CD4 CTLs Research Articles

Loss of immune cell identity with age inferred from large atlases of single cell transcriptomes.

By analyzing two large atlases of almost 4 million cells, we show that immune-senescence involves a gradual loss of cellular identity, reflecting increased cellular heterogeneity, for effector, and cytotoxic immune cells. The effects are largely similar in both males and females and were robustly reproduced in two atlases, one assembled from 35 diverse studies including 678 adults, the other the OneK1K study of 982 adults. Since the mean transcriptional differences among cell-types remain constant across age deciles, there is little evidence for the alternative mechanism of convergence of cell-type identity. Key pathways promoting activation and stemness are down-regulated in aged T cells, while CD8 TEM and CD4 CTLs exhibited elevated inflammatory, and cytotoxicity in older individuals. Elevated inflammatory signaling pathways, such as MAPK and TNF-alpha signaling via NF-kB, also occur across all aged immune cells, particularly amongst effector immune cells. This finding of lost transcriptional identity with age carries several implications, spanning from a fundamental biological understanding of aging mechanisms to clinical perspectives on the efficacy of immunomodulation in elderly people.

The presence of cytotoxic CD4 and exhausted-like CD8+ T-cells is a signature of active tuberculosis

Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups—uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)—were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.

Abstract 1028: Cytotoxic CD4+ T Cells: Unraveling The Residual Risk In Lipid Lowering For Type 2 Diabetes Patients

Type 2 Diabetes (T2D) significantly increases the risk of cardiovascular diseases (CVDs), marked by severe plaque pathology resistant to standard cholesterol-lowering treatments. While the roles of the innate immune system and macrophages in CVDs are well-established, T cell alterations, particularly involving cytotoxic CD4 T cells (CD4 CTLs), remain unexplored in human atherosclerotic plaques. In our study, we aimed to identify the phenotype and function of CD4 CTLs in blood & plaque tissue, in the context of T2D. Using unbiased single-cell RNA sequencing (scRNA-seq) analysis, we examined over 70,000 immune cells from 22 plaques of patients undergoing carotid endarterectomy (T2D: n=12, Non-Diabetic (ND): n=10), & peripheral blood mononuclear cells (PBMCs) from the CHORD study (NCT04369664) (T2D: n=12, ND: n=4). scRNA-seq analysis revealed a diverse population of CD4 T cells, like naïve, central memory (CM), effector memory (EM), tissue resident memory (Trm), T regulatory (Tregs), & two clusters of cytotoxic (CTL) T cells expressing canonical cytotoxicity-related genes. Cluster 5 expressed GZMB, GNLY, PRF1 , whereas Cluster 10 expressed GZMA, GZMK . Cell-cell communication analysis demonstrated that both CTL clusters received signaling from dendritic cells and macrophages, including TREM2+ macrophages. CTL T cells reciprocally communicated with clusters of EM and EMRA CD8 T cells, underscoring their central role in plaque cell-cell communication. Notably, Cluster 5, one of these two CTL clusters, was expanded in T2D plaque samples (p=0.032) and expressed genes involved in chemotaxis, IL-2 signaling, & transcription factors (e.g., RUNX3, TBX21 ) involved in T cell activation. The equivalent of CD4 CTL phenotype was found to be expanded in T2D individuals compared to controls, & these elevated levels persisted in T2D patients following intensive lipid-lowering therapy, indicating resistance to the treatment regimen. In summary, our study emphasizes the crucial role of CD4 CTLs in the immune dynamics of atherosclerotic plaques. The observed resistance of CTLs to lipid-lowering interventions underscores the necessity for targeted therapeutic strategies to address the distinctive immune changes in T2D-associated cardiovascular diseases.

The compensatory role of T cells from lymph nodes in mice with splenectomy.

