Deborah R. Surman

Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells.

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I–restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.

CD8+ T Cell Immunity Against a Tumor/Self-Antigen Is Augmented by CD4+ T Helper Cells and Hindered by Naturally Occurring T Regulatory Cells

CD4+ T cells control the effector function, memory, and maintenance of CD8+ T cells. Paradoxically, we found that absence of CD4+ T cells enhanced adoptive immunotherapy of cancer when using CD8+ T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4+CD25− Th cells (Th cells) with tumor/self-reactive CD8+ T cells and vaccination into CD4+ T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4+ T cells that contained a mixture of Th and CD4+CD25+ T regulatory cells (Treg cells) or Treg cells alone prevented effective adoptive immunotherapy. Maintenance of CD8+ T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2−/− mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring Treg cells to be effective.

IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T cells

IL-15 and IL-2 possess similar properties, including the ability to induce T cell proliferation. However, whereas IL-2 can promote apoptosis and limit CD8+ memory T cell survival and proliferation, IL-15 helps maintain a memory CD8+ T cell population and can inhibit apoptosis. We sought to determine whether IL-15 could enhance the in vivo function of tumor/self-reactive CD8+ T cells by using a T cell receptor transgenic mouse (pmel-1) whose CD8+ T cells recognize an epitope derived from the self/melanoma antigen gp100. By removing endogenous IL-15 by using tumor-bearing IL-15 knockout hosts or supplementing IL-15 by means of exogenous administration, as a component of culture media or as a transgene expressed by adoptively transferred T cells, we demonstrate that IL-15 can improve the in vivo antitumor activity of adoptively transferred CD8+ T cells. These results provide several avenues for improving adoptive immunotherapy of cancer in patients.

Cutting Edge: CD4+ T Cell Control of CD8+ T Cell Reactivity to a Model Tumor Antigen

Neoantigens resulting from the inherent genomic instability of tumor cells generally do not trigger immune recognition. Similarly, transfection of tumors with model Ags often fails to elicit CD8+ T cell responses or alter a tumor’s growth rate or lethality. We report here that the adoptive transfer of activated Th1-type CD4+ T cells specific for a model tumor Ag results in the de novo generation of CD8+ T cells with specificity to that Ag and concomitant tumor destruction. The anti-tumor effects of the CD4+ T cells required the presence of both MHC class I and class II on host cells, as evidenced by experiments in knockout mice, suggesting that CD4+ T cells enhanced the ability of host APC to activate endogenous CD8+ T cells. These results indicate that the apparent inability of tumor cells expressing highly immunogenic epitopes to activate tumor-specific CD8+ T cells can be altered by activated CD4+ T cells.

Poor immunogenicity of a self/tumor antigen derives from peptide–MHC-I instability and is independent of tolerance

Understanding the mechanisms underlying the poor immunogenicity of human self/tumor antigens is challenging because of experimental limitations in humans. Here, we developed a human-mouse chimeric model that allows us to investigate the roles of the frequency and self-reactivity of antigen-specific T cells in determination of the immunogenicity of an epitope (amino acids 209-217) derived from a human melanoma antigen, gp100. In these transgenic mice, CD8+ T cells express the variable regions of a human T cell receptor (hTCR) specific for an HLA-A*0201-restricted gp100(209-217). Immunization of hTCR-transgenic mice with gp100(209-217) peptide elicited minimal T cell responses, even in mice in which the epitope was knocked out. Conversely, a modified epitope, gp100(209-217(2M)), was significantly more immunogenic. Both biological and physical assays revealed a fast rate of dissociation of the native peptide from the HLA-A*0201 molecule and a considerably slower rate of dissociation of the modified peptide. In vivo, the time allowed for dissociation of peptide-MHC complexes on APCs prior to their exposure to T cells significantly affected the induction of immune responses. These findings indicate that the poor immunogenicity of some self/tumor antigens is due to the instability of the peptide-MHC complex rather than to the continual deletion or tolerization of self-reactive T cells.

Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice

Maternal supplementation with folic acid is known to reduce the incidence of neural tube defects (NTDs) by as much as 70%. Despite the strong clinical link between folate and NTDs, the biochemical mechanisms through which folic acid acts during neural tube development remain undefined. The Mthfd1l gene encodes a mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase, termed MTHFD1L. This gene is expressed in adults and at all stages of mammalian embryogenesis with localized regions of higher expression along the neural tube, developing brain, craniofacial structures, limb buds, and tail bud. In both embryos and adults, MTHFD1L catalyzes the last step in the flow of one-carbon units from mitochondria to cytoplasm, producing formate from 10-formyl-THF. To investigate the role of mitochondrial formate production during embryonic development, we have analyzed Mthfd1l knockout mice. All embryos lacking Mthfd1l exhibit aberrant neural tube closure including craniorachischisis and exencephaly and/or a wavy neural tube. This fully penetrant folate-pathway mouse model does not require feeding a folate-deficient diet to cause this phenotype. Maternal supplementation with sodium formate decreases the incidence of NTDs and partially rescues the growth defect in embryos lacking Mthfd1l. These results reveal the critical role of mitochondrially derived formate in mammalian development, providing a mechanistic link between folic acid and NTDs. In light of previous studies linking a common splice variant in the human MTHFD1L gene with increased risk for NTDs, this mouse model provides a powerful system to help elucidate the specific metabolic mechanisms that underlie folate-associated birth defects, including NTDs.

Bedside to bench and back again: how animal models are guiding the development of new immunotherapies for cancer.

