Human Lymphocytes Research Articles

Specific imaging of CD8 + T-Cell dynamics with a nanobody radiotracer against human CD8β.

While immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8+ T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8+ T-cell dynamics. Nanobodies targeting human CD8β were generated by llama immunizations and subsequent biopanning. The lead anti-human CD8β nanobody was characterized on binding, specificity, stability and toxicity. The lead nanobody was labeled with technetium-99m, gallium-68 and copper-64 for non-invasive imaging of human T-cell lymphomas and CD8+ T cells in human CD8 transgenic mice and non-human primates by SPECT/CT or PET/CT. Repeated imaging of CD8+ T cells in MC38 tumor-bearing mice allowed visualization of CD8+ T-cell dynamics. The nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8+ T cells were non-invasively visualized by SPECT and PET imaging in naïve and tumor-bearing mice and in naïve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8+ T cells and/or faster in vivo pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8+ T-cell dynamics already at early timepoints. This study describes the development of a more specific human CD8+ T-cell-targeting immunotracer, allowing follow-up of immunotherapy responses by non-invasive imaging of human CD8+ T-cell dynamics.

Prospects and Methods for Studying the Proliferative Capacity of the Human Peripheral Blood Lymphocyte Subpopulations in Radiation Medicine

Prospects and Methods for Studying the Proliferative Capacity of the Human Peripheral Blood Lymphocyte Subpopulations in Radiation Medicine

CLL-042 Identification and Diagnosis of Possible Mutations in Human p53 Lymphocytes

CLL-042 Identification and Diagnosis of Possible Mutations in Human p53 Lymphocytes

Plasma vitamin D levels are correlated with the pathogenesis of human T-cell leukemia virus type 1-associated diseases.

The active form of vitamin D (VD) exerts hormonal effects by regulating the expression of genes involved in T-cell activity, cell differentiation, and proliferation. Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of life-threatening diseases, adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM). Among ATL patients, hypercalcemia is one of the most serious complications due to bone resorption. In this study, wild-type mice administered UV-irradiated HTLV-1-infected cells showed up to 47% decrease of plasma VD level compared with untreated mice. To clarify the effect of HTLV-1 on plasma VD level, 315 samples registered in nationwide cohort study on ATL onset were measured. The VD level in HAM (14.98 ± 8.5 ng/mL) was significantly lower than those in asymptomatic carriers and ATL (p < 0.05). Upon comparing the VD levels in ATL stratified by disease subtypes, acute ATL showed a lower level (15.81 ± 12.0 ng/mL) than chronic and smoldering types (p < 0.05). In the longitudinal observation, VD levels were significantly higher in untreated spontaneous remission cases than in ATL progression cases, in which the VD levels decreased approximately 40% after onset. In cases of relapse after transplantation, the plasma VD level dropped to 38.7% of the pre-relapse level, while in cases of complete remission, the VD level increased with improvement of the performance status. Taken together, these results suggest that plasma VD level is a potential indicator for the onset and relapse of HTLV-1-associated diseases.

Analysis of Human Papillomavirus-Associated Cervical Cancer Differentially Expressed Genes and Identification of Prognostic Factors using Integrated Bioinformatics Approaches.

Human papillomavirus (HPV)-induced cervical cancer progresses through a series of steps. Despite our limited understanding of the mechanisms driving this progression, identifying the key genes involved could significantly improve early detection and treatment. Two gene expression profiles of GSE9750 and GSE6791, which included cervical cancer HPV-positive and -negative samples, were evaluated using the R limma package with established cut-off criteria of P value < 0.05 and | fold change| ≥ 1. KEGG pathway enrichment was performed to identify potential pathways. Weighted gene co-expression network analysis (WGCNA) was used to discover co-expressed gene modules and trait-module connections. Considering the defined criteria, 115 differentially expressed genes (DEGs) were identified. The DEG's KEGG pathway enrichment analysis revealed enrichment in highly relevant pathways to the HPV infection, including cell cycle, viral carcinogenesis, autophagy-animal, Epstein-Barr virus infection, human T-cell leukemia virus 1 infection, and microRNAs in cancer. WGCNA results in 13 co-expression modules, and the magenta module is identified with significant relations to HPV, cervical cancer stage, and metastasis traits. The survival analysis identified BEX1 and CDC45 as potential prognostic factors in HPV-associated cervical cancer. The innovation of our work lies in identifying essential genes associated with the multi-step process of cervical carcinogenesis. In fact, the current study has the potential to give a distinct viewpoint on the molecular pathways linked to cervical cancer. Considering the potential importance of the hub genes, we recommend conducting in-depth wet lab research to determine their impact on the biological mechanisms of cervical cancer.