The spleen is a vital organ for the immune system, while splenectomy may be necessary for various reasons. However, there is limited research on the impact of splenectomy on T cell function in peripheral lymph nodes as a compensatory mechanism in preventing infections. This study aimed to investigate the characteristics and function of CD8+ and CD4+ T cells in different peripheral lymph nodes during viral infection using a well-established splenectomy model. The results revealed that splenectomy caused an increase in CD8+GP33+ T cells in the mesenteric lymph nodes (MLN). Moreover, we demonstrated that splenectomy resulted in an increase of effector KLRG1+ T cells in the MLN. Additionally, the number of CD4+ cytotoxic T cells (CD4 CTLs) was also elevated in the peripheral lymph nodes of mice with splenectomy. Surprisingly, aged mice exhibited a stronger compensatory ability than adult mice, as evidenced by an increase in effector CD8+ T cells in all peripheral lymph nodes. These findings provide compelling evidence that T cells in MLN play a crucial role in protecting individuals with splenectomy against viral infections. The study offers new insights into understanding the changes in the immune system of individuals with splenectomy and highlights the potential compensatory mechanisms involved by T cells in peripheral lymph nodes.

G Protein-Coupled Receptor 56 Characterizes CTLs and Reflects the Progression of Lung Cancer Patients.

CTLs play important roles in host immune responses to tumors. CD4 CTLs are characterized by their ability to secrete cytotoxic effector molecules, such as granzyme B and perforin, and kill target cells in a MHC class II-restricted manner. However, the cell surface markers of CD4 CTLs remain unknown, which hinders their separation and research on their function. In this study, we performed a bioinformatics analysis and experimental validation that revealed that G protein-coupled receptor 56 (GPR56) is a cell surface marker that can be used to characterize CD4 CTLs. We found that GPR56 and granzyme B were coexpressed in extremely high levels in human peripheral blood T cells, and that anti-GPR56 stimulation significantly upregulated the expression of granzyme B in both CD4+GPR56+ and CD8+GPR56+ T cells. These findings suggest that GPR56 expression and the GPR56 signaling pathway could contribute directly to the toxic function of either CD4+ or CD8+ T cells. We also used GPR56 as a biomarker to investigate the clinical significance of CD4 CTLs. GPR56+ T cell levels were increased in patients with lung cancer, and GPR56 expression was significantly correlated with lung cancer progression. A further analysis revealed an increase in exhausted cell states in lung cancer patients because of upregulation of programmed cell death protein 1 expression in GPR56+ T cells. The findings of this study suggest that GPR56 characterizes the cytotoxic states of either CD4+ or CD8+ T cells.

Single cell mapping identifies expansion of Cytotoxic CD4 T cells in the atherosclerotic plaque of subjects with Type 2 Diabetes

Abstract Patients with Type 2 diabetes (T2D) are at greater risk of atherosclerotic cardiovascular disease (ASCVD) and fail to fully respond to standard lipid lowering drugs. While the role of macrophages in T2D plaque pathology has been investigated, no studies have systematically investigated the heterogeneity of CD4 T cells in human plaques and how they respond to cholesterol reduction. To identify the specific T cell alterations in plaques from patients with T2D, we performed scRNAseq of human carotid plaques from 22 subjects (54.5% T2D) were being treated with cholesterol lowering drugs (average LDL cholesterol of 1.2 mmol/L) undergoing carotid endarterectomy. Data were processed and integrated using Seurat and Slingshot algorithms. We identified a total of 24 immune cell clusters by unbiased clustering of CD45+ cells, including CD4 T cells that presented similar frequencies between T2D (~11%) and non-T2D patients (~10%). CD4 T cells comprised of 13 sub clusters: naïve, central memory (CM), effector memory (EM), T regulatory (Tregs), cytotoxic (CTL), and gamma delta (γδ) T cells. One cluster of CD4 CTLs (cluster 5) expressing TRDC and TRGC2 surface markers along with cytotoxicity markers GZMB, GNLY, and PRF1 was significantly expanded in T2D plaques (p=0.032). This CD4CTL expressed high levels of transcription factors (TFs) involved in T cell differentiation such as RUNX3 and ZEB2 and contained sets of genes involved in cytotoxicity, chemotaxis and IL-1 signaling. Trajectory analysis using Slingshot inferred that CD4 CTLs clusters in the plaques differentiate from CD4 EM T cells. Plaques from patients with T2D are highly enriched in CTL/γδ T cells and suggest that their cytotoxic potential contributes to the increased cardiovascular risk. This Work is supported by AHA SFRN 20SFRN35210252 and the AHA-SFRN Training Program.

