γδ T-Cell Immunophenotype for the Study of Human Aging.

Flow cytometry serves as a crucial tool in immunology, allowing for the detailed analysis of immune cell populations. γδ T cells, a subset of T cells, play pivotal roles in immune surveillance and immune aging. Assessing the phenotype and functional capabilities of γδ T cells isolated from whole blood or tissue within the context of human aging yields invaluable insights into the dynamic changes affecting immune function, tissue homeostasis, susceptibility to infections, and inflammatory responses.

Homology-independent targeted insertion-mediated derivation of M1-biased macrophages harbouring Megf10 and CD3ζ from human pluripotent stem cells.

BACKGROUND: Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified macrophages (CAR-Ms) are challenging. METHODS: Here, we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach, together with a simple differentiation protocol, produced stable and highly effective CAR-Ms without heterogeneity. FINDINGS: These engineered cells phagocytosed cancer cells, leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5Mζ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), ERK, and STAT1 signalling, and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids. INTERPRETATION: Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours. FUNDING: This work was supported by KRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01).

Epigenetic Insights into Autism Spectrum Disorder: DNA Methylation Levels of NR3C1, ASCL1, and FOXO3 in Korean Autism Spectrum Disorder Sibling Pairs.

Objective: Previous research on autism spectrum disorder (ASD) in Koreans has primarily focused on genetic diversity because of its high heritability. However, the emerging recognition of transgenerational epigenetic changes has recently shifted research attention towards epigenetic perspectives. Methods: This study investigated the DNA methylation patterns of the promoter regions of candidate genes such as NR3C1, ASCL1, and FOXO3 in blood samples from ASD probands and their unaffected siblings. The analysis included 54 families (ASD proband group: 54; unaffected biological sibling group: 63). The diagnostic process involved screening the probands and their siblings for ASD based on the Diagnostic and Statistical Manual of Mental Disorders 5th edition. Intelligence, social ability, and medical history were thoroughly assessed using various scales and questionnaires. Genomic DNA from blood samples was analyzed using a methylation-sensitive quantitative polymerase chain reaction to examine the DNA methylation status of candidate genes. Results: Methylation levels in candidate gene promoter regions differed significantly between the proband and sibling groups for all candidate genes. Correlation analysis between the proband and sibling groups revealed strong and significant correlations in NR3C1 and ASCL1 methylation. Additionally, in the analysis of the relationship between DNA and ASD phenotypes, FOXO3 methylation correlated with social quotient in probands, and ASCL1 methylation was associated with nonverbal communication, and daily living skills as measured by the Korean Vineland Adaptive Behavior Scale. Notably, ASCL1 methylation was significantly associated with parental age at pregnancy. Conclusion: This study proposes DNA methylation of NR3C1, ASCL1, and FOXO3 in peripheral blood samples is a potential epigenetic biomarker of ASD.

Comparative Analysis of Extracellular Vesicles from Cytotoxic CD8+ αß T Cells and γδ T Cells.

BACKGROUND: Although belonging to different branches of the immune system, cytotoxic CD8+ αß T cells and γδ T cells utilize common cytolytic effectors including FasL, granzymes, perforin and granulysin. The effector proteins are stored in different subsets of lysosome-related effector vesicles (LREVs) and released to the immunological synapse upon target cell encounter. Notably, in activated cells, LREVs and potentially other vesicles are continuously produced and released as extracellular vesicles (EVs). Presumably, EVs serve as mediators of intercellular communication in the local microenvironment or at distant sites. METHODS: EVs of activated and expanded cytotoxic CD8+ αß T cells or γδ T cells were enriched from culture supernatants by differential and ultracentrifugation and characterized by nanoparticle tracking analyses and Western blotting. For a comparative proteomic profiling, EV preparations from both cell types were isobaric labeled with tandem mass tags (TMT10plex) and subjected to mass spectrometry analysis. RESULTS: 686 proteins were quantified in EV preparations of cytotoxic CD8+ αß T cells and γδ T cells. Both populations shared a major set of similarly abundant proteins, while much fewer proteins presented higher abundance levels in either CD8+ αß T cells or γδ T cells. To our knowledge, we provide the first comparative analysis of EVs from cytotoxic CD8+ αß T cells and γδ T cells.

[Characteristics of Ozone Photochemical Reaction and Emission Reduction Strategies in Summer and Autumn in Shangqiu].

Recently, ozone （O3） pollution in Shangqiu has become increasingly prominent, especially in summer and autumn, crucially affecting the local environmental air quality. Based on the monitoring data of O3 pollution days from the Environmental Monitoring Station in June and September 2022 （representing summer and autumn） in Shangqiu, an observation-based model （OBM） was used to study the causes and photochemical reaction characteristics of O3 pollution in the city and precursor emission reduction strategies were studied. The observation results indicated that during summer in Shangqiu, the ρ（O3） and O3 daily maximum 8 h moving concentrations [ρ（MDA8-O3）] were 149.7 µg·m-3 and 195.4 µg·m-3, whereas in autumn, ρ（O3） and ρ（MDA8-O3） were 119.8 µg·m-3 and 173.9 µg·m-3, respectively； the O3 concentration in summer was significantly higher than that in autumn. Ozone sensitivity research showed that the generation of O3 in summer and autumn in Shangqiu was controlled by volatile organic compounds （VOCs）. Among them, oxygen-containing volatile organic compounds （OVOCs）, aromatic hydrocarbons, and alkenes contributed the most to the ozone generation potential （OFP） and ·OH reactivity （L·OH）, and the control must have been strengthened. The OBM simulation results indicated that the maximum O3 generation rates in summer and autumn were 23.0×10-9 h-1 and 13.6×10-9 h-1, with maximum net generation rates of 17.4×10-9 h-1 and 10.4×10-9 h-1 and the maximum and maximum net generation rates of O3 in summer were 1.68 times higher than those in autumn, indicating that the photochemical reactions in summer were significantly stronger than those in autumn. Compared with that in summer, the generation of O3 in autumn was greatly influenced by regional inputs from other regions or cities, with a maximum input of 14.2×10-9 h-1. The prevention and control of O3 pollution in the summer and autumn seasons in Shangqiu should mainly focus on controlling VOCs. The reduction ratio of VOCs/nitrogen oxides （NOx） in autumn should be greater than that in summer and the reduction ratios of 3∶1 in summer and 4∶1 in autumn could be adopted for control.

[Evolution Characteristics and Typical Pollution Episodes of PM2.5 and O3 Complex Pollution in Bozhou City from 2017 to 2022].

In China, atmospheric pollution exhibits a complex pattern, with simultaneous exceedances of fine particulate matter （PM2.5） and ozone （O3） levels becoming evident. To understand the complex pollution characteristics and evolution patterns of PM2.5 and O3 in Bozhou City, various methods such as weather classification, analysis of typical pollution processes, and investigation of precursor sources were employed to explore the pollution and variations of PM2.5 and O3 in Bozhou City from 2017 to 2022 and subsequently analyze their causes and precursor sources. The results indicated that： ① PM2.5-O3 complex pollution in Bozhou City mostly occurred under high-pressure weather conditions, with daytime high temperatures and low humidity promoting the formation of O3 pollution, whereas nighttime high humidity and atmospheric oxidative conditions promoted the generation of secondary components such as nitrates and ammonium salts in PM2.5. ② During the pollution process, PM2.5 in Bozhou City mainly originated from biomass burning, secondary generation, traffic pollution, coal combustion, and dust sources. Volatile organic compounds （VOCs） primarily emerged from plant sources, traffic pollution, oil and gas evaporation, solvent use, fossil fuel combustion, residential emissions, and industrial emissions. Biomass burning and traffic pollution made significant contributions to the pollution process. ③ Analysis of air mass trajectories and regional pollution situations indicated that the overlay of northern and southern air masses, along with local generation, were the main causes of the PM2.5-O3 complex pollution in Bozhou from October 18th to 27th, 2022.

Vδ1 T cells integrated in full thickness skin equivalents: a model for the role of human skin-resident γδT cells.

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. In contrast to murine skin-resident γδT cells, much less is known regarding their role and function in skin health and disease. Here we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents (SE). We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the SEs. Plated on top of the dermal equivalents (DEs), almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the DE and the epithelium. Vδ1 T cells contributed to epidermal differentiation as indicated by histology, expression of epidermal differentiation markers and RNAseq expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin and skin carcinoma equivalents (SE, SCE) and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

CAR immunotherapy in autoimmune diseases: promises and challenges.

