BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency invivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent

Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4 CD25 FoxP3 regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA. © 2012 Australasian Society for Immunology Inc

Merger of dynamic two-photon and phosphorescence lifetime microscopy reveals dependence of lymphocyte motility on oxygen in solid and hematological tumors

Abstract Background Low availability of oxygen in tumors contributes to the hostility of the tumor microenvironment toward the immune system. However, the dynamic relationship between local oxygen levels and the immune surveillance of tumors by tumor infiltrating T-lymphocytes (TIL) remains unclear. This situation reflects a methodological difficulty in visualizing oxygen gradients in living tissue in a manner that is suitable for spatiotemporal quantification and contextual correlation with individual cell dynamics tracked by typical fluorescence reporter systems. Methods Here, we devise a regimen for intravital oxygen and cell dynamics co-imaging, termed ‘Fast’ Scanning Two-photon Phosphorescence Lifetime Imaging Microscopy (FaST-PLIM). Using FaST-PLIM, we image the cellular motility of T-lymphocytes in relation to the microscopic distribution of oxygen in mouse models of hematological and solid tumors, namely in bone marrow with or without B-cell acute lymphocytic leukemia (ALL), and in lungs with sarcoma tumors. Results Both in bone marrow leukemia and solid tumor models, TILs encountered regions of varying oxygen concentrations, including regions of hypoxia (defined as pO2 below 5 mmHg), especially in advanced-stage ALL and within solid tumor cores. T cell motility was sustained and weakly correlated with local pO2 above 5 mmHg but it was very slow in pO2 below this level. In solid tumors, this relationship was reflected in slow migration of TIL in tumor cores compared to that in tumor margins. Remarkably, breathing 100% oxygen alleviated tumor core hypoxia and rapidly invigorated the motility of otherwise stalled tumor core TILs. Conclusions This study demonstrates a versatile and highly contextual FaST-PLIM method for phosphorescence lifetime-based oxygen imaging in living animal tumor immunology models. The initial results of this method application to ALL and solid lung tumor models highlight the importance of oxygen supply for the maintenance of intratumoral T cell migration, define a 5 mmHg local oxygen concentration threshold for TIL motility, and demonstrate efficacy of supplementary oxygen breathing in TIL motility enhancement coincident with reduction of tumor hypoxia

Suppression of Immunodominant Antitumor and Antiviral CD8⁺ T Cell Responses by Indoleamine 2,3-Dioxygenase

<div><p>Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme known to suppress antitumor CD8⁺ T cells (T_CD8). The role of IDO in regulation of antiviral T_CD8 responses is far less clear. In addition, whether IDO controls both immunodominant and subdominant T_CD8 is not fully understood. This is an important question because the dominance status of tumor- and virus-specific T_CD8 may determine their significance in protective immunity and in vaccine design. We evaluated the magnitude and breadth of cross-primed T_CD8 responses to simian virus 40 (SV40) large T antigen as well as primary and recall T_CD8 responses to influenza A virus (IAV) in the absence or presence of IDO. IDO^−/− mice and wild-type mice treated with 1-methyl-D-tryptophan, a pharmacological inhibitor of IDO, exhibited augmented responses to immunodominant epitopes encoded by T antigen and IAV. IDO-mediated suppression of these responses was independent of CD4⁺CD25⁺FoxP3⁺ regulatory T cells, which remained numerically and functionally intact in IDO^−/− mice. Treatment with L-kynurenine failed to inhibit T_CD8 responses, indicating that tryptophan metabolites are not responsible for the suppressive effect of IDO in our models. Immunodominant T antigen-specific T_CD8 from IDO^−/− mice showed increased Ki-67 expression, suggesting that they may have acquired a more vigorous proliferative capacity <i>in vivo</i>. In conclusion, IDO suppresses immunodominant T_CD8 responses to tumor and viral antigens. Our work also demonstrates that systemic primary and recall T_CD8 responses to IAV are controlled by IDO. Inhibition of IDO thus represents an attractive adjuvant strategy in boosting anticancer and antiviral T_CD8 targeting highly immunogenic antigens.</p></div

The cross-primed T_CD8 response to T Ag is augmented in the absence of IDO.

