Immune checkpoint status and exhaustion-related phenotypes of CD8+ T cells from the tumor-draining regional lymph nodes in breast cancer.

BACKGROUND: Functional status of T cells determines the responsiveness of cancer patients to immunotherapeutic interventions. Even though T cell-mediated immunity is inaugurated in the tumor-adjacent lymph nodes, peripheral blood has been routinely sampled for testing the immunological assays. The purpose of this study is to determine the immune checkpoint molecule expression and the exhaustion-related phenotype of cytotoxic T cells in the regional lymph nodes from breast cancer patients. PATIENTS AND METHODS: Multicolor immunophenotyping was used to determine the expression of PD-1, TIM-3, LAG3, CTLA-4, CCR7, CD45RO, CD127, CD25, CXCR5, and ICOS molecules on CD3+ CD4- CD56- CD8+ cytotoxic T cells freshly obtained from the lymph nodes and the peripheral blood samples of the breast cancer patients. The results were assessed together with the clinical data. RESULTS: A population of cytotoxic T cells was noted with high PD-1 and CXCR5 expression in the lymph nodes of the breast cancer patients. Co-expression of PD-1, CXCR5, TIM-3, and ICOS indicated a follicular helper T cell (Tfh)-like, exhaustion-related immunophenotype in these cytotoxic T cells. Only a minor population with CTLA-4 and LAG3 expression was noted. The PD-1+ CXCR5+ cytotoxic T cells largely displayed CD45RO+ CCR7+ central memory markers. The amount of CXCR5-expressing PD-1- cytotoxic T cells was elevated in the lymph nodes of the patients. CONCLUSION: The regional lymph nodes of breast cancer patients harbor Tfh-like exhausted cytotoxic T lymphocytes with high PD-1 and TIM-3 checkpoint molecule expression. The immunological conditions in the regional lymph nodes should be implicated for immune checkpoint immunotherapy (ICI) of cancer.

Human memory T cell dynamics after aluminum-adjuvanted inactivated whole-virion SARS-CoV-2 vaccination.

This study evaluates the functional capacity of CD4+ and CD8+ terminally-differentiated effector (TEMRA), central memory (TCM), and effector memory (TEM) cells obtained from the volunteers vaccinated with an aluminum-adjuvanted inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac). The volunteers were followed for T cell immune responses following the termination of a randomized phase III clinical trial. Seven days and four months after the second dose of the vaccine, the memory T cell subsets were collected and stimulated by autologous monocyte-derived dendritic cells (mDCs) loaded with SARS-CoV-2 spike glycoprotein S1. Compared to the placebo group, memory T cells from the vaccinated individuals significantly proliferated in response to S1-loaded mDCs. CD4+ and CD8+ memory T cell proliferation was detected in 86% and 78% of the vaccinated individuals, respectively. More than 73% (after a short-term) and 62% (after an intermediate-term) of the vaccinated individuals harbored TCM and/or TEM cells that responded to S1-loaded mDCs by secreting IFN-γ. The expression of CD25, CD38, 4-1BB, PD-1, and CD107a indicated a modulation in the memory T cell subsets. Especially on day 120, PD-1 was upregulated on CD4+ TEMRA and TCM, and on CD8+ TEM and TCM cells; accordingly, proliferation and IFN-γ secretion capacities tended to decline after 4 months. In conclusion, the combination of inactivated whole-virion particles with aluminum adjuvants possesses capacities to induce functional T cell responses.

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors.

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20-25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reduction.

Effects of Particle Geometry for PLGA-Based Nanoparticles: Preparation and In Vitro/In Vivo Evaluation.