Sex differences in constructing dose-response calibration curves for micronuclei using cytokinesis-blocked micronucleus assay for radiation biological dosimetry in the Iranian population

Biological dosimetry, using chromosome damage biomarkers, can be considered as a key measure for radiation overexposure assessment. Therefore, for accurate dose estimation through biodosimetry, it's imperative for each biological dosimetry laboratory to establish its own specific dose-response calibration curve. In this research, the cytokinesis-blocked micronucleus (CBMN) assay was utilized to determine the frequencies of micronuclei (MN) per binucleated cell in human peripheral blood lymphocytes exposed to x-ray radiation using a LINAC (6 MV) at doses up to 4 Gy. The aim was to establish an in vitro dose-response calibration curve in our laboratory for the Iranian demographic by analyzing blood samples of ten participants (5 males and 5 females) through CABAS and Dose Estimate software. Our findings indicate an over-dispersion of the Poisson distribution in the pooled data across both sexes, coupled with a linear-quadratic increase in MN yield with dose which was particularly pronounced in the female group. The constructed dose-response curves for micronuclei yield are represented by equations Y= (0.0102 ± 0.0016) +(0.0296 ± 0.0054) D+(0.0232 ± 0.0017) D2 and Y= (0.0084 ± 0.0010) +(0.0212 ± 0.0034) D+(0.0160 ± 0.0011) D2 for the female groups, respectively. The alpha and beta coefficients, derived from the Dose Estimate software for each male and female group, were closely comparable with those obtained from the CABAS program and previous studies. The analysis of statistical parameters such as the Weighted Chi Squared (χ2) and p-value suggests that using distinct curves for each sex, rather than a unified biodosimetry formula for pooled data, ensures more accurate radiation dose estimates. Consequently, the findings of this study provide us with the assurance to utilize the derived calibration curve of MN for upcoming biological dosimetry needs in Iran. However, to enhance the reliability of results, it's essential to use biodosimetry with diverse assays and consider all clinical and physical parameters, given the limitations of cytogenetic assays.

Immunophenotypic, genetic, and clinical characterization of adult T-cell leukemia/lymphoma: A single tertiary care center experience in the United States.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive mature T-cell neoplasm caused by human T-cell lymphotropic virus type 1 (HTLV-1). Its most common immunophenotype is CD4+/CD7-/CD25+, although unusual immunophenotypes can occur and may lead to misdiagnosis. The immunophenotypes, cytogenetics, molecular features, clinical presentations, treatment, and prognosis of 131 patients with ATLL were retrospectively studied in a large tertiary medical center in the United States. All cases showed loss of CD7 expression. While 82.4% of cases demonstrated CD4+, 17.6% exhibited unusual phenotypes, including CD4+/CD8+ (6.9%), CD4-/CD8- (2.3%), CD5- (3.1%), CD2-, and CD3-. The most common cytogenetics abnormalities included polysomy 3 (34.6%), translocation 1 (23.1%), and abnormalities found on chromosome 11 (30.8%) and chromosome 14 (26.9%). The common gene mutations identified by the next-generation sequencing study were TP53 (16.7%), TBL1XR1 (16.7%), EP300 (14.3%), and NOTCH1 (14.3%). TBL1XR1 mutation is associated with genetic instabilities. There was no significant difference between the clinical presentations of these 2 groups. Adult T-cell leukemia/lymphoma exhibits versatile immunophenotypic, cytogenetic, and molecular features. Simultaneous involvement of blood, lymph nodes, and other organs, along with hypercalcemia in a patient from an endemic area, necessitates HTLV-1 testing to avoid underdiagnosis of this dismal disease that might need aggressive chemotherapy followed by bone marrow transplant.

Urodynamic findings and vibegron effects on neurogenic lower urinary tract dysfunction caused by human T-cell leukemia virus type I-associated myelopathy/tropical spastic paraparesis.

