Selective T-cell depletion in stored whole blood using green-synthesised γ-Fe2O3 nanoparticles with gamma or laser irradiation: A comparative in-vitro study

Allogeneic HSCT for consolidation in pediatric refractory or relapsed ALK-positive anaplastic large cell lymphoma.

Clonal dynamics, tolerance, and adverse events after CD45-ADC-conditioned autologous HSPC transplantation in macaques.

Kyasanur Forest Disease (KFD) is a tick-borne viral haemorrhagic fever endemic to southern India. We present a case series of five PCR-confirmed patients with KFD who developed oral candidiasis. CD4 and CD8 T-lymphocyte counts were measured using flow cytometry alongside detailed clinical assessment. All five patients with candidiasis had CD4 counts <300 cells/µl, and four had CD8 counts <200 cells/µl. None had other known immunosuppressive conditions. CD4 and CD8 lymphopenia are consistent and clinically relevant features among patients with KFD who developed oral candidiasis, suggesting transient mucosal immune suppression as a potential pathogenic mechanism.

Exploration of In Vivo T-cell Depletion protocols in Allogeneic Hematopoietic Stem Cell Transplantation for Hematological Malignancies.

For patients with high-risk acute lymphoblastic leukemia (ALL), allogeneic hematopoietic cell transplantation (HCT) remains standard of care. In the setting of an HLA-matched unrelated donor HCT, in vivo T-cell depletion (TCD) for prophylaxis of graft versus host disease (GVHD) relies on anti-thymocyte globulin (ATG) in Europe and alemtuzumab in the UK. In a retrospective study from the EBMT registry, we pair-matched 90 ALL patients aged ≥40 years transplanted in CR1 according to age (median 56 years) and ALL subtype (37.8% Ph-negative B-ALL, 46.7% Ph-positive B-ALL, 15.6% T-ALL). Reduced-intensity conditioning included fludarabine/melphalan (94.4%) in the alemtuzumab and fludarabine/busulfan (36.7%), fludarabine/total body irradiation (21.1%), fludarabine/melphalan (14.4%) and thiotepa/busulfan/fludarabine (13.3%) in the ATG group. Two-year leukemia-free and overall survival were similar between groups (Alemtuzumab: 56.4% vs ATG: 50.7%, HR 0.82, p = 0.34, and 62.7% vs 62.9%, HR 0.91, p = 0.67), as were cumulative incidence of relapse (23.7% vs 23.9%, HR 0.89, p = 0.69) and non-relapse mortality (19.9% vs 25.4%, HR 0.75, p = 0.32), resulting in similar GVHD- and relapse-free survival (GRFS) of 48.9% vs 42.1%, HR 0.8, p = 0.24. With GVHD and infections as main reasons for death in both groups, we conclude that both IS strategies are both safe for RIC HCT of these ALL patients.

Xenotransplantation has emerged as a promising solution to the critical organ shortage, with encouraging results in preclinical nonhuman primate studies and recent first-in-human transplants. Our group previously performed pig-to-rhesus macaque renal xenotransplants using the clinically available costimulation blockade agent belatacept; however, graft survival was modest (>1 moh). Analysis of rejected xenografts identified natural killer (NK) cells as a predominant infiltrating population. In this study, we investigated whether adding adjuvant αIL-15-an agent known to suppress T-cell subsets and deplete NK cells in rhesus macaques-could improve xenograft survival. αIL-15 was combined with a clinically relevant immunosuppressive regimen comprising T-cell depletion, belatacept, mycophenolate mofetil, and steroids. We found that the addition of αIL-15 significantly improved xenograft survival and function. Longitudinal analysis of NK cell subsets revealed a shift from a predominant cytotoxic CD16 + CD56 - population to a double-negative CD16 - CD56 - phenotype following αIL-15 treatment. These findings deepen our understanding of NK cell subsets and their potential contributions to xenograft injury and suggest that targeted modulation of NK cell populations can enhance xenograft outcomes in a preclinical pig-to-rhesus macaque model of renal xenotransplantation.

