Cell Receptor Stimulation Research Articles

Fibroblast activation protein-targeted-4-1BB ligand agonist amplifies effector functions of intratumoral T cells in human cancer

BackgroundThe costimulatory receptor 4-1BB (CD137, TNFRSF9) plays an important role in sustaining effective T cell immune responses and is investigated as target for cancer therapy. Systemic 4-1BB directed therapies elicit...

Deficiency of STING Promotes Collagen-Specific Antibody Production and B Cell Survival in Collagen-Induced Arthritis.

The levels of interferon-alpha are high in the serum and synovial fluid of rheumatoid arthritis (RA) patients. Activation of the stimulator of type I interferon genes (STING) mediates the productions of type I interferon and promotes chronic inflammation. STING plays a significant role in autoimmune lupus mice. However, the function of STING in collagen-induced arthritis (CIA) model has never been described. This study aimed to test the function of STING in CIA. The Sting-deficient mice developed arthritis comparable to WT mice. The levels of anti-collagen antibody from Sting-deficient mice were significantly higher than the WT mice. The B cells derived from Sting-deficient mice showed better survival than WT mice in response to the B cell receptor (BCR) stimulation. Activation of STING also induced B cell death, especially in activated B cells. This study demonstrated that the inhibition of STING promotes anti-collagen antibodies and B cell survival, which suggested that STING acts as a negative regulator of B cell function in the CIA model.

Inflammescent CX3CR1+CD57+CD8+ T cells are generated and expanded by IL-15.

HIV infection is associated with an increase in the proportion of activated CD8+ memory T cells (Tmem) that express CX3CR1, but how these cells are generated and maintained in vivo is unclear. We demonstrate that increased CX3CR1 expression on CD8+ Tmem in people living with HIV (PLWH) is dependent on coinfection with human CMV, and CX3CR1+CD8+ Tmem are enriched for a putatively immunosenescent CD57+CD28- phenotype. The cytokine IL-15 promotes the phenotype, survival, and proliferation of CX3CR1+CD57+CD8+ Tmem in vitro, whereas T cell receptor stimulation leads to their death. IL-15-driven survival is dependent on STAT5 and Bcl-2 activity, and IL-15-induced proliferation requires STAT5 and mTORC1. Thus, we identify mechanistic pathways that could explain how "inflammescent" CX3CR1+CD57+ CD8+ Tmem dominate the overall memory T cell pool in CMV-seropositive PLWH and that support reevaluation of immune senescence as a nonproliferative dead end.

Novel role for gelatinases in modulating metabolic reprogramming associated with T cell receptor stimulation in CD4+ T cell

Abstract The gelatinases, MMP-2 and MMP-9, are proteolytic enzymes with diverse physiological roles. They are linked with various aspects of the immune response, such as leukocyte trafficking and cytokine activation. However, their dysregulation is associated with various pathologies such as metastasis in cancer progression and blood-brain barrier disruption in multiple sclerosis. Much of what is known about the gelatinases is in context of their extracellular activities. However, recent studies including this study suggest an intracellular role in immune cell activation. Our findings suggest, that gelatinase activity can modulate T cell bioenergetics, affecting both oxidative phosphorylation and the glycolytic pathways with a greater influence on the latter. This results in a mitigated CD4+ T cell response to TCR stimulation. Upon stimulation, CD4+ T cells lacking gelatinase activity, demonstrate a reduced capacity to be activated as determined by the expression of early activation markers, proliferation, and the production of effector cytokines. In addition, RNA-seq analysis of control and gelatinase inhibited CD4+ T cells highlight key proteins associated with both the immune system and metabolic processes as predicted by STRING. We confirm our pharmacological studies with the genetic ablation of the gelatinases and further analysis of these shRNA treated T cells using the Nanostring metabolic panel. In addition, we demonstrate in-vivo relevancy using a murine model for multiple sclerosis known to be initiated by CD4+ T cells; gelatinase inhibition resulted in reduced clinical severity. These results suggest an intracellular role for gelatinases in CD4+ T cell mediated diseases and their potential as therapeutic targets.