In recent years, the use of chimeric antigen receptor (CAR)-T cells has emerged as a promising immunotherapy in multiple diseases. CAR-T cells are T cells genetically modified to express a surface receptor, known as CAR, for the targeting of cognate antigens on specific cells. The effectiveness of CAR-T cell therapy in hematologic malignancies including leukemia, myeloma, and non-Hodgkin's lymphoma has led to consider its use as a potential avenue of treatment for autoimmune diseases. However, broadening the use of CAR-T cell therapy to a large spectrum of autoimmune conditions is challenging particularly because of the possible development of side effects including cytokine release syndrome and neurotoxicity. The design of CAR therapy that include additional immune cells such as double-negative T cells, γδ T cells, T regulatory cells and natural killer cells has shown promising results in preclinical studies and clinical trials in oncology, suggesting a similar potential utility in the treatment of autoimmune diseases. This review examines the mechanisms, efficacy, and safety of CAR approaches with a focus on their use in autoimmune diseases including systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, multiple sclerosis, myasthenia gravis, lupus nephritis and other autoimmune diseases. Advantages and disadvantages as compared to CAR-T cell therapy will also be discussed.

The role of post-translational modifications of cGAS in γδ T cells.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) senses DNA in a sequence-independent manner, triggering cGAMP synthesis, which activates stimulator of interferon genes (STING) and the subsequent expression of type I interferons, tumour necrosis factor alpha (TNF-α) and other proinflammatory factors in two downstream pathways. However, the function of the cGASSTING pathway in γδ T cells remains unclear. The γδ T-cell population differs from the innate-like lymphocyte population, particularly with respect to tissue distribution, indicating the unique potential of γδ T cells in treating infections and cancers. On the basis of accumulating evidence, cGAS activity is modulated by protein posttranslational modifications (PTMs), including phosphorylation, O-GlcNAcylation, acetylation, ubiquitylation and methylation, which affect multiple cGAS functions. Thus, here, we summarize recent research on PTMs of the cGAS protein that modulate γδ T-cell function. An understanding of cGAS features and modulation mechanisms may facilitate the design of therapies for γδ T-cell-related immune diseases and cancer.

Sex-specific divergences in the types and timing of infiltrating immune cells during the intervertebral disc acute injury response are associated with reduced degenerative changes.

OBJECTIVE: Inadequate repair of the intervertebral disc (IVD) contributes to low back pain. Infiltrating immune cells into damaged tissues are critical mediators of repair, yet little is known about the identities, roles, and temporal regulation following IVD injury. By analyzing transcripts of immune cell markers, histopathologic analysis, immunofluorescence, and flow cytometry, we aimed to define the temporal cascade of infiltrating immune cells and their associations with IVD degeneration. METHOD: Caudal IVDs from 12-week-old C57BL/6 mice were injured and monitored for 42 days post-injury. Transcriptional markers identifying myeloid, B, and T immune cells, and angiogenic factors were measured in the IVDs every 2-3 days. Histopathologic degeneration of the IVD was measured throughout. Flow cytometry and immunofluorescence were used to identify and localize cells including the B, T, NKT, monocytes, neutrophils, macrophages, and dendritic cells. RESULTS: The injured IVD revealed distinct phases of inflammation and proliferation. Robust temporal oscillation in the myeloid and T cell transcripts was observed in females. Cd3+ T cells were more abundant in females than in males. The Cd3+Cd4-Cd8- T cells that dominate the female cascade contain rare γδ T cells. Injury-mediated degeneration was prevalent in both sexes but more severe in males. CONCLUSIONS: This study defines the coordinated infiltration of immune cells in the IVD following injury. We report the discovery of γδ T cells in the female IVD, and this was associated with less severe degeneration. γδ T cells have potent anti-inflammatory roles and may suppress degeneration following IVD injury.

