Hypoxia and antitumor CD8(+) T cells: An incompatible alliance?

T Lymphocytes face pathologically low O2 tensions within the tumor bed at which they will have to function in order to impact on the malignancy. Recent studies highlighting the importance of O2 and hypoxia-inducible factors for CD8(+) T-cell function and fate must now be integrated into tumor immunology concepts if immunotherapies are to progress. Here, we discuss, reinterpret, and reconcile the many apparent contradictions in these data and we propose that O2 is a master regulator of the CD8(+) T-cell response. Certain T cell functions are enhanced, others suppressed, but on balance, hypoxia is globally detrimental to the antitumor response

Anti-Ly6G binding and trafficking mediate positive neutrophil selection to unleash the anti-tumor efficacy of radiation therapy.

The anti-Ly6G antibody is used to deplete Ly6G <sup>pos</sup> neutrophils and study their role in diverse pathologies. However, depletion is never absolute, as Ly6G <sup>low</sup> neutrophils resistant to depletion rapidly emerge. Studying the functionality of these residual neutrophils is necessary to interpret anti-Ly6G-based experimental designs. In vitro, we found anti-Ly6G binding induced Ly6G internalization, surface Ly6G paucity, and primed the oxidative burst of neutrophils upon TNF α co-stimulation. In vivo, we found neutrophils resistant to anti-Ly6G depletion exhibited anti-neutrophil-cytoplasmic-antibodies. In the pre-clinical Kras <sup>Lox-STOP-Lox-G12D/WT</sup> ; Trp53 <sup>Flox/Flox</sup> mouse lung tumor model, abnormal neutrophil accumulation and aging was accompanied with an N2-like SiglecF <sup>pos</sup> polarization and ly6g downregulation. Consequently, SiglecF <sup>pos</sup> neutrophils exposed to anti-Ly6G reverted to Ly6G <sup>low</sup> and were resistant to depletion. Noting that anti-Ly6G mediated neutrophil depletion alone had no anti-tumor effect, we found a long-lasting rate of tumor regression (50%) by combining anti-Ly6G with radiation-therapy, in this model reputed to be refractory to standard anticancer therapies. Mechanistically, anti-Ly6G regulated neutrophil aging while radiation-therapy enhanced the homing of anti-Ly6G-boundSiglecF <sup>neg</sup> neutrophils to tumors. This anti-tumor effect was recapitulated by G-CSF administration prior to RT and abrogated with an anti-TNFα antibody co-administration. In summary, we report that incomplete depletion of neutrophils using targeted antibodies can intrinsically promote their oxidative activity. This effect depends on antigen/antibody trafficking and can be harnessed locally using select delivery of radiation-therapy to impair tumor progression. This underutilized aspect of immune physiology may be adapted to expand the scope of neutrophil-related research

Cystathionine-gamma-lyase overexpression in T cells enhances antitumor effect independently of cysteine autonomy.

T cells could be engineered to overcome the aberrant metabolic milieu of solid tumors and tip the balance in favor of a long-lasting clinical response. Here, we explored the therapeutic potential of stably overexpressing cystathionine-gamma-lyase (CTH, CSE, or cystathionase), a pivotal enzyme of the transsulfuration pathway, in antitumor CD8 <sup>+</sup> T cells with the initial aim to boost intrinsic cysteine metabolism. Using a mouse model of adoptive cell transfer (ACT), we found that CTH-expressing T cells showed a superior control of tumor growth compared to control T cells. However, contrary to our hypothesis, this effect was not associated with increased T cell expansion in vivo or proliferation rescue in the absence of cysteine/cystine in vitro. Rather than impacting methionine or cysteine, ACT with CTH overexpression unexpectedly reduced glycine, serine, and proline concentration within the tumor interstitial fluid. Interestingly, in vitro tumor cell growth was mostly impacted by the combination of serine/proline or serine/glycine deprivation. These results suggest that metabolic gene engineering of T cells could be further investigated to locally modulate amino acid availability within the tumor environment while avoiding systemic toxicity

GL261 MHCI, ICAM-1 and PDL1 expression is impacted by decitabine.

<p>In the left panels, a representative histogram of GL261-OVA GDC (GDCs) and GL261-OVA GIC (GICs) expression of MHCI (H-2Kb) (A), ICAM-1 (B), and PDL1 (C) is shown (DAC used at 10μM, 48h incubation, both isotype control and antibody staining are shown). Live cells were gated. In the right panels, expression of each molecule (calculated as MFIR) by GL261-OVA GDCs (GDCs) and GL261-OVA GICs (GICs) after 48h DAC treatment is shown. Error bars represent SD. One-way Anova test (Dunnet’s multiple comparison test) was performed to compare the expression of MHCI, ICAM-1 or PDL1 by the same cell type at different DAC concentrations; unpaired t-test was performed to compare the expression of MHCI, ICAM-1 or PDL1 between the two cell types; p<0.05;**p<0.01;***p<0.001, n = 3.</p

Decitabine treatment of GL261 glioma increases CTL-mediated killing and enhances CTL reactivation.

<p>(A) 4h killing induced by OT1 or Pmel-1 CTLs on GL261-OVA GDCs (GDCs) and GL261-OVA GICs (GICs). Target cells were plated for 48h with DAC (10μM) and then labeled with CFSE. When target cells were incubated with Pmel-1 CTLs, they were pulsed 1h with 1μg/ml gp100 peptide before the assay. The Effector:Target (E:T) ratio used was 10:1. Live cells were discriminated by LIVE/DEAD fixable yellow dead cell stain. Error bars represent SD. *p<0.05;**p<0.01;***p<0.001, paired t-test, n = 6. (B) 20h killing induced by Pmel-1 CTLs on GL261-OVA GDCs (GDCs) and GL261-OVA GICs (GICs). Target cells were pulsed 1h with 1μg/ml gp100 peptide before the assay. The protocol is described in (A). Error bars represent SD. *p<0.05;**p<0.01;***p<0.001, Paired t-test performed between untreated and treated groups; unpaired t-test performed between GICs and GDCs, n = 3 (C) OT1 and Pmel-1 CTL increase in cell division number after incubation with irradiated GL261-OVA GDCs (GDCs) or GL261-OVA GICS (GICs) calculated as difference in cell division number compared with CTLs incubated alone. CTLs were labeled with CFSE and co-cultured with irradiated GDCs or GICs that were previously treated with Decitabine (10μM, 48h). The number of T cell divisions was assessed by cytofluorometry (CFSE fluorescent peaks of gated events). CTLs were identified by CD8 staining. As positive control, anti-CD3/CD28-coated Dynabeads (Beads αCD3/CD28) were used. Error bars represent SD.*p<0.05;**p<0.01;***p<0.001, paired t-test, n = 3 (D) IFN-γ secretion by OT1 CTLs after incubation with GL261-OVA GDCs (GDCs) or GL261-OVA GICS (GICs). Values were normalised to positive controls (181 ng/ml +/- 84.9). Error bars represent SD.*p<0.05;**p<0.01;***p<0.001, paired t-test, n = 3.</p

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma