IKAROS Family Transcription Factors in Lymphocyte Differentiation and Function.

The IKAROS family of transcription factors comprises four zinc-finger proteins (IKAROS, HELIOS, AIOLOS, and EOS), which over the last decades have been established to be critical regulators of the development and function of lymphoid cells. These factors act as homo- or heterodimers and are involved both in gene activation and repression. Their function often involves cross-talk with other regulatory circuits, such as the JAK/STAT, NF-κB, and NOTCH pathways. They control lymphocyte differentiation at multiple stages and are notably critical for lymphoid commitment in multipotent hematopoietic progenitors and for T and B cell differentiation downstream of pre-TCR and pre-BCR signaling. They also control many aspects of effector functions in mature B and T cells. They are dysregulated or mutated in multiple pathologies affecting the lymphoid system, which range from leukemia to immunodeficiencies. In this chapter, we review the molecular and physiological function of these factors in lymphocytes and their implications in human pathologies.

Isolation and immunosuppressive functions of myeloid-derived suppressor cell-derived exosomes.

Myeloid-derived suppressor cells (MDSCs) are an integral part of the tumor microenvironment (TME). MDSC's involvement in the TME starts as soon as the primary tumor starts to get its blood supply causing an immunosuppressive environment and tumor cell invasion, and then at the formation of premetastatic niche through full-blown metastasis in distal organs. All of these functions don't require physical interaction of MDSC as some of the MDSC's functions can be replicated by secreted exosomes (MDSC-derived exosomes), which can alter the microenvironment through cellular interaction by fusion with the plasma membrane and subsequent release of their cargo, consisting of proteins, soluble factors, lipids, DNAs, microRNAs (miRNAs), and RNAs. In this method paper, we explained how to isolate MDSC exosomes and how to use the exosome to observe immunosuppressive function. We also discussed how to measure the number of exosomes by nanoparticle tracking analysis. Additionally, we outlined how to measure the protein of exosomes as well as the types of protein by Bradford assay and membrane cytokine array respectively. We also provided instructions on how to utilize MDSC-derived exosomes to get knowledge about in vitro immune cell migration, scratch assay with the tumor cells, and in vivo effect of MDSC exosome along with T cell function and proliferation.

IL-7 producing immunotherapy improves ex vivo T cell functions of immunosenescent patients, especially post hip fracture.

Following acute stress such as trauma or sepsis, most of critically ill elderly patients become immunosuppressed and susceptible to secondary infections and enhanced mortality. We have developed a virus-based immunotherapy encoding human interleukin-7 (hIL-7) aiming at restoring both innate an adaptative immune homeostasis in these patients. We assessed the impact of this encoded hIL-7 on the ex vivo immune functions of T cells from PBMC of immunosenescent patients with or without hip fracture. T-cell ex vivo phenotyping was characterized in terms of senescence (CD57), IL-7 receptor (CD127) expression, and T cell differentiation profile. Then, post stimulation, activation status, and functionality (STAT5/STAT1 phosphorylation and T cell proliferation assays) were evaluated by flow cytometry. Our data show that T cells from both groups display immunosenescence features, express CD127 and are activated after stimulation by virotherapy-produced hIL-7-Fc. Interestingly, hip fracture patients exhibit a unique functional ability: An important T cell proliferation occurred compared to controls following stimulation with hIL-7-Fc. In addition, stimulation led to an increased naïve T cell as well as a decreased effector memory T cell proportions compared to controls. This preliminary study indicates that the produced hIL-7-Fc is well recognized by T cells and initiates IL-7 signaling through STAT5 and STAT1 phosphorylation. This signaling efficiently leads to T cell proliferation and activation and enables a T cell "rejuvenation." These results are in favor of the clinical development of the hIL-7-Fc expressing virotherapy to restore or induce immune T cell responses in immunosenescent hip fracture patients.

Positive regulators of T cell functions as predictors of prognosis and microenvironment characteristics of low-grade gliomas.

