Unraveling the Complex Interplay Between T Cell Metabolism and Function.

Metabolism drives function, on both an organismal and a cellular level. In T cell biology, metabolic remodeling is intrinsically linked to cellular development, activation, function, differentiation, and survival. After naive T cells are activated, increased demands for metabolic currency in the form of ATP, as well as biomass for cell growth, proliferation, and the production of effector molecules, are met by rewiring cellular metabolism. Consequently, pharmacological strategies are being developed to perturb or enhance selective metabolic processes that are skewed in immune-related pathologies. Here we review the most recent advances describing the metabolic changes that occur during the T cell lifecycle. We discuss how T cell metabolism can have profound effects on health and disease and where it might be a promising target to treat a variety of pathologies.

Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease.

BACKGROUND: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large patient cohorts, we identified increased numbers of CD56brightPerforin- regulatory-like NK cells (NKreg-like) associated with cGvHD suppression. Thus, we hypothesized that NKreg-like cells may be a potential candidate for cGvHD cell therapy. AIM: To expand NKreg-like cells while maintaining regulatory phenotype and function. METHODS: Total NK cells were first expanded with IL-2, which was then combined with rapamycin, Transforming Growth Factor Beta 1 (TGF-ß1), NECA (Adenosine A2A receptor (A2AR) agonist), metformin, or dexamethasone, to prevent change in cell phenotype/function. The functional characteristics were evaluated via T cell suppression assays and the phenotype was measured using flow cytometry. The optimal expansion protocol was compared in terms of function and metabolism for three NK expansion media, and cells from cord vs. peripheral blood. Further, expanded NKreg-like cell gene expression was characterized using bulk RNA sequencing. Finally, NKreg-like cells were differentiated from CD34+ hematopoietic stem and progenitor cells (HSPCs) and compared in terms of proliferation and function. RESULTS: The expansion of total NK cells found the addition of TGF-ß1 and/or NECA with the pulsing of rapamycin in IL-2 containing media to prevent NKreg-like differentiation (up to 200-fold expansion). Expanded NKreg-like cells maintained a phenotype, transcriptome, and T cell suppression similar to freshly isolated NKreg-like cells. NKreg-like expansion was greatest in the Immunocult media (up to 300-fold), and NKreg-like cells from peripheral blood demonstrated significantly greater proliferation than cells isolated from cord blood (65-fold). The metabolic profile of NKreg-like and cytolytic NK cells appeared similar at baseline, though rapamycin induced a shift to oxidative over glycolytic metabolism. Further, we demonstrated that suppressive NKreg-like cells may alternatively be expanded from CD34+ cells isolated from cord blood, reaching an average 340-fold expansion. CONCLUSIONS: In conclusion, our studies have optimized two alternative expansion approaches for deriving functional NKreg-like cells. Additionally, evaluating the transcriptomic and metabolic characteristics provides useful information regarding NKreg-like cell function and differentiation. With further optimization and in vivo validation, we may work towards preparing these cells as a therapy for cGvHD.

Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma.

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc - transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659.

PAX3-FOXO1 induces up-regulation of Noxa sensitizing alveolar rhabdomyosarcoma cells to apoptosis.

Alveolar rhabdomyosarcoma (ARMS) has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR), which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a γ-secretase inhibitor (GSI1, Z-LLNle-CHO), the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.

Genomic stability and functional activity may be lost in telomerase-transduced human CD8+ T lymphocytes.

To obtain the large amount of T cells required for adoptive immunotherapy in a clinical setting, T-cell lifespan extension by human telomerase reverse transcriptase (hTERT) transduction is of particular interest. However, constitutive expression of hTERT is associated with malignant transformation and thus warrants a detailed evaluation of the safety of hTERT-transduced T cells before clinical application. In view of this, we performed an extensive cytogenetic analysis of hTERT-transduced MART-1 (melanoma antigen recognized by T cell 1)-and human papillomavirus type 16 (HPV16) E7-specific human CD8+ cytotoxic T lymphocytes (CTLs), reactive against melanoma and cervical carcinoma, respectively. Our results, obtained by (spectral) karyotyping and array comparative genomic hybridization, showed the development of minor chromosomal aberrations in an hTERT-transduced MART-1-specific CTL clone, whereas severe clonal aberrations were detected in an hTERT-transduced HPV16 E7-specific CTL clone. Furthermore, hTERT transduction did not protect CTLs from immunosenescence, because the HPV16 E7-specific, hTERT-transduced CTL clone showed a decreased functional activity on prolonged culture. Although the general frequency of major chromosomal aberrations in hTERT-transduced CTLs and the in vivo significance of our observations remain still unclear at this point, the currently available data suggest that clinical application of hTERT-transduced CTLs should proceed with caution.