<p>WT and IDO^−/− mice were injected <i>i.p.</i> with allogeneic T Ag⁺ KD2SV cells. Nine days later, the frequencies (A) and absolute numbers (B) of T Ag-specific T_CD8 recognizing site IV, total T Ag-specific T_CD8 that synthesize IFN-γ after incubation with C57SV cells, and total alloreactive T_CD8 that produce IFN-γ after incubation with KD2SV cells were determined by ICS as described in Materials and Methods. Background obtained from wells receiving no peptide was subtracted and values are presented as mean ± SEM of 8 mice/group pooled from independent experiments.</p

nTreg cells do not mediate the suppressive effect of IDO on the site IV-specific response.

<p>(A) Splenocytes from indicated numbers of naïve WT and IDO^−/− mice were stained for surface CD4 and intracellular FoxP3. In separate experiments, WT and IDO^−/− mice were inoculated with C57SV cells followed, 9 days later, by cytofluorimetric determination of their splenic nTreg cell frequencies, which were used to calculate the absolute number of nTreg cells within each spleen. Representative FACS plots are shown in addition to mean nTreg cell frequencies and absolute numbers ± SEM for each group. (B) WT CD4⁺CD25⁻ conventional T cells were co-cultured with γ-irradiated bone marrow-derived DCs and stimulated with an anti-CD3 mAb in the presence of varying numbers of CD4⁺CD25⁺ nTreg cells magnetically purified from WT and IDO^−/− mice. T cell proliferation was measured by tritiated thymidine incorporation after 72 hours. (C) WT and IDO^−/− mice were injected with an anti-CD25 mAb (clone PC61) or PBS 3 days before they were inoculated with C57SV cells. Nine days later, site IV-specific T_CD8 were enumerated by ICS for IFN-γ. Values are presented as mean ± SEM for indicated numbers of mice per group pooled from 3 independent experiments.</p

Ki-67 expression by T Ag-specific T_CD8 and non-specific proliferative responses of IDO^−/− T cells.

<p>(A) Splenocytes from WT and IDO^−/− mice inoculated with C57SV cells were restimulated for 5 hours with synthetic peptides corresponding to site I or site IV. Cells were then stained for surface CD8, intracellular IFN-γ and intracellular Ki-67 as described in Materials and Methods. Representative FACS plots after live gating on CD8⁺ events are shown. Quadrants’ positions were set based on staining with an isotype control. (B) WT and IDO^−/− splenocytes were left untreated or stimulated with PHA, ConA or a mitogenic anti-CD3 mAb. T cell proliferation was measured by tritiated thymidine incorporation after 72 hours.</p

Genetic deficiency of IDO enhances the T_CD8 response to T Ag’s most immunodominant epitope.

<p>WT and IDO^−/− mice were injected <i>i.p.</i> with syngeneic, SV40-transformed C57SV cells. Nine days later, splenic T_CD8 were examined <i>ex vivo</i> for IFN-γ accumulation following brief restimulation (of 5 hours in duration) with C57SV cells used at 2×10⁵ cells/well (A) or synthetic peptides corresponding to T Ag-derived epitopes (B). Background obtained from wells receiving no peptide was subtracted and values are expressed as mean ± standard error of the mean (SEM) of multiple mice per group (n = 12 and n = 10 for WT and IDO^−/− mice, respectively) pooled from independent experiments yielding similar results.</p

Pharmacological inhibition of IDO amplifies the T_CD8 response to T Ag’s most immunodominant epitope.

<p>WT mice were treated with 1-D-MT (10 mg/mouse total) or vehicle and injected <i>i.p.</i> with C57SV cells. Nine days later, splenic T_CD8 were examined <i>ex vivo</i> for IFN-γ accumulation following restimulation with C57SV cells (A and C) or synthetic peptides corresponding to T Ag epitopes (B and D). T Ag-specific T_CD8 frequencies were determined after subtracting background and expressed as mean ± SEM of 7 mice per group (A and B). These values were used to calculate the absolute number of T Ag-specific T_CD8 present within each spleen (C and D).</p