Human T-cell lymphotropic virus type 1 infection can cause HTLV-1-associated myelopathy/tropical spastic paraparesis, characterized by spastic paralysis of both lower limbs. More than 90% of HAM/TSP patients show lower urinary tract symptoms and dysfunction. A 27-year-old woman diagnosed with HAM/TSP presented with overactive bladder. A urodynamic study revealed detrusor overactivity. OAB remained despite improvements in spastic paralysis of the lower limbs after starting prednisolone. The addition of solifenacin likewise failed to improve OAB or DO in this patient. Switching from solifenacin to vibegron improved OAB symptoms. In patients for whom OAB and DO do not improve with steroid treatment and solifenacin, treatment with vibegron may improve OAB symptoms.

Biological variability of human intraepithelial lymphocytes throughout the human gastrointestinal tract in health and coeliac disease.

Intraepithelial lymphocytes are the first line of defence of the human intestinal immune system. Besides, their composition is altered on patients with coeliac disease (CD), so they are considered as biomarkers with utility on their diagnose and/or monitoring. Our aim is to address their variability through the human gastrointestinal tract in health and characterized them in further depth in the coeliac duodenum. Intraepithelial lymphocytes were isolated from human gastric, duodenal, ileal and colonic biopsies, then stained with specific antibodies and acquired by flow cytometry. Our results confirmed that the profile of Intraepithelial lymphocytes change through the length of the human gastrointestinal tract. Besides and given the central role that Interleukin-15 (IL-15) elicits on CD pathogenesis; we also assessed the expression of its receptor revealing that there was virtually no functional IL-15 receptor on duodenal Intraepithelial lymphocytes. Nevertheless and contrary to our expectations, the active IL-15 receptor was not increased either on Intraepithelial lymphocytes from CD patients. IL-15 might require additional stimulus to activate intraepithelial lymphocytes. These findings may provide novel tools to aid on a CD diagnosis and/or monitoring, at the time that provide the bases to perform functional studies in order of getting a deeper insight in the specific function that Intraepithelial lymphocytes elicit on CD pathogenesis.

The Injected Plasma of Myasthenia Gravis Patient with A Low T-reg Level Caused Clinical Myasthenic Syndromes in Swiss-Webster Mice

BACKGROUND: Myasthenia gravis (MG) is a rare autoimmune disease affecting neuromuscular junction involvement. The finding that T-reg level in MG patients was lower than that in normal persons leads to the idea that the primary pathology of the disease is T-reg dependent. The T-reg level of MG patients seems to be decreasing compared to that of normal persons. The study was conducted to observe the contribution of T-reg level in plasma injected into Swiss-Webster mice to develop clinically and pathologically myasthenic syndromes.METHODS: Swiss-Webster mice were grouped into three groups: the groups received plasma with normal, low, and high T-reg levels, respectively. The T-reg levels of the mice were measured with flow cytometry analysis and a human regulatory T-cell cocktail for T-cell surface cell marker. The motor function, interleukin (IL)-2, interferon (IFN)-γ, and thymus weight of mice were measured after the injection. Histopathological examination was performed to analyze mice’s muscles and thymus.RESULTS: The result identified that the motor function (2-week treatment group: p=0.021 and 3-week treatment group: p=0.032) and muscle width (p=0.014, p=0.032 and p≤0.001) were significantly lower in the low T-reg level plasma group compared to control and high T-reg level plasma groups. The thymus showed an increase in weight without an increase in the cortex-medulla ratio of the thymus, indicating hyperplasia. Both IL-2 and IFN-γ levels were lower in the low and high T-reg level groups compared with the control group, indicating the autoimmune process.CONCLUSION: Low T-reg level was associated with lower motor function, muscle width, increased thymus weight, as well as lower IL-2 and IFN-γ levels. T-reg level contributed to clinical myasthenic syndromes but not pathological findings. This research method is expected to be a basis for the development of animal models with Swiss-Webster mice.KEYWORDS: animal model, Myasthenia gravis, Swiss-Webster mice

Expansion of human γδ T cells in periphery: Lessons learned from development, infections, and compromised thymic function.

γδ T cells predominantly develop in the fetal period. Post birth they respond swiftly to environmental insults, pathogens and tumors, especially when other immune effector cells are less ready to function. Most of our understanding of γδ T-cell development, peripheral adaptation, and function derives from murine studies. The recent advancement of immunological methods allows now to decipher human γδ T-cell biology in patient cohorts and tissue samples, and to manipulate them using in vitro systems. In this review, we summarize γδ T-cell development in the human thymus, their functional adaptation to the microbial environment from birth until old age, and their capacity to expand and fill up the peripheral niche under conditions of perturbations of conventional T-cell development.

