Prevalence of active cytomegalovirus infection at diagnosis of ovarian cancer and during chemotherapy and subsequent changes in cognitive functioning.

PURPOSE: One of the most frequently reported effects of cancer and its treatments is cancer-related cognitive impairment (CRCI). Viral infections may affect inflammation and immune function and therefore may influence patient symptoms, including CRCI. The goal of this study was to describe the prevalence of cytomegalovirus (CMV) infections at diagnosis, during, and after chemotherapy in individuals with ovarian cancer and explore CMV infection at diagnosis with cancer-related cognitive impairment (CRCI) following chemotherapy. METHODS: We recruited adults newly diagnosed with ovarian, primary peritoneal or fallopian tube cancer at a single academic cancer center into two prospective studies. In Study 1 (N = 71), participants provided blood samples at diagnosis. In Study 2 (N = 18), participants provided blood samples and completed symptom surveys before, during and after front-line adjuvant chemotherapy. Serum CMV DNA levels were assessed using digital PCR; >100 copies/mL of serum was considered positive for active CMV infection (CMV+). CRCI was measured using the Functional Assessment of Cancer Therapy - Cognitive Function (FACT-Cog) questionnaire. Changes in FACT-Cog scores were compared by CMV status at diagnosis using t-tests at each time point. RESULTS: At diagnosis, 29.2% were CMV+ (28.2% in Study 1, 33.3% in Study 2). Following three cycles of chemotherapy (Study 2), CMV positivity rose to 60.0% and then back down to 31.3% after chemotherapy. We observed significant differences in CRCI following chemotherapy by CMV status at diagnosis. CONCLUSION: Our data suggest that active CMV infection is common among patients undergoing treatment for ovarian cancer and may contribute to symptoms of CRCI.

Reverse Translation Identifies the Synergistic Role of Immune Checkpoint Blockade and IL15 to Enhance Immunotherapy of Ovarian Cancer.

Immune checkpoint blockade (ICB) has changed the standard of care for many patients with cancer, yet no ICB is approved for ovarian cancer. We hypothesized that maintenance therapy with an IL15 "superagonist" (N-803) and ICB in combination could induce potent immune activation in ovarian cancer. Using flow cytometry, cytometry by time of flight analysis, and cytotoxicity assays, we analyzed patient samples from women with advanced epithelial ovarian cancer treated with N-803 for indications of PD-1/PD-L1 upregulation with this treatment. In addition, ICB and N-803 were evaluated in preclinical studies to determine the functional impact of combination therapy on natural killer (NK) cells in vitro and in vivo. We observed that N-803 stimulated initial NK-cell expansion in patient samples; however, proliferation was not sustained beyond 2 weeks despite continued treatment. This result was reverse translated back to the laboratory to determine the functional relevance of this finding. The addition of ICB with an antibody-dependent cellular cytotoxicity IgG1 antibody against PD-L1 (avelumab) or an IgG4 antibody against PD-1 (pembrolizumab) enhanced N-803 induced NK-cell function in vitro. Using models of human ovarian cancer and NK-cell adoptive transfer in mice, we showed enhanced antitumor control with N-803 and ICB, as well as a combination effect that enhanced NK-cell persistence and expansion in vivo. This work suggests that PD-1/PD-L1 blockade combined with IL15 signaling may overcome resistance to cytokine therapy in ovarian cancer.

DGKζ exerts greater control than DGKα over CD8+ T cell activity and tumor inhibition.

Two isoforms of diacylglycerol kinases (DGKs), DGKα and DGKζ, are primarily responsible for terminating DAG-mediated activation of Ras and PKCθ pathways in T cells. A direct comparison of tumor growth between mice lacking each isoform has not been undertaken. We evaluated the growth of three syngeneic tumor cell lines in mice lacking either DGKα or DGKζ in the presence or absence of treatment with anti-PD1 and determined that (i) mice deficient in DGKζ conferred enhanced control of tumor relative to mice deficient in DGKα and (ii) deficiency of DGKζ acted additively with anti-PD1 in tumor control. Consistent with this finding, functional and RNA-sequencing analyses revealed greater changes in stimulated DGKζ-deficient T cells compared with DGKα-deficient T cells, which were enhanced relative to wildtype T cells. DGKζ also imparted greater regulation than DGKα in human T cells. Together, these data support targeting the ζ isoform of DGKs to therapeutically enhance T cell anti-tumor activity.

Cytomegalovirus and systemic inflammation at time of surgery is associated with worse outcomes in serous ovarian cancer.