Dysfunction of γδ T cells in pediatric chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) is a progressive and life-threatening disease characterized by persistent or recurrent EBV activation. It has been reported that, γδ T cells, a type of cytotoxic lymphocyte, play a critical role in restricting EBV. However, the functional status of γδ T cells in pediatric CAEBV patients has not yet been described. In this study, flow cytometry analysis was conducted to explore the cytokine production capacity of γδ T cells in CAEBV patients. A diminished frequency of γδ T cells and decreased expression of cytolytic molecule granzyme B were found in CAEBV patients, suggesting a dysfunction in the immune regulatory function of γδ T cells in this disease.

IL-17A-producing γδ T cells: A novel target in stroke immunotherapy.

The activation of the immune system is crucial for the fate of the ischemic brain tissue and neurological outcome in experimental stroke. Rapidly after stroke γδ (γδ17), T cells release IL-17A in the ischemic brain and thereby amplify the early detrimental immune response. Notably, IL-17A levels in γδ17 T cells are modulated by the intestinal microbiota which is, in turn, shaped by the diet. Importantly, besides their proinflammatory effects, meningeal γδ17 T cells have been recently implicated in regulating neuronal signaling, behavior, and cognition under homeostatic and pathological conditions at the brain-meningeal interface. Against this background, we propose that a dietary intervention represents a promising treatment option to improve poststroke outcomes by the modulation of the microbiota composition and IL-17A levels in γδ T cells.

Progress of research on γδ T cells in colorectal cancer (Review).

Colorectal cancer (CRC) ranks as the third most prevalent malignancy and second leading cause of cancer­related fatalities worldwide. Immunotherapy alone or in combination with chemotherapy has a favorable survival benefit for patients with CRC. Unlike αß T cells, which are prone to drug resistance, γδ T cells do not exhibit major histocompatibility complex restriction and can target tumor cells through diverse mechanisms. Recent research has demonstrated the widespread involvement of Vδ1T, Vδ2T, and γδ T17 cells in tumorigenesis and progression. In the present review, the influence of different factors, including immune checkpoint molecules, the tumor microenvironment and microorganisms, was summarized on the antitumor/protumor effects of these cells, aiming to provide insights for the development of more efficient and less toxic immunotherapy­based anticancer drugs.

[Simulation and Analysis of Ozone Pollution Process in Shijiazhuang Based on CMAQ-ISAM Model].

In Shijiazhuang City, ozone （O3） pollution occurs frequently in June every year. In June 2023, the average O3 8 h concentration （O3-8h） pollution exceeded 80% of the days in the month, and O3 was the primary pollutant, accounting for 100%. For an O3 heavy pollution process from June 11 to 18, the air quality model WRF-CMAQ was used for simulation, and the average error data MFB and MFE were -10.47% and 17.96%, respectively, which was within the ideal error range. The CMAQ process analysis module was used to simulate the physical and chemical processes in Shijiazhuang City, and the dry deposition （DDEP） contribution concentration was -23.88 µg·m-3, which was the main process of O3 consumption, whereas the transport process （TRAN） was the main source of O3, among which the contribution was more significant in vertical transport （VTRA）. At the same time, the source analysis module （ISAM） was used to analyze the O3 contribution of local and surrounding areas in Shijiazhuang City. The results showed that the contribution rate of local industry sources in Shijiazhuang City was as follows： traffic source （12.54%） > industrial source （6.94%） > residential source （6.56%） > power source （4.75%）. The long-distance transmission source （BCON） continued to be in the first place with a high contribution rate of 63.31%. In the heavy pollution period under stable weather, the contribution concentration of BCON in the D02 layer of the nested domain to Shijiazhuang City was lower than the sum of the marked area. Among the surrounding cities, Baoding City had the highest contribution rate under stable weather, accounting for 26.21%. In the late period, the contribution concentration of Xingtai City increased rapidly under the action of high-value southwest wind. To effectively reduce O3 pollution, it is necessary to reduce emissions in the city and to control the upwind cities in advance, and the implementation of inter-regional joint prevention and control is the key.

[Spatiotemporal Variation Characteristics of Ozone and Identification of Key Influencing Factors Based on Random Forest Model： A Case Study of Chuzhou City].

