Search Results (23)

Introduction: Prostate cancer has been generally resistant to immunotherapy approaches. Radiation can be immunostimulatory, but the extent to which standard prostate cancer treatments induce immune activation has not been well described. The bone-targeted radiopharmaceutical Radium223 (Ra223) has been proposed to enrich immune function, but clinical studies have not fully delineated whether this is true, or by what mechanisms. Enzalutamide has been shown to increase PD-L1 expression on dendritic cells, which could impact immune activation, though the extent to which this is associated with other evidence of immune activation remains uncertain, and combination strategies remain of interest. We performed a randomized phase II trial to evaluate whether Radium223 (Ra223) added to enzalutamide would induce greater immune activation and clinical responses compared to enzalutamide alone in men with metastatic castration-resistant prostate cancer (mCRPC). Methods: Eligible patients were randomized 2:1 to Arm A (enzalutamide 160 mg PO daily + Ra223 55 kBq/kg IV q4 weeks × 6 doses) or Arm B (enzalutamide 160 mg PO daily). Blood was collected at treatment start and during treatment to measure soluble immune checkpoint biomarkers (BTLA, TIM3, HVEM, GITR, LAG3, PD-1, CTLA-4, PD-L1, PD-L2, ICOS). Immunophenotyping by mass cytometry time of flight (CyTOF) was performed to measure peripheral blood mononuclear cell populations before and after treatment. CyTOF was used to determine changes in circulating immune cell population subsets before and after treatment. Biopsies were performed of an active bone metastatic lesion prior to study treatment and after at least 3 months. IHC was subsequently performed to examine changes in immune cell population subsets before and after treatment, and changes in pSTAT3 levels. Results: In total, 30 patients were enrolled, with median age 68. The median duration of follow up was 36 months. PSA responses, PFS, and OS were not significantly different between the two arms; however, the study was not powered for clinical endpoints. Peripheral blood and bone biopsy specimens were analyzed for immune correlatives. Soluble receptor concentrations showed significantly increased expression of PDL-2 in the combination arm, but this was not seen on CyTOF. Otherwise, there were no significant differences in markers of immune activation/exhaustion or immune cell population subsets in the combination arm and enzalutamide monotherapy arm. IHC also did not show a significant difference in immune cell population subsets in bone biopsy specimens before and after treatment in both arms. However, treatment with the combination arm did show significantly increased levels of pSTAT3 (p = 0.04), which was not seen in the enzalutamide monotherapy arm. Conclusions: Our study showed an overall lack of evidence for immune activation or cytokine induction with the combination, which does not make a strong case for combinatorial immunotherapy approaches. However, the combination did induce higher levels of pSTAT3, which has been implicated in radio-resistance. Therefore, the addition of a STAT3 inhibitor to the combination may be of interest to improve efficacy. Full article

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and presents a continuously growing incidence and high mortality rates worldwide. Besides advances in diagnosis and promising results of pre-clinical studies, established curative therapeutic options for HCC are not currently available. Recent progress in understanding the tumor microenvironment (TME) interactions has turned the scientific interest to immunotherapy, revolutionizing the treatment of patients with advanced HCC. However, the limited number of HCC patients who benefit from current immunotherapeutic options creates the need to explore novel targets associated with improved patient response rates and potentially establish them as a part of novel combinatorial treatment options. Glucocorticoid-induced TNFR-related protein (GITR) belongs to the TNFR superfamily (TNFRSF) and promotes CD8⁺ and CD4⁺ effector T-cell function with simultaneous inhibition of Tregs function, when activated by its ligand, GITRL. GITR is currently considered a potential immunotherapy target in various kinds of neoplasms, especially with the concomitant use of programmed cell-death protein-1 (PD-1) blockade. Regarding liver disease, a high GITR expression in liver progenitor cells has been observed, associated with impaired hepatocyte differentiation, and decreased progenitor cell-mediated liver regeneration. Considering real-world data proving its anti-tumor effect and recently published evidence in pre-clinical models proving its involvement in pre-cancerous liver disease, the idea of its inclusion in HCC therapeutic options theoretically arises. In this review, we aim to summarize the current evidence supporting targeting GITR/GITRL signaling as a potential treatment strategy for advanced HCC. Full article