The physicochemical properties (size, shape, zeta potential, porosity, elasticity, etc.) of nanocarriers influence their biological behavior directly, which may result in alterations of the therapeutic outcome. Understanding the effect of shape on the cellular interaction and biodistribution of intravenously injected particles could have fundamental importance for the rational design of drug delivery systems. In the present study, spherical, rod and elliptical disk-shaped PLGA nanoparticles were developed for examining systematically their behavior in vitro and in vivo. An important finding is that the release of the encapsulated human serum albumin (HSA) was significantly higher in spherical particles compared to rod and elliptical disks, indicating that the shape can make a difference. Safety studies showed that the toxicity of PLGA nanoparticles is not shape dependent in the studied concentration range. This study has pioneering findings on comparing spherical, rod and elliptical disk-shaped PLGA nanoparticles in terms of particle size, particle size distribution, colloidal stability, morphology, drug encapsulation, drug release, safety of nanoparticles, cellular uptake and biodistribution. Nude mice bearing non-small cell lung cancer were treated with 3 differently shaped nanoparticles, and the accumulation of nanoparticles in tumor tissue and other organs was not statistically different (p > 0.05). It was found that PLGA nanoparticles with 1.00, 4.0 ± 0.5, 7.5 ± 0.5 aspect ratios did not differ on total tumor accumulation in non-small cell lung cancer.

A novel combination for the treatment of small cell lung cancer: Active targeted irinotecan and stattic co-loaded PLGA nanoparticles.

Polymeric nanoparticles are widely used drug delivery systems for cancer treatment due to their properties such as ease of passing through biological membranes, opportunity to modify drug release, specifically targeting drugs to diseased areas, and potential of reducing side effects. Here, we formulated irinotecan and Stattic co-loaded PLGA nanoparticles targeted to small cell lung cancer. Nanoparticles were successfully conjugated with CD56 antibody with a conjugation efficiency of 84.39 ± 1.01%, and characterization of formulated nanoparticles was conducted with in-vitro and in-vivo studies. Formulated particles had sizes in the range of 130-180 nm with PDI values smaller than 0.3. Encapsulation and active targeting of irinotecan and Stattic resulted in increased cytotoxicity and anti-cancer efficiency in-vitro. Furthermore, it was shown with ex-vivo biodistribution studies that conjugated nanoparticles were successfully targeted to CD56-expressing SCLC cells and distributed mainly to tumor tissue and lungs. Compliant with our hypothesis and literature, the STAT3 pathway was successfully inhibited with Stattic solution and Stattic loaded nanoparticles. Additionally, intravenous injection of conjugated co-loaded nanoparticles resulted in decreased side effects and better anti-tumor activity than individual solutions of drugs in SCLC tumor-bearing mice. These results may indicate a new treatment option for clinically aggressive small cell lung cancer.

Tailored modulation of stemness and drug resistance marker characteristics in K-Ras mutant lung cancer cells via PD-L1 gene suppression.

AIMS: We aimed to analyze the association of tumor cell-specific Programmed Cell Death Ligand 1 (PD-L1) expression with stemness markers and multi-drug resistance genes in non-small cell lung cancer. MAIN METHODS: ATP Binding Cassette Subfamily G Member 2 (ABCG2), Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), CD44, Epithelial cell adhesion molecule (EPCAM), Kruppel-like factor 4 (KLF4), MYC Proto-Oncogene (MYC), Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX2) as well as ATP Binding Cassette Subfamily C Member (ABCC) 1-6, ABCC10-12 with ATP Binding Cassette Subfamily B Member (ABCB) 1 and ABCB4-9 belongs to ABC transporters family were analyzed and their correlation with PD-L1 was evaluated using Cancer Cell Line Encyclopedia and The Cancer Genome Atlas data sets. We validated potential lung cancer stemness markers harboring ABCG2, CD44, ALDH1A1 and SOX-2, which are affected as a result of siRNA-mediated suppression of PD-L1 via flow cytometry. K-Ras downstream signaling proteins as well as multidrug resistance proteins were also investigated. KEY FINDINGS: PD-L1 was found to be positively correlated with CD44, whereas negatively correlated with ALDH1A1 (Pearson r = 0.44, r = -0.48; respectively) in 45 K-Ras mutated NSCLC cells based on CCLE database. While ABCC5 was dominantly decreased in K-Ras mutant lung cancer cells affected by PD-L1 gene suppression, expression changes were observed in the activation of distinct signaling pathways in a cell line-dependent manner. SIGNIFICANCE: The evaluation of markers in lung adenocarcinoma with different types of K-Ras mutations in terms of both stemness and drug resistance markers will contribute to the development of personalized immunotherapy regimens.

