Tissue-resident FOLR2+ macrophages associate with CD8+ T cell infiltration in human breast cancer.

Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2+ tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2+ macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8+ T cells. FOLR2+ macrophages efficiently prime effector CD8+ T cells ex vivo. The density of FOLR2+ macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies.

Treg-targeted IL-2/anti-IL-2 complex controls graft-versus-host disease and supports anti-tumor effect in allogeneic hematopoietic stem cell transplantation.

Modulating an immune response in opposite directions represents the holy grail in allogeneic hematopoietic stem cell transplantation (allo-HSCT) to avoid insufficient reactivity of donor T cells and hematologic malignancy relapse while controlling the potential development of graft-versus-host disease (GVHD), in which donor T cells attack the recipient's tissues. IL-2/anti-IL-2 complexes (IL-2Cx) represent a therapeutic option to selectively accentuate or dampen the immune response. In dedicated experimental models of allo-HSCT, including also human cells injected in immunodeficient NSG mice, we evaluated side-by-side the therapeutic effect of two IL-2Cx designed either to boost regulatory T cells (Treg) or alternatively to activate effector T cells (Teff), on GVHD occurrence and tumor relapse. We also evaluated the effect of the complexes on the phenotype and function of immune cells in vivo. Unexpectedly, both pro-Treg and pro-Teff IL-2Cx prevented GVHD development. They both induced Treg expansion and reduced CD8+ T-cell numbers, compared to untreated mice. However, only mice treated with the pro-Treg IL-2Cx, showed a dramatic reduction of exhausted CD8+ T cells, consistent with a potent anti-tumor effect. When evaluated on human cells, pro-Treg IL-2Cx also preferentially induced Treg expansion in vitro and in vivo, while allowing the development of a potent anti-tumor effect in NSG mice. Our results demonstrate the clinical relevance of using a pro-Treg, but not a pro-Teff IL2Cx to modulate alloreactivity after HSCT, while promoting a graft-versus-leukemia effect.

The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity.

Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.

CBX3 antagonizes IFNγ/STAT1/PD-L1 axis to modulate colon inflammation and CRC chemosensitivity.

As an important immune stimulator and modulator, IFNγ is crucial for gut homeostasis and its dysregulation links to diverse colon pathologies, such as colitis and colorectal cancer (CRC). Here, we demonstrated that the epigenetic regulator, CBX3 (also known as HP1γ) antagonizes IFNγ signaling in the colon epithelium by transcriptionally repressing two critical IFNγ-responsive genes: STAT1 and CD274 (encoding Programmed death-ligand 1, PD-L1). Accordingly, CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated STAT1 and CD274 expressions. Chromatin immunoprecipitation indicated that CBX3 tethers to STAT1 and CD274 promoters to inhibit their expression. Reversely, IFNγ significantly reduces CBX3 binding to these promoters and primes gene expression. This antagonist effect between CBX3 and IFNγ on STAT1/PD-L1 expression was also observed in CRC. Strikingly, CBX3 deletion heightened CRC cells sensitivity to IFNγ, which ultimately enhanced their chemosensitivity under IFNγ stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Overall, this work reveals that by negatively tuning IFNγ-stimulated immune genes' transcription, CBX3 participates in modulating colon inflammatory response and CRC chemo-resistance.

CD74 supports accumulation and function of regulatory T cells in tumors.

Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observe that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs reveals that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intratumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they are associated with accelerated tumor rejection in preclinical models in female mice. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a target to interfere with Treg anti-tumor activity.

Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Breast cancer remotely imposes a myeloid bias on haematopoietic stem cells by reprogramming the bone marrow niche.

