Fasting reshapes tissue-specific niches to improve NK cell-mediated anti-tumor immunity.

Fasting is associated with improved outcomes in cancer. Here, we investigated the impact of fasting on natural killer (NK) cell anti-tumor immunity. Cyclic fasting improved immunity against solid and metastatic tumors in an NK cell-dependent manner. During fasting, NK cells underwent redistribution from peripheral tissues to the bone marrow (BM). In humans, fasting also reduced circulating NK cell numbers. NK cells in the spleen of fasted mice were metabolically rewired by elevated concentrations of fatty acids and glucocorticoids, augmenting fatty acid metabolism via increased expression of the enzyme CPT1A, and Cpt1a deletion impaired NK cell survival and function in this setting. In parallel, redistribution of NK cells to the BM during fasting required the trafficking mediators S1PR5 and CXCR4. These cells were primed by an increased pool of interleukin (IL)-12-expressing BM myeloid cells, which improved IFN-γ production. Our findings identify a link between dietary restriction and optimized innate immune responses, with the potential to enhance immunotherapy strategies.

Factor of time in dendritic cell (DC) maturation: short-term activation of DCs significantly improves type 1 cytokine production and T cell responses.

Dendritic cells (DCs) have been intensively studied in correlation to tumor immunology and for the development DC-based cancer vaccines. Here, we present the significance of the temporal aspect of DC maturation for the most essential subsequent timepoint, namely at interaction with responding T cells or after CD40-Ligand restimulation. Mostly, DC maturation is still being achieved by activation processes which lasts 24 h to 48 h. We hypothesized this amount of time is excessive from a biological standpoint and could be the underlying cause for functional exhaustion. Indeed, shorter maturation periods resulted in extensive capacity of monocyte-derived DCs to produce inflammatory cytokines after re-stimulation with CD40-Ligand. This effect was most evident for the primary type 1 polarizing cytokine, IL-12p70. This capacity reached peak at 6 h and dropped sharply with longer exposure to initial maturation stimuli (up to 48 h). The 6 h maturation protocol reflected superiority in subsequent functionality tests. Namely, DCs displayed twice the allostimulatory capacity of 24 h- and 48 h-matured DCs. Similarly, type 1 T cell response measured by IFN-γ production was 3-fold higher when CD4+ T cells had been stimulated with shortly matured DC and over 8-fold greater in case of CD8+ T cells, compared to longer matured DCs. The extent of melanoma-specific CD8+ cytotoxic T cell induction was also greater in case of 6 h DC maturation. The major limitation of the study is that it lacks in vivo evidence, which we aim to examine in the future. Our findings show an unexpectedly significant impact of temporal exposure to activation signals for subsequent DC functionality, which we believe can be readily integrated into existing knowledge on in vitro/ex vivo DC manipulation for various uses. We also believe this has important implications for DC vaccine design for future clinical trials.

Quantifying in vivo murine antigen-specific T cell responses without requirement for prior knowledge of antigen identity.

Extracorporeal Photochemotherapy (ECP) is a widely applied anti-cancer immunotherapy for patients with cutaneous T cell lymphoma (CTCL). By using apoptotic malignant cells as a source of patient-specific tumor antigen, it enables clinically relevant and curative anti-CTCL immunity, with potential efficacy in other tumors. Currentmethods to track patient-specific responses are tedious, and new methods are needed to assess putative global immunity. We developed a clinically practical method to assess antigen-specific T cell activation that does not rely on knowledge of the particular antigen, thereby eliminating the requirement for patient-specific reagents. In the OT-I transgenic murine system, we quantified calcium flux to reveal early T cell engagement by antigen presenting cells constitutively displaying a model antigenic peptide, ovalbumin (OVA)-derived SIINFEKL. We detected calcium flux in OVA-specific T cells, triggered by specific T cell receptor engagement by SIINFEKL peptide-loaded DC. This approach led to sensitive detection of antigen-specific calcium flux (ACF) down to a peptide-loading concentration of ∼10-3uM and at a frequency of ∼0.1% OT-I cells among wild-type (WT), non-responding cells. Antigen-specific T cells were detected in spleen, lymph nodes, and peripheral blood after adoptive transfer into control recipient mice. Methods like this for assessing therapeutic response are lacking in patients currently on immune-based therapies, such as ECP, where assessment of clinical response is made by delayed measurement of the size of the malignant clone. These findings suggest an early, practical way to measure therapeutically-induced anti-tumor responses in ECP-treated patients that have been immunized against their malignant cells.

Proteobacteria impair anti-tumor immunity in the omentum by consuming arginine.

Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ T cells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E. coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ T cell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.

In pancreatic cancer patients, chemotherapy reshapes the gene expression profile and antigen receptor repertoire of T lymphocytes and enhances their effector response to tumor-associated antigens.

