Beyond the anti-PD-1/PD-L1 era: promising role of the BTLA/HVEM axis as a future target for cancer immunotherapy.

Recent introduction of monoclonal antibodies targeting immune checkpoints to harness antitumor immunity has revolutionized the cancer treatment landscape. The therapeutic success of immune checkpoint blockade (ICB)-based therapies mainly relies on PD-1/PD-L1 and CTLA-4 blockade. However, the limited overall responses and lack of reliable predictive biomarkers of patient´s response are major pitfalls limiting immunotherapy success. Hence, this reflects the compelling need of unveiling novel targets for immunotherapy that allow to expand the spectrum of ICB-based strategies to achieve optimal therapeutic efficacy and benefit for cancer patients. This review thoroughly dissects current molecular and functional knowledge of BTLA/HVEM axis and the future perspectives to become a target for cancer immunotherapy. BTLA/HVEM dysregulation is commonly found and linked to poor prognosis in solid and hematological malignancies. Moreover, circulating BTLA has been revealed as a blood-based predictive biomarker of immunotherapy response in various cancers. On this basis, BTLA/HVEM axis emerges as a novel promising target for cancer immunotherapy. This prompted rapid development and clinical testing of the anti-BTLA blocking antibody Tifcemalimab/icatolimab as the first BTLA-targeted therapy in various ongoing phase I clinical trials with encouraging results on preliminary efficacy and safety profile as monotherapy and combined with other anti-PD-1/PD-L1 therapies. Nevertheless, it is anticipated that the intricate signaling network constituted by BTLA/HVEM/CD160/LIGHT involved in immune response regulation, tumor development and tumor microenvironment could limit therapeutic success. Therefore, in-depth functional characterization in different cancer settings is highly recommended for adequate design and implementation of BTLA-targeted therapies to guarantee the best clinical outcomes to benefit cancer patients.

Immunoglobulin-like transcript 2 blockade restores antitumor immune responses in glioblastoma.

Glioblastoma stands as the most frequent primary brain tumor. Despite the multimodal therapy for glioblastoma patients, the survival rate is very low, highlighting the need for novel therapies that improve patient outcomes. Immune checkpoint blockade strategies are achieving promising results in a myriad of tumors and several studies have reported its efficacy in glioblastoma at a preclinical level. ILT2 is a novel immune checkpoint that exerts an inhibitory effect via the interaction with classical and non-classical HLA class-I molecules. Herein, we report that ILT2 blockade promotes antitumor responses against glioblastoma. In silico and immunohistochemical analyses revealed that the expression of ILT2 and its ligands HLA-A, -B, -C, and -E are highly expressed in patients with glioblastoma. Disruption of ILT2 with blocking monoclonal antibodies increased natural killer cell-mediated IFN-γ production and cytotoxicity against glioblastoma, partially reverting the immunosuppression linked to this malignancy. In addition, co-treatment with temozolomide strengthened the antitumor capacity of anti-ILT2-treated immune cells. Collectively, our results establish the basis for future studies regarding the clinical potential of ILT2 blockade alone or in combination regimens in glioblastoma.

BTLA dysregulation correlates with poor outcome and diminished T cell-mediated antitumor responses in chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) progressively develop marked immunosuppression, dampening innate and adaptive-driven antitumor responses. However, the underlying mechanisms promoting immune exhaustion are largely unknown. Herein, we provide new insights into the role of BTLA/HVEM axis promoting defects in T cell-mediated responses against leukemic cells. Increased expression of BTLA, an inhibitory immune checkpoint, was detected on the surface of CD4 + and CD8 + T lymphocytes in patients with CLL. Moreover, high levels of BTLA on CD4 + T cells correlated with diminished time to treatment. Signaling through BTLA activation led to decreased IL-2 and IFN-γ production ex vivo, whereas BTLA/HVEM binding disruption enhanced IFN-γ + CD8 + T lymphocytes. Accordingly, BTLA blockade in combination with bispecific anti-CD3/anti-CD19 antibody promoted CD8 + T cell-mediated anti-leukemic responses. Finally, treatment with an anti-BLTA blocking monoclonal antibody alone or in combination with ibrutinib-induced leukemic cell depletion in vitro. Altogether, our data reveal that BTLA dysregulation has a prognostic role and is limiting T cell-driven antitumor responses, thus providing new insights about immune exhaustion in patients with CLL.

NKG2D Signaling: The Immune Subversive Side of HDAC3.