Transforming growth factor beta induced PD-L1 expression promotes pro-fibrotic signaling

Abstract Immune checkpoint proteins such as programmed death-ligand 1 (PD-L1) retain general immune homeostasis and self-tolerance. PD-L1 binds to programmed cell death protein 1 (PD-1) and inhibits T cell activation or protects tumor cells from T cell-mediated killing. PD-L1 has recently been shown to be up-regulated in invasive lung fibroblasts by p53 and focal adhesion kinase (FAK), any potential role of transforming growth factor-beta (TGFβ) in PD-L1 action is unknown in fibrosis. TGFβ is a mysterious protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, fibrotic disorders, and immune modulation, et al. In the current study, we showed that TGFβ induces the expression of the immunoinhibitory molecule PD-L1 in human and murine fibroblasts in a Smad2/3- and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFβ-dependent induction of extracellular matrix proteins, including collagen Ia1 (colIa1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFβ signaling, TGFβ stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T-cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.

Maturation State of HIV Infected CD4 T Cells Impacts the Inducible Response of the Latent Viral Reservoir

Abstract A structured microenvironment is necessary for proper immune cell function. While it is rarely possible to use ex vivo experimental models of human tissue (e.g. tonsil), culture of multiple immune cell types together in close contact may benefit cell survival and function. To study HIV latency in primary CD4 T cells, we use an in vitro model, which produces an infected cell hierarchy closely resembling that found in vivo: HIV frequency in effector memory (TEM) > central memory (TCM) > naïve (TN) cells. To enable mechanistic investigations, we developed a method to track CD4 cell phenotype origins following long-term culture and T cell receptor (TCR) stimulation. TN, TCM and TEM subsets were isolated by sorting and stained with live-cell tracking dyes: carboxyfluorescein (CFSE), Yellow and Violet dye, respectively. The tagged subsets were pooled with unstained CD4 cells to help preserve a healthy microenvironment. This mixture was co-cultured with autologous productively infected, TCR-activated cells to facilitate cell-to-cell HIV transmission and establishment of latency. After 3 days, dye-labeled cells were removed by sorting and rested an additional 3 days to complete provirus integration. Latent HIV reactivation by TCR stimulation was measured with HIV RNA expression by PrimeFlow analysis. Residual dye staining provided inference of initial subset origin on a single cell basis. Using this design improved viability and survival for all maturation subsets, compared to isolating subsets prior to stimulation. Results showed that HIV provirus in TN cells was the most responsive to TCR activation. In addition, our study highlights a novel approach that can easily be adapted to other rare cell subsets in biologically relevant settings.

A novel and human-specific CD46-KLF/SP interaction mediates gene expression required for successful Th1 induction

Abstract Signals mediated by autocrine activation of the human-specific complement receptor CD46 during T cell receptor (TCR) stimulation are vital to Th1 induction in human CD4+ T cells, but how exactly CD46 in a molecular level mediates this role is currently undefined. CD46 is expressed in different isoforms that can bear either one of two distinct cytoplasmic tails: CYT-1 or CYT-2. Nuclear translocation of CYT-1 is a critical requirement for the expression of genes coding for nutrient-influx-channels and mTORC1 activity that mediate metabolic adaptations needed for Th1 responses. The lack of a DNA binding domain in CD46-CYT-1 precludes it from acting directly as a transcription factor (TF) and we hence hypothesized that CYT-1 regulates gene expression via direct interaction with specific TF activator and/or repressor complexes. Indeed, CUT&RUN experiments performed using our novel antibody raised against cleaved CYT-1 identified key members of the KLF/SP TFs gene family as potential interacting partners of CD46-CYT-1. Subsequent ELISA and MST experiment confirmed strong, dose-dependent CYT-1/KLF/SP TFs interactions and also demonstrated that CYT-1 fostered KLF/SP TFs binding to appropriate DNA motifs. Genome-wide comparison of the KLF/SP TFs and CYT1 bound genes in T cells revealed their enrichment in crucial basic cell-physiological pathways. Moreover, the CUT&RUN data in conjunction with ATAC-seq analyses indicated that this novel CYT-1/KLF/SP axis may control general chromatin remodeling – a notion we are currently exploring. These data define a novel and critical human-specific pathway of gene regulation and further underpin the vital role of intracellular/autocrine complement in the regulation of normal cellular activity.

Role of C-C Motif Chemokine Ligand 20 in the Regulation of Epidermal γδ T Cells

Abstract The epidermis provides a barrier that protects against environmental damage and infection by pathogens. Epidermal T cells and keratinocytes work together to maintain skin homeostasis upon damage. During tissue repair, epidermal γδ T cells become activated to secrete cytokines, chemokines and growth factors to initiate inflammatory responses. Previous work in our lab has shown that during obesity and type 2 diabetes, Chemokine Receptor 6 (CCR6) is downregulated by murine epidermal T cells. The ligand for CCR6, C-C Motif Chemokine Ligand 20 (CCL20), is produced by keratinocytes and is responsible for cellular migration. Little is known about how CCR6 and CCL20 ligation affects epidermal γδ T cells. In this study, murine epidermal γδ T cells were harvested and stimulated in the absence or presence of CCL20 to discover how CCL20 affects activation and function of the cells. Epidermal γδ T cells that have been stimulated with anti-CD3ɛ and CCL20 downregulate their T cell receptor and upregulate CD25 showing cell activation. Interestingly, CCL20 costimulates T cell receptor stimulation to activate epidermal γδ T cells. Immunofluorescent microscopy was used to visualize the impact of CCL20 on activated cells verses non-activated cells in situ. Epidermal γδ T cells treated with CCL20 did not migrate within or out of the epidermis unlike previous studies on dermal γδ T cells. Flow cytometric analysis and Luminex assays were performed to determine which cytokines and chemokines are produced in response to CCL20. We show that CCL20 skews epidermal γδ T cells to produce pro-inflammatory cytokines and chemokines. Future studies will determine how CCL20 and CCR6 ligation affects murine models with obesity and type 2 diabetes.

The Swing of Lipids at Peroxisomes and Endolysosomes in T Cell Activation.

The immune synapse (IS) is a well-known intercellular communication platform, organized at the interphase between the antigen presenting cell (APC) and the T cell. After T cell receptor (TCR) stimulation, signaling from plasma membrane proteins and lipids is amplified by molecules and downstream pathways for full synapse formation and maintenance. This secondary signaling event relies on intracellular reorganization at the IS, involving the cytoskeleton and components of the secretory/recycling machinery, such as the Golgi apparatus and the endolysosomal system (ELS). T cell activation triggers a metabolic reprogramming that involves the synthesis of lipids, which act as signaling mediators, and an increase of mitochondrial activity. Then, this mitochondrial activity results in elevated reactive oxygen species (ROS) production that may lead to cytotoxicity. The regulation of ROS levels requires the concerted action of mitochondria and peroxisomes. In this review, we analyze this reprogramming and the signaling implications of endolysosomal, mitochondrial, peroxisomal, and lipidic systems in T cell activation.

SAXS‐enabled Macromolecular Engineering at SIBYLS

Macromolecular engineering provides novel opportunities in many fields. However, progress can be slow when structural analysis relies on low‐throughput structural techniques. Employing unique and high‐throughput X‐ray scattering (SAXS) tools, we have engineered a Self‐Assembling Protein Nanoparticle to display Antibodies (SAPNA) with application in CAR T Cell therapy. Adoptive CAR T Cell therapy is a highly effective, but extremely expensive new cancer treatment with costs in the range of ~$1 million per patient (hospital fees included). The therapy requires >100 million CAR T cells per infusion per patient. A key step in the CAR T manufacturing process is polyclonal expansion of the patient‐ or donor‐derived T cells. This is currently achieved through CD3/CD28 T cell receptor stimulation by agonist antibodies (either soluble or chemically linked to magnetic beads). We have shown our engineered SAPNA solution is highly effective relative to existing technologies at expanding CAR T cells with potential application in fighting cancer. High‐Throughput SAXS (HT‐SAXS) was an integral technique that enabled structural insights into engineered constructs. A crucial step was monitoring macromolecular changes in response to mutations and solution conditions. We also utilized Size Exclusion Chromatography SAXS (SEC‐SAXS) to assess the antibody display, while providing important information on the structural dynamics of the antibodies themselves. Herein, we detail the SAXS platform (sibyls.als.lbl.gov) we used to rapidly converge on our functional SAPNA and SAPNA’s potential application in CAR T Cell therapy.Support or Funding InformationUS DOE‐BER, Integrated Diffraction Analysis Technologies (IDAT) program. Additional support from NIGMS project ALS‐ENABLE (P30 GM124169) and a High‐End Instrumentation Grant S10OD018483.

B Cell Development and T-Dependent Antibody Response Are Regulated by p38γ and p38δ.

p38MAP kinase (MAPK) signal transduction pathways are important regulators of inflammation and the immune response; their involvement in immune cell development and function is still largely unknown. Here we analysed the role of the p38 MAPK isoforms p38γ and p38δ in B cell differentiation in bone marrow (BM) and spleen, using mice lacking p38γ and p38δ, or conditional knockout mice that lack both p38γ and p38δ specifically in the B cell compartment. We found that the B cell differentiation programme in the BM was not affected in p38γ/δ-deficient mice. Moreover, these mice had reduced numbers of peripheral B cells as well as altered marginal zone B cell differentiation in the spleen. Expression of co-stimulatory proteins and activation markers in p38γ/δ-deficient B cells are diminished in response to B cell receptor (BCR) and CD40 stimulation; p38γ and p38δ were necessary for B cell proliferation induced by BCR and CD40 but not by TLR4 signaling. Furthermore, p38γ/δ-null mice produced significantly lower antibody responses to T-dependent antigens. Our results identify unreported functions for p38γ and p38δ in B cells and in the T-dependent humoral response; and show that the combined activity of these kinases is needed for peripheral B cell differentiation and function.

Ca2+ signaling in T lymphocytes: the interplay of the endoplasmic reticulum, mitochondria, membrane potential, and CRAC channels on transcription factor activation

T cell receptor stimulation initiates a cascade of reactions that cause an increase in intracellular calcium (Ca2+) concentration mediated through inositol 1,4,5-trisphosphate (IP3). To understand the basic mechanisms by which the immune response in T cells is activated, it is useful to understand the signaling pathways that contain important targets for drugs in a quantitative fashion. A computational model helps us to understand how the selected elements in the pathways interact with each other, and which component plays the crucial role in systems. We have developed a mathematical model to explore the mechanism for controlling transcription factor activity, which regulates gene expression, by the modulation of calcium signaling triggered during T cell activation. The model simulates the activation and modulation of Ca2+ release-activated Ca2+ (CRAC) channels by mitochondrial dynamics and depletion of endoplasmic reticulum (ER) store, and also includes membrane potential in T-cells. The model simulates the experimental finding that increases in Ca2+ current enhances the activation of transcription factors and the Ca2+ influx through CRAC is also essential for the NFAT and NFκB activation. The model also suggests that plasma membrane Ca2+-ATPase (PMCA) controls a majority of the extrusion of Ca2+ and modulates the activation of CRAC channels. Furthermore, the model simulations explain how the complex interaction of the endoplasmic reticulum, membrane potential, mitochondria, and ion channels such as CRAC channels control T cell activation.