Prognostic Impact of Human Lymphocyte Antigen (HLA) Class I Expression in Patients With Breast Cancer.

Various biomarkers are utilized in the field of breast cancer. Human lymphocyte antigen (HLA) class I molecules have a critical role in cancer immune surveillance. Therefore, this study aimed to assess the HLA class I expression and analyze the correlation with clinicopathologic factors in breast cancer. We investigated the clinical pathology archives of 150 consecutive patients with breast cancer who underwent a curative operation at the Sapporo Medical University, Japan, from January 2012 to December 2014. Immunohistochemical staining was used to evaluate HLA class I expression and CD8-positive T cell infiltration. The Pearson χ2 test was used to assess HLA class I expression level and clinicopathological parameters. The Kaplan-Meier method was used to evaluate survival and the log-rank test to analyze the differences between survival curves. Patients with dull/negative HLA class I had significantly poor disease-free survival (DFS) compared with those with positive HLA class I (p=0.0073). Univariate analyses revealed that pT, pN, positive lymphatic invasion, and dull/negative HLA class I were significantly associated with DFS. Multivariate analyses revealed dull/negative HLA class I as an independent poor prognostic factor (hazard ratio=2.75, 95% confidence interval=1.30-5.80, p=0.008). HLA class I expression level may have a very sensitive prognostic effect on patients with breast cancer.

A Systematical Review on ART Use in HTLV Infection: Clinical, Virological, and Immunological Outcomes.

Human T-cell lymphotropic virus (HTLV) infection affects over ten million people worldwide, but there is no effective treatment so far. This review describes the virological, immunological, and clinical outcomes of antiretroviral therapy (ART) in people with HTLV infection. This systematic review followed PRISMA reporting guidelines and was registered in PROSPERO: CRD42022350076. The Newcastle-Ottawa Scale, adapted for cross-sectional studies, and Rob-2 were used to assess the methodological quality of these studies. Systematic searches were conducted in the Medline (PubMed), Scopus (Elsevier), Cochrane Library, and Web of Science (Clarivate Analytics) databases. We retrieved data from eight methodologically diverse articles on treatment of patients infected by HTLV-1 or HTLV-2 alone, or coinfected by HIV-1, who received Raltegravir, Tenofovir, Lamivudine, or Zidovudine. The proviral load decreased in three out of seven studies over 4 to 48 weeks of antiretroviral use. Cellular immune response (CD4, CD8, CD25, CD69, and CD71 cells) was evaluated in six studies. While no significant clinical improvement was observed, all studies reported clinical stability during treatment. Despite the demonstrated antiviral activity of ART, in vitro, clinical improvement was not proven. Most studies showed disease stability during ART use, suggesting potential clinical benefits. There is a need of larger, well-controlled trials to define the role of ART in the treatment of HTLV infection.

Peptides with immunomodulatory properties (Imuno TF®) increase the frequency of the CD8+ T cell population in vitro

Imuno TF® is a food supplement composed of oligo- and polypeptides with roles in the immune system. It has been previously demonstrated that Imuno TF® positively regulated Th1 cytokines while decreasing Th2 cytokines (reduced secretion of IL-10 and increased secretion of IL-6 and TNF-α). Here we aimed to investigate the actions of Imuno TF® on the frequency of stimulated CD8+ and CD4+ T-cell populations and their cytokine productions. Human lymphocyte cultures were used for that, and IL-2, IFN-γ, IL-4, IL-5, IL-7, IL-13, and IL-35 were quantified by ELISA and RT qPCR. The frequency of CD4+ and CD8+ populations was investigated through flow cytometry. We observed an increased frequency of CD8+ T-cells after the combined stimulation of cells with Imuno TF® and ConA compared to controls. No differences were observed regarding the frequency of CD4+ T-cells. In addition, a significant increase in IL-2, IL-7, and IFN-γ levels was observed, while IL-4, IL-5, and IL-13 presented reduced levels. No alterations were observed in IL-35 levels. Our results suggest that the Imuno TF® can potentially increase the CD8+ T-cell population by positively regulating cytokines associated with Th1 response and increased IL-7 levels while reducing Th2 cytokine-mediated immune responses.

