Uncoupling protein 2 regulates metabolic reprogramming and fate of antigen-stimulated CD8+ T cells.

Adoptive cell therapy (ACT) employing ex vivo-generated tumor antigen-specific CD8+ T cells shows tumor efficacy when the transferred cells possess both effector and memory functions. New strategies based on understanding of mechanisms that balance CD8+ T cell differentiation toward effector and memory responses are highly desirable. Emerging information confirms a central role for antigen-induced metabolic reprogramming in CD8+ T cell differentiation and clonal expansion. The mitochondrial protein uncoupling protein 2 (UCP2) is induced by antigen stimulation of CD8+ T cells; however, its role in metabolic reprogramming underlying differentiation and clonal expansion has not been reported. Employing genetic (siRNA) and pharmacologic (Genipin) approaches, we note that antigen-induced UCP2 expression reduces glycolysis, fatty acid synthesis and production of reactive oxygen species to balance differentiation with survival of effector CD8+ T cells. Inhibition of UCP2 promotes CD8+ T cell terminal differentiation into short-lived effector cells (CD62L(lo)KLRG1(Hi)IFNγ(Hi)) that undergo clonal contraction. These findings are the first to reveal a role for antigen-induced UCP2 expression in balancing CD8+ T cell differentiation and survival. Targeting UCP2 to regulate metabolic reprogramming of CD8+ T cells is an attractive new approach to augment efficacy of tumor therapy by ACT.

Structure-based design of altered MHC class II-restricted peptide ligands with heterogeneous immunogenicity.

Insights gained from characterizing MHC-peptide-TCR interactions have held the promise that directed structural modifications can have predictable functional consequences. The ability to manipulate T cell reactivity synthetically or through genetic engineering might thus be translated into new therapies for common diseases such as cancer and autoimmune disorders. In the current study, we determined the crystal structure of HLA-DR4 in complex with the nonmutated dominant gp100 epitope gp10044-59, associated with many melanomas. Altered peptide ligands (APLs) were designed to enhance MHC binding and hence T cell recognition of gp100 in HLA-DR4(+) melanoma patients. Increased MHC binding of several APLs was observed, validating this approach biochemically. Nevertheless, heterogeneous preferences of CD4(+) T cells from several HLA-DR4(+) melanoma patients for different gp100 APLs suggested highly variable TCR usage, even among six patients who had been vaccinated against the wild-type gp100 peptide. This heterogeneity prevented the selection of an APL candidate for developing an improved generic gp100 vaccine in melanoma. Our results are consistent with the idea that even conservative changes in MHC anchor residues may result in subtle, yet crucial, effects on peptide contacts with the TCR or on peptide dynamics, such that alterations intended to enhance immunogenicity may be unpredictable or counterproductive. They also underscore a critical knowledge gap that needs to be filled before structural and in vitro observations can be used reliably to devise new immunotherapies for cancer and other disorders.

Early lymphocyte recovery after intensive timed sequential chemotherapy for acute myelogenous leukemia: peripheral oligoclonal expansion of regulatory T cells.

Few published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4(+) and included a greatly expanded population of CD3(+)CD4(+)CD25(+)Foxp3(+) T cells. Recovering CD3(+)CD4(+)CD25(+)Foxp3(+) T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.

Immunologic Signatures of Peripheral Blood T Cells Reveal the Outcome of p53MVA Vaccine and Pembrolizumab Treatment in Patients with Advanced Ovarian Cancer.