Anti-thymocyte globulin (ATG Thymoglobulin®) and anti-T-lymphocyte globulin (ATLG Grafalon®) are commonly used in hematopoietic stem cell transplantation (HSCT) for in vivo T-cell depletion, aiming to reduce graft failure and graft-versus-host disease (GvHD). Despite their widespread use, the precise mechanisms by which ATG/ATLG affect immune recovery post-transplantation remain partially understood. In this study, in 289 pediatric HSCT patients treated with ATG (n=213) or ATLG (n=76), the relationship between longitudinal serum levels of the active T-lymphocyte-binding component of ATG/ATLG and immune reconstitution was evaluated. High levels of active ATG/ATLG did not prevent the recovery of neutrophils or NK-cells; however, T-cell recovery was delayed until active ATG/ATLG concentrations fell below 1AU/mL. This is in apparent contradiction with the fact that ATG/ATLG are known not only to contain antibodies against antigens present on T-cells but also to antigens present on other immune and non-immune cells, including B-, NK-cells, granulocytes, monocytes and hematopoietic stem and progenitor cells (HSPC). To address this, we assessed the binding capacities of ATG and ATLG to immune cell subsets and HSPC both in vitro directly from the vial and in vivo from patients serum samples taken at multiple time points pre- and post-HSCT. Shortly after infusion, we observed a rapid reduction of ATG and ATLG binding to all cell types except for T-cells. This real-life specificity of ATG and ATLG explains their selective impact on T-cell reconstitution. These findings enhance our understanding of the mechanisms of action of ATG and ATLG and may contribute to optimization of these therapies.

Severe Omicron cases present profound lymphocytopenia, suggesting variant-specific immune injury. We identify CD63 as a conserved T-cell host factor supporting ACE2-independent SARS-CoV-2 entry. Despite lower intracellular viral loads than the ancestral strain, Omicron elicits enhanced T-cell apoptosis largely through a bystander mechanism. Omicron-stimulated epithelial cells secrete GDF15, which upregulates the pro-apoptotic protein BCL2L13 in T cells and thereby remotely accelerates apoptosis in uninfected bystanders. Functionally, recombinant GDF15 increases BCL2L13 and apoptosis, while genetic dampening of BCL2L13 blunts Omicron-specific high-intensity bystander death. In clinical samples, plasma GDF15 associates with mortality, SOFA scores, and lower lymphocyte counts, bridging the epithelial-immune axis to patient outcomes. Our data delineate a two-track model of Omicron immune injury-CD63-enabled T-cell entry plus GDF15-BCL2L13-driven bystander apoptosis-that reconciles lower epithelial cytopathicity with deeper T-cell depletion in critical disease. These findings nominate the GDF15-BCL2L13 axis as a mechanistic marker and potential point of intervention.

Background: Left ventricular assist device (LVAD) implantation is an important treatment for end-stage heart failure. However, its impact on immune cell dynamics is still unclear, which potentially contributes to the high postoperative infection rate. Materials and Methods: We conducted a longitudinal multimodal analysis integrating clinical blood tests (n = 139, 2-year follow-up), single-cell RNA sequencing (scRNA-seq, n = 7, 5 time points), and flow cytometry (n = 20, 5 time points) to characterize the effects of LVAD implantation on immune cell counts, subset composition, and functional changes. Patient-derived peripheral blood mononuclear cells were used in in vitro experiments to validate the quantitative and functional alterations induced by the LVAD. Results: LVAD implantation induced a dramatic decrease in lymphocytes in the first week, which returned to preoperative levels by 2 months post-operation. Furthermore, flow cytometry and scRNA-seq data reveal that T cells are the lymphocytes with the most significant decrease after LVAD implantation. Notably, CD8-Tem-GZMB, a group of effector memory T cells that primarily perform cell killing and cytotoxic functions, declined significantly in the 2 weeks post-operation. Functional ELISpot assay indicates that the reduction in T-cell numbers manifests as an overall decrease in cytotoxic function. Mechanistically, apoptosis induced by shear stress exceeding the physiological range may contribute to T-cell depletion following LVAD implantation. An approximately 1.5-fold increase in the monocyte counts was observed in the first week post-operation and recovered by the first month, with decreased HLA-DR expression and antigen presentation function at 2 weeks postoperatively. NK cell counts and percentage declined significantly after LVAD implantation and did not recover until 3 months postoperatively. Conclusion: This study reveals a short-term impairment of cellular and innate immunity after LVAD implantation, which is associated with reduction in immune cell numbers and functional decline. This study provides a theoretical basis for monitoring the immune status and preventing potential infections after LVAD implantation.

