-positive murine thymocytes and Lyt-2-positive splenic T cells to give rise to CTL. rIL-6 alone could not induce CTL generation, the presence of IL-2 during the early phase of culture period was found to be essential for the IL-6 activity in the induction of CTL. The effect of rIL-6 was not mediated by the induction of IL-2 inasmuch as rIL-6 did not augment IL-2 production in MLC and anti-IL-2 antibody could not neutralize IL-6 activity. rIL-6 augmented CTL generation even when added 72 h after the initiation of cultures. The enhancing activity of rIL-6 could be neutralized with anti-IL-6 antibodies even when added 72 h after the initiation of cultures. The present data indicate that IL-6 acts in the late phase of CTL generation.
Growth and differentiation of thymocytes and mature T lymphocytes is regulated by cellular interactions that are in part mediated by soluble factors. We identify IL-6, formerly called B cell stimulating factor (BSF-2). IFN-beta 2, or hybridoma-plasmacytoma growth factor (HPGF) as a novel T cell costimulant rIL-6 induced a six-to seven-fold increase in proliferation of human thymocytes stimulated with suboptimal doses of PHA. A similar effect with added IL-6 could be observed using peripheral blood T lymphocytes, but only if the cultures were first rigorously depleted of monocytes that release high levels of IL-6. Analysis of the mechanism of the IL-6 effect on thymocytes and T lymphocytes showed that IL-6 did not lead to an increase in IL-2-R expression. Concentrations of antibody to IL-2-R inhibiting IL-2 effects did not block the IL-6-induced proliferation, indicating that the IL-6 effect was relatively IL-2 independent. These results identify IL-6 as a novel costimulant of human thymocytes and mature T lymphocytes, and suggest that IL-6 is also an important regulatory of cellular immunity.
Recent studies have investigated how defined peptides influence T cell development. Using a T cell receptor-transgenic beta2-microglobulin-deficient model, we have examined T cell maturation in fetal thymic organ cultures in the presence of various peptides containing single-alanine substitutions of the strong peptide agonist, p33. Cocultivation with the peptide A4Y, which contains an altered T cell contact residue, resulted in efficient positive selection. Several in vitro assays demonstrated that A4Y was a moderate agonist relative to p33. Although A4Y promoted positive selection over a wide concentration range, high doses of this peptide could not induce clonal deletion. Thymocytes maturing in the presence of A4Y were no longer able to respond to A4Y, but could proliferate against p33. These studies demonstrate that (a) peptides that induce efficient positive selection at high concentrations are not exclusively antagonists; (b) some agonists do not promote clonal deletion; (c) positive selection requires a unique T cell receptor-peptide-major histocompatibility complex interaction; and (d) interactions with selecting peptides during T cell ontogeny may define the functional reactivity of mature T cells.
Using a tumor model of spontaneously arising insulinomas expressing a defined tumor-associated antigen, we investigated whether tumor growth promotes cross-presentation and tolerance of tumor-specific T cells. We found that an advanced tumor burden enhanced cross-presentation of tumor-associated antigens to high avidity tumor-specific T cells, inducing T cell proliferation and limited effector function in vivo. However, contrary to other models, tumor-specific T cells were not tolerized despite a high tumor burden. In fact, in tumor-bearing mice, persistence and responsiveness of adoptively transferred tumor-specific T cells were enhanced. Accordingly, a potent T cell–mediated antitumor response could be elicited by intravenous administration of tumor-derived peptide and agonistic anti-CD40 antibody or viral immunization and reimmunization. Thus, in this model, tumor growth promotes activation of high avidity tumor-specific T cells instead of tolerance. Therefore, the host remains responsive to T cell immunotherapy.