PURPOSE: Our previous studies indicated that p53-reactive T cells were associated with clinical benefit in patients with advanced ovarian cancer who were treated with p53-expressing modified vaccinia Ankara (p53MVA) vaccine and gemcitabine chemotherapy. To replace chemotherapy with an approach that will enhance vaccine efficacy and antitumor immunity, we treated patients with p53MVA in combination with PD-1 checkpoint blocker, pembrolizumab. We also attempted to further characterize the activation status of T cells prior to vaccination and during treatment. EXPERIMENTAL DESIGN: Patients received up to three triweekly vaccinations concurrent with pembrolizumab, followed by pembrolizumab monotherapy at 3-week intervals. Correlative studies analyzed peripheral blood T-cell phenotypes and profiles of immune function gene expression. RESULTS: We observed 6/28 (21%) patients with a clinical benefit to therapy, including 3 partial responses (PR) and 3 patients with stable disease (SD) for 6+ months. The median progression-free survival was 1.8 months (95% confidence interval: 1.7-3.8) and median overall survival was 15.1 months (9.4-30.4). Two patients remain progression-free at 28 and 33 months. Of the 18 patients evaluable in correlative studies, 6 were immunologic responders of whom 5 had clinical benefit (3 PR, 2 SD). Immunologic non-responders expressed in pretreatment peripheral blood mononuclear cell samples high levels of mRNA for multiple molecules associated with terminally differentiated T cells. CONCLUSIONS: p53MVA/pembrolizumab immunotherapy showed promising antitumor activity in patients who demonstrated functionally competent peripheral blood T cells. Detection of markers of terminally differentiated T cells before treatment may identify patients unlikely to respond to p53MVA/pembrolizumab. SIGNIFICANCE: The activity of a combination immunotherapy of p53 vaccine and PD-1 checkpoint blockade in patients with platinum-resistant ovarian cancer was evaluated in a phase II trial. Clinical benefit was correlated with the responsive immune status of patients before and during the treatment, defining potential predictive markers for immune therapy.

Human papillomavirus 16-associated cervical intraepithelial neoplasia in humans excludes CD8 T cells from dysplastic epithelium.

High-grade cervical dysplasia caused by human papillomavirus (HPV) type 16 is a lesion that should be susceptible to an HPV-specific immune response; disease initiation and persistence is predicated on expression of two viral Ags, E6 and E7. In immune-competent subjects, at least 25% of HPV16(+) high-grade cervical dysplasia lesions undergo complete regression. However, in the peripheral blood, naturally occurring IFN-γ T cell responses to HPV E6 and E7 are weak, requiring ex vivo sensitization to detect, and are not sufficiently sensitive to predict regression. In this study, we present immunologic data directly assessing cervical lymphocytes from this cohort. We found that nearly all cervical tissue T cells express the mucosal homing receptor, α(4)ß(7) surface integrin. T cells isolated from dysplastic mucosa were skewed toward a central memory phenotype compared with normal mucosal resident T cells, and dysplastic lesions expressed transcripts for CCL19 and CCL21, raising the possibility that the tissue itself sustains a response that is not detectable in the blood. Moreover, lesion regression in the study window could retrospectively be predicted at study entry by the ability of CD8(+) T cells to gain access to lesional epithelium. Vascular endothelial expression of mucosal addressin cell adhesion molecule-1, the ligand that supports entry of α(4)ß(7)(+) T cells into tissues, colocalized tightly with the distribution of CD8 T cells and was not expressed in persistent dysplastic epithelium. These findings suggest that dysregulated expression of vascular adhesion molecules plays a role in immune evasion very early in the course of HPV disease.

Naturally occurring systemic immune responses to HPV antigens do not predict regression of CIN2/3.

Essentially all squamous cervical cancers and their precursor lesions, high grade cervical intraepithelial neoplasia (CIN2/3), are caused by persistent human papillomavirus (HPV) infection. However, not all CIN2/3 lesions progress to cancer. In a brief, observational study window monitoring subjects with CIN2/3 from protocol entry (biopsy diagnosis) to definitive therapy (cervical conization) at week 15, in a cohort of 50 subjects, we found that 26% of CIN2/3 lesions associated with HPV16, the genotype most commonly associated with disease, underwent complete histologic regression. Nonetheless, HPV16-specific T cell responses measured in peripheral blood obtained at the time of study entry and at the time of conization were marginally detectable directly ex vivo, and did not correlate with lesion regression. This finding suggests that, in the setting of natural infection, immune responses which are involved in elimination of cervical dysplastic epithelium are not represented to any great extent in the systemic circulation.

A phase I trial of a human papillomavirus DNA vaccine for HPV16+ cervical intraepithelial neoplasia 2/3.