Sepsis triggers both excessive inflammation and immunosuppression, the latter partly characterized by CD4+ T-cell depletion. The mechanisms underlying this depletion, especially its interplay with cytokine storms driven by inflammatory factors such as interleukin (IL)-6, remain unclear. This study aimed to elucidate the molecular mechanisms contributing to CD4+ T-cell depletion in sepsis, focusing specifically on the IL-6/Janus kinases (JAKs)/signal transducer and activator of transcription 3 (STAT3) signaling axis and programmed cell death. Prospective cohort study. Adult ICUs at a university hospital. Adult sepsis and septic shock patients without any documented immune comorbidity. None. A prospective analysis enrolled 151 patients (93 sepsis, 58 septic shock) and 20 controls. Flow cytometry and enzyme-linked immunosorbent assay were used to assess immune cell populations and cytokine profiles, with multivariate analyses exploring their interrelationships. An additional 30 sepsis patients and ten controls were recruited to investigate mechanisms. Peripheral blood mononuclear cells (PBMCs) underwent RNA sequencing (RNA-seq). Isolated CD4+ T cells were stimulated with IL-6 in vitro, followed by treatment with specific inhibitors targeting pyroptosis, apoptosis, necroptosis, the JAKs/STAT3 pathway, or receptor-interacting protein kinase 1 (RIPK1). Western blotting, flow cytometry, immunofluorescence, Cell Counting Kit-8 assays, and interferon-gamma staining evaluated cell death pathways, PANoptosome (a complex mediating apoptosis, pyroptosis and necroptosis)-assembly, and function. Significant CD4+ T-cell loss occurred in both sepsis and septic shock groups, strongly correlating with elevated IL-6 levels. Sepsis PBMC RNA-seq revealed activated IL-6/JAKs/STAT3 signaling and upregulated apoptosis/pyroptosis/necroptosis genes. In vitro, IL-6 induced pyroptosis, apoptosis, and necroptosis (PANoptosis) in CD4+ T cells via IL-6/JAKs/STAT3-dependent RIPK1-PANoptosome assembly. Inhibiting JAKs/STAT3 or RIPK1 significantly reduced PANoptosis, partially restored CD4+ T-cell viability and functional capacity. PANoptosis has been observed to be a form of CD4+ T-cell death in sepsis patients. Evidence suggests that IL-6 may be associated with the exhaustion process, mechanistically involving the activation of the JAKs/STAT3 pathway. It is also hypothesized that this process might be linked to RIPK1-PANoptosome-mediated PANoptosis.

Head and neck squamous cell carcinoma (HNSCC) continues to be a deadly cancer with heterogeneous molecular characteristics and poor survival outcomes, particularly in HPV- patients. This study aimed to identify key overexpressed genes that drive HNSCC progression, evaluate their prognostic value, and explore associations with HPV status, promoter methylation, and changes in the immune microenvironment. Using bioinformatics tools (UALCAN, HPA, TISIDB), we analyzed TCGA-HNSC data to evaluate gene expression, survival correlations, HPV subgroup differences, promoter methylation, and immune infiltration patterns. Statistical significance was defined as p < 0.05. Among the top 50 overexpressed genes in HNSCC, eight (LAMC2, CDKN2A, MFAP2, CTHRC1, CXCL13, FST, SPP1, PLAU) exhibited significant survival associations (p < 0.05). HPV- tumors demonstrated marked upregulation of LAMC2, CTHRC1, FST, SPP1, and PLAU, alongside downregulation of CDKN2A and CXCL13. Promoter hypomethylation in tumor tissues correlated with overexpression of LAMC2, CTHRC1, CXCL13, FST, SPP1, and PLAU, whereas CDKN2A showed hypermethylation. Immune infiltration analysis revealed strong correlations between these genes and immunosuppressive Tregs or cytotoxic T-cell depletion. This study identifies eight prognostic biomarkers in HNSCC linked to HPV-driven heterogeneity. These genes could be potential targets for therapy in combination with immunotherapy and epigenetic regulators, helping to overcome tumor resistance in HNSCC with different HPV status.