Identification and Diagnosis of Possible Mutations in Human p53 Lymphocytes

Identification and Diagnosis of Possible Mutations in Human p53 Lymphocytes

A novel high-performance rapid screening test for the detection of total HTLV-I and HTLV-II antibodies in HTLV-I/II infected patients

Rapid diagnosis of human T-cell lymphotropic virus (HTLV) type-I and -II infections are essential for timely and cost-effective disease interventions. MP Diagnostics ASSURE HTLV-I/II Rapid Test was developed for the rapid detection of anti-HTLV-I/II antibodies in patients’ serum, plasma, and whole blood specimens. ASSURE HTLV-I/II Rapid Test employed MP Biomedicals’ proprietary HTLV-I/II Trifusion recombinant antigen conjugated with gold nanoparticles and HTLV-I / HTLV-II recombinant antigens immobilized on the nitrocellulose membrane to detect total HTLV-I and HTLV-II antibodies. The overall performance of the ASSURE HTLV-I/II Rapid Test was found to be 99.42% sensitivity (95% Confidence Interval, 98.32–99.88%) and 100% specificity (95% Confidence Interval, 99.58–100.00%) in the tested clinical samples, including a total of 518 HTLV-I/II positive specimens (396 HTLV-I infection, 97 HTLV-II infection and 25 HTLV-I/II dual infection) and 872 HTLV negative clinical specimens consisting of 691 healthy donor samples, 116 potentially cross-reactive samples, and 65 samples with interfering substances. The ASSURE HTLV-I/II Rapid Test can effectively be deployed as a screening tool in any prevalence studies, blood banks or organ transplant centres.

B-cell depletion limits HTLV-1-infected T-cell expansion and ameliorate HTLV-1-associated myelopathy.

Human T-cell leukemia virus type 1-associated myelopathy (HAM) is a chronic, progressive, inflammatory disease with unclear pathogenesis and no effective treatments. We aimed to investigate a novel mechanistic theory and treat HAM patients with rituximab, which can deplete CD20+ B lymphocytes in circulation. Single-cell RNA sequencing (scRNA-seq) data was analyzed to identify HTLV-1-associated B cells and their effect on Tcells. An observational analysis of our HAM cohort was conducted to elucidate changes in the immunological microenvironment of these patients. Peripheral blood mononuclear cells (PBMC) from HAM patients were isolated to explore the efficacy of B cell depletion in vitro. To assess the effect of B-cell depletion on HAM patients, eligible participants in our cohort received rituximab therapy (NCT04004819). ScRNA-seq results suggest a significant effect of HTLV-1-associated B cells on Tcells. Additionally, HTLV-1 was found to infect B cells and depletion of B cells inhibited the proliferation of Tcells. Number of B cells in HAM patients had positive correlation with the proviral load and infected cell counts. Depletion of B cells led to a reduction in HTLV-1 proviral load in vitro. Furthermore, in clinical trial, 14 HAM patients were enrolled. Three patients (21.4%) who received rituximab failed to achieve remission, compared to 24 (85.7%) patients received any other therapy that failed to achieve remission. With a low level of circulating B cells, the proportion of Ki67-positive cells in CD4+ Tcells fell. This study provided evidence that depleting B-lymphocytes is an innovative strategy for treating patients with HAM and broadens the understanding of the role of B cells in infectious immunity.

Bleomycin-induced chromosomal aberrations in Epstein-Barr virus-transformed human lymphoblastoid cells

We have evaluated the induction of complete (i.e., without open ends) and incomplete (i.e., with non-rejoined or open ends) chromosomal aberrations by the radiomimetic antibiotic bleomycin (BLM) in human lymphoblastoid cells immortalized with the Epstein-Barr virus (EBV). An EBV-induced lymphoblastoid cell line (T-37) was exposed to BLM (10–200 µg/mL) for 2 h at 37ºC, and chromosomal aberrations were analyzed 24 h after treatment, using PNA-FISH with pan-telomeric and pan-centromeric probes. Both complete (multicentrics, rings, compound acentric fragments, and interstitial deletions) and incomplete (incomplete chromosomes or IC, and terminal acentric fragments or TAF) chromosomal aberrations increased significantly in BLM-exposed cells, although the concentration-response relationship was non-linear. Of the acentric fragments (ace) induced by BLM, 40 % were compound fragments (CF, ace +/+). TAF (ace, +/-) and interstitial fragments (IAF, ace -/-) were induced at similar frequencies (30 %). 230 ICE were induced by BLM, of which 52 % were IC and 48 % TAF. The average ratio between total incomplete chromosome elements (ICE) and multicentrics was 1.52. These findings suggest that human lymphoblastoid cells exhibit less repair capacity than human lymphocytes, with respect to BLM-induced ICE, and that chromosomal incompleteness is a common event following exposure of these cells to BLM.