In vivo and in vitro differences in CD8+ T cell metabolism

CD8+ T cells activated in vivo display different metabolic profiles than those stimulated in vitro, underscoring the key role of the environment in dictating metabolic pathways downstream of T cell receptor stimulation.

Functions of human liver CD69+CD103-CD8+ T cells depend on HIF-2α activity in healthy and pathologic livers

Human liver CD69+CD8+ T cells are ~95% CD103- and ~5% CD103+. Although CD69+CD103+CD8+ T cells show tissue residency and robustly respond to antigens, CD69+CD103-CD8+ T cells are not yet well understood. Liver perfusate and paired peripheral blood were collected from healthy living donors and recipients with cirrhosis during liver transplantation. Liver tissues were obtained from patients with acute hepatitis A. Phenotypic and functional analyses were performed by flow cytometry. Gene expression profiles were determined by microarray and quantitative reverse transcription PCR. PT-2385 was used to inhibit hypoxia-inducible factor (HIF)-2α. Human liver CD69+CD103-CD8+ T cells exhibited HIF-2α upregulation with a phenotype of tissue residency and terminal differentiation. CD103- cells comprised non-hepatotropic virus-specific T cells as well as hepatotropic virus-specific T cells, but CD103+ cells exhibited only hepatotropic virus specificity. Although CD103- cells were weaker effectors on a per cell basis than CD103+ cells, following T cell receptor or interleukin-15 stimulation, they remained the major CD69+CD8+ effector population in the liver, surviving with less cell death. An HIF-2α inhibitor suppressed the effector functions and survival of CD69+CD103-CD8+ T cells. In addition, HIF-2α expression in liver CD69+CD103-CD8+ T cells was significantly increased in patients with acute hepatitis A or cirrhosis. Liver CD69+CD103-CD8+ T cells are tissue resident and terminally differentiated, and their effector functions depend on HIF-2α. Furthermore, activation of liver CD69+CD103-CD8+ T cells with HIF-2α upregulation is observed during liver pathology. The immunologic characteristics and the role of CD69+CD103-CD8+ T cells, which are a major population of human liver CD8+ T cells, remain unknown. Our study shows that these Tcells have a terminally differentiated tissue-resident phenotype, and their effector functions depend on a transcription factor, HIF-2α. Furthermore, these T cells were activated and expressed higher levels of HIF-2α in liver pathologies, suggesting that they play an important role in immune responses in liver tissues and the pathogenesis of human liver disease.

Lupus-associated endogenous retroviral LTR polymorphism and epigenetic imprinting promote HRES-1/RAB4 expression and mTOR activation.

Overexpression and long terminal repeat (LTR) polymorphism of the HRES‑1/Rab4 human endogenous retrovirus locus have been associated with T cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is diminished overall in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we determined how lupus-associated polymorphic rs451401 alleles of the LTR regulate transcription from the HRES-1/Rab4 promoter and thus affect T cell activation. The results showed that cytosine-119 is hypermethylated while cytosine-51 of the promoter and the LTR enhancer are hypomethylated in SLE. Pharmacologic or genetic inactivation of DNA methyltransferase 1 augmented the expression of HRES-1/Rab4. The minimal promoter was selectively recognized by metabolic stress sensor NRF1 when cytosine-119 but not cytosine-51 was methylated, and NRF1 stimulated HRES-1/Rab4 expression in human T cells. In turn, IRF2 and PSIP1 bound to the LTR enhancer and exerted control over HRES-1/Rab4 expression in rs451401 genotype- and methylation-dependent manners. The LTR enhancer conferred markedly greater expression of HRES-1/Rab4 in subjects with rs451401CC over rs451401GG alleles that in turn promoted mechanistic target of rapamycin (mTOR) activation upon T cell receptor stimulation. HRES-1/Rab4 alone robustly activated mTOR in human T cells. These findings identify HRES-1/Rab4 as a methylation- and rs451401 allele-dependent transducer of environmental stress and controller of T cell activation.

Transforming growth factor beta induces fibroblasts to express and release the immunomodulatory protein PD-L1 into extracellular vesicles.

