Comprehensive TCR repertoire analysis of CD4+ T-cell subsets in rheumatoid arthritis.

The pathogenesis of rheumatoid arthritis (RA), a systemic autoimmune disease characterized by autoreactive T-cell accumulation and pro-inflammatory cytokine overproduction, is unclear. Systematically addressing T-cell receptor (TCR) repertoires of different CD4+ T-cell subsets could help understand RA pathogenesis. Here, peripheral CD4+ T cells from treatment-naïve RA patients and healthy controls were sorted into seven subsets including naïve, effector, central memory, effector memory (EMT), Th1, Th17, and regulatory T cells. T-cell receptor ß chain repertoires were then analyzed by next-generation sequencing. We identified T-cell clonal expansion in EMT and Th17 cells of RA patients, with highly similar TCR repertoires. Ex vivo experiments demonstrated the preferred differentiation from EMT to Th17 cells in RA. Notably, we showed that TCR diversity and abundance of differentiated T cells of Th17 were significantly correlated with RA disease activity. Based on these observations, we propose that abnormal differentiation from EMT to Th17 and expansion of Th17 play pivotal role in RA pathogenesis.

Chemokine receptor CCR5 correlates with functional CD8+ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation.

C-C chemokine receptor 5 (CCR5) plays an essential role in HIV pathogenesis as the major coreceptor on CD4+ T cells used by HIV, yet the function of CCR5 on CD8 T cells is not well understood. Furthermore, the immunologic effects of the CCR5 inhibitor maraviroc (MVC), despite approval for clinical use, have not yet been well evaluated for their potential effects on cytotoxic T-cell responses. In this study, we characterized the development and function of CCR5+CD8+ T cells in rhesus macaques with or without Simian immunodeficiency virus (SIV) infection. We also investigated the effects of the CCR5 antagonist MVC on functional CCR5+CD8+ T-cell responses in vitro. The data show that CCR5+CD8+ T cells have an effector memory phenotype and increase with age in systemic and mucosal lymphoid tissues as a heterogeneous population of polyfunctional CD8 T cells. In addition, CCR5 is highly expressed on SIV gag-specific (CM9+) CD8+ T cells in SIV-infected macaques, yet CCR5+CD8+ T cells are significantly reduced in mucosal lymphoid tissues with disease progression. Furthermore, in vitro MVC treatment reduced activation and cytokine secretion of CD8+ T cells via a CCR5-independent pathway. These findings suggest that surface CCR5 protein plays an important role in differentiation and activation of CD8+ T cells. Although MVC may be helpful in reducing chronic inflammation and activation, it may also inhibit virus-specific CD8+ T-cell responses. Thus optimal use of CCR5 antagonists either alone or in combination with other drugs should be defined by further investigation.-Wang, X., Russell-Lodrigue, K. E., Ratterree, M. S., Veazey, R. S., Xu, H. Chemokine receptor CCR5 correlates with functional CD8+ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation.

T-cell subset differentiation and antibody responses following antiretroviral therapy during simian immunodeficiency virus infection.

Antiretroviral therapy (ART) for the treatment of human immunodeficiency virus (HIV) infection represents a major breakthrough in the treatment of HIV/acquired immune-deficiency syndrome. However, it remains unclear how ART influences virus-specific immune responses and understanding this is important for developing novel cure and eradication interventions for HIV-1. In the present study, we evaluate how ART impacts T-cell and antibody responses in simian immunodeficiency virus (SIV) -infected rhesus macaques. We evaluated CD4 and CD8 T-cell responses by multiparameter flow cytometry, viral loads by quantitative RT-PCR by a two-step process using SIV-specific primers and antibody neutralization function by luciferase-based TZM-bl assays. We demonstrate that macaques treated with ART exhibit phenotypic and qualitative effects on T-cell and antibody responses. Macaques on ART exhibited low numbers of virus-specific T-cell responses, and these responses appeared to be partially biased towards central memory subsets. More importantly, there were significantly reduced neutralizing antibody responses in macaques treated with ART. Collectively, these data improve the understanding of how virus-specific immune responses are generated during ART, and suggest the potential importance of therapeutic vaccines to maintain adaptive immunity during treated infection.

Ethylenecarbodiimide-fixed splenocytes carrying whole islet antigens decrease the incidence of diabetes in NOD mice via down-regulation of effector memory T cells and autoantibodies.