PURPOSE: To evaluate the safety and immunogenicity of a therapeutic human papillomavirus (HPV)16 DNA vaccine administered to women with HPV16+cervical intraepithelial neoplasia (CIN)2/3. EXPERIMENTAL DESIGN: This phase I trial incorporated the standard '3+3'' dose-escalation design with an additional 6 patients allocated to the maximally tolerated dose. Healthy adult women with colposcopically directed, biopsy-proven HPV16+ CIN2/3 received 3 i.m. vaccinations (0.5, 1, or 3 mg) of a plasmid expressing a Sig-E7(detox)-heat shock protein 70 fusion protein on days 0, 28, and 56, and underwent standard therapeutic resection of the cervical squamocolumnar junction at day 105 (week 15). The safety and immunogenicity of the vaccine and histologic outcome based on resection at week 15 were assessed. RESULTS: Fifteen patients were evaluable (3 each at 0.5 and 1mg, 9 at 3 mg). The vaccine was well tolerated: most adverse events were mild, transient injection-site discomfort; no dose-limiting toxicities were observed. Although HPVE7-specific T-cell responses to E7 detected by enzyme-linked immunospot assays (IFN-gamma) were of low frequency and magnitude, detectable increases in response subsequent to vaccination were identified in subjects in the second and third cohorts. Complete histologic regression occurred in 3 of 9 (33%; 7-70% confidence interval) individuals in the highest-dose cohort. Although the difference is not significant, it is slightly higher than would be expected in an unvaccinated cohort (25%). CONCLUSIONS: This HPV16 DNA vaccine was safe and well tolerated. Whereas it seems possible to elicit HPV-specific T-cell responses in patients with established dysplastic lesions, other factors are likely to play a role in lesion regression.

p53-Reactive T Cells Are Associated with Clinical Benefit in Patients with Platinum-Resistant Epithelial Ovarian Cancer After Treatment with a p53 Vaccine and Gemcitabine Chemotherapy.

Purpose: To conduct a phase I trial of a Modified Vaccinia Ankara vaccine delivering wild-type human p53 (p53MVA) in combination with gemcitabine chemotherapy in patients with platinum-resistant ovarian cancer.Experimental Design: Patients received gemcitabine on days 1 and 8 and p53MVA vaccine on day 15, during the first 3 cycles of chemotherapy. Toxicity was classified using the NCI Common Toxicity Criteria and clinical response assessed by CT scan. Peripheral blood samples were collected for immunophenotyping and monitoring of anti-p53 immune responses.Results: Eleven patients were evaluated for p53MVA/gemcitabine toxicity, clinical outcome, and immunologic response. TOXICITY: there were no DLTs, but 3 of 11 patients came off study early due to gemcitabine-attributed adverse events (AE). Minimal AEs were attributed to p53MVA vaccination. Immunologic and clinical response: enhanced in vitro recognition of p53 peptides was detectable after immunization in both the CD4+ and CD8+ T-cell compartments in 5 of 11 and 6 of 11 patients, respectively. Changes in peripheral T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC) did not correlate significantly with vaccine response or progression-free survival (PFS). Patients with the greatest expansion of p53-reactive T cells had significantly longer PFS than patients with lower p53-reactivity after therapy. Tumor shrinkage or disease stabilization occurred in 4 patients.Conclusions: p53MVA was well tolerated, but gemcitabine without steroid pretreatment was intolerable in some patients. However, elevated p53-reactive CD4+ and CD8+ T-cell responses after therapy correlated with longer PFS. Therefore, if responses to p53MVA can be enhanced with alternative agents, superior clinical responses may be achievable. Clin Cancer Res; 24(6); 1315-25. ©2018 AACR.

Complete regression of cutaneous metastases with systemic immune response in a patient with triple negative breast cancer receiving p53MVA vaccine with pembrolizumab.

A heavily pretreated patient with triple negative breast cancer distinguished by cutaneous metastases received p53MVA vaccine in combination with pembrolizumab. Her cutaneous metastases regressed and after 2 cycles of therapy, a skin biopsy showed a complete pathological response. Systemic response was confirmed with restaging CT and bone scans. Activation of p53-specific T cell responses and elevation of multiple immune response genes in peripheral blood correlated with the rapid clinical response which lasted for 6 months after the initiation of combined therapy.

Sustained CD4+ T cell-driven lymphopenia without a compensatory IL-7/IL-15 response among high-grade glioma patients treated with radiation and temozolomide.