Immunotherapy using adoptive cell transfer is a promising approach that can result in the regression of bulky, invasive cancer in some patients. However, currently available therapies remain less successful than desired. To study the mechanisms of action and possible improvements in cell‐transfer therapies, we use a murine model system with analogous components to the treatment of patients. T cell receptor transgenic CD8+ T cells (pmel‐1) specifically recognizing the melanocyte differentiation antigen gp100 are adoptively transferred into lympho‐depleted mice bearing large, established, 14‐day subcutaneous B16 melanoma (0.5–1 cm in diameter) on the day of treatment. Adoptive cell transfer in combination with interleukin interleukin‐2 or interleukin‐15 cytokine administration and vaccination using an altered form of the target antigen, gp100, can result in the complete and durable regression of large tumor burdens. Complete responders frequently develop autoimmunity with vitiligo at the former tumor site that often spreads to involve the whole coat. These findings have important implications for the design of immunotherapy trials in humans.

Bile acid and cigarette smoke enhance the aggressive phenotype of esophageal adenocarcinoma cells by downregulation of the mitochondrial uncoupling protein-2

Limited information is available regarding mechanisms that link the known carcinogenic risk factors of gastro-esophageal reflux and cigarette smoking to metabolic alterations in esophageal adenocarcinoma (EAC). In the present study, we utilized a novel in-vitro model to examine whether bile acid and cigarette smoke increase the aggressiveness of EAC and whether these changes are associated with metabolic changes. EAC cells (EACC) were exposed to 10 μg/ml cigarette smoke condensate (CSC) and/or 100 μM of the oncogenic bile acid, deoxycholic acid (DCA), for 5 days. These exposure conditions were chosen given their lack of effect on proliferation or viability. DCA and CSC increased invasion, migration, and clonogenicity in EAC cells. These changes were associated with concomitant increases in ATP, ROS, and lactate production indicative of increased mitochondrial respiration as well as glycolytic activity. DCA and CSC exposure significantly decreased expression of uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein implicated in regulation of the proton gradient. Knockdown of UCP2 in EACC phenocopied DCA and CSC exposure as evidenced by increased cell migration, invasion, and clonogenicity, whereas over-expression of UCP2 had an inverse effect. Furthermore, over-expression of UCP2 abrogated DCA and CSC-mediated increases in lactate and ATP production in EACC. DCA and CSC promote the aggressive phenotype of EACC with concomitant metabolic changes occurring via downregulation of UCP2. These results indicate that UCP2 is integral to the aggressive phenotype of EACC. This mechanism suggests that targeting alterations in cellular energetics may be a novel strategy for EAC therapy.

Metabolomic and BH3 profiling of esophageal cancers: novel assessment methods for precision therapy

BackgroundEsophageal cancers accounted for nearly 16,000 deaths in 2016. The number of patients with esophageal cancers increases every year. Neoadjuvant chemoradiotherapy (nCRT) prior to esophagectomy is a standard treatment for esophageal cancers. The patients who have no residual tumor (pathological complete response (pCR)) at surgery are the most likely to experience long term survival. Accurately determining which patients will have a pCR will improve prognostic information for patients and families, confirm lack of response to nCRT, or avoid surgery if no residual tumor is present. Imaging, endoscopy, and liquid biomarkers have all failed to detect pCR without performing an esophagectomy.MethodsIn this study, we are enrolling patients with esophageal adenocarcinoma and squamous cell carcinoma. Patients will undergo standard evaluation including CT scans, laboratory tests, endoscopy with biopsies, and evaluation by a thoracic surgeon. Tissue biopsy is required for enrollment that will be sent for BH3 profiling and metabolomics. Patients will be treated with standard nCRT followed by surgery. Patients with metastatic disease are not eligible. Surgery at the National Cancer Institute will be minimally-invasive robotic surgery. Patients will remain on study indefinitely with regular clinic visits and imaging tests.DiscussionThe mitochondria are critically involved in the intrinsic pathway apoptosis. Bcl-2 homology domain 3 (BH3) profiling is a technique to measure a cell’s susceptibility to apoptosis. BH3 profiling measures the relative interactions of proteins that induce or block apoptosis. The collective balance of these proteins determines whether a cell is near the threshold to undergo apoptosis. If the cell is near this threshold, then the tumor may be more likely to die when treated with nCRT. The mitochondria secrete metabolites that may be detectable as biomarkers. Metabolomics is a global assessment of all metabolite changes that has been performed for detection, monitoring, prognosis, and treatment response in cancers. Stratification of patients based on whether pCR occurs or not may elucidate metabolomic signatures that may be associated with response. We are asking whether BH3 profiling or a metabolomic signature will correlate with tumor death after nCRT for esophageal cancer.Trial registrationNCT03223662; Clinicaltrials.gov. July 21, 2017.

Acquisition of Terminal Effector Functions In Vitro Impairs In Vivo Anti-Tumor Efficacy

Tet). However, in tumors, two separate CD8+ cell populations were observed, namely CD8, Tet and CD8, Tet. The ratio between these two populations was found to differ according to the route of T cell administration. CD69 and CD25 were found to be up-regulated in the CD8, Tet population, as would be expected for recently activated T cells. In conclusion, adoptive transfer resulted in accumulation of high numbers of tumor-specific T cells in the tumor tissue irrespective of the injection route. In the spleen, intraperitoneal injection resulted in a significantly higher number of adoptively transferred cells on day 5 and 7, compared to intravenous injection.