Type 1 diabetes mellitus (T1DM) is a syndrome of loss of glucose homeostasis caused by the loss of ß cell chronic autoimmunity against islet cells. Islet-specific epitopes coupled antigen presenting cells by Ethylenecarbodiimide (ECDI) is a promising strategy to induce antigen-specific tolerance. However, single epitope induced tolerance is insufficient to prevent the onset of T1DM. The aim of this study is to evaluate the efficacy of whole islet antigens in preventing the onset and progression of T1DM and identify the underlying immune mechanism in NOD mice. In this study, the whole islet antigens, derived from islet lysate isolated from BALB/c mice, were coupled to splenocytes of BALB/c mice by ECDI fixation (SP-Islet lysate), and then intravenously administrated to NOD mice. The results showed that, compared with control group, SP-Islet lysate group significantly decreased T1DM incidence and improved the survival of NOD mice. SP-Islet lysate treated mice had reduced insulitis score and autoantibody levels, and improved glucose tolerance and insulin/glucagon production. Furthermore, the effector memory T cells (TEMs) were downregulated and regulatory T cells (Tregs) were upregulated by the SP-Islet lysate treatment, with reduced populations of Th1&Th17 cells. In conclusion, ECDI-fixed splenocytes carrying whole islet antigens effectively prevented the onset of T1DM in NOD mice, via suppressing the production of autoantibodies and inducing anergy of autoreactive T cells.

Cytomegalovirus- and Epstein-Barr Virus-Induced T-Cell Expansions in Young Children Do Not Impair Naive T-cell Populations or Vaccination Responses: The Generation R Study.

BACKGROUND: Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) induce effector memory T-cell expansions, which are variable and potentially depend on the age at primary exposure and coinfections. We evaluated the T-cell compartment and herpesvirus infections in 6-year-old children. METHODS: T-cell subsets and immunoglobulin G seropositivity for CMV, EBV, herpes-simplex virus 1, and varicella-zoster virus were studied in 1079 6-year-old children. A random subgroup of 225 children was evaluated for CMV and EBV seropositivity before 2 years of age and for vaccination responses against measles and tetanus. RESULTS: CMV and EBV infections were associated with significant expansions of CD27(-) and CD27(+) effector memory T cells, respectively. These expansions were enhanced in CMV-EBV-coinfected children and were independent of varicella-zoster virus or herpes-simplex virus 1 coinfection. Naive and central memory T-cell numbers were not affected, nor were anti-tetanus and anti-measles immunoglobulin G levels. Children infected before 2 years of age showed smaller effector memory T-cell expansions than those infected between 2 and 6 years of age. CONCLUSIONS: CMV- and EBV-related T-cell expansions do not impair naive T-cell numbers or maintenance of protective responses against nonrelated pathogens. Duration of infection was not directly related to larger expansions of effector memory T cells in children, suggesting that other mechanisms affect these expansions at later age.

Lymph node targeting strategy using a hydrogel sustained-release system to load effector memory T cells improves the anti-tumor efficacy of anti-PD-1.

Communication between tumors and lymph nodes carries substantial significance for antitumor immunotherapy. Remodeling the immune microenvironment of tumor-draining lymph nodes (TdLN) plays a key role in enhancing the anti-tumor ability of immunotherapy. In this study, we constructed a biomimetic artificial lymph node structure composed of F127 hydrogel loading effector memory T (TEM) cells and PD-1 inhibitors (aPD-1). The biomimetic lymph nodes facilitate the delivery of TEM cells and aPD-1 to the TdLN and the tumor immune microenvironment, thus realizing effective and sustained anti-tumor immunotherapy. Exploiting their unique gel-forming and degradation properties, the cold tumors were speedily transformed into hot tumors via TEM cell supplementation. Meanwhile, the efficacy of aPD-1 was markedly elevated compared with conventional drug delivery methods. Our finding suggested that the development of F127@TEM@aPD-1 holds promising potential as a future novel clinical drug delivery technique. STATEMENT OF SIGNIFICANCE: F127@TEM@aPD-1 show unique advantages in cancer treatment. When injected subcutaneously, F127@TEM@aPD-1 can continuously supplement TEM cells and aPD-1 to tumor draining lymph nodes (TdLN) and the tumor microenvironment, not only improving the efficacy of ICB therapy through slow release, but also exhibiting dual regulatory effects on the tumor and TdLN.

Peripheral T Cell Subpopulations as a Potential Surrogate Biomarker during Atezolizumab plus Bevacizumab Treatment for Hepatocellular Carcinoma.