Prolonged lymphopenia correlating with decreased survival commonly occurs among glioma patients undergoing radiation therapy (RT) and temozolomide (TMZ) treatment. To better understand the pathophysiology of this phenomenon, we prospectively monitored serum cytokine levels and lymphocyte subsets in 15 high-grade glioma patients undergoing combined radiation and TMZ (referred to as RT/TMZ) treatment. Sufficient data for analysis were acquired from 11 of the patients initially enrolled. Lymphocyte phenotyping data were obtained using cytofluorometric analysis and serum cytokine levels were measured using the a multiplex bead-based assays. Total lymphocyte counts (TLCs) were > 1000 cells per µL peripheral blood in 10/11 patients at baseline, but dropped significantly after treatment. Specifically, after RT/TMZ therapy, the TLCs were found to be < 500 cells/µL in 2/11 patients, 500-1000 cells/µL in 7/11 patients, and > 1000 cells/µL in the remaining 2 patients. Among residual mononuclear blood cells, we observed a proportional drop in B and CD4+ T cells but not in CD8+ T lymphocytes. Natural killer cells remained to near-to-baseline levels and there was a transient and slight (insignificant) increase in regulatory T cells (Tregs). The circulating levels of IL-7 and IL-15 remained low despite marked drops in both the total and CD4+ T lymphocyte counts. Thus, patients with malignant glioma undergoing RT/TMZ treatment exhibit a marked decline in TLCs, affecting both CD4+ T cells and B lymphocytes, in the absence of a compensatory increase in interleukin-7 levels. The failure to mount an appropriate homeostatic cytokine response may be responsible for the prolonged lymphopenia frequently observed in these patients.

HLA class I-restricted responses to vaccinia recognize a broad array of proteins mainly involved in virulence and viral gene regulation.

We have analyzed by ex vivo ELISPOT the anti-vaccinia cytotoxic T lymphocyte responses of peripheral blood mononuclear cells from humans vaccinated with Dryvax vaccine. More than 6,000 peptides from 258 putative vaccinia ORFs predicted to bind the common molecules of the HLA A1, A2, A3, A24, B7, and B44 supertypes were screened with peripheral blood mononuclear cells of 31 vaccinees. A total of 48 epitopes derived from 35 different vaccinia antigens were identified, some of which (B8R, D1R, D5R, C10L, C19L, C7L, F12, and O1L) were recognized by multiple donors and contain multiple epitopes recognized in the context of different HLA types. The antigens recognized tend to be >100 residues in length and are expressed predominantly in the early phases of infection, although some late antigens were also recognized. Viral genome regulation and virulence factor were recognized most frequently, whereas few structural proteins were immunogenic. Finally, most epitopes were highly conserved among vaccinia virus Western Reserve, variola major and modified vaccinia Ankara, supporting their potential use in vaccine and diagnostic applications.

Costimulation of T cell receptor-triggered IL-2 production by Jurkat T cells via fibroblast growth factor receptor 1 upon its engagement by CD56.

Recent studies have demonstrated that neural cell adhesion molecule (NCAM) is involved in multiple adhesive interactions with several different classes of ligands on the cell surface and in the extracellular matrix. One of these ligands is fibroblast growth factor receptor (FGFR) that is expressed on neural cells. While it is known that CD56 is a molecular isoform of NCAM expressed on human NK cells and a subset of T cells, it remains poorly characterized, with its ligand unidentified. Therefore, we were prompted to examine if CD56 molecules on NK cells interact with FGFR expressed on T cells. We demonstrate that ligation of FGFR1 beta on J.C2-14 Jurkat T cells by CD56 on fixed NK-92 cells costimulates TCR/CD3-triggered IL-2 production. CD56-binding mAbs inhibited the costimulatory effect of NK-92 cells in 50-75%. Flow cytometric analysis and cell adhesion assays showed that FGFR1 beta/Fc and FGFR2 beta/Fc chimeric proteins bind to NK-92 cells. The binding of FGFR1 beta/Fc protein to CD56 molecules was verified by immunoprecipitation of CD56 with anti-CD56 mAb followed by Western blotting with FGFR1 beta/Fc. These findings suggest that ligation of FGFR1 by CD56 may contribute to the interaction between NK cells and T cells that we have postulated in our previous studies.