Natural killer (NK) cells are alerted to infected and transformed cells by local upregulation of ligands for the NK-activating receptor NKG2D. In a recent report, Greene et al. unveil a new mechanism that induces the expression of the NKG2D ligand retinoic acid early-inducible (RAE-1) in response to murine cytomegalovirus (MCMV) infection through inhibition of casein kinase 2 (CK2), an activator of the repressor histone deacetylase HDAC3.

Mechanisms of Apoptosis Resistance to NK Cell-Mediated Cytotoxicity in Cancer.

Natural killer (NK) cells are major contributors to immunosurveillance and control of tumor development by inducing apoptosis of malignant cells. Among the main mechanisms involved in NK cell-mediated cytotoxicity, the death receptor pathway and the release of granules containing perforin/granzymes stand out due to their efficacy in eliminating tumor cells. However, accumulated evidence suggest a profound immune suppression in the context of tumor progression affecting effector cells, such as NK cells, leading to decreased cytotoxicity. This diminished capability, together with the development of resistance to apoptosis by cancer cells, favor the loss of immunogenicity and promote immunosuppression, thus partially inducing NK cell-mediated killing resistance. Altered expression patterns of pro- and anti-apoptotic proteins along with genetic background comprise the main mechanisms of resistance to NK cell-related apoptosis. Herein, we summarize the main effector cytotoxic mechanisms against tumor cells, as well as the major resistance strategies acquired by tumor cells that hamper the extrinsic and intrinsic apoptotic pathways related to NK cell-mediated killing.

Phytochemical Study of the Genus Salpichroa (Solanaceae), Chemotaxonomic Considerations, and Biological Evaluation in Prostate and Breast Cancer Cells.

Twelve Salpichroa taxa have been phytochemically analyzed. From the aerial parts of S. scandens, four known salpichrolides A, C, I, S, and an unreported withanolide named salpichrolide V (1), were isolated. In S. dependens, S. gayi, S. glandulosa subsp. glandulosa, S. glandulosa subps. weddellii, S. leucantha, S. micrantha, S. microloba, S. proboscidea, S. ramosissima, S. tristis var. tristis, and S. weberbauerii, no withanolides were found. The chemical content of ca. 85% of the Salpichroa taxa is in agreement with molecular studies, which suggest that Salpichroa and Jaborosa, a genus considered morphologically close to Salpichroa, are distant in the systematic of the Solanoideae subfamily. Moreover, the in vitro cytotoxic activity of a set of natural salpichrolides and derivatives was examined against two prostate carcinoma cell lines (PC3 and LNCaP) and two human breast cancer cell lines (MCF-7 and T47D). Several compounds showed moderate activity (IC50  = 64.91 - 29.97 µm).

Analysis of clinical prognostic variables for Chronic Lymphocytic Leukemia decision-making problems.

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is a disease with highly heterogeneous clinical course. A key goal is the prediction of patients with high risk of disease progression, which could benefit from an earlier or more intense treatment. In this work we introduce a simple methodology based on machine learning methods to help physicians in their decision making in different problems related to CLL. MATERIAL AND METHODS: Clinical data belongs to a retrospective study of a cohort of 265 Caucasians who were diagnosed with CLL between 1997 and 2007 in Hospital Cabueñes (Asturias, Spain). Different machine learning methods were applied to find the shortest list of most discriminatory prognostic variables to predict the need of Chemotherapy Treatment and the development of an Autoimmune Disease. RESULTS: Autoimmune disease occurrence was predicted with very high accuracy (>90%). Autoimmune disease development is currently an unpredictable severe complication of CLL. Chemotherapy Treatment has been predicted with a lower accuracy (80%). Risk analysis showed that the number of false positives and false negatives are well balanced. CONCLUSIONS: Our study highlights the importance of prognostic variables associated with the characteristics of platelets, reticulocytes and natural killers, which are the main targets of the autoimmune haemolytic anemia and immune thrombocytopenia for autoimmune disease development, and also, the relevance of some clinical variables related with the immune characteristics of CLL patients that are not taking into account by current prognostic markers for predicting the need of chemotherapy. Because of its simplicity, this methodology could be implemented in spreadsheets.

NKG2D signaling in cancer immunosurveillance.

The immune system is able to detect and eliminate transformed cells. The activating receptor NKG2D is particularly relevant for cancer immunosurveillance. NKG2D ligand expression renders tumor cells more susceptible to be killed by NK and T cells, and correlates with the clinical outcome of the disease. However, tumors develop mechanisms to overcome the NKG2D-mediated immune response, which has been associated with poor prognosis and impairment of the clinical benefits of immunotherapy in many human cancers. The highly specific pattern of expression displayed by the NKG2D ligands, mainly confined to tumor cells, together with the strong immune response triggered by this receptor clearly supports the idea that the NKG2D-mediated pathway may be a powerful target for the treatment of cancer. This review draws together the most recent discoveries concerning the biology of the NKG2D signaling and their therapeutic relevance in the context of cancer.