The therapeutic benefits of the immunotherapeutic combination of atezolizumab and bevacizumab (Atez/Bev) in hepatocellular carcinoma (HCC) vary. Therapeutic biomarkers might help improve outcomes for HCC patients receiving Atez/Bev therapy. The role of systemic immune profiles in HCC progression also remains unclear. This study aimed to evaluate the status and dynamics of peripheral T cell subpopulations in HCC patients receiving Atez/Bev treatment and to explore biomarkers predictive of a therapeutic response. We enrolled 83 unresectable advanced HCC patients who commenced Atez/Bev treatment at our hospital between October 2020 and June 2022. Peripheral T cell subpopulations in peripheral blood mononuclear cells at baseline and 3 weeks post-treatment were investigated using flow cytometry and compared with those in control samples from 18 healthy individuals. We retrospectively analyzed the association between peripheral T cell subpopulation profiles and clinical outcomes. Baseline peripheral T cell subpopulations could be profiled in 70 patients with sufficient cell counts, among whom 3-week subpopulations could be evaluated in 51 patients. Multivariate analysis showed that a high baseline proportion of CD8+ central memory T (TCM) cells was independently associated with longer progression-free survival (PFS). Further, overall survival (OS) was significantly prolonged in patients with increased CD8+ effector memory T (TEM) cell proportions. In conclusion, TCM proportion at baseline might be a good indicator of the efficacy of Atez/Bev therapy. Furthermore, observation of increasing TEM proportions might be an early predictor of the potential clinical benefits of treatment.

Directly selecting cell-type marker genes for single-cell clustering analyses.

In single-cell RNA sequencing (scRNA-seq) studies, cell types and their marker genes are often identified by clustering and differentially expressed gene (DEG) analysis. A common practice is to select genes using surrogate criteria such as variance and deviance, then cluster them using selected genes and detect markers by DEG analysis assuming known cell types. The surrogate criteria can miss important genes or select unimportant genes, while DEG analysis has the selection-bias problem. We present Festem, a statistical method for the direct selection of cell-type markers for downstream clustering. Festem distinguishes marker genes with heterogeneous distribution across cells that are cluster informative. Simulation and scRNA-seq applications demonstrate that Festem can sensitively select markers with high precision and enables the identification of cell types often missed by other methods. In a large intrahepatic cholangiocarcinoma dataset, we identify diverse CD8+ T cell types and potential prognostic marker genes.

DSP-0509, a systemically available TLR7 agonist, exhibits combination effect with immune checkpoint blockade by activating anti-tumor immune effects.

TLR7 is an innate immune receptor that recognizes single-stranded RNAs, and its activation leads to anti-tumor immune effects. Although it is the only approved TLR7 agonist in cancer therapy, imiquimod is allowed to be administered with topical formulation. Thus, systemic administrative TLR7 agonist is expected in terms of expanding applicable cancer types. Here, we demonstrated the identification and characterization of DSP-0509 as a novel small-molecule TLR7 agonist. DSP-0509 is designed to have unique physicochemical features that could be administered systemically with a short half-life. DSP-0509 activated bone marrow-derived dendritic cells (BMDCs) and induced inflammatory cytokines including type I interferons. In the LM8 tumor-bearing mouse model, DSP-0509 reduced tumor growth not only in subcutaneous primary lesions but also in lung metastatic lesions. DSP-0509 inhibited tumor growth in several syngeneic tumor-bearing mouse models. We found that the CD8+ T cell infiltration of tumor before treatment tended to be positively correlated with anti-tumor efficacy in several mouse tumor models. The combination of DSP-0509 with anti-PD-1 antibody significantly enhanced the tumor growth inhibition compared to each monotherapy in CT26 model mice. In addition, the effector memory T cells were expanded in both the peripheral blood and tumor, and rejection of tumor re-challenge occurred in the combination group. Moreover, synergistic anti-tumor efficacy and effector memory T cell upregulation were also observed for the combination with anti-CTLA-4 antibody. The analysis of the tumor-immune microenvironment by using the nCounter assay revealed that the combination of DSP-0509 with anti-PD-1 antibody enhanced infiltration by multiple immune cells including cytotoxic T cells. In addition, the T cell function pathway and antigen presentation pathway were activated in the combination group. We confirmed that DSP-0509 enhanced the anti-tumor immune effects of anti-PD-1 antibody by inducing type I interferons via activation of dendritic cells and even CTLs. In conclusion, we expect that DSP-0509, a new TLR7 agonist that synergistically induces anti-tumor effector memory T cells with immune checkpoint blockers (ICBs) and can be administered systemically, will be used in the treatment of multiple cancers.