Introduction: Pancreatic Ductal Adenocarcinoma (PDA) is one of the most aggressive malignancies with a 5-year survival rate of 13%. Less than 20% of patients have a resectable tumor at diagnosis due to the lack of distinctive symptoms and reliable biomarkers. PDA is resistant to chemotherapy (CT) and understanding how to gain an anti-tumor effector response following stimulation is, therefore, critical for setting up an effective immunotherapy. Methods: Proliferation, and cytokine release and TCRB repertoire of from PDA patient peripheral T lymphocytes, before and after CT, were analyzed in vitro in response to four tumor-associated antigens (TAA), namely ENO1, FUBP1, GAPDH and K2C8. Transcriptional state of PDA patient PBMC was investigated using RNA-Seq before and after CT. Results: CT increased the number of TAA recognized by T lymphocytes, which positively correlated with patient survival, and high IFN-γ production TAA-induced responses were significantly increased after CT. We found that some ENO1-stimulated T cell clonotypes from CT-treated patients were expanded or de-novo induced, and that some clonotypes were reduced or even disappeared after CT. Patients that showed a higher number of effector responses to TAA (high IFN-γ/IL-10 ratio) after CT expressed increased fatty acid-related transcriptional signature. Conversely, patients that showed a higher number of regulatory responses to TAA (low IFN-γ/IL-10 ratio) after CT significantly expressed an increased IRAK1/IL1R axis-related transcriptional signature. Conclusion: These data suggest that the expression of fatty acid or IRAK1/IL1Rrelated genes predicts T lymphocyte effector or regulatory responses to TAA in patients that undergo CT. These findings are a springboard to set up precision immunotherapies in PDA based on the TAA vaccination in combination with CT.

Development and Optimal Immune Strategy of an Alum-Stabilized Pickering emulsion for Cancer Vaccines.

Therapeutic cancer vaccines are considered as one of the most cost-effective ways to eliminate cancer cells. Although many efforts have been invested into improving their therapeutic effect, transient maturation and activations of dendritic cells (DCs) cause weak responses and hamper the subsequent T cell responses. Here, we report on an alum-stabilized Pickering emulsion (APE) that can load a high number of antigens and continue to release them for extensive maturation and activations of antigen-presenting cells (APCs). After two vaccinations, APE/OVA induced both IFN-γ-secreting T cells (Th1) and IL-4-secreting T cells (Th2), generating effector CD8+ T cells against tumor growth. Additionally, although they boosted the cellular immune responses in the spleen, we found that multiple administrations of cancer vaccines (three or four times in 3-day intervals) may increase the immunosuppression with more PD-1+ CD8+ and LAG-3+ CD8+ T cells within the tumor environment, leading to the diminished overall anti-tumor efficacy. Combining this with anti-PD-1 antibodies evidently hindered the suppressive effect of multiple vaccine administrations, leading to the amplified tumor regression in B16-OVA-bearing mice.

The CD40 agonist HERA-CD40L results in enhanced activation of antigen presenting cells, promoting an anti-tumor effect alone and in combination with radiotherapy.

Introduction: The ability to modulate and enhance the anti-tumor immune responses is critical in developing novel therapies in cancer. The Tumor Necrosis Factor (TNF) Receptor Super Family (TNFRSF) are potentially excellent targets for modulation which result in specific anti-tumor immune responses. CD40 is a member of the TNFRSF and several clinical therapies are under development. CD40 signaling plays a pivotal role in regulating the immune system from B cell responses to myeloid cell driven activation of T cells. The CD40 signaling axis is well characterized and here we compare next generation HERA-Ligands to conventional monoclonal antibody based immune modulation for the treatment of cancer. Methods & results: HERA-CD40L is a novel molecule that targets CD40 mediated signal transduction and demonstrates a clear mode of action in generating an activated receptor complex via recruitment of TRAFs, cIAP1, and HOIP, leading to TRAF2 phosphorylation and ultimately resulting in the enhanced activation of key inflammatory/survival pathway and transcription factors such asNFkB, AKT, p38, ERK1/2, JNK, and STAT1 in dendritic cells. Furthermore, HERA-CD40L demonstrated a strong modulation of the tumor microenvironment (TME) via the increase in intratumoral CD8+ T cells and the functional switch from pro-tumor macrophages (TAMs) to anti-tumor macrophages that together results in a significant reduction of tumor growth in a CT26 mouse model. Furthermore, radiotherapy which may have an immunosuppressive modulation of the TME, was shown to have an immunostimulatory effect in combination with HERA-CD40L. Radiotherapy in combination with HERA-CD40L treatment resulted in an increase in detected intratumoral CD4+/8+ T cells compared to RT alone and, additionally, the repolarization of TAMs was also observed, resulting in an inhibition of tumor growth in a TRAMP-C1 mouse model. Discussion: Taken together, HERA-CD40L resulted in activating signal transduction mechanisms in dendritic cells, resulting in an increase in intratumoral T cells and manipulation of the TME to be pro-inflammatory, repolarizing M2 macrophages to M1, enhancing tumor control.

B Cell Orchestration of Anti-tumor Immune Responses: A Matter of Cell Localization and Communication.