Myeloid cell infiltration of solid tumours generally associates with poor patient prognosis and disease severity1-13. Therefore, understanding the regulation of myeloid cell differentiation during cancer is crucial to counteract their pro-tumourigenic role. Bone marrow (BM) haematopoiesis is a tightly regulated process for the production of all immune cells in accordance to tissue needs14. Myeloid cells differentiate during haematopoiesis from multipotent haematopoietic stem and progenitor cells (HSPCs)15-17. HSPCs can sense inflammatory signals from the periphery during infections18-21 or inflammatory disorders22-27. In these settings, HSPC expansion is associated with increased myeloid differentiation28,29. During carcinogenesis, the elevation of haematopoietic growth factors supports the expansion and differentiation of committed myeloid progenitors5,30. However, it is unclear whether cancer-related inflammation also triggers demand-adapted haematopoiesis at the level of multipotent HSPCs. In the BM, HSPCs reside within the haematopoietic niche which delivers HSC maintenance and differentiation cues31-35. Mesenchymal stem cells (MSCs) are a major cellular component of the BM niche and contribute to HSC homeostasis36-41. Modifications of MSCs in systemic disorders have been associated with HSC differentiation towards myeloid cells22,42. It is unknown if MSCs are regulated in the context of solid tumours and if their myeloid supportive activity is impacted by cancer-induced systemic changes. Here, using unbiased transcriptomic analysis and in situ imaging of HSCs and the BM niche during breast cancer, we show that both HSCs and MSCs are transcriptionally and spatially modified. We demonstrate that breast tumour can distantly remodel the cellular cross-talks in the BM niche leading to increased myelopoiesis.

Selective STING stimulation in dendritic cells primes antitumor T cell responses.

T cells that recognize tumor antigens are crucial for mounting antitumor immune responses. Induction of antitumor T cells in immunogenic tumors depends on STING, the intracellular innate immune receptor for cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and related cyclic dinucleotides (CDNs). However, the optimal way to leverage STING activation in nonimmunogenic tumors is still unclear. Here, we show that cGAMP delivery by intratumoral injection of virus-like particles (cGAMP-VLP) led to differentiation of circulating tumor-specific T cells, decreased tumor regulatory T cells (Tregs), and antitumoral responses that synergized with PD1 blockade. By contrast, intratumoral injection of the synthetic CDN ADU-S100 led to tumor necrosis and systemic T cell activation but simultaneously depleted immune cells from injected tumors and induced minimal priming of circulating tumor-specific T cells. The antitumor effects of cGAMP-VLP required type 1 conventional dendritic cells (cDC1), whereas ADU-S100 eliminated cDC1 from injected tumors. cGAMP-VLP preferentially targeted STING in dendritic cells at a 1000-fold smaller dose than ADU-S100. Subcutaneous administration of cGAMP-VLP showed synergy when combined with PD1 blockade or a tumor Treg-depleting antibody to elicit systemic tumor-specific T cells and antitumor activity, leading to complete and durable tumor eradication in the case of tumor Treg depletion. These findings show that cell targeting of STING stimulation shapes the antitumor T cell response and identify a therapeutic strategy to enhance T cell-targeted immunotherapy.

CD39+ conventional CD4+ T cells with exhaustion traits and cytotoxic potential infiltrate tumors and expand upon CTLA-4 blockade.

Conventional CD4+ T (Tconv) lymphocytes play important roles in tumor immunity; however, their contribution to tumor elimination remains poorly understood. Here, we describe a subset of tumor-infiltrating Tconv cells characterized by the expression of CD39. In several mouse cancer models, we observed that CD39+ Tconv cells accumulated in tumors but were absent in lymphoid organs. Compared to tumor CD39- counterparts, CD39+ Tconv cells exhibited a cytotoxic and exhausted signature at the transcriptomic level, confirmed by high protein expression of inhibitory receptors and transcription factors related to the exhaustion. Additionally, CD39+ Tconv cells showed increased production of IFNγ, granzyme B, perforin and CD107a expression, but reduced production of TNF. Around 55% of OVA-specific Tconv from B16-OVA tumor-bearing mice, expressed CD39. In vivo CTLA-4 blockade induced the expansion of tumor CD39+ Tconv cells, which maintained their cytotoxic and exhausted features. In breast cancer patients, CD39+ Tconv cells were found in tumors and in metastatic lymph nodes but were less frequent in adjacent non-tumoral mammary tissue and not detected in non-metastatic lymph nodes and blood. Human tumor CD39+ Tconv cells constituted a heterogeneous cell population with features of exhaustion, high expression of inhibitory receptors and CD107a. We found that high CD4 and ENTPD1 (CD39) gene expression in human tumor tissues correlated with a higher overall survival rate in breast cancer patients. Our results identify CD39 as a biomarker of Tconv cells, with characteristics of both exhaustion and cytotoxic potential, and indicate CD39+ Tconv cells as players within the immune response against tumors.