Global transcriptomic network analysis of the crosstalk between microbiota and cancer-related cells in the oral-gut-lung axis.

The diagnosis and treatment of lung, colon, and gastric cancer through the histologic characteristics and genomic biomarkers have not had a strong impact on the mortality rates of the top three global causes of death by cancer. Twenty-five transcriptomic analyses (10 lung cancer, 10 gastric cancer, and 5 colon cancer datasets) followed our own bioinformatic pipeline based on the utilization of specialized libraries from the R language and DAVID´s gene enrichment analyses to identify a regulatory metafirm network of transcription factors and target genes common in every type of cancer, with experimental evidence that supports its relationship with the unlocking of cell phenotypic plasticity for the acquisition of the hallmarks of cancer during the tumoral process. The network's regulatory functional and signaling pathways might depend on the constant crosstalk with the microbiome network established in the oral-gut-lung axis. The global transcriptomic network analysis highlighted the impact of transcription factors (SOX4, TCF3, TEAD4, ETV4, and FOXM1) that might be related to stem cell programming and cancer progression through the regulation of the expression of genes, such as cancer-cell membrane receptors, that interact with several microorganisms, including human T-cell leukemia virus 1 (HTLV-1), the human papilloma virus (HPV), the Epstein-Barr virus (EBV), and SARS-CoV-2. These interactions can trigger the MAPK, non-canonical WNT, and IFN signaling pathways, which regulate key transcription factor overexpression during the establishment and progression of lung, colon, and gastric cancer, respectively, along with the formation of the microbiome network. The global transcriptomic network analysis highlights the important interaction between key transcription factors in lung, colon, and gastric cancer, which regulates the expression of cancer-cell membrane receptors for the interaction with the microbiome network during the tumorigenic process.

Phase I Study of Fianlimab, a Human Lymphocyte Activation Gene-3 (LAG-3) Monoclonal Antibody, in Combination With Cemiplimab in Advanced Melanoma.

Coblockade of lymphocyte activation gene-3 (LAG-3) and PD-1 receptors could provide significant clinical benefit for patients with advanced melanoma. Fianlimab and cemiplimab are high-affinity, human, hinge-stabilized IgG4 monoclonal antibodies, targeting LAG-3 and PD-1, respectively. We report results from a first-in-human phase-I study of fianlimab and cemiplimab safety and efficacy in various malignancies including advanced melanoma. Patients with advanced melanoma were eligible for enrollment into four cohorts: three for patients without and one for patients with previous anti-PD-1 therapy in the advanced disease setting. Patients were treated with fianlimab 1,600 mg and cemiplimab 350 mg intravenously once every 3 weeks for up to 51 weeks, with an optional additional 51 weeks if clinically indicated. The primary end point was objective response rate (ORR) per RECIST 1.1 criteria. ORRs were 63% for patients with anti-PD-1-naïve melanoma (cohort-6; n = 40; median follow-up 20.8 months), 63% for patients with systemic treatment-naïve melanoma (cohort-15; n = 40; 11.5 months), and 56% for patients with previous neo/adjuvant treatment melanoma (cohort-16; n = 18, 9.7 months). At a median follow-up of 12.6 months for the combined cohorts (6 + 15 + 16), the ORR was 61.2% and the median progression-free survival (mPFS) 13.3 months (95% CI, 7.5 to not estimated [NE]). In patients (n = 13) with previous anti-PD-1 adjuvant therapy, ORR was 61.5% and mPFS 12 months (95% CI, 1.4 to NE). ORR in patients with previous anti-PD-1 therapy for advanced disease (n = 15) was 13.3% and mPFS 1.5 months (95% CI, 1.3 to 7.7). Treatment-emergent and treatment-related adverse events ≥grade 3 (G3) were observed in 44% and 22% of patients, respectively. Except for increased incidence of adrenal insufficiency (12%-G1-4, 4%-G3-4), no new safety signals were recorded. The current results show a promising benefit-risk profile of fianlimab/cemiplimab combination for patients with advanced melanoma, including those with previous anti-PD-1 therapy in the adjuvant, but not advanced, setting.