Tolerance induction in memory CD4 T cells is partial and reversible

Memory T cells respond rapidly in part because they are less reliant on heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of re‐activating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggest they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re‐infection. These data indicate that antigen specific tolerogenic strategies must examine multiple parameters of T cell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells

Adora2b Adenosine Receptor Engagement Enhances Regulatory T Cell Abundance during Endotoxin-Induced Pulmonary Inflammation

Anti-inflammatory signals play an essential role in constraining the magnitude of an inflammatory response. Extracellular adenosine is a critical tissue-protective factor, limiting the extent of inflammation. Given the potent anti-inflammatory effects of extracellular adenosine, we sought to investigate how extracellular adenosine regulates T cell activation and differentiation. Adenosine receptor activation by a pan adenosine-receptor agonist enhanced the abundance of murine regulatory T cells (Tregs), a cell type critical in constraining inflammation. Gene expression studies in both naïve CD4 T cells and Tregs revealed that these cells expressed multiple adenosine receptors. Based on recent studies implicating the Adora2b in endogenous anti-inflammatory responses during acute inflammation, we used a pharmacologic approach to specifically activate Adora2b. Indeed, these studies revealed robust enhancement of Treg differentiation in wild-type mice, but not in Adora2b−/− T cells. Finally, when we subjected Adora2b-deficient mice to endotoxin-induced pulmonary inflammation, we found that these mice experienced more severe inflammation, characterized by increased cell recruitment and increased fluid leakage into the airways. Notably, Adora2b-deficient mice failed to induce Tregs after endotoxin-induced inflammation and instead had an enhanced recruitment of pro-inflammatory effector T cells. In total, these data indicate that the Adora2b adenosine receptor serves a potent anti-inflammatory role, functioning at least in part through the enhancement of Tregs, to limit inflammation

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.

BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches

Characterization of a Spontaneous 9.5-Kilobase-Deletion Mutant of Murine Gammaherpesvirus 68 Reveals Tissue-Specific Genetic Requirements for Latency

Murine gammaherpesvirus 68 (γHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While γHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of γHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available γHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency

Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function

Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies

Relative expression levels of adenosine receptor genes in T cell subsets.

<p>Real-time PCR analysis of mRNA for the four adenosine receptor genes in FACS purified CD4 T cell subsets of naïve CD4 T cells or Tregs, with Tregs isolated from FoxP3GFP or DEREG mice. Values were standardized to bulk spleen mRNA, with each value showing expression relative to actin. High level expression of FoxP3 mRNA is consistent with a highly purified population of Tregs. Data representative of two to three independent experiments, analyzing at least three independently isolated populations for both naïve CD4 T cells and Tregs. Data depict mean ± SEM for each transcript. Statistically significant differences were calculated by unpaired t test comparing expression in naïve CD4 T cells relative to Tregs, as indicated.</p

The Ikaros transcription factor regulates responsiveness to IL-12 and expression of IL-2 receptor alpha in mature, activated CD8 T cells.

The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation

An Adora2b-specific agonist enhances Treg abundance in vitro following activation of murine T cells.

<p>Bulk splenocytes from either <i>Adora2b</i>+/+ (C57BL/6J) or <i>Adora2b</i>−/− mice were cultured with soluble anti-CD3 for three days with or without the Bay60-6583 compound, at which time the relative abundance of Tregs was assessed. (A) Fold change in Treg abundance relative to <i>Adora2b</i>+/+ cultures without the Bay compound control, where Tregs were defined as viable, CD4+ FoxP3+ cells by flow cytometry. (B) Flow cytometry plots shown from <i>Adora2b</i>+/+ cultured splenocytes. Bay60-6583, an Adora2b-specific agonist was added to a final concentration of 4 nM. Data from two independent experiments, each containing 1–3 independent replicates. The numbers present on each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate, with FoxP3 expressing cells defined relative to isotype control-stained samples (not shown). Statistically significant differences are indicated and were calculated by one-way ANOVA followed by Bonferroni's post-test correction. ns indicates a comparison that is not statistically significantly different.</p