Memory T cell responses in seronegative older adults following SARS-CoV-2 vaccination.

Generating memory T cell responses besides humoral immune responses is essential when it comes to the efficacy of a vaccine. In this study, the presence of memory T cell responses after aluminum-adjuvanted inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac) in seronegative and seropositive elderly individuals were examined. CD4+ and CD8+ memory T cell proliferation and IFN-γ production capacities were evaluated. Additionally, clinical frailty scale (CFS) and FRAIL scales of the individuals were scored. CD4+ memory T cell responses more prominent than CD8+ memory T cells. In seronegative individuals, 80% of them had memory CD4+ and IFN-γ, whereas 50% of them had memory CD4+ and all of them had IFN-γ responses. Additionally, 40% of seronegative patients and 50% of seropositive patients had memory CD8+ responses. To sum up, humoral immune responses are not associated with memory T cell responses, and in seronegative individuals, memory T cell responses can be detected.

Small cell lung cancer stem cells display mesenchymal properties and exploit immune checkpoint pathways in activated cytotoxic T lymphocytes.

Small cell lung cancer (SCLC) is an aggressive tumor type with early dissemination and distant metastasis capacity. Even though optimal chemotherapy responses are observed initially in many patients, therapy resistance is almost inevitable. Accordingly, SCLC has been regarded as an archetype for cancer stem cell (CSC) dynamics. To determine the immune-modulatory influence of CSC in SCLC, this study focused on the characterization of CD44+CD90+ CSC-like subpopulations in SCLC. These cells displayed mesenchymal properties, differentiated into different lineages and further contributed to CD8+ cytotoxic T lymphocytes (CTL) responses. The interaction between CD44+CD90+ CSC-like cells and T cells led to the upregulation of checkpoint molecules PD-1, CTLA-4, TIM-3, and LAG3. In the patient-derived lymph nodes, CD44+ SCLC metastases were also observed with T cells expressing PD-1, TIM-3, or LAG3. Proliferation and IFN-γ expression capacity of TIM-3 and LAG3 co-expressing CTLs are adversely affected over long-time co-culture with CD44+CD90+ CSC-like cells. Moreover, especially through IFN-γ secreted by the T cells, the CSC-like SCLC cells highly expressed PD-L1 and PD-L2. Upon a second encounter with immune-experienced, IFN-γ-stimulated CSC-like SCLC cells, both cytotoxic and proliferation capacities of T cells were hampered. In conclusion, our data provide evidence for the superior potential of the SCLC cells with stem-like and mesenchymal properties to gain immune regulatory capacities and cope with cytotoxic T cell responses. With their high metastatic and immune-modulatory assets, the CSC subpopulation in SCLC may serve as a preferential target for checkpoint blockade immunotherapy .

Functional responsiveness of memory T cells from COVID-19 patients.

The presence of memory T cells in COVID-19 patients has been acknowledged, however the functional potency of memory responses is critical for protection. In this study, naïve, effector, effector memory, and central memory CD4+ and CD8+ T cells obtained from the COVID-19 survivors were re-exposed to autologous monocyte-derived DCs that were loaded with SARS-CoV-2 spike glycoprotein S1. Proliferation capacity, CD25, 4-1BB, and PD-1 expression, and IFN-γ, IL-6, granzyme, granulysin, and FasL secretion were enhanced in CD4+ and CD8+ effector memory and central memory T cells. Albeit being at heterogeneous levels, the memory T cells from the individuals with COVID-19 history possess functional capacities to reinvigorate anti-viral immunity against SARS-CoV-2.

Human splenic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are strategically located immune regulatory cells in cancer.

