PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Myeloid antigen-presenting cell niches sustain antitumor T cells and license PD-1 blockade via CD28 costimulation.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia.

Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule.

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor ß (TGF-ß) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis.

Ampk regulates IgD expression but not energy stress with B cell activation.

Ampk is an energy gatekeeper that responds to decreases in ATP by inhibiting energy-consuming anabolic processes and promoting energy-generating catabolic processes. Recently, we showed that Lkb1, an understudied kinase in B lymphocytes and a major upstream kinase for Ampk, had critical and unexpected roles in activating naïve B cells and in germinal center formation. Therefore, we examined whether Lkb1 activities during B cell activation depend on Ampk and report surprising Ampk activation with in vitro B cell stimulation in the absence of energy stress, coupled to rapid biomass accumulation. Despite Ampk activation and a controlling role for Lkb1 in B cell activation, Ampk knockout did not significantly affect B cell activation, differentiation, nutrient dynamics, gene expression, or humoral immune responses. Instead, Ampk loss specifically repressed the transcriptional expression of IgD and its regulator, Zfp318. Results also reveal that early activation of Ampk by phenformin treatment impairs germinal center formation but does not significantly alter antibody responses. Combined, the data show an unexpectedly specific role for Ampk in the regulation of IgD expression during B cell activation.

Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures.

Copy number alteration (CNA) profiling of human tumors has revealed recurrent patterns of DNA amplifications and deletions across diverse cancer types. These patterns are suggestive of conserved selection pressures during tumor evolution but cannot be fully explained by known oncogenes and tumor suppressor genes. Using a pan-cancer analysis of CNA data from patient tumors and experimental systems, here we show that principal component analysis-defined CNA signatures are predictive of glycolytic phenotypes, including 18F-fluorodeoxy-glucose (FDG) avidity of patient tumors, and increased proliferation. The primary CNA signature is enriched for p53 mutations and is associated with glycolysis through coordinate amplification of glycolytic genes and other cancer-linked metabolic enzymes. A pan-cancer and cross-species comparison of CNAs highlighted 26 consistently altered DNA regions, containing 11 enzymes in the glycolysis pathway in addition to known cancer-driving genes. Furthermore, exogenous expression of hexokinase and enolase enzymes in an experimental immortalization system altered the subsequent copy number status of the corresponding endogenous loci, supporting the hypothesis that these metabolic genes act as drivers within the conserved CNA amplification regions. Taken together, these results demonstrate that metabolic stress acts as a selective pressure underlying the recurrent CNAs observed in human tumors, and further cast genomic instability as an enabling event in tumorigenesis and metabolic evolution.

BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy.

Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma.