Effector Cell Subsets Research Articles

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

Uveal melanoma (UM) is a rare melanoma originating in the eye’s uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases.

Uveal melanoma (UM) is a rare melanoma originating in the eye's uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Harnessing Immune Cell Metabolism to Modulate Alloresponse in Transplantation.

Immune cell metabolism plays a pivotal role in shaping and modulating immune responses. The metabolic state of immune cells influences their development, activation, differentiation, and overall function, impacting both innate and adaptive immunity. While glycolysis is crucial for activation and effector function of CD8 T cells, regulatory T cells mainly use oxidative phosphorylation and fatty acid oxidation, highlighting how different metabolic programs shape immune cells. Modification of cell metabolism may provide new therapeutic approaches to prevent rejection and avoid immunosuppressive toxicities. In particular, the distinct metabolic patterns of effector and suppressive cell subsets offer promising opportunities to target metabolic pathways that influence immune responses and graft outcomes. Herein, we review the main metabolic pathways used by immune cells, the techniques available to assay immune metabolism, and evidence supporting the possibility of shifting the immune response towards a tolerogenic profile by modifying energetic metabolism.

Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation

While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required forTreg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.

Development of Innate-Immune-Cell-Based Immunotherapy for Adult T-Cell Leukemia-Lymphoma.

γδ T cells and natural killer (NK) cells have attracted much attention as promising effector cell subsets for adoptive transfer for use in the treatment of malignant and infectious diseases, because they exhibit potent cytotoxic activity against a variety of malignant tumors, as well as virus-infected cells, in a major histocompatibility complex (MHC)-unrestricted manner. In addition, γδ T cells and NK cells express a high level of CD16, a receptor required for antibody-dependent cellular cytotoxicity. Adult T-cell leukemia-lymphoma (ATL) is caused by human T-lymphotropic virus type I (HTLV-1) and is characterized by the proliferation of malignant peripheral CD4+ T cells. Although several treatments, such as chemotherapy, monoclonal antibodies, and allogeneic hematopoietic stem cell transplantation, are currently available, their efficacy is limited. In order to develop alternative therapeutic modalities, we considered the possibility of infusion therapy harnessing γδ T cells and NK cells expanded using a novel nitrogen-containing bisphosphonate prodrug (PTA) and interleukin (IL)-2/IL-18, and we examined the efficacy of the cell-based therapy for ATL in vitro. Peripheral blood samples were collected from 55 patients with ATL and peripheral blood mononuclear cells (PBMCs) were stimulated with PTA and IL-2/IL-18 for 11 days to expand γδ T cells and NK cells. To expand NK cells alone, CD3+ T-cell-depleted PBMCs were cultured with IL-2/IL-18 for 10 days. Subsequently, the expanded cells were examined for cytotoxicity against ATL cell lines in vitro. The proportion of γδ T cells in PBMCs was markedly low in elderly ATL patients. The median expansion rate of the γδ T cells was 1998-fold, and it was 12-fold for the NK cells, indicating that γδ T cells derived from ATL patients were efficiently expanded ex vivo, irrespective of aging and HTLV-1 infection status. Anti-CCR4 antibodies enhanced the cytotoxic activity of the γδ T cells and NK cells against HTLV-1-infected CCR4-expressing CD4+ T cells in an antibody concentration-dependent manner. Taken together, the adoptive transfer of γδ T cells and NK cells expanded with PTA/IL-2/IL-18 is a promising alternative therapy for ATL.

Cryopreservation Changes the Immune Effector Cell Composition of Peripheral Blood Stem Cell Grafts: An Analysis from the DKMS and NMDP Graft Composition Study

The impact of the cellular composition of PBSC grafts on the immune reconstitution as well as graft-versus-host and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (alloHCT) is only poorly understood. DKMS and NMDP joined forces to comprehensively characterize the cellular composition of 2,000 PBSC products using a 34-color flow cytometry panel to define immune effector cell subsets of interest and hematopoietic stem cells. In parallel, we have created a biobank of unstimulated donor PBMC and PBSC graft samples for future analyses. As part of this larger effort, we investigated the effect of cryopreservation on the cellular composition of PBSC products. Cryopreservation of PBSC grafts was used frequently during the COVID19-pandemic and facilitated more flexible timelines for transplantation. Still, one third of the US patients and 15% of the German patients continue to receive cryopreserved PBSC grafts. Ongoing studies are investigating the impact of cryopreservation on immune-effector cell function and the outcome of hematopoietic stem cell transplantation. Several observational studies have reported delayed neutrophil and platelet engraftment with cryopreserved products. More recently, less chronic GVHD was reported for patients who had received cryopreserved PBSC products. Cryopreservation affects the cellular composition and hence has the potential to change immune effector cell functions of the graft, but available data on the alterations of cell population frequencies are scarce. We analyzed the cellular composition of 20 fresh grafts and compared them to their frozen and thawed counterparts. Cryopreservation of product samples followed routine clinical procedures using a 10% DMSO containing cryo-medium and a controlled rate freezing. After thawing at 37°C, cells were washed, stained with a 34-parameter phenotyping panel, fixed and analyzed immediately on a full spectrum flow cytometer. Following spectral unmixing and manual spillover correction, proportions of immune cells were assessed using manual gating. First, we investigated the relative frequencies of 10 non-overlapping individual cell populations relative to viable CD45+ cells, including several lymphocyte populations, monocytes, granulocytes, hematopoietic stem cells (HSC) and dendritic cells. Granulocytes showed the largest decrease after cryopreservation and thawing (18.2 % vs 3.5 %; p<0.001). The loss of granulocytes results in an increase of relative frequencies of many other immune cell populations including monocytes (35.4 % vs 44.7 %; p=0.018), B cells (5.8 % vs 9.6 %; p=0.006), and HSC (1.0 % vs 1.5 %; p<0.001). We therefore also calculated changes relative to the viable non-granulocyte cells. By doing so, a significant relative increase of cells was observed for HSC (1.2 % vs 1.6 %; p<0.001), whereas monocyte and B cell frequencies changed only marginally. This suggests that HSC are more resilient to freezing than other cell types. When comparing the proportions of 40 other immune cell (sub-) populations relative to their parent population in the gating hierarchy, we found a profound and significant decrease of the relative frequencies for CD16+ myeloid dendritic cells (mDCs, 68 % vs 30 % of all mDCs; p<0.001) and granulocytic myeloid derived suppressor cells (gMDSCs, 2 % vs 1 % of all MDSCs; p<0.001). In an independent set of analyses performed at Roswell Park Comprehensive Cancer Center, Buffalo, comparable results were obtained with a significant increase of HSC, a significant decrease of CD16+ mDCs, and a trend of a decrease of gMDSCs. As CD16+ mDCs may have a role in the development of chronic GVHD after alloHCT, this decrease may be meaningful. In summary, we found that cryopreservation of PBSC allografts resulted in a decreased relative frequency of granulocytes, and CD16+ mDCs, as well as an increased relative frequency of HSCs. The results provide some insight into the impact of cryopreservation on PBSC graft composition and warrant further efforts to explore the potential mechanisms for the differential outcomes observed using fresh vs cryopreserved PBSCs.

Targeting GD2 after allogeneic SCT: effector cell composition defines the optimal use of ch14.18 and the bispecific antibody construct NG-CU (GD2-CD3).

We investigated whether T cell-recruiting bispecific anti-CD3/GD2 antibody NG-CU might be an alternative to therapeutic anti-GD2 monoclonal antibody (mAb) ch14.18, mediating complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) through natural killer (NK) cells for immunotherapy in high-risk/relapsed neuroblastoma after autologous/allogeneic stem cell transplantation (auto/alloSCT). Different antibody concentrations and effector-to-target ratios (E:T) were evaluated using xCELLigence RTCA system, peripheral blood mononuclear cells (PBMCs) (healthy donors and patients after alloSCT), and neuroblastoma cell lines (LS/LAN-1). Mean specific lysis of LS cells utilizing PBMCs from healthy donors and ch14.18 (1µg/ml) was 40/66/75% after 12/24/48 h compared to 66/93/100% in the presence of NG-CU (100 ng/ml). NG-CU showed enhanced cytotoxicity compared to ch14.18, even at lower concentrations and E:T ratios, and completely eradicated LS cells after 72 h. To decipher the influence of effector cell subsets on lysis, different ratios of T and NK cells were tested. At a ratio of 1:1, ch14.18 was more effective than NG-CU. Using patient PBMCs taken at different time points posttransplant, significant lysis with both constructs was detectable depending on percentages and total numbers of T and NK cells; in the early posttransplant phase, NK cells were predominant and ch14.18 was superior, whereas later on, T cells represented the majority of immune cells and NG-CU was more effective. Our study highlights the importance of analyzing effector cell subsets in patients before initiating antibody-based therapy. Consequently, we propose an adjusted administration of both antibody constructs, considering the state of posttransplant immune recovery, to optimize anti-tumor activity.

Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates

CD8+ Tcells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ Tcells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ Tcells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.

The clinical and experimental treatment of Juvenile Idiopathic Arthritis.

Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children and comprises of multiple subtypes. The most relevant disease subtypes, grouped upon current insight in disease mechanisms, are nonsystemic (oligo- and polyarticular) JIA and systemic JIA (sJIA). In this review, we summarize some of the main proposed mechanisms of disease in both nonsystemic and sJIA and discuss how current therapeutic modalities target some of the pathogenic immune pathways. Chronic inflammation in nonsystemic JIA is the result of a complex interplay between effector and regulatory immune cell subsets, with adaptive immune cells, specifically T-cell subsets and antigen-presenting cells, in a central role. There is, however, also innate immune cell contribution. SJIA is nowadays recognized as an acquired chronic inflammatory disorder with striking autoinflammatory features in the first phase of the disease. Some sJIA patients develop a refractory disease course, with indications for involvement of adaptive immune pathways as well. Currently, therapeutic strategies are directed at suppressing effector mechanisms in both non-systemic and sJIA. These strategies are often not yet optimally tuned nor timed to the known active mechanisms of disease in individual patients in both non-systemic and sJIA. We discuss current treatment strategies in JIA, specifically the 'Step-up' and 'Treat to Target approach' and explore how increased insight into the biology of disease may translate into future more targeted strategies for this chronic inflammatory disease at relevant time points: preclinical disease, active disease, and clinically inactive disease.

Role of MHC class I pathways in Mycobacterium tuberculosis antigen presentation.

MHC class I antigen processing is an underappreciated area of nonviral host-pathogen interactions, bridging both immunology and cell biology, where the pathogen's natural life cycle involves little presence in the cytoplasm. The effective response to MHC-I foreign antigen presentation is not only cell death but also phenotypic changes in other cells and stimulation of the memory cells ready for the next antigen reoccurrence. This review looks at the MHC-I antigen processing pathway and potential alternative sources of the antigens, focusing on Mycobacterium tuberculosis (Mtb) as an intracellular pathogen that co-evolved with humans and developed an array of decoy strategies to survive in a hostile environment by manipulating host immunity to its own advantage. As that happens via the selective antigen presentation process, reinforcement of the effective antigen recognition on MHC-I molecules may stimulate subsets of effector cells that act earlier and more locally. Vaccines against tuberculosis (TB) could potentially eliminate this disease, yet their development has been slow, and success is limited in the context of this global disease's spread. This review's conclusions set out potential directions for MHC-I-focused approaches for the next generation of vaccines.

Human natural killer cells: Form, function, and development

Human natural killer (NK) cells are innate lymphoid cells that mediate important effector functions in the control of viral infection and malignancy. Their ability to distinguish "self" from "nonself" and lyse virally infected and tumorigenic cells through germline-encoded receptors makes them important players in maintaining human health and a powerful tool for immunotherapeutic applications and fighting disease. This review introduces our current understanding of NK cell biology, including key facets of NK cell differentiation and the acquisition and execution of NK cell effector function. Further, it addresses the clinical relevance of NK cells in both primary immunodeficiency and immunotherapy. It is intended to provide an up-to-date and comprehensive overview of this important and interesting innate immune effector cell subset.

The Role of Neutrophils in Oncolytic Orf Virus-Mediated Cancer Immunotherapy.

Neutrophils are innate leukocytes with diverse effector functions that allow them to respond to pathogens rapidly. Accumulating evidence has highlighted these cells’ complex roles in the host’s response to viral infections and tumor progression. Oncolytic virotherapy is emerging as a promising treatment modality in the armamentarium of cancer therapeutics. Oncolytic viruses preferentially kill cancer cells and stimulate tumor-associated inflammation, resulting in tumor regression. Assessing the activity of individual effector cell subsets following oncolytic virotherapy is important in identifying their contribution to antitumor immunity. In this study, we investigated the role of neutrophils in oncolytic Orf-virus-mediated immunotherapy in a murine model of pulmonary melanoma metastases. The systemic administration of the Orf virus stimulated a dramatic increase in the number of leukocytes in circulation and within the tumor microenvironment, most of which were neutrophils. Analysis of tumor-burdened lungs shortly after therapy revealed significant numbers of phenotypically immature neutrophils, with the enhanced expression of molecules affiliated with activation, migration, and cytotoxicity. Neutrophils stimulated by Orf virus therapy were directly tumoricidal through tumor necrosis factor-α-mediated effects and were required for optimal antitumor efficacy following Orf virus therapy. Taken together, these data reveal neutrophils as a crucial innate effector to consider when investigating oncolytic virotherapy.

Enhanced development of functional human NK cells in NOD-scid-IL2rgnull mice expressing human IL15.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.

Immunity parameters in adults with measles compared with healthy persons

Measles is a highly contagious viral infection transmitted by airborne droplets, characterized by fever, intoxication and specific rashes on the skin and mucous membranes. Despite the availability of highly effective vaccines and many years of efforts by the world medical community with active immunization of the worlds population against this infection under the auspices of WHO, measles still remains a serious problem. The aim of this work was to investigate the effect of measles infection in adults upon the wide range of lymphocyte subsets and blood cytokine profile in comparison with healthy controls.
 The venous blood samples from 50 adult measles patients aged 20 to 55 years, were taken 61 days after the onset of skin rash, being compared with blood samples from 50 healthy adults of similar age group. The 200 L plasma aliquotes resulting from spontaneous sedimentation of the formed elements in an Eppendorf tube were taken, frozen at -30 C and used within 3 months for the cytokine profile assays. 15 cytokines were tested by multiplex technique (MagPix, BioRad, USA). Mononuclear cells were isolated by gradient centrifugation and immunophenotyped using four-color staining by means of equipment and reagents from BD Biosciences (USA).
 In the group of measles patients, activation of innate immunity was revealed, i.e., the IL-1, IL-6, IL-23, IL-31 cytokines and TNF, which belong to early pro-inflammatory cytokines, were significantly increased. In measles patients, a significant increase in cytokines was found, suggesting active participation of epithelial cells in immune response to the measles virus. They produce danger signals (IL-25 and IL-33), inducing the development of adaptive immunity, activate their protective abilities via IL-17F production, and are involved in repair under the influence of IL-22. Some cells of adaptive immunity are infected with the measles virus and die, others actively respond to the viral infection and proliferate, thus leading to changing ratio of their subsets. Hence, the patients showed a significant decrease in T lymphocytes due to a decrease in CD4+ cells, an increased percentage of cells in senescent and exhaustion state, a significant decrease in TEMRO subpopulations, both among CD4+ and CD8+ lymphocytes, and an increase in CD8+TCM. The levels of B cell subpopulations (Bm, B1, Breg) in measles patients did not differ from healthy ones, and the level of plasmablasts was significantly increased. The level of CD4+ lymphocyte subpopulations and production of their cytokine markers varied greatly. In the patient group, a shift in the type of immune response towards Th2 and Th17 was found, activation of Tfh and Treg was detected, and increased expression of HLA-DR and CD38 activation markers was found.
 In response to measles infection, there are several independent, multidirectional processes observed in the patients. On the one hand, the measles virus attacks epithelial cells of mucous membranes and skin and immunocompetent cells, exerting a cytopathic effect and leading to lymphopenia and selective decrease in various lymphocyte subsets. On the other hand, the measles virus initiates activation of both innate and adaptive immunity, thus causing production of the corresponding cytokines, expression of activation markers, and an increase in effector cell subsets. Better understanding of the immunopathogenesis of measles infection and associated immunosuppression will help us to improve vaccination outcomes against this infection and prevent measles-related mortality.

THEMIS enhances the magnitude of normal and neuroinflammatory type 1 immune responses by promoting TCR-independent signals.

Signals that determine the differentiation of naïve CD4+ T helper (TH) cells into specific effector cell subsets are primarily stimulated by cytokines, but additional signals are required to adjust the magnitude of TH cell responses and set the balance between effective immunity and immunological tolerance. By inducing the post-thymic deletion of the T cell lineage signaling protein THEMIS, we showed that THEMIS promoted the development of optimal type 1 immune responses to foreign antigens but stimulated signals that favored encephalitogenic responses to self-neuroantigens. THEMIS was required to stimulate the expression of the gene encoding the transcriptional regulator T-BET and the production of the cytokine interferon-γ (IFN-γ), and it enhanced the ability of encephalitogenic CD4+ T cells to migrate into the central nervous system. Consistently, analysis of THEMIS expression in polarized CD4+ T cells showed that THEMIS was selectively increased in abundance in TH1 cells. The stimulation of predifferentiated effector CD4+ T cells with antigen-presenting cells revealed a stimulatory function for THEMIS on type 1 cytokine responses, similar to those observed ex vivo after immunization. In contrast, THEMIS exerted opposing effects on naïve CD4+ T cells in vitro by inhibiting the T cell receptor (TCR)-mediated signals that lead to TH1 cell responses. These data suggest that THEMIS exerts TCR-independent functions in effector T cells, which increase the magnitude of normal and pathogenic TH1 cell-mediated responses.

Total glucosides of paeony inhibit breast cancer growth by inhibiting TAMs infiltration through NF-κB/CCL2 signaling

PurposeThe high density of tumor-associated macrophages (TAMs) and inflammatory factors are crucial elements leading to tumor immune tolerance. Previously, we found that total glucosides of paeony (TGP) have strong inhibitory effects on the release of various inflammatory factors; however, it is unclear whether the inhibitory effects can improve the inflammatory microenvironment of tumors. Therefore, in the present study, we investigated the mechanism via which TGP depresses tumor growth and metastasis via modulation of TAM infiltration in breast cancer. MethodsWe assessed the effects of TGP on various mouse models of tumor. Lung metastasis was detected using hematoxylin and eosin staining. T cell (CD3+CD4+ and CD3+CD8+) effector and memory subsets, and TAM (CD45+CD11b+F4/80+) populations in the tumor microenvironment (TME) were examined using flow cytometry. Lipopolysaccharide (LPS)-stimulated macrophage experiments were used to investigate the TGP anti-inflammatory effects in vitro. Furthermore, conditional medium (CM) was added to detect 4T1 breast cancer cell growth using a Real-Time Cell Analyzer (RTCA) xCELLigence system. Inflammatory cytokine and chemokine levels were measured using cytometric bead array (CBA) kits and quantitative polymerase chain reaction (qPCR). NF-κB expression in the nucleus was examined by immunofluorescence and Western blot analysis. ResultsTGP suppressed tumor growth and lung metastasis, decreased CD45+CD11b+F4/80+ (TAMs) population obviously, and increased CD44LowCD62LHi (T memory stem cells) and CD44HiCD62LHi (central memory cells) populations in the tumor-infiltrating CD4+ and CD8+ T cells. In addition, TGP reduced inflammatory factor levels in tumors, thus inhibiting the infiltration of TAMs to improve the inflammation immunosuppressive microenvironment. In the in vitro experiment, TGP inhibited IL-10 and C-C Motif Chemokine Ligand 2 (CCL2) secretion and mRNA expression in LPS-stimulated macrophages to inhibit 4T1 cell growth and restrain macrophages M2 polarization. In addition, TGP can directly inhibit 4T1 cell proliferation by restraining autocrine CCL2 and IL-10. Further mechanistic studies reavealed that TGP inhibited CCL2 secretion by inhibiting NF-κB accumulation in the nucleus in macrophages. ConclusionTGP reduced TAM recruitment mainly through the NF-κB/CCL2 signaling pathway, thereby promoting T cell infiltration in the TME. TGP has a unique advantage in balancing the inflammatory response. Furthermore, our results present novel insights on the mechanisms underlying TAM infiltration that were inhibited by TGP, with potential application in development of novel therapies targeting CCL2 pathways.

Role of CNSs Conserved Distal Cis-Regulatory Elements in CD4 + T Cell Development and Differentiation.

Naïve CD4+ T cells differentiate into diverse subsets of effector cells and perform various homeostatic and immune functions. The differentiation and maintenance of these different subsets are controlled through the upregulation and silencing of master genes. Mechanistic studies of the regulation of these master genes identified conserved and distal intronic regulatory elements, which are accessible subsets of conserved non-coding sequences (CNSs), acting as cis-regulatory elements in a lineage-specific manner that controls the function of CD4+ T cells. Abnormal CNS activity is associated with incorrect expression of master genes and development of autoimmune diseases or immune suppression. Here, we describe the function of several conserved, distal cis-regulatory elements at the Foxp3, Rorc, Il-4, Il-10 and Il-17 gene locus were shown to play important roles in CD4+ T cells differentiation. Together, this review briefly outlines currently known CNSs, with a focus on their regulations and functions in complexes modulating the differentiation and maintenance of various CD4+ T cells subsets, in health and disease contexts, as well as during the conversion of T regulatory cells to T helper 17 cells. This article will provide a comprehensive view of CNSs conserved distal cis-regulatory elements at a few loci that control aspects of CD4+ T cells function.

A phase 2 study of anti-PD-L1 antibody (atezolizumab) in grade 2 and 3 chondrosarcoma.

11528 Background: Chondrosarcoma is one of the most common bone malignancies in adults, and the third most common in pediatric patients (pts). The most prevalent subtype, conventional chondrosarcoma, is a slow growing tumor that is historically known to be refractory to chemotherapy. Anecdotal reports indicated a role for anti-PD-(L)1 in the treatment of this disease. This is the first prospective report on the efficacy of the PD-L1-targeting agent, atezolizumab, in this rare disease. Methods: Patients (pts) ages 2 and older with unresectable grade 2 or 3 conventional chondrosarcoma were eligible. No prior anti-PD-(L)1 treatment was allowed, otherwise pts were eligible irrespective of prior therapies as long as protocol-specified washout period requirements were met. Pts received atezolizumab 1200 mg (15 mg/kg with 1200 mg cap in pediatric pts) once every 21 days. Imaging was carried out at end of cycle 3, and then every two cycles. Research biopsies were collected from adult pts prior to C1D1, prior to C3D1, and at progression. Immuno-pharmacodynamic (IO-PD) studies were performed on paired tumor samples and circulating immune cells to help elucidate signaling pathways mediating the immune response, with focus on subsets of effector cells in the tumor microenvironment. Results: A total of 9 pts (7 males, 2 females) were enrolled in 6 centers across the US and Canada. Six pts were Caucasian/White, 1 Asian, 1 Hispanic, and 1 unknown. Median age was 49 years (42-72). No objective responses were seen. Three pts (33%) experienced disease stability (SD) per RECIST 1.1, for a median duration of 21 weeks as of data cutoff (January 2022). A patient with SD remains on active treatment (tx) for 35 weeks. Three patients had no tx-related adverse events (AEs). Six pts (67%) experienced at least one tx-related AE. Two patients experienced > G2 AEs, but only one was considered tx-related (lymphopenia). Immune-related AEs were all G1/2 and included hepatitis (2), hypothyroidism (1), hyperthyroidism (1), and maculopapular rash (1). IO-PD studies are ongoing and will be reported at the conference if available. Conclusions: Atezolizumab was well-tolerated but demonstrated limited activity in this cohort of pts with few treatment options. Ongoing IO-PD studies will provide insight into atezolizumab’s effect upon immune cell content and activation in the tumor microenvironment that will help design future immunotherapy trials in this disease and other sarcoma types. The study was funded by NCI Contract HHSN261201500003I. Clinical trial information: NCT04458922.

CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model

BackgroundCD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model.MethodsThe contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments.ResultsWe showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells.ConclusionsThe study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted invivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.