Cytotoxin-Associated Gene A-Negative Helicobacter pylori Promotes Gastric Mucosal CX3CR1+CD4+ Effector Memory T Cell Recruitment in Mice.

BACKGROUND: Helicobacter pylori can cause many kinds of gastric disorders, ranging from gastritis to gastric cancer. Cytotoxin-associated gene A (CagA)+ H. pylori is more likely to cause gastric histopathologic damage than CagA- H. pylori. However, the underlying mechanism needs to be further investigated. MATERIALS AND METHODS: Mice were intragastrically administered equal amounts of CagA+ or CagA- H. pylori. Four weeks later, 24 chemokines in stomachs were measured using a mouse chemokine array, and the phenotypes of the recruited gastric CD4+ T cells were analyzed. The migration pathway was evaluated. Finally, the correlation between each pair among the recruited CD4+ T cell sub-population, H. pylori colonization level, and histopathologic damage score were determined by Pearson correlation analysis. RESULTS: The concentration of chemokines, CCL3 and CX3CL1, were significantly elevated in CagA- H. pylori-infected gastric mucosa than in CagA+ H. pylori-infected gastric mucosa. Among them, CX3CL1 secreted by gastric epithelial cells, which was elicited more effectively by CagA- H. pylori than by the CagA+ strain, dramatically promoted mucosal CD4+ T cell migration. The expression of CX3CR1, the only known receptor of CX3CL1, was upregulated on the surface of gastric CD4+ T cells in CagA- H. pylori-infected stomach. In addition, most of the CX3CR1-positive gastric CD4+ T cells were CD44+CD69-CCR7- effector memory T cells (Tem). Pearson correlation analysis showed that the recruited CX3CR1+CD4+ Tem cell population was negatively correlated with H. pylori colonization level and histopathologic damage score. CONCLUSION: CagA- H. pylori promotes gastric mucosal CX3CR1+CD4+ Tem recruitment in mice.

Imaging Effector Memory T-Cells Predicts Response to PD1-Chemotherapy Combinations in Colon Cancer.

Often, patients fail to respond to immune checkpoint inhibitor (ICI) treatment despite favourable biomarker status. Numerous chemotherapeutic agents have been shown to promote tumour immunogenicity when used in conjunction with ICIs; however, little is known about whether such combination therapies lead to a lasting immune response. Given the potential toxicity of ICI-chemotherapy combinations, identification of biomarkers that accurately predict how individuals respond to specific treatment combinations and whether these responses will be long lasting is of paramount importance. In this study, we explored [18F]AlF-NOTA-KCNA3P, a peptide radiopharmaceutical that targets the Kv1.3 potassium channel overexpressed on T-effector memory (TEM) cells as a PET imaging biomarker for lasting immunological memory response. The first-line colon cancer chemotherapies oxaliplatin and 5-fluorouracil were assessed in a syngeneic colon cancer model, either as monotherapies or in combination with PD1, comparing radiopharmaceutical uptake to memory-associated immune cells in the tumour. [18F]AlF-NOTA-KCNA3P reliably separated tumours with immunological memory responses from non-responding tumours and could be used to measure Kv1.3-expressing TEM cells responsible for durable immunological memory response to combination therapy in vivo.

Phenotyping of CAR T cells.

Adaptive immunotherapy has been growing as a therapeutic alternative for cancer and other diseases treatment; chimeric antigen receptor (CAR) T cells comprise a type of adaptive immunotherapy, with recombinant receptor constructs expressed in T cells to target cells expressing specific antigens, is a potential new treatment for blood cancers and solid tumors. The phenotypic, activation, and functional profile of infused CAR-T cells have been linked to the efficacy of CAR-T cell treatment. As a result, immunophenotypic characterization of CAR-T cells during the bioprocess is critical for controlling cell quality and, eventually, improving antitumor effectiveness. Therefore, we propose a flow cytometry based immunophenotyping analysis of CAR-T cells in this chapter to define the phenotype of CAR-T subsets.

Childhood Adiposity Associated With Expanded Effector Memory CD8+ and Vδ2+Vγ9+ T Cells.