Cytotoxic CD4+ T cells eliminate senescent cells by targeting cytomegalovirus antigen

Senescent cell accumulation has been implicated in the pathogenesis of aging-associated diseases, including cancer. The mechanism that prevents the accumulation of senescent cells in aging human organs is unclear. Here, we demonstrate that a virus-immune axis controls the senescent fibroblast accumulation in the human skin. Senescent fibroblasts increased in old skin compared with young skin. However, they did not increase with advancing age in the elderly. Increased CXCL9 and cytotoxic CD4+ Tcells (CD4 CTLs) recruitment were significantly associated with reduced senescent fibroblasts in the old skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), becoming direct CD4 CTL targets. Skin-resident CD4 CTLs eliminated HCMV-gB+ senescent fibroblasts in an HLA-II-dependent manner, and HCMV-gB activated CD4 CTLs from the human skin. Collectively, our findings demonstrate HCMV reactivation in senescent cells, which CD4 CTLs can directly eliminate through the recognition of the HCMV-gB antigen.

VEGF-A enhances the cytotoxic function of CD4+ cytotoxic T cells via the VEGF-receptor 1/VEGF-receptor 2/AKT/mTOR pathway

BackgroundCD4+ cytotoxic T cells (CD4 CTLs) are CD4+ T cells with major histocompatibility complex-II-restricted cytotoxic function. Under pathologic conditions, CD4 CTLs hasten the development of autoimmune disease or viral infection by enhancing cytotoxicity. However, the regulators of the cytotoxicity of CD4 CTLs are not fully understood.MethodsTo explore the potential regulators of the cytotoxicity of CD4 CTLs, bulk RNA and single-cell RNA sequencing (scRNA-seq), enzyme-linked immunosorbent assay, flow cytometry, quantitative PCR, and in-vitro stimulation and inhibition assays were performed.ResultsIn this study, we found that VEGF-A promoted the cytotoxicity of CD4 CTLs through scRNA-seq and flow cytometry. Regarding the specific VEGF receptor (R) involved, VEGF-R1/R2 signaling was activated in CD4 CTLs with increased cytotoxicity, and the VEGF-A effects were inhibited when anti-VEGF-R1/R2 neutralizing antibodies were applied. Mechanistically, VEGF-A treatment activated the AKT/mTOR pathway in CD4 CTLs, and the increases of cytotoxic molecules induced by VEGF-A were significantly reduced when the AKT/mTOR pathway was inhibited.ConclusionIn conclusion, VEGF-A enhances the cytotoxicity of CD4 CTLs through the VEGF-R1/VEGF-R2/AKT/mTOR pathway, providing insights for the development of novel treatments for disorders associated with CD4 CTLs.

Abstract 13609: Heterogeneous Populations of Cytotoxic CD4 T Cells Are Expanded in Atherosclerotic Plaques of Patients With Type 2 Diabetes

Introduction: Atherosclerotic cardiovascular disease (ACVD) is the main cause of morbidity and mortality in patients with type 2 diabetes (T2D). ACVD is more severe and diffuse in T2D than in subjects with no diabetes (ND). Moreover, cholesterol reduction in T2D leads to less plaques resolution. While the role of macrophages in T2D plaque pathology has been investigated, no studies have systematically investigated the heterogeneity of CD4 T cells in human plaques. Hypothesis: Plaques of patients with T2D have a greater CD4 T cell heterogeneity and pathogenic phenotypes compared with plaques of ND subjects. Methods: To identify the specific T cell alterations in plaques we analyzed scRNA sequencing data of >80,000 plaque immune cells from 22 patients undergoing carotid endarterectomy with or without T2D (54.5% T2D). Results: Unbiased clustering of CD45+ cells identified a total of 24 immune cell clusters, including CD4 T cells that presented similar cell frequencies between T2D (~11%) and ND patients (~10%). CD4 T cells comprise 7 clusters: naïve, proliferating, central memory (CM), effector memory (EM), T regulatory (Tregs), cytotoxic (CTL), and gamma delta (γδ) T cells. CD4 CTLs expressed GZMB, GNLY, PRF1 , and could be divided into TRAC +EM (cluster 9) and CTLγδ T cells expressing both TRDC and TRGC2 (cluster 5). CTLγδ T cells were significantly expanded in T2D plaques (p=0.032), and expressed high levels of GZMA and transcription factors involved in T cell differentiation such as RUNX and TBX21 . These CTLγδ T cells are enriched with sets of genes involved in T-cell activation, numerous pathways of apoptosis, cytotoxicity, chemotaxis and cytokine signaling (i.e. IL1, IL2, IL9 ). Conclusions: Our data show that plaques from patients with T2D are highly enriched in unique CTL/γδ T cells and suggest that their cytotoxic potential may contribute to the increased cardiovascular risk of subjects with T2D.