In contrast to the mouse, functional assets of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in the human spleen remain to be better elucidated. Here, we report that the spleen in gastric and pancreatic cancer adopts an immune regulatory character, harbors excessive amount of PMN-MDSC, and anatomically enables their interaction with T cells. Compared to the peripheral blood, the spleen from cancer patients contained significantly higher levels of low-density PMN-MDSC, but not early-stage MDSC (e-MDSC) and monocytic-MDSC (M-MDSC). Low-density fraction of polymorphonuclear (PMN) cells was enriched in immature myeloid cells and displayed higher levels of CD10, CD16, and ROS than their blood-derived counterparts. They were also positive for PD-L1, LOX-1, and pSTAT3. The white pulp and periarteriolar lymphoid sheath (PALS) were strategically surrounded by PMN cells that were in contact with T cells. Unlike those from the blood, both low-density and normal-density PMN cells from the human spleen suppressed T cell proliferation and IFN-γ production. Independent of clinical grade, high PMN-MDSC percentages were associated with decreased survival in gastric cancer. In summary, our results outline the immune regulatory role of the spleen in cancer where neutrophils acquire MDSC functions and feasibly interact with T cells.

Aggregation of chitosan nanoparticles in cell culture: Reasons and resolutions.

Nanoparticles are promising drug delivery systems which are flexible for targeting specific tissues to reduce therapeutic doses and minimize side effects. Nanoparticles should be maintained with high stability and uniformity; however, aggregation is a major challenge which commonly impairs stability and efficacy of nanocarriers. In this study, we revisited the factors that influence the stability of chitosan (Protasan™ UP CL113) nanoparticles prepared with ionotropic gelation, widely recognized to be prone to aggregation, and proposed a model to overcome the negative influence of aggregation while testing in vitro efficacy. Decrease in pH due to cell proliferation, 37 °C cell culture temperature, serum in culture media, and incubation time were considered as factors causing chitosan nanoparticles' aggregation which deteriorates cell culture assay readouts, increases optical density values and leads to false-positive results. Size and stability studies were not sufficient to avoid misleading results in cell culture. The chitosan nanoparticle aggregation was almost inevitable under standard culture conditions; nevertheless, the removal of nanoparticles before aggregation but after an incubation period long enough for efficient cellular uptake was determined as a feasible and inexpensive method for testing the in vitro efficacy of polymeric nanoformulations. This approach was used with blank and gemcitabine-loaded chitosan nanoparticles on pancreatic cancer cells and proved to be useful for reliable cytotoxicity results.

Protocol to assess the suppression of T-cell proliferation by human MDSC.

Inhibition of T-cell proliferation is the most common approach to assess human myeloid-derived suppressor cell (MDSC) functions. However, diverse methodologies hinder the comparison of results obtained in different laboratories. In this chapter, we present a T-cell proliferation assay procedure based on allogeneic MDSC and T-cells that is potentially suitable to multi-center studies. The T-cells are isolated from non-cancerous donors and frozen for later use in different research groups. We observed that pure thawed T-cells showed poor proliferative capacities. To retain proliferation, T-cell-autologous mature dendritic cells are supplemented after thawing. MDSC are isolated from clinical samples and represent the sole variant between assays. Flow cytometry is used to assess T-cell proliferation by the dilution of a tracking dye.

Development and in vitro evaluation of a new adjuvant system containing Salmonella Typhi porins and chitosan.

In order to improve the immunogenicity of the highly purified vaccine antigens, addition of an adjuvant to formulation, without affecting the safety of the vaccine, has been the key aim of the vaccine formulators. In recent years, adjuvants which are composed of a delivery system and immunopotentiators have been preferred to induce potent immune responses. In this study, we have combined Salmonella Typhi porins and chitosan to develop a new adjuvant system to enhance the immunogenicity of the highly purified antigens. Cationic gels, microparticle (1.69 ± 0.01 µm) and nanoparticles (337.7 ± 1.7 nm) based on chitosan were prepared with high loading efficiency of porins. Cellular uptake was examined by confocal laser scanning microscopy, and the macrophage activation was investigated by measuring the surface marker as well as the cytokine release in vitro in J774A.1 macrophage murine cells. Porins alone were not taken up by the macrophage cells whereas in combination with chitosan a significant uptake was obtained. Porins-chitosan combination systems were found to induce CD80, CD86 and MHC-II expressions at different levels by different formulations depending on the particle size. Similarly, TNF-α and IL-6 levels were found to increase with porins-chitosan combination. Our results demonstrated that combination of porins with chitosan as a particulate system exerts enhanced adjuvant effect, suggesting a promising adjuvant system for subunit vaccines with combined immunostimulating activity.