The cause of ozone pollution is a complex scientific problem. Studying the spatiotemporal variation characteristics of O3 at different time scales and analyzing the key influencing factors of O3 concentration is of great significance for the precise formulation of urban air pollution control measures and the improvement of urban air quality. Based on the analysis of the spatiotemporal variation characteristics of O3 concentration in Chuzhou City, we studied the 12 ozone-influencing factors of meteorology and pollutants at multiple time scales using Spearman correlation analysis and a random forest model. The results showed that： ① The O3 pollution level of Chuzhou City showed an aggravating trend, and the O3 concentration distribution showed a spatial pattern of "high in the southeast and low in the northwest." ② From February to May, SO2 concentration had a strong impact on the increase in O3 concentration. From June to September, PM2.5 and PM10 were significantly positively correlated with ozone and had a greater impact. ③ Relative humidity, temperature, and wind speed had a significant impact on O3, whereas barometric pressure and hourly rainfall had a weak impact. ④ The O3 pollution mechanism in Chuzhou City changed from "pollutant-controlled" to "meteorology-controlled." ⑤ Among meteorological and pollutant factors, the three influencing factors that had the greatest influence on O3 concentration were temperature, wind speed, and relative humidity, with PM10 concentration, PM2.5 concentration, and SO2 concentration also contributing. All of the above six influencing factors had a significant nonlinear relationship with the O3 concentration.

[Effect of Meteorological Elements and Air Pollutants on Ozone in Yinchuan City Park].

To examine the underlying determinants of ozone （O3） in Yinchuan's urban park during varying seasons and to ascertain the role played by meteorological events and air contaminants in influencing O3 concentrations at high altitudes, data on O3, meteorological factors, and air pollutants were collected through prolonged positional observations carried out at the Ningxia Yinchuan National Urban Ecosystem Research Station. Pearson correlation analysis and a structural equation model were utilized to investigate the spatio-temporal distribution patterns, trends, and the primary factors influencing O3. The findings demonstrated a notable seasonal variability in O3 levels in Yinchuan's urban park, displaying an "unimodal type" with the O3 concentration peaking in summer （131.18 µg·m-3） and bottoming out in winter （71.45 µg·m-3）. Among the meteorological factors, the highest impact on O3 was attributed to temperature and wind speed （temperature mainly through direct effects and wind speed mainly through indirect effects）. Conversely, air pollutants such as NOx and SO2 greatly affected O3 primarily through direct effects. Wind speed was identified as the primary influencing factor on O3 during spring and summer, potentially contributing 29% and 24.7%, respectively. Conversely, NO2 was implicated as the primary factor during autumn and winter, with an estimated contribution of 26.6% and 29.7%, respectively. Thus, a structural equation model can efficiently reveal the primary determinants behind O3 variations throughout various seasons, which could furnish a scientifically rigorous foundation and technical aid for mitigating and managing O3 levels in high-altitude regions.

Innate T-cell-derived IL-17A/F protects from bleomycin-induced acute lung injury but not bleomycin or adenoviral TGF-ß1-induced lung fibrosis in mice.

The pathobiology of IL-17 in lung fibrogenesis is controversial. Here we examined the role of IL-17A/F in bleomycin (BLM) and adenoviral TGF-ß1-induced lung fibrosis in mice. In both experimental models, WT and IL17af-/- mice showed increased collagen contents and remodeled lung architecture as assessed by histopathological examination, suggesting that IL-17A/F is dispensable for lung fibrogenesis. However, IL17af-/- mice responded to the BLM challenge with perturbed lung leukocyte subset recruitment. More specifically, bleomycin triggered angiocentric neutrophilic infiltrations of the lung accompanied by increased mortality of IL17af-/- but not WT mice. WT bone marrow transplantation failed to correct this phenotype in BLM-challenged IL17af-/- mice. Conversely, IL17a/f-/- bone marrow transplantation → WT did not perturb lung leukocytic responses upon BLM. At the same time, IL17af-/- mice treated with recombinant IL-17A/F showed an attenuated lung inflammatory response to BLM. Together, the data show that the degree of BLM-driven acute lung injury was critically dependent on the presence of IL-17A/F, while in both models, the fibrotic remodeling process was not.

The role of Interleukin-38 in modulating T cells in chronic Colitis: A mouse model study.