CD4+ Cytotoxic T cells – Phenotype, Function and Transcriptional Networks Controlling Their Differentiation Pathways

The two major subsets of peripheral T cells are classically divided into the CD4+ T helper cells and the cytotoxic CD8+ T cell lineage. However, the appearance of some effector CD4+ T cell populations displaying cytotoxic activity, in particular during viral infections, has been observed, thus breaking the functional dichotomy of CD4+ and CD8+ T lymphocytes. The strong association of the appearance of CD4+ cytotoxic T lymphocytes (CD4 CTLs) with viral infections suggests an important role of this subset in antiviral immunity by controlling viral replication and infection. Moreover, CD4 CTLs have been linked with anti-tumor activity and might also cause immunopathology in autoimmune diseases. This raises interest into the molecular mechanisms regulating CD4 CTL differentiation, which are poorly understood in comparison to differentiation pathways of other Th subsets. In this review, we provide a brief overview about key features of CD4 CTLs, including their role in viral infections and cancer immunity, and about the link between CD4 CTLs and immune-mediated diseases. Subsequently, we will discuss the current knowledge about transcriptional and epigenetic networks controlling CD4 CTL differentiation and highlight recent data suggesting a role for histone deacetylases in the generation of CD4 CTLs.

Prognostic Impact of Cytotoxic CD4 T Cells in Tumor Immune Microenvironment of Patients with Breast Cancer.

Prognostic Impact of Cytotoxic CD4 T Cells in Tumor Immune Microenvironment of Patients with Breast Cancer.

Single-cell transcriptome and TCR profiling reveal activated and expanded T cell populations in Parkinson\u2019s disease

Given the chronic inflammatory nature of Parkinson’s disease (PD), T cell immunity may be important for disease onset. Here, we performed single-cell transcriptome and TCR sequencing, and conducted integrative analyses to decode composition, function and lineage relationship of T cells in the blood and cerebrospinal fluid of PD. Combined expression and TCR-based lineage tracking, we discovered a large population of CD8+ T cells showing continuous progression from central memory to terminal effector T cells in PD patients. Additionally, we identified a group of cytotoxic CD4+ T cells (CD4 CTLs) remarkably expanded in PD patients, which derived from Th1 cells by TCR-based fate decision. Finally, we screened putative TCR–antigen pairs that existed in both blood and cerebrospinal fluid of PD patients to provide potential evidence for peripheral T cells to participate in neuronal degeneration. Our study provides valuable insights and rich resources for understanding the adaptive immune response in PD.

35P Prognostic impact of cytotoxic CD4 T cells (CD4 CTL) in tumour immune microenvironment of breast cancer patients

CD4 CTLs are a subset of CD4 T cells that infiltrate the tumor immune environment (TIME) and have cytotoxic activity against malignancies. A gene signature defining the CD4 CTLs was recently developed (Cell. 2020; 181(7):1612-1625.e13). The prognostic value of CD4 CTLs in breast cancer TIME is unknown.

Clinical Relevance of CD4 Cytotoxic T Cells in High-Risk Neuroblastoma.

Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy, and the survival of these patients remains poor for the last three decades. To effectively treat these extremely unfavorable neuroblastomas, innovative immunotherapy approaches would be the most promising. In this article, we discuss the identity of tumor-infiltrating effector cells and immunosuppressive cells in high-risk neuroblastoma. Neuroblastoma is unique in that it expresses little or no classical HLA Class I and II. In contrast, high-risk neuroblastomas express the stress-responsive non-classical Class I, HLA-E molecule. HLA-E is the ligand of activating receptors NKG2C/E that are expressed on memory NK cells, CD8+T cells and CD4 CTLs. By examining a comprehensive RNA-seq gene expression dataset, we detected relatively high levels of CD4 expression in high-risk neuroblastoma tissues. The majority of CD4+ cells were CD3+, and thus they were likely tumor-associated CD4+T cells. In addition, high-level of both CD4 and NKG2C/E expression was associated with prolonged survival of the high-risk neuroblastoma patients, but CD8 levels were not, further suggesting that the CD4+ NKG2C/E+ T cells or CD4 CTL conferred cytotoxicity against the neuroblastoma cells. However, this T cell mediated- “protective effect” declined over time, in part due to the progressive formation of immunosuppressive tumor microenvironment. These observations suggest that to improve survival of high-risk neuroblastoma patients, it is essential to gain insights into how to enhance CD4 CTL cytotoxicity and control the immunosuppressive tumor microenvironment during the course of the disease.

Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians

Supercentenarians, people who have reached 110 y of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells (CD4 cytotoxic T lymphocytes [CTLs]) as a signature of supercentenarians. Furthermore, single-cell T cell receptor sequencing of 2 supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15 to 35% of the entire CD4 T cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, CD4 CTLs extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.

Differential Requirement of Cd8 Enhancers E8I and E8VI in Cytotoxic Lineage T Cells and in Intestinal Intraepithelial Lymphocytes.

CD8 expression in T lymphocytes is tightly regulated by the activity of at least six Cd8 enhancers (E8I-E8VI), however their complex developmental stage-, subset-, and lineage-specific interplays are incompletely understood. Here we analyzed ATAC-seq data on the Immunological Genome Project database and identified a similar developmental regulation of chromatin accessibility of a subregion of E8I, designated E8I-core, and of E8VI. Loss of E8I-core led to a similar reduction in CD8 expression in naïve CD8+ T cells and in IELs as observed in E8I−/− mice, demonstrating that we identified the core enhancer region of E8I. While E8VI−/− mice displayed a mild reduction in CD8 expression levels on CD8SP thymocytes and peripheral CD8+ T cells, CD8 levels were further reduced upon combined deletion of E8I-core and E8VI. Moreover, activated E8I-core−/−E8VI−/− CD8+ T cells lost CD8 expression to a greater degree than E8I-core−/− and E8VI−/− CD8+ T cells, suggesting that the combined activity of both enhancers is required for establishment and maintenance of CD8 expression before and after TCR activation. Finally, we observed a severe reduction of CD4 CTLs among the TCRβ+CD4+ IEL population in E8I-core−/− but not E8VI−/− mice. Such a reduction was not observed in Cd8a−/− mice, indicating that E8I-core controls the generation of CD4 CTLs independently of its role in Cd8a gene regulation. Further, the combined deletion of E8I-core and E8VI restored CD4 CTL subsets, suggesting an antagonistic function of E8VI in the generation of CD4 CTLs. Together, our study demonstrates a complex utilization and interplay of E8I-core and E8VI in regulating CD8 expression in cytotoxic lineage T cells and in IELs. Moreover, we revealed a novel E8I-mediated regulatory mechanism controlling the generation of intestinal CD4 CTLs.

CD4 CTL, a Cytotoxic Subset of CD4+ T Cells, Their Differentiation and Function.

CD4+ T cells with cytotoxic activity (CD4 CTL) have been observed in various immune responses. These cells are characterized by their ability to secrete granzyme B and perforin and to kill the target cells in an MHC class II-restricted fashion. Although CD4 CTLs were once thought to be an in vitro artifact associated with long-term culturing, they have since been identified in vivo and shown to play important roles in antiviral and antitumor immunity, as well as in inflammation. Functional characterization of CD4 CTL suggests their potential significance for therapeutic purposes. However, in order to develop effective CD4 CTL therapy it is necessary to understand the differentiation and generation of these cells. Although the mechanisms regulating development of various CD4+ Th subsets have been clarified in terms of the cytokine and transcription factor requirement, the CD4 CTL differentiation mechanism remains elusive. These cells are thought to be most closely related to Th1 cells secreting IFNγ and regulated by eomesodermin and/or T-bet transcription factors for their differentiation. However, our studies and those of others have identified CD4 CTLs within other CD4+ T cell subsets, including naïve T cells. We have identified class I-restricted T cell-associated molecule as a marker of CD4 CTL and, by using this marker, we detected a subset of naïve T cells that have the potential to differentiate into CD4 CTL. CD4 CTL develops at sites of infections as well as inflammation. In this review, we summarize recent findings about the generation of CD4 CTL and propose a model with several differentiation pathways.