Prospective trial of breast MRI versus 2D and 3D ultrasound for evaluation of response to neoadjuvant chemotherapy.

BACKGROUND: Preoperative imaging to assess response to neoadjuvant chemotherapy in breast cancer is routine but no single imaging modality is standard of practice. Our hypothesis is that ultrasound (US) is comparable to magnetic resonance imaging (MRI) in the prediction of residual disease. METHODS: A single-institution, Institutional Review Board-approved prospective trial of primary invasive ductal breast cancer patients receiving neoadjuvant chemotherapy enrolled women from 2008 to 2012. Two-dimensional (2D) and three-dimensional (3D) US, as well as MRI images of pre- and post-neoadjuvant tumors were obtained. Skin involvement or inadequate images were excluded. Residual tumor on imaging was compared with surgical pathology. Differences of tumor volume on imaging and pathology were compared using the non-parametric Wilcoxon signed-rank test. US to MRI agreement was determined by the kappa coefficient. Tumor volumes in estrogen receptor (ER), progesterone receptor (PR), and Her2neu subgroups were compared using the Kruskal-Wallis test. ER/PR staining <5 % was considered negative; Her2neu status was determined by in situ hybridization. RESULTS: Forty-two patients were enrolled in the study; 39 had evaluable post-treatment data. Four patients were Her2neu positive, and 17 (46 %) patients had triple-negative tumors. Among 11 (28 %) patients with pathologic complete response (pCR), US correctly predicted pCR in six (54.5 %) patients compared with eight (72.7 %) patients when MRI was used. This is a substantial agreement between US and MRI in predicting pCR (kappa = 0.62). There was no difference between 2D and 3D US modalities. For the 39 patients, US and MRI had no significant difference in volume estimation of pathology, even stratified by receptor status. CONCLUSION: The estimation of residual breast tumor volume by US and MRI achieves similar results, including prediction of pCR.

Epithelial-mesenchymal transition induces an antitumor immune response mediated by NKG2D receptor.

Epithelial-mesenchymal transition (EMT) is a morphogenetic process characterized by the acquisition of mesenchymal properties linked with an invasive phenotype and metastasis of tumor cells. NK group 2, member D (NKG2D) is an NK cell-activating receptor crucially involved in cancer immunosurveillance. In this study, we show that induction of EMT by TGF-ß stimulation of human keratinocytes, by glycogen synthase kinase-3ß inhibition in several epithelial tumor cell lines, and by Snail1 overexpression in colorectal cancer cells strongly upregulated the expression of NKG2D ligands (NKG2DLs), MHC class I chain-related molecules A and B (MICA/B) and ULBP1-3. Overexpression of Snail1 and inhibition of glycogen synthase kinase-3ß in colorectal tumor cells markedly induced the activity of Sp1 transcription factor, which plays a key role in the upregulation of NKG2DL expression during EMT. The stimulation of MICA/B expression by TGF-ß treatment was independent of Sp1, but it involved posttranslational mechanisms mediated by mammalian target of rapamycin pathway. Accordingly, with the increased expression of NKG2DLs, triggering of EMT rendered cancer cells more susceptible to NKG2D-mediated killing by NK cells. In agreement, MICA/B were expressed in vivo in well-differentiated colorectal tumors with retained epithelial characteristics, whereas no expression of MICA/B was detected in poorly differentiated and invasive colorectal tumors that have lost epithelial characteristics. This decrease of MICA/B expression was associated with a dramatic increase of NKG2D(+)-tumor infiltrating lymphocytes. Overall, our findings indicate that EMT is a relevant checkpoint in the control of tumor progression through NKG2D-mediated immune responses.

Withanolides with antibacterial activity from Nicandra john-tyleriana.

Eleven new withanolides (1-11) were isolated and characterized from the aerial parts of Nicandra john-tyleriana. Five of these withanolides have an unmodified skeleton (1-5), two are acnistins (6, 7), and four are withajardins (8-11). These new isolates were fully characterized using a combination of spectroscopic techniques (including multidimensional NMR) and mass spectrometry. All compounds were evaluated for their antibacterial activity against Bacillus, Enterococcus, Escherichia, Listeria, Pseudomonas, and Staphylococcus strains.

Clinical differences between men and women with psoriatic arthritis: relevance of the analysis of genes and polymorphisms in the major histocompatibility complex region and of the age at onset of psoriasis.