Background: Low-grade gliomas (LGG) are one of the most prevalent types of brain cancers. The efficacy of immunotherapy in LGG is limited compared to other cancers. Immunosuppression in the tumor microenvironment (TME) of LGG is one of the main reasons for the low efficacy of immunotherapy. Recent studies have identified 33 positive regulators of T cell functions (TPRs) that play a critical role in promoting the proliferation, activity, and functions of multiple immunocytes. However, their role in the TME of LGG has not been investigated. This study aimed to construct a risk model based on these TPRs and to detect the significance of immunotypes in predicting LGG prognosis and immunotherapy efficacy. Methods: A total of 688 LGGs and 202 normal brain tissues were extracted from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and Genotype-Tissue Expression (GTEx) databases. The NMF R package was used to identify TRP-related subtypes. The TPR prognostic model was established using the least absolute shrinkage and selection operator (LASSO) algorithm to predict the overall survival of LGG samples. Results: The Subtype 2 patients had worse survival outcomes, suppressed immune function, and higher immune cell infiltration. A risk regression model consisting of 14 TPRs was established, and its performance was validated in CGGA325 cohorts. The low-risk group exhibited better overall survival, immune microenvironment, and immunotherapy response, as determined via the TIDE algorithm, indicating that increasing the level of immune infiltration can effectively improve the response to immunotherapy in the low-risk group. The risk score was determined to be an independent hazard factor (p<0.001) although other clinical features (age, sex, grade, IDH status, 1p19q codel status, MGMT status, and accepted radiotherapy) were considered. Lastly, high-risk groups in both cohorts revealed optimal drug responses to rapamycin, paclitaxel, JW-7-52-1, and bortezomib. Conclusions: Our study identified two distinct TPR subtypes and built a TPR signature to elucidate the characteristics of T cell proliferation in LGG and its association with immune status and prognosis. These findings shed light on possible immunotherapeutic strategies for LGGs.

Emerging Next-Generation Target for Cancer Immunotherapy Research: The Orphan Nuclear Receptor NR2F6.

Additional therapeutic targets suitable for boosting anti-tumor effector responses have been found inside effector CD4+ and CD8+ T cells. It is likely that future treatment options will combine surface receptor and intracellular protein targets. Utilizing germline gene ablation as well as CRISPR/Cas9-mediated acute gene mutagenesis, the nuclear receptor NR2F6 (nuclear receptor subfamily 2 group F member 6, also called Ear-2) has been firmly characterized as such an intracellular immune checkpoint in effector T cells. Targeting this receptor appears to be a strategy for improving anti-tumor immunotherapy responses, especially in combination with CTLA-4 and PD-1. Current preclinical experimental knowledge firmly validates the immune checkpoint function of NR2F6 in murine tumor models, which provides a promising perspective for immunotherapy regimens in humans in the near future. While the clinical focus remains on the B7/CD28 family members, protein candidate targets such as NR2F6 are now being investigated in laboratories around the world and in R&D companies. Such an alternative therapeutic approach, if demonstrated to be successful, could supplement the existing therapeutic models and significantly increase response rates of cancer patients and/or expand the reach of immune therapy regimens to include a wider range of cancer entities. In this perspective review, the role of NR2F6 as an emerging and druggable target in immuno-oncology research will be discussed, with special emphasis on the unique potential of NR2F6 and its critical and non-redundant role in both immune and tumor cells.

T cell responses to Chlamydia.

Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection.

Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy.

BACKGROUND: NR2F6 has been proposed as an alternative cancer immune checkpoint in the effector T cell compartment. However, a realistic assessment of the in vivo therapeutic potential of NR2F6 requires acute depletion. METHODS: Employing primary T cells isolated from Cas9-transgenic mice for electroporation of chemically synthesized sgRNA, we established a CRISPR/Cas9-mediated acute knockout protocol of Nr2f6 in primary mouse T cells. RESULTS: Analyzing these Nr2f6CRISPR/Cas9 knockout T cells, we reproducibly observed a hyper-reactive effector phenotype upon CD3/CD28 stimulation in vitro, highly reminiscent to Nr2f6-/- T cells. Importantly, CRISPR/Cas9-mediated Nr2f6 ablation prior to adoptive cell therapy (ACT) of autologous polyclonal T cells into wild-type tumor-bearing recipient mice in combination with PD-L1 or CTLA-4 tumor immune checkpoint blockade significantly delayed MC38 tumor progression and induced superior survival, thus further validating a T cell-inhibitory function of NR2F6 during tumor progression. CONCLUSIONS: These findings indicate that Nr2f6CRISPR/Cas9 knockout T cells are comparable to germline Nr2f6-/- T cells, a result providing an independent confirmation of the immune checkpoint function of lymphatic NR2F6. Taken together, CRISPR/Cas9-mediated acute Nr2f6 gene ablation in primary mouse T cells prior to ACT appeared feasible for potentiating established PD-L1 and CTLA-4 blockade therapies, thereby pioneering NR2F6 inhibition as a sensitizing target for augmented tumor regression. Video abstract.