Long peptide vaccination can lead to lethality through CD4+ T cell-mediated cytokine storm.

The optimization of anticancer therapeutic vaccines can lead to better efficacy but also to stronger adverse effects. In a mouse model of antitumor vaccination using a long peptide (LP), which included MHC class I- and II-restricted male (H-Y) epitopes, we observed unexpected mortality. Mice with an increased frequency of anti-H-Y CD4 T cells were primed with LP+CpG and boosted 10 d later. Within hours of boost, they displayed shock-like signs with high mortality. Serum cytokine levels were high. TNF-alpha secreted by the CD4 T cells was identified as the key effector molecule. Priming with a short peptide (SP), which included the MHC class II-restricted epitope, was a more efficient primer than LP, but did not lead to mortality when used as boost. The high mortality induced by LP compared with SP was probably related to its specific ability to be presented by B cells. Finally, targeting the LP sequence to dendritic cells allowed tumor protection without side effects. Our data: 1) confirm that the immune system can be very dangerous; 2) caution against the use of systemic activation of high-frequency Ag-specific T cells as induced by high doses of LP; and 3) underline the benefit of targeting Ag to dendritic cells.

Innate lymphoid cells: NK and cytotoxic ILC3 subsets infiltrate metastatic breast cancer lymph nodes.

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality.

CD8+T cell responsiveness to anti-PD-1 is epigenetically regulated by Suv39h1 in melanomas.

Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an "epigenetic checkpoint" for tumor immunity.

Pseudocowpox virus, a novel vector to enhance the therapeutic efficacy of antitumor vaccination.

Objective: Antitumor viral vaccines, and more particularly poxviral vaccines, represent an active field for clinical development and translational research. To improve the efficacy and treatment outcome, new viral vectors are sought, with emphasis on their abilities to stimulate innate immunity, to display tumor antigens and to induce a specific T-cell response. Methods: We screened for a new poxviral backbone with improved innate and adaptive immune stimulation using IFN-α secretion levels in infected PBMC cultures as selection criteria. Assessment of virus effectiveness was made in vitro and in vivo. Results: The bovine pseudocowpox virus (PCPV) stood out among several poxviruses for its ability to induce significant secretion of IFN-α. PCPV produced efficient activation of human monocytes and dendritic cells, degranulation of NK cells and reversed MDSC-induced T-cell suppression, without being offensive to activated T cells. A PCPV-based vaccine, encoding the HPV16 E7 protein (PCPV-E7), stimulated strong antigen-specific T-cell responses in TC1 tumor-bearing mice. Complete regression of tumors was obtained in a CD8+ T-cell-dependent manner after intratumoral injection of PCPV-E7, followed by intravenous injection of the cancer vaccine MVA-E7. PCPV also proved active when injected repeatedly intratumorally in MC38 tumor-bearing mice, generating tumor-specific T-cell responses without encoding a specific MC38 antigen. From a translational perspective, we demonstrated that PCPV-E7 effectively stimulated IFN-γ production by T cells from tumor-draining lymph nodes of HPV+-infected cancer patients. Conclusion: We propose PCPV as a viral vector suitable for vaccination in the field of personalised cancer vaccines, in particular for heterologous prime-boost regimens.