The immune system plays a crucial role in cancer development either by fostering tumor growth or destroying tumor cells, which has open new avenues for cancer immunotherapy. It was only over the last decade that the role of B cells in controlling anti-tumor immune responses in the tumor milieu has begun to be appreciated. B and plasma cells can exert anti-tumor effects through antibody-dependent cell cytotoxicity (ADCC) and activation of the complement cascade, even though their effector functions extend beyond the classical humoral immunity. In tumor tissues, B cells can be found in lymphoid aggregates, known as tertiary lymphoid structures (TLSs), well-organized non-encapsulated structures composed of immune and stromal cells. These structures reflect a process of lymphoid neogenesis occurring in peripheral tissues upon long-lasting exposure to inflammatory signals. The TLS provides an area of intense B cell antigen presentation that can lead to optimal T cell activation and effector functions, as well as the generation of effector B cells, which can be further differentiated in either antibody-secreting plasma cells or memory B cells. Of clinical interest, the crosstalk between B cells and antigen-experienced and exhausted CD8+ T cells within mature TLS was recently associated with improved response to immune checkpoint blockade (ICB) in melanoma, sarcoma and lung cancer. Otherwise, B cells sparsely distributed in the tumor microenvironment or organized in immature TLSs were found to exert immune-regulatory functions, inhibiting anti-tumor immunity through the secretion of anti-inflammatory cytokines. Such phenotype might arise when B cells interact with malignant cells rather than T and dendritic cells. Differences in the spatial distribution likely underlie discrepancies between the role of B cells inferred from human samples or mouse models. Many fast-growing orthotopic tumors develop a malignant cell-rich bulk with reduced stroma and are devoid of TLSs, which highlights the importance of carefully selecting pre-clinical models. In summary, strategies that promote TLS formation in close proximity to tumor cells are likely to favor immunotherapy responses. Here, the cellular and molecular programs coordinating B cell development, activation and organization within TLSs will be reviewed, focusing on their translational relevance to cancer immunotherapy.

A long way to the battlefront: CAR T cell therapy against solid cancers.

Chimeric antigen receptors (CARs) are engineered synthetic receptors that redirect and reprogram T cells to tumor surface antigens for subsequent eradication. The unprecedented efficacy of CD19-CAR T cells against B-cell malignancies has inspired oncologists to extend these efforts for the treatment of solid tumors. However, limited success has been achieved so far, partially due to some of the formidable challenges, e.g. suppression of full activation, inhibition of T cell localization, lacking of ideal targets, inefficient trafficking and infiltration, immunosuppression of microenvironment, and the probability of off targets and associated side effects. Significant progresses have being made recently. Thus, an updated summary is urgently needed. Here in this review, we discuss the advantages and some of the key hurdles encountered by CAR T cell therapy in solid tumors as well as the strategies adopted to improve therapeutic outcomes of this approach. Continuing efforts to increase therapeutic potential and decrease the adverse effects of adaptive cell transfer are suggested as well.

In vivo cancer vaccination: Which dendritic cells to target and how?

The field of cancer immunotherapy has been revolutionized with the use of immune checkpoint blockade antibodies such as anti-programmed cell death 1 protein (PD-1) and chimeric antigen receptor T cells. Significant clinical benefits are observed in different cancer types with these treatments. While considerable efforts are made in augmenting tumor-specific T cell responses with these therapies, other immunotherapies that actively stimulate endogenous anti-tumor T cells and generating long-term memory have received less attention. Given the high cost of cancer immunotherapies especially with chimeric antigen receptor T cells, not many patients will have access to such treatments. The next-generation of cancer immunotherapy could entail in vivo cancer vaccination to activate both the innate and adaptive anti-tumor responses. This could potentially be achieved via in vivo targeting of dendritic cells which are an indispensable link between the innate and adaptive immunities. Dendritic cells highly expressed toll-like receptors for recognizing and eliminating pathogens. Synthetic toll-like receptors agonists could be synthesized at a low cost and have shown promise in preclinical and clinical trials. As different subsets of human dendritic cells exist in the immune system, activation with different toll-like receptor agonists could exert profound effects on the quality and magnitude of anti-tumor T cell responses. Here, we reviewed the different subsets of human dendritic cells. Using published preclinical and clinical cancers studies available on PubMed, we discussed the use of clinically approved and emerging toll-like receptor agonists to activate dendritic cells in vivo for cancer immunotherapy. Finally, we searched www.clinicaltrials.gov and summarized the active cancer trials evaluating toll-like receptor agonists as an adjuvant.

The Potential of Intralesional Rose Bengal to Stimulate T-Cell Mediated Anti-Tumor Responses.

Rose Bengal (RB) is a red synthetic dye that was initially used in the garment industry and has been used safely for decades as a corneal stain by ophthalmologists. Antineoplastic properties of RB have also been observed, though the mechanism of action remained to be elucidated. Recently, interest in RB as a therapeutic cancer treatment has increased due to significant anti-tumor responses with direct tumor injection in human clinical trials for metastatic melanoma. In these patients, there has been the implication that RB may mount a T-cell mediated anti-tumor response and impart antigen-specific responses in distant bystander lesions. This article serves to evaluate the potential of intralesional rose bengal to stimulate T-cell mediated anti-tumor responses in in-vitro, pre-clinical, and clinical studies.