Autoimmunity: CD4(+) CTLs drive IgG4-related disease.

Autoimmunity: CD4(+) CTLs drive IgG4-related disease.

Vaccination Produces CD4 T Cells with a Novel CD154-CD40-Dependent Cytolytic Mechanism.

The discovery of new vaccines against infectious diseases and cancer requires the development of novel adjuvants with well-defined activities. The TLR4 agonist adjuvant GLA-SE elicits robust Th1 responses to a variety of vaccine Ags and is in clinical development for both infectious diseases and cancer. We demonstrate that immunization with a recombinant protein Ag and GLA-SE also induces granzyme A expression in CD4 T cells and produces cytolytic cells that can be detected in vivo. Surprisingly, these in vivo CTLs were CD4 T cells, not CD8 T cells, and this cytolytic activity was not dependent on granzyme A/B or perforin. Unlike previously reported CD4 CTLs, the transcription factors Tbet and Eomes were not necessary for their development. CTL activity was also independent of the Fas ligand-Fas, TRAIL-DR5, and canonical death pathways, indicating a novel mechanism of CTL activity. Rather, the in vivo CD4 CTL activity induced by vaccination required T cell expression of CD154 (CD40L) and target cell expression of CD40. Thus, vaccination with a TLR4 agonist adjuvant induces CD4 CTLs, which kill through a previously unknown CD154-dependent mechanism.

Continuous Therapy for Elderly Newly Diagnosed Multiple Myeloma

e28 Immunomodulatory drugs (IMiDs) directly trigger multiple myeloma (MM) cell apoptosis, abrogate adhesion to bone marrow (BM), modulate cytokines, inhibit angiogenesis, and augment T-cell, NK-cell, andNK-T cell function while downregulating Tregs. Binding to cereblon is implicated in both their direct cytotoxic and immunerelated effects. Importantly, they can augment immune therapies in MM, evidenced by increasing antibody-dependent cellular cytotoxicity and clinical activity of the mAbs elotuzumab, daratubumab, and SAR650984, and immunotoxin indatuximab ravtansine. MM cells and accessory cells that augment MM cell growth and suppress host immune function, including myeloid derived suppressor cells (MDSCs) and plasmacytoid dendritic cells (pDCs), express PD-L1; immune effector T and NK cells express PD-1. Moreover, PD-L1 expression increases with progression from normal to MGUS to active MM, and with progression from newly diagnosed to relapsed and refractory disease. Preclinical studies show that anti-PD-1 Ab, -PD-L1 Ab, or both can induce autologous effector cell-mediated cytotoxicity. Anti-PD-L1 Ab can induce MM-cell specific CD4 and CD8 CTLs and NK cell cytotoxicity, even in the presence of MDSCs and pDCs, thereby inhibiting MM cell growth in the BM. PD-L1 is induced on MM and accessory cells in the BM, as is PD-1 on immune effector cells. Importantly, IMiDs downregulate PDL-1 expression on MM and accessory cells and PD-1 on immune effector cells, thereby enhancing CD4, CD8, and NK-cell mediated MM cytotoxicity in the presence of BM accessory cells. Peptide-based vaccine strategies are also being evaluated in smouldering MM to block progression to active disease, as are MM cell/DC fusion vaccines to treat minimal residual disease posttransplant and improve outcome. In both cases, CD4 and CD8 MM-specific immune responses have been triggered by vaccination; this can be increased by lenalidomide (LEN) and/or checkpoint inhibition. A randomized trial is now comparing LEN with LEN plus MM cell/DC fusion vaccine posttransplant; it is likely that LEN and checkpoint inhibition will be combined, to generate sustained autologous memory anti-MM immunity. Immune approach combinations including mAbs, vaccines, IMiDs, and checkpoint inhibitors offer great potential to overcome effects of immune suppressive cytokines and accessory cells in the BM microenvironment, restore host CD4, CD8, and NK cell antitumour immunity, and so improve patient outcome in MM.