Polyfunctional KLRG-1+CD57+ Senescent CD4+ T Cells Infiltrate Tumors and Are Expanded in Peripheral Blood From Breast Cancer Patients.

Senescent T cells have been described during aging, chronic infections, and cancer; however, a comprehensive study of the phenotype, function, and transcriptional program of this T cell population in breast cancer (BC) patients is missing. Compared to healthy donors (HDs), BC patients exhibit an accumulation of KLRG-1+CD57+ CD4+ and CD8+ T cells in peripheral blood. These T cells infiltrate tumors and tumor-draining lymph nodes. KLRG-1+CD57+ CD4+ and CD8+ T cells from BC patients and HDs exhibit features of senescence, and despite their inhibitory receptor expression, they produce more effector cytokines and exhibit higher expression of Perforin, Granzyme B, and CD107a than non-senescent subsets. When compared to blood counterparts, tumor-infiltrating senescent CD4+ T cells show similar surface phenotype but reduced cytokine production. Transcriptional profiling of senescent CD4+ T cells from the peripheral blood of BC patients reveals enrichment in genes associated with NK or CD8+-mediated cytotoxicity, TCR-mediated stimulation, and cell exhaustion compared to non-senescent T cells. Comparison of the transcriptional profile of senescent CD4+ T cells from peripheral blood of BC patients with those of HDs highlighted marked similarities but also relevant differences. Senescent CD4+ T cells from BC patients show enrichment in T-cell signaling, processes involved in DNA replication, p53 pathways, oncogene-induced senescence, among others compared to their counterparts in HDs. High gene expression of CD4, KLRG-1, and B3GAT1 (CD57), which correlates with increased overall survival for BC patients, underscores the usefulness of the evaluation of the frequency of senescent CD4+ T cells as a biomarker in the follow-up of patients.

Long-lasting cross-presentation of tumor antigen in human DC.

DC cross-present exogenous antigens on MHC class I molecules, a process required for the onset of anti-tumor immune responses. In order to study the cross-presentation of tumor antigens by human DC, we compared the pathways of cross-presentation of long peptides requiring internalization and intracellular processing with the direct presentation of short peptides, which does not require intracellular processing. We found that, after brief incubations with DC, short peptides were presented to CD8(+) T cells with higher efficiencies than long peptides. After longer times of chase in the absence of peptide, however, the efficiency of presentation of the two types of peptides was reversed. After 2-3 days, DC pulsed with long peptides still activated T cells efficiently, while DC pulsed with short peptides failed to do so. Long-lasting presentation of the long peptides was, at least in part, due to a stored persistent pool of antigen, which was still available for loading on MHC class I molecules after several days of chase. These results show that the use of long synthetic peptides allows the efficient, long-lasting, presentation of tumor antigens, suggesting that long peptides represent an interesting approach for active anti-tumor vaccination.

Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients.

Tumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy.

Peptide-TLR-7/8a conjugate vaccines chemically programmed for nanoparticle self-assembly enhance CD8 T-cell immunity to tumor antigens.

Personalized cancer vaccines targeting patient-specific neoantigens are a promising cancer treatment modality; however, neoantigen physicochemical variability can present challenges to manufacturing personalized cancer vaccines in an optimal format for inducing anticancer T cells. Here, we developed a vaccine platform (SNP-7/8a) based on charge-modified peptide-TLR-7/8a conjugates that are chemically programmed to self-assemble into nanoparticles of uniform size (~20 nm) irrespective of the peptide antigen composition. This approach provided precise loading of diverse peptide neoantigens linked to TLR-7/8a (adjuvant) in nanoparticles, which increased uptake by and activation of antigen-presenting cells that promote T-cell immunity. Vaccination of mice with SNP-7/8a using predicted neoantigens (n = 179) from three tumor models induced CD8 T cells against ~50% of neoantigens with high predicted MHC-I binding affinity and led to enhanced tumor clearance. SNP-7/8a delivering in silico-designed mock neoantigens also induced CD8 T cells in nonhuman primates. Altogether, SNP-7/8a is a generalizable approach for codelivering peptide antigens and adjuvants in nanoparticles for inducing anticancer T-cell immunity.