BACKGROUND: Interleukin (IL)-38 belongs to the IL-36 subfamily within the IL-1 family. Patients with inflammatory bowel diseases (IBD) exhibit higher levels of IL-38 in their intestinal tissue compared to healthy controls, suggesting that IL-38 may play a role in the pathogenesis of IBD. However, IL-38's impact on T cell-mediated immune responses in gastrointestinal inflammation has not been investigated. Therefore, the objective of this work was to elucidate the role of IL-38 in modulating T cells in a mouse model of dextran sulfate sodium (DSS)-induced chronic colitis. METHODS: Recombinant IL-38 (rIL-38) was administered intraperitoneally (i.p.) to mice with chronic colitis induced by DSS. Clinical symptoms, length of colon, and histologic alterations were assessed. Cytokine production was quantified using ELISA, and helper T (Th) cell subsets were evaluated via flow cytometry. RESULTS: Administration of recombinant IL-38 (rIL-38) alleviated DSS-induced chronic colitis. In addition, animals with chronic colitis treated with rIL-38 exhibited a significant decrease in the spontaneous production of inflammatory cytokines by neutrophils in the lamina propria. Furthermore, rIL-38 treatment increased the absolute numbers and percentages of regulatory T cells (Tregs) but decreased the absolute numbers and percentages of Th1 and Th17 cells. Moreover, rIL-38 treatment also decreased IL-17A-producing γδT cells substantially. CONCLUSION: This study's results show that IL-38 reduces the effects of chronic colitis caused by DSS by boosting Treg reactions and reducing Th1/Th17 reactions and IL-17A-producing γδT cells in LPL. Therefore, we propose that IL-38 has the potential to be utilized as a biological therapy agent for IBD.

Hb SKMC and an unprecedented γδß-thalassemia: first report from Iraq.

BACKGROUND: Thalassemias are genetic disorders of globin chain synthesis. In Iraq, ß-thalassemia is more prevalent than α-thalassemia. This study identifies two unpredicted globin gene mutations, a rare α-globin gene mutation (Hb SKMC) and a novel γδß-thalassemia deletion. METHODS: Over 2 years, the Genetics unit at PAR hospital in Erbil, northern Iraq processed 137 ß-thalassemia and 97 α-thalassemia genetic testing requests. Three symptomatic thalassemia cases with unreported genotypes were identified. Proband-1α and proband-2α had Hb H disease, while proband-1ß had severe transfusion-dependent ß-thalassemia (TDT). Molecular studies included multiplex PCR, reverse hybridization, multiplex ligation-dependent probe amplification (MLPA), and globin gene sequencing. RESULTS: The α-thalassemia probands exhibited moderate microcytic hypochromic anemia with irregular transfusions and splenomegaly. Hb H disease was confirmed by positive Hb H tests and high-performance liquid chromatography (HPLC). Molecular analysis revealed heterozygous -MED deletion in proband-1α and α2Poly-A2 mutation in proband-2α. Sequencing identified the Hb SKMC (HBA1:c.283_300+3dup) mutation in both probands. The ß-thalassemia proband showed anemia and regular transfusions. Molecular studies detected the IVS1.110 G>A mutation and a novel γδß-thalassemia deletion in compound heterozygous form. The maternal sample showed the IVS1.110 G>A mutation, and MLPA confirmed the γδß-thalassemia deletion in the paternal sample. CONCLUSION: These findings highlight the genetic diversity of thalassemias in the region and emphasize the importance of advanced molecular diagnostics in detecting rare mutations.

γδ T-cells in human malignancies: insights from single-cell studies and analytical considerations.

γδ T-cells are a rare population of T-cells with both adaptive and innate-like properties. Despite their low prevalence, they have been found to be implicated various human diseases. γδ T-cell infiltration has been associated with improved clinical outcomes in solid cancers, prompting renewed interest in understanding their biology. To date, their biology remains elusive due to their low prevalence. The introduction of high-resolution single-cell sequencing has allowed various groups to characterize key effector subsets in various contexts, as well as begin to elucidate key regulatory mechanisms directing the differentiation and activity of these cells. In this review, we will review some of insights obtained from single-cell studies of γδ T-cells across various malignancies and highlight some important questions that remain unaddressed.