PD-L2+ wound zone macrophage-like cells display M1/M2-mixed activation and restrain the effector Th1 responses.

Depending on the microenvironment conditions, macrophages display phenotypic and functional heterogeneity. This study characterized the programmed cell death-ligand 2 (PD-L2)-expressing macrophage-like cells drained from surgical wound zones, and investigated their influence on helper T (Th) cell responses. Although all CD14+ myeloid cells possessed macrophage-like features, CD206+ and CD163+ cells constituted a specific subpopulation with high PD-L2 expression. There was a modest correlation between the PD-L2 levels on CD206+ macrophages and the amount of interferon (IFN)-γ in the drainage fluid. The adhesion-independent macrophages simultaneously presented both classically-activated M1 and alternatively-activated M2 characteristics. CD206+ and PD-L2+ cells were identified with high granularity and size, expressed arginase-1 and costimulatory molecules, had enhanced phagocytic activity and produced reactive oxygen species. The genes associated with macrophage differentiation (MERTK, AXL and TYRO3) were also upregulated. These cells provided costimulation to Th cells; yet, when PD-L2 was blocked, T-cell proliferation and IFNγ production were enhanced. Under defined conditions devoid of activation stimuli and matrix adhesion, ex vivo-generated monocyte-derived macrophages displayed limited capacity to stimulate T cells. Upon exposure to IFNγ, they significantly upregulated programmed death 1 ligands, especially PD-L2. These cells did not completely abrogate T-cell differentiation; however, PD-L2 checkpoint blockade restored Th1 proliferation and secretion of interleukin-2, tumor necrosis factor-α and IFNγ. In conclusion, upregulation of PD-L2 on the wound zone macrophages may constitute a negative feedback loop that restrains the Th1 effector responses and avoids exacerbation of inflammation during tissue healing.

Myeloid maturation potentiates STAT3-mediated atypical IFN-γ signaling and upregulation of PD-1 ligands in AML and MDS.

Interferon (IFN)-γ is the major mediator of anti-tumor immune responses; nevertheless, cancer cells use intrigue strategies to alter IFN-γ signaling and avoid elimination. Understanding the immune regulatory mechanisms employed by acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells upon exposure to IFN-γ is critical for development of immunotherapy and checkpoint blockade therapy approaches. This study aims to explore the influence of myeloid maturation on IFN-γ-induced PD-L1 and PD-L2 expression and on pro-leukemogenic transcription factor STAT3 signaling in AML and MDS. Stimulation of myeloid blasts' maturation by all-trans retinoic acid (ATRA) or 1α,25-dihydroxyvitamin D3 (vitamin D) increased the CD11b+ fraction that expressed PD-1 ligands in response to IFN-γ. Intriguingly, STAT3 pathway was potently induced by IFN-γ and strengthened upon prolonged exposure. Nonetheless, STAT3-mediated atypical IFN-γ signaling appeared as a negligible factor for PD-L1 and PD-L2 expression. These negative influences of IFN-γ could be alleviated by a small-molecule inhibitor of STAT3, stattic, which also inhibited the upregulation of PD-L1. In conclusion, induction of myeloid maturation enhances the responsiveness of AML and MDS cells to IFN-γ. However, these malignant myeloid cells can exploit both STAT3 pathway and PD-1 ligands to survive IFN-γ-mediated immunity and maintain secondary immune resistance.

A Novel Missense LIG4 Mutation in a Patient With a Phenotype Mimicking Behçet's Disease.