It has been shown that males with spondyloarthritis tend to suffer from more severe spinal disease while females are more likely to have peripheral joint involvement. Nevertheless, gender-related differences have not been thoroughly explored in psoriatic arthritis (PsA). In PsA, males accumulate more peripheral and axial joint damage compared to women. However, it is not clear whether these findings are secondary to differences in occupational physical activity, hormonal changes, or other factors. The present study analyzed the differences in clinical expression of PsA between men and women. We have also evaluated the possible existence of gender-linked differences in the distribution of genes and polymorphisms within the major histocompatibility complex and whether patients' age at the onset of psoriasis established any differences in these aspects. Women suffered more polyarthritis, greater functional impairment, and a larger number of swollen joints during followup. We appreciated a differential expression of certain MHC genes according to gender and age at onset of psoriasis. Our results point to the need to include patient's age at the onset of psoriasis and gender as key stratification elements in future studies of genetic associations in PsA.

Disulphide-isomerase-enabled shedding of tumour-associated NKG2D ligands.

Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.

Quest for antibacterial alkaloids from Rauhia multiflora through bioassay complementary-guided fractionation.

The clinical efficacy of many existing antibiotics is currently threatened by the emergence of microbial resistance. This recognized worldwide situation prompts to greater efforts to discover antimicrobial agents of natural origin, including plant sources. The objective of this work was to evaluate the antimicrobial activities of extracts, fractions and pure compounds from Rauhia multiflora using a bioguided complementary fractionation, contributing also to explain some traditional uses of this genus. Some subfractions showed antimicrobial activity against the Gram-negative and Gram-positive bacteria. Galantamine was identified and isolated as the main alkaloid, together with two additional structures of the same skeleton. Characterization by GC-MS revealed the presence of twelve galantamine-type and four crinane-type compounds. The tentative structure of one of the galantamine-type skeletons is proposed here for the first time. Altogether, these results support the use of Rauhia genus to inhibit bacterial growth.

Chemo-Immunotherapy: A New Trend in Cancer Treatment.

Chemotherapy has been the basis of advanced cancer treatment for decades. This therapy has largely been considered immunosuppressive, yet accumulated preclinical and clinical evidence shows that certain chemotherapeutic drugs, under defined conditions, may stimulate antitumor immunity and potentiate immune checkpoint inhibitor (ICI)-based therapy. Its effectiveness has been highlighted by recent regulatory approvals of various combinations of chemotherapy with ICIs in several tumors, particularly in some difficult-to-treat cancers. This review discusses the immune modulatory properties of chemotherapy and how they may be harnessed to develop novel chemo-immunotherapy combinations. It also highlights the key determinants of the success of chemo-immunotherapy and provides an overview of the combined chemo-immunotherapies that have been clinically approved.

Expression of ERp5 and GRP78 on the membrane of chronic lymphocytic leukemia cells: association with soluble MICA shedding.

MICA is a ligand of the activating receptor NKG2D, expressed by NK and T cells. MICA expression is induced in cancer cells favoring their elimination by the immune system; however, many advanced tumors shed soluble MICA (sMICA), which impairs NKG2D-mediated cytotoxicity. ERp5 and GRP78 are endoplasmic reticulum-resident proteins that are translocated to the surface of epithelial tumor cells where they interact with MICA and are involved in sMICA shedding. In this study, we analyze the role of ERp5 and GRP78 in sMICA shedding in chronic lymphocytic leukemia (CLL). Immunofluorescence and flow cytometry analyses showed that ERp5 and GRP78 were significantly expressed on the surface of B cells and leukemia cells, but they were not expressed on T cells. The expression of ERp5 and GRP78 was significantly higher in leukemia cells than in B cells from controls. ERp5 and GRP78 co-localized with MICA on the surface of leukemia cells and the levels of expression of ERp5 and GRP78 correlated with the level of expression of membrane-bound MICA in CLL patients. Associated with higher expression of membrane-bound ERp5 and GRP78, serum sMICA levels were approximately threefold higher in patients than in controls. Elevated sMICA levels in CLL patients were associated with the down-modulation of NKG2D surface expression on CD8 T cells. Finally, pharmacological inhibition of B cell lines and stimulated leukemia cells showed that ERp5 activity is involved in sMICA shedding in CLL. In conclusion, these results uncover a molecular mechanism which regulates MICA protein shedding and immune evasion in CLL.

Patients with psoriatic arthritis may show differences in their clinical and genetic profiles depending on their age at psoriasis onset.