With the aim to advance the understanding of immune regulation in MCL and to identify targetable T-cell subsets, we set out to combine image analysis and spatial omic technology focused on both early and late differentiation stages of T cells. MCL patient tissue (n = 102) was explored using image analysis and GeoMx spatial omics profiling of 69 proteins and 1812 mRNAs. Tumor cells, T helper (T_H) cells and cytotoxic (T_C) cells of early (CD57−) and late (CD57+) differentiation stage were analyzed. An image analysis workflow was developed based on fine-tuned Cellpose models for cell segmentation and classification. T_C and CD57+ subsets of T cells were enriched in tumor-rich compared to tumor-sparse regions. Tumor-sparse regions had a higher expression of several key immune suppressive proteins, tentatively controlling T-cell expansion in regions close to the tumor. We revealed that T cells in late differentiation stages (CD57+) are enriched among MCL infiltrating T cells and are predictive of an increased expression of immune suppressive markers. CD47, IDO1 and CTLA-4 were identified as potential targets for patients with T-cell-rich MCL TIME, while GITR might be a feasible target for MCL patients with sparse T-cell infiltration. In subgroups of patients with a high degree of CD57+ T_C-cell infiltration, several immune checkpoint inhibitors, including TIGIT, PD-L1 and LAG3 were increased, emphasizing the immune-suppressive features of this highly differentiated T-cell subset not previously described in MCL. Full article

Regulatory T cells (Tregs) are essential to maintain immune homeostasis by promoting self-tolerance. Reduced Treg numbers or functionality can lead to a loss of tolerance, increasing the risk of developing autoimmune diseases. An overwhelming variety of human Tregs has been described, based on either specific phenotype, tissue compartment, or pathological condition, yet the bulk of the literature only addresses CD25-positive and CD127-negative cells, coined by naturally occurring Tregs (nTregs), most of which express the transcription factor Forkhead box protein 3 (FOXP3). While the discovery of FOXP3 was seminal to understanding the origin and biology of nTregs, there is evidence in humans that not all T cells expressing FOXP3 are regulatory, and that not all Tregs express FOXP3. Namely, the activation of human T cells induces the transient expression of FOXP3, irrespective of whether they are regulatory or inflammatory effectors, while some induced T cells that may be broadly defined as Tregs (e.g., Tr1 cells) typically lack demethylation and do not express FOXP3. Furthermore, it is unknown whether and how many nTregs exist without FOXP3 expression. Several other candidate regulatory molecules, such as GITR, Lag-3, GARP, GPA33, Helios, and Neuropilin, have been identified but subsequently discarded as Treg-specific markers. Multiparametric analyses have uncovered a plethora of Treg phenotypes, and neither single markers nor combinations thereof can define all and only Tregs. To date, only the functional capacity to inhibit immune responses defines a Treg and distinguishes Tregs from inflammatory T cells (Teffs) in humans. This review revisits current knowledge of the Treg universe with respect to their heterogeneity in phenotype and function. We propose that it is unavoidable to characterize human Tregs by their phenotype in combination with their function, since phenotype alone does not unambiguously define Tregs. There is an unmet need to align the expression of specific markers or combinations thereof with a particular suppressive function to coin functional Treg entities and categorize Treg diversity. Full article

Background: The immune checkpoint blockade remains obscure in osteosarcoma (OS). We aim to explore the clinical significance of soluble immune checkpoint (ICK)-related proteins in OS. Methods: We profiled 14 soluble ICK-related proteins (BTLA, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, CD28, CD80, CD137, CD27, and CTLA-4) in the plasma of 76 OS patients and matched controls. We evaluated the associations between the biomarkers and the risk of OS using unconditional multivariate logistic regression. The multivariate Cox model was utilized to develop the prediction model of OS. Immune subtypes were established from the identified biomarkers. Transcriptional data from GEO were analyzed to elucidate potential mechanisms. Results: We found that sTIM3, sCD137, sIDO, and sCTLA4 were significantly correlated with OS risk (all p < 0.05). sBTLA, sPDL2, and sCD27 were significantly associated with the risk of lung metastasis, whereas sBTLA and sTIM3 were associated with the risk of disease progression. We also established an immune subtype based on sBTLA, sPD1, sTIM3, and sPDL2. Patients in the sICK-type2 subtype had significantly decreased progression-free survival (PFS) and lung metastasis-free survival (LMFS) than those in the sICK-type1 subtype (log-rank p = 2.8 × 10⁻², 1.7 × 10⁻², respectively). Interestingly, we found that the trend of LMFS and PFS in the subtypes of corresponding ICK genes’ expression was opposite to the results in the blood (log-rank p = 2.6 × 10⁻⁴, 9.5 × 10⁻⁴, respectively). Conclusion: Four soluble ICK-related proteins were associated with the survival of OS patients. Soluble ICK-related proteins could be promising biomarkers for the outcomes and immunotherapy of OS patients, though more research is warranted. Full article

The success of cancer immunotherapy is largely associated with immunologically hot tumors. Approaches that promote the infiltration of immune cells into tumor beds are urgently needed to transform cold tumors into hot tumors. Oncolytic viruses can transform the tumor microenvironment (TME), resulting in immunologically hot tumors. Cytokines are good candidates for arming oncolytic viruses to enhance their function in this transformation. Here, we used the oncolytic vaccinia virus (oVV) to deliver interleukin-9 (IL-9) into the tumor bed and explored its antitumor effects in colon and lung tumor models. Our data show that IL-9 prolongs viral persistence, which is probably mediated by the up-regulation of IL-10. The vvDD-IL-9 treatment elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, granzyme B, and perforin. IL-9 expression increased the percentages of CD4⁺ and CD8⁺ T cells in the TME and decreased the percentage of oVV-induced immune suppressive myeloid-derived suppressor cells (MDSC), leading to potent antitumor effects compared with parental virus treatment. The vvDD-IL-9 treatment also increased the percentage of regulatory T cells (Tregs) in the TME and elevated the expression of immune checkpoint molecules such as PD-1, PD-L1, and CTLA-4, but not GITR. The combination therapy of vvDD-IL-9 and the anti-CTLA-4 antibody, but not the anti-GITR antibody, induced systemic tumor-specific antitumor immunity and significantly extended the overall survival of mice, indicating a potential translation of the IL-9-expressing oncolytic virus into a clinical trial to enhance the antitumor effects elicited by an immune checkpoint blockade for cancer immunotherapy. Full article

The hormonally active vitamin D₃ metabolite, calcitriol, functions as an important modulator of the immune system. We assumed that calcitriol exerts different effects on immune cells and cytokine production, depending on the age of the animal; therefore, we analyzed its effects on regulatory T lymphocytes and regulatory B lymphocytes in healthy young and old female C57Bl/6/Foxp3GFP mice. In the lymph nodes of young mice, calcitriol decreased the percentage of Tregs, including tTregs and pTregs, and the expression of GITR, CD103, and CD101; however, calcitriol increased the level of IL-35 in adipose tissue. In the case of aged mice, calcitriol decreased the percentages of tTregs and CD19+ cells in lymph nodes and the level of osteopontin in the plasma. Additionally, increases in the levels of IgG and the lowest levels of IFN-γ, IL-10, and IL-35 were observed in the adipose tissue of aged mice. This study showed that calcitriol treatment had different effects, mainly on Treg phenotypes and cytokine secretion, in young and old female mice; it seemed that calcitriol enhanced the immunosuppressive properties of the lymphatic organs and adipose tissue of healthy young mice but not of healthy aged mice, where the opposite effects were observed. Full article

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer’s resilient defenses. Full article

The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αβ T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of ‘off-the-shelf’ CAR-T cell products for successful allogeneic adoptive immunotherapy. Full article

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc−scOX40L, Fc−sc4-1BBL, Fc−scCD27L (CD70) and Fc−scGITRL. Without intentional cross-linking, these soluble Fc−scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4⁺ and CD8⁺ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc−scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc−scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins. Full article

Identifying patients’ immune system status has become critical to managing SARS-CoV-2 infection and avoiding the appearance of secondary infections during a hospital stay. Despite the high volume of research, robust severity and outcome markers are still lacking in COVID-19. We recruited 87 COVID-19 patients and analyzed, by unbiased automated software, 356 parameters at baseline emergency department admission including: high depth immune phenotyping and immune checkpoint expression by spectral flow cytometry, cytokines and other soluble molecules in plasma as well as routine clinical variables. We identified 69 baseline alterations in the expression of immune checkpoints, Ig-like V type receptors and other immune population markers associated with severity (O₂ requirement). Thirty-four changes in these markers/populations were associated with secondary infection appearance. In addition, through a longitudinal sample collection, we described the changes which take place in the immune system of COVID-19 patients during secondary infections and in response to corticosteroid treatment. Our study provides information about immune checkpoint molecules and other less-studied receptors with Ig-like V-type domains such as CD108, CD226, HVEM (CD270), B7H3 (CD276), B7H5 (VISTA) and GITR (CD357), defining these as novel interesting molecules in severe and corticosteroids-treated acute infections. Full article

Regulatory T cells (Tregs) control immune system activity and inhibit inflammation. While, in mice, short-chain fatty acids (SCFAs) are known to be essential regulators of naturally occurring and in vitro induced Tregs (iTregs), data on their contribution to the development of human iTregs are sparse, with no reports of the successful SCFAs-augmented in vitro generation of fully functional human iTregs. Likewise, markers undoubtedly defining human iTregs are missing. Here, we aimed to generate fully functional human iTregs in vitro using protocols involving SCFAs and to characterize the underlying mechanism. Our target was to identify the potential phenotypic markers best characterizing human iTregs. Naïve non-Treg CD4⁺ cells were isolated from the peripheral blood of 13 healthy adults and cord blood of 12 healthy term newborns. Cells were subjected to differentiation toward iTregs using a transforming growth factor β (TGF-β)-based protocol, with or without SCFAs (acetate, butyrate, or propionate). Thereafter, they were subjected to flow cytometric phenotyping or a suppression assay. During differentiation, cells were collected for chromatin-immunoprecipitation (ChIP)-based analysis of histone acetylation. The enrichment of the TGF-β-based protocol with butyrate or propionate potentiated the in vitro differentiation of human naïve CD4⁺ non-Tregs towards iTregs and augmented the suppressive capacity of the latter. These seemed to be at least partly underlain by the effects of SCFAs on the histone acetylation levels in differentiating cells. GITR, ICOS, CD39, PD-1, and PD-L1 were proven to be potential markers of human iTregs. Our results might boost the further development of Treg-based therapies against autoimmune, allergic and other chronic inflammatory disorders. Full article

Altered regulatory T cell (Treg) function could contribute to MS. The expression of activating and inhibitory receptors influences the activity of Tregs. Our aim was to investigate T cell phenotypes in relapsing–remitting MS (RRMS) patients at an early phase of the disease. We examined the influence of demographic parameters on the distribution of CD4+ and CD8+ T cell subclasses by generalized linear modeling. We also studied the expression of the following markers—CTLA-4, GITR, PD-1, FoxP3, Helios, CD28, CD62L, CD103—on T cell subsets from peripheral blood with a 14-color flow cytometry panel. We used an antibody array to define the profiles of 34 Th1/Th2/Th17 cytokines in the serum. Expression of PD-1 and GITR on CD4+ and CD8+ Tregs was decreased in RRMS patients. The proinflammatory factors IFN-γ, IL-17, IL-17F, TGFβ-1, TGFβ-3, IL-1SRII, IL-12 p40, sgp130, IL-6sR were significantly increased in RRMS patients. Therefore, a deficiency of PD-1 and GITR immune checkpoints on CD4+ and CD8+ Tregs is a feature of RRMS and might underlie impaired T cell control. Full article

The Tumor Necrosis Factor Receptor Superfamily (TNFRSF) is a large and important immunoregulatory family that provides crucial co-stimulatory signals to many if not all immune effector cells. Each co-stimulatory TNFRSF member has a distinct expression profile and a unique functional impact on various types of cells and at different stages of the immune response. Correspondingly, exploiting TNFRSF-mediated signaling for cancer immunotherapy has been a major field of interest, with various therapeutic TNFRSF-exploiting anti-cancer approaches such as 4-1BB and CD27 agonistic antibodies being evaluated (pre)clinically. A further application of TNFRSF signaling is the incorporation of the intracellular co-stimulatory domain of a TNFRSF into so-called Chimeric Antigen Receptor (CAR) constructs for CAR-T cell therapy, the most prominent example of which is the 4-1BB co-stimulatory domain included in the clinically approved product Kymriah. In fact, CAR-T cell function can be clearly influenced by the unique co-stimulatory features of members of the TNFRSF. Here, we review a select group of TNFRSF members (4-1BB, OX40, CD27, CD40, HVEM, and GITR) that have gained prominence as co-stimulatory domains in CAR-T cell therapy and illustrate the unique features that each confers to CAR-T cells. Full article

The prognosis of patients with advanced cutaneous melanoma has radically changed in the past decade. Nevertheless, primary or acquired resistance to systemic treatment occurs in many cases, highlighting the need for novel treatment strategies. This review has the purpose of summarizing the current area of interest for the treatment of metastatic or unresectable advanced cutaneous melanoma, including data from recently completed or ongoing clinical trials. The main fields of investigation include the identification of new immune checkpoint inhibitors (anti-LAG3, GITR agonist and anti-TIGIT), adoptive cell therapy, vaccines, engineered TCR therapy, IL-2 agonists, novel targets for targeted therapy (new MEK or RAF inhibitors, HDAC, IDO, ERK, Axl, ATR and PARP inhibitors), or combination strategies (antiangiogenetic agents plus immune checkpoint inhibitors, intra-tumoral immunotherapy in combination with systemic therapy). In many cases, only preliminary efficacy data from early phase trials are available, which require confirmation in larger patient cohorts. A more in-depth knowledge of the biological effects of the molecules and identifying predictive biomarkers remain crucial for selecting patient populations most likely to benefit from novel emerging treatment strategies. Full article