The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3−class II−CD11b+CD80/86+ plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4+CD25highFoxP3+ regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.

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.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature hematopoietic precursors known to suppress immune responses in infection, chronic inflammation, cancer, and autoimmunity. In this paper, we review recent findings detailing their mode of action and discuss recent reports that suggest that MDSC are also expanded during transplantation and that modulation of MDSC can participate in preventing graft rejection as well as graft-versus-host disease.

T-cells play a critical role in tumor immunity. Indeed, the presence of tumor-infiltrating lymphocytes is a predictor of favorable patient prognosis for many indications and is a requirement for responsiveness to immune checkpoint blockade therapy targeting programmed cell death 1. For tumors lacking immune infiltrate, or for which antigen processing and/or presentation has been downregulated, a promising immunotherapeutic approach is chimeric antigen receptor (CAR) T-cell therapy. CARs are hybrid receptors that link the tumor antigen specificity and affinity of an antibody-derived single-chain variable fragment with signaling endodomains associated with T-cell activation. CAR therapy targeting CD19 has yielded extraordinary clinical responses against some hematological tumors. Solid tumors, however, remain an important challenge to CAR T-cells due to issues of homing, tumor vasculature and stromal barriers, and a range of obstacles in the tumor bed. Protumoral immune infiltrate including T regulatory cells and myeloid-derived suppressor cells have been well characterized for their ability to upregulate inhibitory receptors and molecules that hinder effector T-cells. A critical role for metabolic barriers in the tumor microenvironment (TME) is emerging. High glucose consumption and competition for key amino acids by tumor cells can leave T-cells with insufficient energy and biosynthetic precursors to support activities such as cytokine secretion and lead to a phenotypic state of anergy or exhaustion. CAR T-cell expansion protocols that promote a less differentiated phenotype, combined with optimal receptor design and coengineering strategies, along with immunomodulatory therapies that also promote endogenous immunity, offer great promise in surmounting immunometabolic barriers in the TME and curing solid tumors.

CD8+ T cells controlling pathogens or tumors must function at sites where oxygen tension is frequently low, and never as high as under atmospheric culture conditions. However, T-cell function in vivo is generally analyzed indirectly, or is extrapolated from in vitro studies under nonphysiologic oxygen tensions. In this study, we delineate the role of physiologic and pathologic oxygen tension in vitro during reactivation and differentiation of tumor-specific CD8 + T cells. Using CD8 + T cells from pmel-1 mice, we observed that the generation of CTLs under 5% O 2 , which corresponds to physioxia in lymph nodes, gave rise to a higher effector signature than those generated under atmospheric oxygen fractions (21% O 2 ). Hypoxia (1% O 2 ) did not modify cytotoxicity, but decreasing O 2 tensions during CTL and CD8 + tumor-infiltrating lymphocyte reactivation dose-dependently decreased proliferation, induced secretion of the immunosuppressive cytokine IL-10, and upregulated the expression of CD137 (4-1BB) and CD25. Overall, our data indicate that oxygen tension is a key regulator of CD8 + T-cell function and fate and suggest that IL-10 release may be an unanticipated component of CD8 + T cell-mediated immune responses in most in vivo microenvironments. Keywords: CD8+ T cell r Oxygen r Hypoxia r IL-10 r T-cell reactivation Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionIn a healthy physiologic context, oxygen tensions in mammalian tissues are tightly regulated, but still show significant variation both between tissues and within the same tissue (Supporting Information Fig. 1A) [1][2][3]. However, oxygen tensions are far below physiologic values in different pathologic conditions, mostly involving inflammation [4], solid tumors [5,6], and infections [7]. In contrast to sufficient oxygen supply (i.e. normoxia), oxygen deprivation (i.e. hypoxia) leads to cellular responses involving Correspondence: Dr. Paul R. Walker e-mail: Paul.Walker@hcuge.ch stabilization of HIFs (hypoxia-inducible factors), transcription factors that are composed of an inducible α-subunit (i.e. HIF-1α, HIF-2α, or HIF-3α) together with a constitutive β-subunit (i.e. HIF-1β, HIF-2β, or HIF-3β) [8]. Upon stabilization, HIFs transcribe various genes with HIF-responsive elements in their promoter; these include those that increase angiogenesis (e.g. VEGF) and glucose metabolism (e.g. glucose transporters) [9]. Interestingly, HIF-1α and HIF-2α can have unique, redundant, or opposing roles [9,10]. Furthermore, while HIF-1α is described to be stabilized and active below 2% O 2 in an acute manner, HIF-2α has been shown to be stable below 5% O 2 in a chronic manner [11,12]. However, although HIFs are central to many hypoxia responses, certain effects have been shown to be HIF-independent [13][14][15]. Intriguingly, HIF-1α can also be involved in immune cell activation Eur. J. Immunol. 2015Immunol. . 45: 2263Immunol. -2275 under atmospheric oxygen fractions (AtO 2 ; i.e. 21%), as rep...

SummaryLymphocyte-activation gene-3 (LAG-3, CD223) is a marker for recently activated effector T cells. Activated T lymphocytes are of major importance in many autoimmune diseases and organ transplant rejection. Therefore, specifically depleting LAG-3 + T cells might lead to targeted immunosuppression that would spare resting T cells while eliminating pathogenic activated T cells. We have shown previously that anti-LAG-3 antibodies sharing depleting as well as modulating activities inhibit heart allograft rejection in rats. Here, we have developed and characterized a cytotoxic LAG-3 chimeric antibody (chimeric A9H12), and evaluated its potential as a selective therapeutic depleting agent in a non-human primate model of delayed-type hypersensitivity (DTH). Chimeric A9H12 showed a high affinity to its antigen and depleted both cytomegalovirus (CMV)-activated CD4+ and CD8 + human T lymphocytes in vitro. In vivo, a single intravenous injection at either 1 or 0·1 mg/kg was sufficient to deplete LAG-3 + -activated T cells in lymph nodes and to prevent the T helper type 1 (Th1)-driven skin inflammation in a tuberculininduced DTH model in baboons. T lymphocyte and macrophage infiltration into the skin was also reduced. The in vivo effect was long-lasting, as several weeks to months were required after injection to restore a positive reaction after antigen challenge. Our data confirm that LAG-3 is a promising therapeutic target for depleting antibodies that might lead to higher therapeutic indexes compared to traditional immunosuppressive agents in autoimmune diseases and transplantation.

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.

The anti-Ly6G antibody is used to deplete Ly6G pos neutrophils and study their role in diverse pathologies. However, depletion is never absolute, as Ly6G low 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 Lox-STOP-Lox-G12D/WT ; Trp53 Flox/Flox mouse lung tumor model, abnormal neutrophil accumulation and aging was accompanied with an N2-like SiglecF pos polarization and ly6g downregulation. Consequently, SiglecF pos neutrophils exposed to anti-Ly6G reverted to Ly6G low 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 neg 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.