OBJECTIVES: The age of psoriasis onset has an important impact on the clinical expression and heritability of psoriasis. Psoriasis characteristics according to the age at disease onset have been extensively studied. However, the impact of the age of psoriasis onset on psoriatic arthritis (PsA) features has not been analysed in depth. The aim of the present paper is to analyse whether the age of psoriasis onset may have an impact on the clinical and genetic characteristics in a cohort of PsA patients. METHODS: The study included 110 PsA patients classified in accordance with the CASPAR criteria. Patients were divided into early (onset age <30 years) and late (onset age >30 years) onset psoriasis, and clinical features were studied in accordance to this stratification. Distribution of several genes within the MHC region were analysed in accordance with the prior stratification, and their frequencies compared to that of 110 healthy matched blood donors. RESULTS: Compared to patients with late-onset disease, PsA patients with early-onset psoriasis showed more frequently: a longer psoriasis-arthritis latency period (9.9±6 years vs. 3.8±4 years, p=0.0001), a positive family history of disease (60.3% vs. 20.5%, OR 6.1, 95% CI: 2.5-15.0, p=0.0001), severe psoriasis (PASI 8.2±4 vs. 3.6±2.2, p=0.0001), clinical enthesitis (37.7% vs. 22.4%, OR 2.09, 95% CI: 0.9-4.9, p=0.08), and oligoarthritis (47.5% vs. 28.6%, OR 2.26, 95% CI: 1.02-5.02, p=0.04). MICA-A9 was associated with susceptibility in both early-onset (60.7% vs. 30%, p=0.0002) and late-onset patients (59.2% vs. 30%, p=0.0008). However, HLA-Cw*0602 was significantly increased in patients with early-onset psoriasis (73.8% vs. 17%, p<0.0001), whereas the allele 384 of the microsatellite C1_4_4, located 34 kb telomeric to HLA-C locus, was increased only in late-onset cases (49% vs. 21%, p=0.001). CONCLUSIONS: Clinical and genetic features of PsA may differ depending on the age at psoriasis onset. This type of stratification should be considered in future genetic and epidemiological studies of PsA.

The origin of the bacterial immune response.

Bacteriophages are probably the oldest viruses, having appeared early during bacterial evolution. Therefore, bacteria and bacteriophages have a long history of co-evolution in which bacteria have developed multiple resistance mechanisms against bacteriophages. These mechanisms, that are very diverse and are in constant evolution, allow the survival of the bacteria. Bacteriophages have adapted to bacterial defense systems, devised strategies to evade these anti-phage mechanisms and restored their infective capacity. In this chapter, we review the bacterial strategies that hinder the phage infection as well as the counter-defense mechanisms developed.

The Emerging Role of NK Cells in Immune Checkpoint Blockade.

Natural killer (NK) cells are innate cytotoxic immune cells that play a fundamental role in anti-tumor immunity, particularly in hematological cancers, disseminated cancers, and metastasis [...].

Involvement of CD4+ and CD8+ T-lymphocytes in the modulation of nociceptive processing evoked by CCL4 in mice.

AIMS: To explore the mechanisms involved in the transformation of analgesia produced by low doses of CCL4 (pg/kg) to hyperalgesia when higher doses (ng/kg) are administered to mice. MAIN METHODS: The unilateral hot plate test was used to assess thermal nociception. CD3+, CD4+ or CD8+ blood cells were depleted with selective antibodies. Expression of CCR5 and IL-16 in lymphocytes was studied by flow cytometry and IL-16 blood levels were measured by ELISA. IL-16 and CD8 were detected by immunofluorescence. KEY FINDINGS: IL-16 and CCR5 expression were demonstrated in CD4+ and CD8+ T-lymphocytes by flow cytometry. Furthermore, CCL4-induced hyperalgesia was abolished by reducing circulating T-lymphocyte levels or by selectively depleting CD4+ lymphocytes. In contrast, when the anti-CD4 antibody was acutely administered, CCL4 induced analgesia instead of hyperalgesia. A similar response was obtained when administering A-770041, that prevents CD4-mediated CCR5 desensitization by inhibiting p56lck kinase. As occurred with the analgesic effect evoked by low doses of CCL4, analgesia evoked by combining CCL4 and A-770041 was reverted by naloxone, naltrindole or an anti-met-enk antibody. Interestingly, flow cytometry assays showed that the number of CD8+, but not CD4+, T-cells expressing IL-16 is reduced after the acute administration of CCL4, a result compatible with the description that CD8+-lymphocytes can rapidly release preformed IL-16. Accordingly, the rise in IL-16 blood concentration evoked by CCL4 was prevented after CD8+ lymphocyte depletion. SIGNIFICANCE: CCL4-evoked hyperalgesia is related to the desensitization of CCR5 in CD4+ T-cells and to the release of IL-16 from CD8+ lymphocytes.