OBJECTIVES: Cytomegalovirus (CMV) is a common infection that establishes latency in healthy people. CMV has been associated with alterations of the immune compartment leading to improved responses, while inflammation has been shown to adversely impact outcomes. We investigated whether CMV serostatus predicts outcomes in ovarian cancer in the presence or absence of inflammation. METHODS: A total of 106 patients with serous ovarian cancer from 2006 to 2009 were analyzed. CMV and systemic inflammation was measured using CMV immunoglobulin G (IgG) and C-reactive protein (CRP), respectively, in serum collected prior to cytoreduction. Patients were stratified by CMV IgG (non-reactive, reactive/borderline) and CRP (≤10, >10 mg/L) status. Overall survival (OS) and recurrence-free survival (RFS) were compared by group using log-rank tests and Cox proportional hazards regression models adjusting for age at surgery. RESULTS: Of 106 eligible patients, 40 (37.7%) were CMV+/CRP+, 24 (22.6%) CMV+/CRP-, 19 (17.9%) CMV-/CRP+, and 23 (21.7%) CMV-/CRP-. CRP+ had higher CA-125 levels (P = 0.05) and higher rates of suboptimal debulking (P = 0.03). There were no other significant differences in demographic, surgical, or pathologic factors between groups. CMV+/CRP+ patients median RFS and OS were 16.9 months (95% CI: 9.0-21.1) and 31.7 months (95% CI: 25.0-48.7), respectively, with a significantly worse RFS (aHR: 1.85, 95% CI: 1.05-3.24, P = 0.03) and OS (aHR: 2.12, 95% CI: 1.17-3.82, P = 0.01) compared to CMV-/CRP- (RFS = 31.2 months (95% CI: 16.0-56.4) and OS = 63.8 months (95% CI: 50.7-87.0)). CMV+/CRP- group displayed the longest OS (89.3 months). CONCLUSIONS: Previous exposure to CMV and high CRP at surgery portended worse RFS and OS compared to women who tested negative. The CMV+/CRP- group had the longest OS, indicating that CMV status alone, in the absence of inflammation, may be protective.

Diacylglycerol kinase ζ (DGKζ) and Casitas b-lineage proto-oncogene b-deficient mice have similar functional outcomes in T cells but DGKζ-deficient mice have increased T cell activation and tumor clearance.

Targeting negative regulators downstream of the T cell receptor (TCR) represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout, DKO) in T cells, and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFNγ, and generation of granzyme B when compared with WT T cells. DKO T cells demonstrated enhanced function above that observed with single knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both non-treated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared to Cbl-b-deficient mice. This represents the first direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.

T Cells Deficient in Diacylglycerol Kinase ζ Are Resistant to PD-1 Inhibition and Help Create Persistent Host Immunity to Leukemia.

Efforts to improve the efficacy of adoptive T-cell therapies and immune checkpoint therapies in myelogenous leukemia are desired. In this study, we evaluated the antileukemia activity of adoptively transferred polyclonal cancer antigen-reactive T cells deficient in the regulator diacylglycerol kinase zeta (DGKζ) with or without PD-1/PD-L1 blockade. In the C1498 mouse model of myeloid leukemia, we showed that leukemia was eradicated more effectively in DGKζ-deficient (DGKζ-/-) mice than wild-type mice. T cells transferred from DGKζ-deficient mice to wild-type tumor-bearing recipients conferred this benefit. Leukemia clearance was similar to mice treated with anti-PD-L1. Strikingly, we found that the activity of adoptively transferred DGKζ-/- T cells relied partly on induction of sustainable host T-cell immunity. Transferring DGKζ-deficient T cells increased the levels of IFNγ and other cytokines in recipient mice, especially with coadministration of anti-PD-L1. Overall, our results offered evidence that targeting DGKζ may leverage the efficacy of adoptive T-cell and immune checkpoint therapies in leukemia treatment. Furthermore, they suggest that DGKζ targeting might decrease risks of antigen escape or resistance to immune checkpoint blockade. Cancer Res; 77(20); 5676-86. ©2017 AACR.

TCR signaling intensity controls CD8+ T cell responsiveness to TGF-ß.

DGK-ζ is a negative regulator of TCR signaling that causes degradation of the second messenger DAG, terminating DAG-mediated activation of Ras and PKCθ. Cytotoxic T cells deficient in DGK-ζ demonstrate enhanced effector functions in vitro and antitumor activity in vivo, perhaps because of insensitivity to inhibitory cytokines. We sought to determine whether the enhanced responsiveness of DGK-ζ-deficient T cells renders them insensitive to the inhibitory cytokine TGF-ß and to determine how the loss of DGK-ζ facilitates this insensitivity. We identified decreased transcriptional and functional responses to TGF-ß in CD8(+) DGK-ζ(-/-) T cells but preserved TGF-ß-mediated conversion of naïve DGK-ζ(-/-) CD4(+) T cells to a regulatory T cell phenotype. Decreased CD8(+) T cell responsiveness to TGF-ß did not result from impaired canonical TGF-ß signal transduction, because similar levels of TGF-ß-R and intracellular Smad components were identified in WT and DGK-ζ(-/-) CD8(+) T cells, and TGF-ß-mediated activation of Smad2 was unchanged. Instead, an enhanced TCR signal strength was responsible for TGF-ß insensitivity, because (i) loss of DGK-ζ conferred resistance to TGF-ß-mediated inhibition of Erk phosphorylation, (ii) TGF-ß insensitivity could be recapitulated by exogenous addition of the DAG analog PMA, and (iii) TGF-ß sensitivity could be observed in DGK-ζ-deficient T cells at limiting dilutions of TCR stimulation. These data indicate that enhanced TCR signal transduction in the absence of DGK-ζ makes T cells relatively insensitive to TGF-ß, in a manner independent of Smads, a finding with practical implications in the development of immunotherapies that target TGF-ß.

Integrin binding by Borrelia burgdorferi†P66 facilitates dissemination but is not required for infectivity.

P66, a Borrelia burgdorferi surface protein with porin and integrin-binding activities, is essential for murine infection. The role of P66 integrin-binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202-208). Neither change affected surface localization or channel-forming activity of P66, but both significantly reduced binding to αv ß3 . Integrin-binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post-subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202-208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post-inoculation, but bacterial burdens recovered to wild-type levels at 4 weeks post-inoculation. The delay in tissue colonization correlated with reduced migration of the Del202-208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.