TLR3 Activation of Intratumoral CD103+ Dendritic Cells Modifies the Tumor Infiltrate Conferring Anti-tumor Immunity.

An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103+ cDC1 and, to a much lesser extent CD11b+ cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8+ Granzyme B+ T cells, (lower Treg/CD8+ ratio) and an important expansion of tumor-antigen specific CD8+ T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field.

CD16+NKG2Ahigh Natural Killer Cells Infiltrate Breast Cancer-Draining Lymph Nodes.

Tumor-draining lymph nodes (TD-LNs) are the first site of metastasis of breast cancer. Natural killer (NK) cells that infiltrate TD-LNs [including noninvaded (NI) or metastatic (M)-LNs from breast cancer patients] and NK cells from healthy donor (HD)-LNs were characterized, and their phenotype analyzed by flow cytometry. Low percentages of tumor cells invaded M-LNs, and these cells expressed ULBP2 and HLA class I molecules. Although NK cells from paired NI and M-LNs were similar, they expressed different markers compared with HD-LN NK cells. Compared with HD-LNs, TD-LN NK cells expressed activating DNAM-1, NKG2C and inhibitory NKG2A receptors, and exhibited elevated CXCR3 expression. CD16, NKG2A, and NKp46 expression were shown to be increased in stage IIIA breast cancer patients. TD-LNs contained a large proportion of activated CD56brightCD16+ NK cells with high expression of NKG2A. We also showed that a subset of LN NK cells expressed PD-1, expression of which was correlated with NKp30 and NKG2C expression. LN NK cell activation status was evaluated by degranulation potential and lytic capacity toward breast cancer cells. NK cells from TD-LNs degranulated after coculture with breast cancer cell lines. Cytokine-activated TD-LN NK cells exerted greater lysis of breast cancer cell lines than HD-LN NK cells and preferentially lysed the HLA class Ilow MCF-7 breast cancer cell line. TD-LNs from breast cancer patients, thus, contained activated lytic NK cells. The expression of inhibitory receptor NKG2A and checkpoint PD-1 by NK cells infiltrating breast cancer-draining LNs supports their potential as targets for immunotherapies using anti-NKG2A and/or anti-PD-1.

IL2/Anti-IL2 Complex Combined with CTLA-4, But Not PD-1, Blockade Rescues Antitumor NK Cell Function by Regulatory T-cell Modulation.

High-dose IL2 immunotherapy can induce long-lasting cancer regression but is toxic and insufficiently efficacious. Improvements are obtained with IL2/anti-IL2 complexes (IL2Cx), which redirect IL2 action to CD8+ T and natural killer (NK) cells. Here, we evaluated the efficacy of combining IL2Cx with blockade of inhibitory immune pathways. In an autochthonous lung adenocarcinoma model, we show that the IL2Cx/anti-PD-1 combination increases CD8+ T-cell infiltration of the lung and controls tumor growth. In the B16-OVA model, which is resistant to checkpoint inhibition, combination of IL2Cx with PD-1 or CTLA-4 pathway blockade reverses that resistance. Both combinations work by reinvigorating exhausted intratumoral CD8+ T cells and by increasing the breadth of tumor-specific T-cell responses. However, only the IL2Cx/anti-CTLA-4 combination is able to rescue NK cell antitumor function by modulating intratumoral regulatory T cells. Overall, association of IL2Cx with PD-1 or CTLA-4 pathway blockade acts by different cellular mechanisms, paving the way for the rational design of combinatorial antitumor therapies.