CONTEXT: Adult obesity is associated with chronic low-grade inflammation and may give rise to future chronic disease. However, it is unclear whether adiposity-related inflammation is already apparent in childhood. OBJECTIVE: To study associations between child adiposity measures with circulating monocytes and naive and memory subsets in CD4, CD8, and γδ T cell lineages. METHODS: Ten-year-old children (n = 890) from the Generation R Cohort underwent dual-energy x-ray absorptiometry and magnetic resonance imaging for body composition (body mass index [BMI], fat mass index [FMI], android-to-gynoid fat mass ratio, visceral fat index, liver fat fraction). Blood samples were taken for detailed immunophenotyping of leukocytes by 11-color flow cytometry. RESULTS: Several statistically significant associations were observed. A 1-SD increase in total FMI was associated with +8.4% (95% CI 2.0, 15.2) Vδ2+Vγ9+ and +7.4% (95% CI 2.4, 12.5) CD8+TEMRO cell numbers. A 1-SD increase in visceral fat index was associated with +10.7% (95% CI 3.3, 18.7) Vδ2+Vγ9+ and +8.3% (95% CI 2.6, 14.4) CD8+TEMRO cell numbers. Higher android-to-gynoid fat mass ratio was only associated with higher Vδ2+Vγ9+ T cells. Liver fat was associated with higher CD8+TEMRO cells but not with Vδ2+Vγ9+ T cells. Only liver fat was associated with lower Th17 cell numbers: a 1-SD increase was associated with -8.9% (95% CI -13.7, -3.7) Th17 cells. No associations for total CD8+, CD4+ T cells, or monocytes were observed. BMI was not associated with immune cells. CONCLUSION: Higher Vδ2+Vγ9+ and CD8+TEMRO cell numbers in children with higher visceral fat index could reflect presence of adiposity-related inflammation in children with adiposity of a general population.

The p53 effector Perp mediates the persistence of CD4+ effector memory T-cell undergoing lymphopenia-induced proliferation.

Under lymphopenic conditions, the rapid spontaneous proliferation produces cells that robustly differentiate into effector memory T (TEM) cells, and the aberrant expansion is preferentially driven by self-antigens. The pool size of effector memory T-cell is governed by a complex homeostatic balance between proliferation and death. Perp is a critical effector involved in the p53-dependent apoptotic pathway and widely expressed in mammalian tissues. We have previously shown that Perp has a prominent role in activation-induced cell death of peripheral Th17 cells. Here, we show that Peripheral Perp-/-CD4+ TEM cells outcompete wild type TEM cells for access to splenic niches in vivo. The skewing of the Perp-/- TEM cells compartment was not the result of a difference in lymphopenia-induced proliferation, but the resistance to apoptosis, particularly after anti-Fas treatment. Data presented in this work indicate that Perp mediates the persistence of CD4+ TEM cells in irradiation-induced lymphopenic settings.

Memory CD4+ T Cells in Immunity and Autoimmune Diseases.

CD4+ T helper (Th) cells play central roles in immunity in health and disease. While much is known about the effector function of Th cells in combating pathogens and promoting autoimmune diseases, the roles and biology of memory CD4+ Th cells are complex and less well understood. In human autoimmune diseases such as multiple sclerosis (MS), there is a critical need to better understand the function and biology of memory T cells. In this review article we summarize current concepts in the field of CD4+ T cell memory, including natural history, developmental pathways, subsets, and functions. Furthermore, we discuss advancements in the field of the newly-described CD4+ tissue-resident memory T cells and of CD4+ memory T cells in autoimmune diseases, two major areas of important unresolved questions in need of answering to advance new vaccine design and development of novel treatments for CD4+ T cell-mediated autoimmune diseases.

CTLA-4 immunotherapy exposes differences in immune response along with different tumor progression in colorectal cancer.

Tumor growth is accompanied by a changing tumor microenvironment and mutations that increase the resistance to therapy. Here, we used syngeneic models to evaluate the drug response of tumors of the same type of different sizes. We used the in vivo efficacy and Ki-67 immunohistochemistry (IHC) assay to assess the difference in responses in response to treatment with the same concentration of anti-CTLA-4. Flow cytometry analysis revealed changes in the immune subpopulations changes the spleen, peripheral blood, lymph node, and tumor tissue across different tumor growth phases. For example, naive CD4+T, CD4+TCM, CD8+TEM, T, B, Treg, CD8+TCM exhibited different percentages depending on the specific immune organ. To further expose the changes in the immune microenvironment, the level of expression of PD-1 and CTLA-4 showed statistically significant difference in related subsets for each four immune tissues in different tumor sizes. In addition, the ratios of CD4 + Teff/ CD4 + Treg and CD8 + T/Treg in corresponding immune tissue were also associated with statistically significant differences alongside tumor growth in different animal models. These results reveal the ongoing changes in the immune microenvironment during tumor progression and anti-CTLA-4 antibody immunotherapy effect depends on the expression level of immune factors.