Recurrent implantation failure (RIF), characterized by repeated failure to achieve clinical pregnancy after embryo transfer, is often associated with abnormal endometrial conditions. However, unexplained RIF is unique in that its underlying cause remains largely unknown, posing a challenge for both clinicians and patients. We performed single-cell RNA sequencing and reported that the endometria of patients with unexplained RIF exhibited increased the proliferation and activation of cytotoxic CD8+ T-cells, which hinder embryo implantation. Additionally, the glandular epithelium, which has the highest metabolic activity, showed abnormal glycolysis and reduced lactate production in RIF. Specifically, the endometrial expression of genes associated with glucose uptake (SLC2A1), glucose metabolism (ALDOA), lactate production (LDHA), and lactate output (SLC16A3) were lower in the patients with unexplained RIF than in the controls. Through uterine horn injection experiments in mice, we demonstrated that inhibiting lactate production in the endometrium prevents embryo implantation and that this effect could be reversed by lactate supplementation. Moreover, lactate inhibitors did not affect implantation in mice with CD8+ T-cells depletion. In vitro experiments also confirmed that lactate inhibition affects the proliferation and activation of CD8+ T-cells. We propose that the endometria of patients with unexplained RIF fail to establish proper immune balance toward the embryo, likely due to abnormal glycolysis and reduced lactate production in the glandular epithelium.

HIV infection profoundly impacts both the immune and hematologic systems. This retrospective study aimed to assess the effect of CD4+ T-cell depletion on peripheral immune and hematologic cell populations in 1293 people living with HIV (PLWH). Patients with CD4+ counts < 200 cells/µL exhibited significant reductions in neutrophils (4000 ± 1200 cells/µL), eosinophils (150 ± 50 cells/µL), basophils (30 ± 10 cells/µL), monocytes (300 ± 100 cells/µL), erythrocytes (4.3 ± 0.6 × 106/µL), and platelets (268 ± 78 × 103/µL). As evaluated in 93 PLWH cases, B cells showed a similar trend (150 ± 50 cells/µL), while the decrease of NK cells was not significant (200 ± 70 cells/µL),. In parallel, we observed a compensatory increase in CD8+ T lymphocytes (850 ± 300 cells/µL). These findings highlight the complex interplay between CD4+ T-cell depletion and both immune and hematologic cells in the context of HIV infection, which emphasizes the need for integrated monitoring to inform targeted interventions and optimize clinical outcomes.

CCR4 expression defines a targetable subset of T-cell acute lymphoblastic leukemia.

The local effect of radiotherapy (RT) is enhanced by CD8+ T-cell responses elicited through dendritic cell (DC)-mediated cross-presentation of tumor antigens, facilitated by RT-induced damage-associated molecular patterns. The abscopal effect-regression of non-irradiated tumors-has been observed clinically, particularly in combination with immune checkpoint blockade, although it remains uncommon. To better understand how to enhance this effect, we investigated two RT/α-programmed death 1 (PD-1)-based triple combinations incorporating either α-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or CD122-targeted interleukin (IL)-2 complexes (IL-2c). We tested these regimens in B16 melanoma and C51 colon carcinoma models in mice with one irradiated and one non-irradiated tumor on opposite flanks. In both models, RT/αPD-1/αCTLA-4 elicited a stronger abscopal response than RT/αPD-1/IL-2c. In the C51 model, RT/αPD-1/αCTLA-4 achieved a 61.5% abscopal cure rate, dependent on both CD8+ and CD4+ T cells. In contrast, the less effective RT/αPD-1/IL-2c response required only CD8+ T cells. The enhanced abscopal effect with RT/αPD-1/αCTLA-4 was associated with increased numbers, effector function, and reduced exhaustion of tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) and of CD4+ TILs, along with elevated CD80+CD86+ DCs in abscopal tumors, as shown by flow cytometry; immunofluorescence confirmed increased T-cell infiltration. CD4+ T-cell depletion during RT/αPD-1/αCTLA-4 treatment impaired abscopal but not irradiated tumor control, reducing infiltration of tumor-specific CD8+ T cells and conventional (c) DC1s, and diminishing cDC1-mediated cross-presentation in abscopal tumors. Activated CD4+ T cells upregulated CD80/CD86 on cDC1s and enhanced cross-presentation, partly via interferon-γ and tumor necrosis factor. Adoptively transferred tumor-specific CD8+ T cells from tumor-irradiated donors localized to unirradiated tumors and draining lymph nodes in αPD-1/αCTLA-4-treated recipients, but not in untreated or CD4+ T cell-depleted mice. These results demonstrate that an RT-based combination therapy that robustly induces CD4+ T cells alongside CD8+ T cells can elicit a strong abscopal response and suggest that CD4+ effector T cells act at abscopal sites by promoting DC-mediated cross-presentation of tumor antigens to CD8+ T cells originating from the irradiated tumor.