Transforming growth factor-beta (TGFβ) is an enigmatic protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGFβ is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in various malignancies, characterizing the pathways and players mediating its action is fundamental. In the current study, we found that TGFβ induces the expression of the immunoinhibitory molecule Programed death-ligand 1 (PD-L1) in human and murine fibroblasts in a Smad2/3- and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFβ-dependent induction of extracellular matrix proteins, including collagen Iα1 (colIα1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFβ signaling, TGFβ stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.

TcellSubC: An Atlas of the Subcellular Proteome of Human T Cells.

We have curated an in-depth subcellular proteomic map of primary human CD4+ T cells, divided into cytosolic, nuclear and membrane fractions generated by an optimized fractionation and HiRIEF-LC-MS/MS workflow for limited amounts of primary cells. The subcellular proteome of T cells was mapped under steady state conditions, as well as upon 15 min and 1 h of T cell receptor (TCR) stimulation, respectively. We quantified the subcellular distribution of 6,572 proteins and identified a subset of 237 potentially translocating proteins, including both well-known examples and novel ones. Microscopic validation confirmed the localization of selected proteins with previously known and unknown localization, respectively. We further provide the data in an easy-to-use web platform to facilitate re-use, as the data can be relevant for basic research as well as for clinical exploitation of T cells as therapeutic targets.

Physiological functions of mitochondrial Na+-Ca2+ exchanger, NCLX, in lymphocytes

Roles of mitochondrial Na+-Ca2+ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca2+ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca2+ recycling via NCLX and sarco/endoplasmic reticulum Ca2+-ATPase SERCA, and interaction with store-operated Ca2+ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.

The Proportion of CD52/GPI Negative T Cells Early Following Alemtuzumab Conditioned Haematopoetic Stem Cell Transplantation Is an Independent Risk Factor for Acute GvHD

T cell depletion with in vivo alemtuzumab is used to ameliorate the graft versus host response and permit donor engraftment in allogeneic haematopoietic stem cell transplantation (allo-HSCT). Previous reports have shown that T cells lacking CD52 are often detectable during the period of early immune reconstitution with this protocol, but the clinical relevance of this cellular population is not understood. In a cohort of 67 consecutive patients undergoing allo-HSCT between 2013-2016 we investigated the phenotype and function of the CD52-negative T cell fraction and related their presence to clinical outcome. 47 patients (70%) had a myeloid disease (AML or MDS) while 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10mg/day from day -5 for 5 days). 63 (94%) received reduced intensity conditioning chemotherapy, while 4 (6%) received a myeloablative regimen. All patients received post-transplant ciclosporin A for GvHD prophylaxis. 6 (9%) also received methotrexate, while 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68% and relapse free survival was 48%. 29% patients experienced acute GvHD (grade 2 or above) and 15% developed chronic GvHD. CD52-negative T cells demonstrated low binding of FLAER, indicating downregulation of the glycophosphatidylinositol (GPI) anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with PNH. CD52-negative T cells were almost exclusively CD4+ and exhibited a dominant memory phenotype with only small numbers of CD25+ CD127lowFoxp3+ regulatory T cells. They exhibited enhanced cytotoxic responses to T cell receptor stimulation in vitro. Early after allo-HSCT, the presence of a significant population of CD52 negative T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that CD52/GPI-negative T cells arise from the selective pressure of alemtuzumab in the setting of lymphopenia and homeostatic proliferation. To our knowledge this is the first study to show that CD52 negative T cells have a reduced regulatory T cell fraction, and have an association with acute GvHD. This suggests that the CD52-negative T cell fraction may represent a 'footprint' of the early alloreactive response focused within the CD4+ population, or contribute to an immune environment with reduced T cell tolerance. These data help to delineate the nature of T cell escape from alemtuzumab surveillance providing new insights into the early alloreactive immune response. Furthermore, this study informs the use of alemtuzumab in conditioning regimens for upcoming cellular immunotherapies. Disclosures No relevant conflicts of interest to declare.

PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGF-β Inhibitors

Programmed cell death (PD)-1/PD-Ligand 1(PD-L1) blockade that reinvigorates exhausted T cells has been approved for the treatment of various solid tumors and hematological malignancies. However, in a clinical trial of multiple myeloma (MM) patients, anti-PD-1 monotherapy did not result in a clinical response. Furthermore, clinical trials of combining PD-1 blockade with immunomodulatory drugs or anti-CD38 monoclonal antibody failed to demonstrate clinical benefits in MM patients. To overcome the limitation of anti-PD-1 therapy in MM, the phenotype and differentiation of CD8+ T cells need to be characterized in the bone marrow (BM) of MM patients, particularly by analyzing myeloma antigen-specific CD8+ T cells. In addition, the role of immunosuppressive factors abundant in the MM microenvironment should be considered, including TGF-β. First, we confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8+ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors including Tim-3, LAG-3, and TIGIT. We also investigated the expression of T-cell transcription factors, such as T-bet, and EOMES, which are related to T-cell differentiation. In BM from MM patients, PD-1+CD8+ T cells had a higher percentage of EomeshiT-betlo cells than PD-1-CD8+ T cells. These data demonstrate that PD-1-expressing CD8+ T cells from the BM of MM patients exhibit a terminally differentiated phenotype with co-expression of multiple immune checkpoint inhibitory receptors. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. Next, we investigated proliferation and cytokine production of BM CD8+ T cells from MM patients. BM CD8+ T cells from MM patients exhibited reduced proliferation and cytokine production upon T cell receptor (TCR) stimulation, compared to BM CD8+ T cells from other control group such as of undetermined significance. However, both anti-PD-1 alone and combined blockade of PD-1 with other immune checkpoint receptors, such as Tim-3, Lag-3, or TIGIT, did not increase the proliferation of BM CD8+ T cells from MM patients. Likewise, anti-PD-1 treatment failed to induce reinvigoration of BM CD8+ T cells stimulated with HLA-A*0201-restricted myeloma antigen peptides, including NY-ESO-1157-165 and HM1.2422-30 peptides. These data demonstrate that blocking PD-1 is not sufficient to restore the function of BM CD8+ T cells from MM patients. It has been known that TGF-β, which is actively secreted by malignant plasma cells and BM stromal cells, can inhibit T-cell responses. We confirmed that the major source of TGF- β1 is plasma cells including myeloma cells among BMMCs from MM patients, and the number of TGF- β1-producing plasma cells, including myeloma cells, is increased in the BM of MM patients. We investigated whether blocking TGF-β signaling enhances reinvigoration of BM CD8+ T cells from MM patients. The combined blockade of PD-1 and TGF- β significantly increased the proliferation of BM CD8+ T cells from MM patients in the presence of TCR stimulation. The production of IFN-γ and TNF by BM CD8+ T cells was also rescued by combined blockade of PD-1 and TGF-β. Moreover, combination of anti-PD-1 antibody and TGF-β inhibitors increased proliferative responses of BM CD8+ T cells from HLA-A2+ MM patients stimulated with a mixture of HLA-A*0201-restricted myeloma antigen peptides (NY-ESO-1157-165 and HM1.2422-30 peptides). Thus, PD-1 blockade reinvigorates BM CD8+ T cells from MM patients in the presence of TGF-β inhibitors. Taken together, BM CD8+ T cells and myeloma antigen-specific CD8+ T cells express increased levels of PD-1 and have a terminally exhausted phenotype in MM patients. Under TGF-β inhibition, anti-PD-1 reinvigorates BM CD8+ T cells from MM patients, but PD-1 blockade alone does not restore the function of BM CD8+ T cells. Blocking both TGF-β and PD-1 can be a promising therapeutic strategy for the treatment of MM. Disclosures No relevant conflicts of interest to declare.