DNA ligase IV (LIG4) syndrome is a rare autosomal recessive disorder, manifesting with variable immune deficiency, growth failure, predisposition to malignancy, and cellular sensitivity to ionizing radiation. The facial features are subtle and variable, as well. Herein, we described an 18-year-old boy, the first child of consanguineous parents who presented with Behçet's disease (BD)-like phenotype, developmental delay, and dysembryoplastic neuroepithelial tumor (DNET). Whole-exome sequencing revealed a homozygous p.Arg871His (c.2612G > A) mutation in LIG4. To date, 35 cases have been reported with LIG4 syndrome. Peripheral blood mononuclear cells of the patient displayed notable sensitivity to ionizing radiation. Flow cytometric annexin V-propidium iodide (PI) and eFluor670 proliferation assays showed accelerated radiation-induced apoptosis and diminished proliferation, respectively. To our knowledge, this is the first case presenting with a BD-like phenotype. This case provides further evidence that rare monogenic defects could be the underlying cause of atypical presentations of some well-described disorders. Moreover, this clinical report further expands the phenotypical spectrum of LIG4 deficiency.

Serial immunological parameters in a phase II trial of exemestane and low-dose oral cyclophosphamide in advanced hormone receptor-positive breast cancer.

BACKGROUND AND PURPOSE: Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant challenge. Prior studies have shown that low-dose oral cyclophosphamide can transiently deplete regulatory T cells (Tregs) and improve anti-tumor immunity. We investigated the combination of exemestane with cyclophosphamide in patients with advanced HR-positive breast cancer and assessed changes in circulating immune cell subsets. METHODS: This was a single-arm phase II trial of exemestane with cyclophosphamide in patients with metastatic HR-positive/HER2-negative breast cancer who had progressed on prior endocrine therapy (ClinicalTrials.gov: NCT01963481). Primary endpoint was progression-free survival (PFS) at 3 months (RECIST 1.1). Secondary objectives included median PFS, objective response rate, duration of response, and safety. Circulating Tregs (FOXP3+Helios+) and other immune cell subsets were monitored during treatment and compared with healthy controls. RESULTS: Twenty-three patients were enrolled. Treatment was well tolerated, without grade 4/5 toxicities. Objective responses were seen in 6/23 patients (26.1%; 95% CI 10.2-48.4%) and were durable (median 11.6 months). Three-month PFS rate was 50.1% (95% CI 33.0-76.0%); median PFS was 4.23 months (95% CI 2.8-11.7). No treatment-related decrease in Tregs was observed. However, elevated baseline levels of Naïve Tregs [greater than 2.5 (the median of the naïve Tregs)] were associated with relative risk of disease progression or death [hazard ratio 11.46 (95% CI 2.32-56.5)]. In addition, the baseline levels of Naïve Tregs (adj-p = 0.04), Memory Tregs (adj-p = 0.003), CD4 + Central Memory T cells (adj-p = 0.0004), PD-1 + CD4 + Central Memory T cells (adj-p = 0.008), and PD-1 + CD4 + Effector Memory T cells (adj-p = 0.009) were significantly greater in the patients than in the healthy controls; the baseline levels of  %CD4 + Naïve T cells (adj-p = 0.0004) were significantly lower in patients compared with healthy controls (n = 40). CONCLUSION: Treg depletion was not observed with low-dose cyclophosphamide when assessed by the specific marker FOXP3 + Helios +; however, baseline naïve Tregs were associated with 3-month PFS. Exemestane/cyclophosphamide combination had favorable safety profile with evidence of clinical activity in heavily pretreated patients.

Functional exhaustion of CD4+ T cells induced by co-stimulatory signals from myeloid leukaemia cells.

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T-cell exhaustion. T-cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co-stimulatory molecules to T-cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia-derived co-stimulatory signals on CD4+ T helper (Th) cell exhaustion, which may limit anti-tumour immunity. Here, CD86 and inducible T-cell co-stimulator ligand (ICOS-LG) co-stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co-cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro-generated exhausted Th cells were defined by up-regulation of programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG3) and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) inhibitory receptors. They were reluctant to proliferate upon re-stimulation and produced reduced amounts of interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Nonetheless, IL-2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co-stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD-1, TIM-3 and/or LAG3 was identified together with CD86+ and/or ICOS-LG+ myeloid blasts. Collectively, co-stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.