Immunophenotypic Analysis of CAR-T Cells.

CAR-T cell immunotherapy is a promising therapeutic modality for cancer patients. The success of CAR-T cell therapy has been associated with the phenotype, activation and functional profiling of infused CAR-T cells. Therefore, immunophenotypic characterization of CAR-T cells during bioprocess is crucial for cell quality control and ultimately for improved antitumor efficacy. In this chapter, we propose a flow cytometry panel to characterize the immunophenotype of the CAR-T subsets.

Improved Expansion and In Vivo Function of Patient T Cells by a Serum-free Medium.

Improvements to T cell culture systems that promote long-term engraftment and function of adoptively transferred T cells will likely result in superior clinical benefit to more individuals. To this end, we recently developed a chemically defined cell culture medium that robustly expands all T cell subsets in the absence of human serum. Using a humanized mouse model, we observed that T cells expanded in the absence of human serum provided durable control of tumors, whereas T cells expanded in medium supplemented with human serum only mediated transient control of tumor growth. Importantly, our new medium effectively expanded more differentiated T cells from multiple myeloma patients in the absence of serum. These patient-derived T cells were also able to provide durable control of B cell tumors in vivo, and this long-term control of cancer was lost when T cells were expanded in the presence of serum. Thus, engineered T cells expanded in an optimized medium in the absence of serum may have improved therapeutic potential.

Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments.

T cells compete with malignant cells for limited nutrients within the solid tumor microenvironment. We found that effector memory CD4 T cells respond distinctly from other T cell subsets to limiting glucose and can maintain high levels of interferon-γ (IFN-γ) production in a nutrient-poor environment. Unlike naive (TN) or central memory T (TCM) cells, effector memory T (TEM) cells fail to upregulate fatty acid synthesis, oxidative phosphorylation, and reductive glutaminolysis in limiting glucose. Interference of fatty acid synthesis in naive T cells dramatically upregulates IFN-γ, while increasing exogenous lipids in media inhibits production of IFN-γ by all subsets, suggesting that relative ratio of fatty acid metabolism to glycolysis is a direct predictor of T cell effector activity. Together, these data suggest that effector memory T cells are programmed to have limited ability to synthesize and metabolize fatty acids, which allows them to maintain T cell function in nutrient-depleted microenvironments.

An Archaeosome-Adjuvanted Vaccine and Checkpoint Inhibitor Therapy Combination Significantly Enhances Protection from Murine Melanoma.

Archaeosomes constitute archaeal lipid vesicle vaccine adjuvants that evoke a strong CD8⁺ T cell response to antigenic cargo. Therapeutic treatment of murine B16-ovalbumin (B16-OVA) melanoma with archaeosome-OVA eliminates small subcutaneous solid tumors; however, they eventually resurge despite an increased frequency of circulating and tumor infiltrating OVA-CD8⁺ T cells. Herein, a number of different approaches were evaluated to improve responses, including dose number, interval, and the combination of vaccine with checkpoint inhibitors. Firstly, we found that tumor protection could not be enhanced by repetitive and/or delayed boosting to maximize the CD8⁺ T cell number and/or phenotype. The in vivo cytotoxicity of vaccine-induced OVA-CD8⁺ T cells was impaired in tumor-bearing mice. Additionally, tumor-infiltrating OVA-CD8⁺ T cells had an increased expression of programmed cell death protein-1 (PD-1) compared to other organ compartments, suggesting impaired function. Combination therapy of tumor-bearing mice with the vaccine archaeosome-OVA, and α-CTLA-4 administered concurrently as well as α-PD-1 and an α-PD-L1 antibody administered starting 9 days after tumor challenge given on a Q3Dx4 schedule (days 9, 12, 15 and 18), significantly enhanced survival. Following multi-combination therapy ~70% of mice had rapid tumor recession, with no detectable tumor mass after >80 days in comparison to a median survival of 17-22 days for untreated or experimental groups receiving single therapies. Overall, archaeosomes offer a powerful platform for delivering cancer antigens when used in combination with checkpoint inhibitor immunotherapies.