Human immunodeficiency virus type 1 (HIV-1) causes immune deficiency, particularly CD4+T-cell depletion. This cross-sectional study explored circulating cell-free DNA (ccfDNA) as a biomarker for immune function and disease progression in HIV-1-infected individuals. We measured ccfDNA levels, CD4+T-cell counts, immune activation/apoptosis markers, and viral load in 79 patients receiving antiretroviral therapy (37 immune responders, 42 non-responders) and 35 controls. Correlation and receiver operating characteristic (ROC) curve analysis were performed to assess relationships between ccfDNA and immunological/virological parameters. ccfDNA negatively correlated with CD4+T-cell counts (r = - 0.78, P < 0.01) and positively correlated with viral load (r = 0.58, P < 0.01) and T-cell immune activation/apoptosis markers (r = 0.78 ~ 0.89, P < 0.01). Multivariate regression showed CD4+T-cell counts independently associated with lower ccfDNA, while CD4+AnnexinV+, CD8+PD1+, and CD8+AnnexinV+T cells correlated with higher ccfDNA. ROC analysis (area under curve = 0.799) indicated ccfDNA's potential as a complementary biomarker for monitoring HIV-1 patients. Preliminary evidence from this study indicates that ccfDNA levels are closely linked to immune dysfunction and may have potential as a complementary biomarker for monitoring immune status in HIV-1-infected patients.

Non-hepatotropic viruses (NHVs), as a category of pathogens not primarily targeting the liver, can also cause hepatic injury. Liver injury associated with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections, in particular, often attracts significant clinical attention. This retrospective cohort study analyzed the seroprevalence and clinical features of EBV and CMV infections among patients in Beijing from 2020 to 2024, with a focus on the impact of immunosuppression status on liver injury patterns. The CMV IgM positivity rate was 4.00% (646/16,201), and the EBV IgM positivity rate was 7.84% (1007/12,838), both showing significant upward annual trends (p < 0.001). Analysis of 236 IgM-positive inpatients revealed a "bifurcation phenomenon": the non-immunosuppressed group exhibited more severe hepatocellular injury (e.g., ALT levels 6.5- to 10.9-fold higher) and cholestatic damage (e.g., CMV group: TBIL increased 7.8-fold), yet had better clinical outcomes (adverse outcome rate: 0-4.8%) compared to the immunosuppressed group (adverse outcome rate: 18.4-27.8%, p < 0.05). Further analysis of 125 patients with confirmed liver injury demonstrated that the immunosuppressed group had severe CD4 + T-cell depletion and inverted CD4 + /CD8+ ratios. During EBV and CMV co-infection, the immunosuppressed group showed higher CMV DNA detection rates (66.7% vs. 20.0%, p = 0.0097) and viral loads (median 2675 vs. 625 copies/mL, p = 0.002). Within the immunosuppressed group, patients with CD4 + T-cell counts > 300 cells/μL had higher ALT and AST levels, supporting an immune-mediated injury mechanism. These findings indicate that immune status and virus type jointly shape the clinical spectrum of EBV/CMV-related liver injury. The dissociation between severe liver injury and favorable prognosis in non-immunosuppressed patients underscores the role of immune pathology, while poorer outcomes in immunosuppressed patients are driven by CD4 + T-cell depletion, impaired viral clearance, and extrahepatic complications.

The discontinuation of the CS2 version of the semi-automated CliniMACS Plus device in 2024 prompted validation of a CD34+ immunoselection process using the fully automated CliniMACS Prodigy (Miltenyi Biotec). This transition was leveraged to evaluate Prodigy's performance on thawed apheresis products. Results were compared with fresh donor apheresis previously processed on the CliniMACS Plus and, within this validation, with fresh products processed on Prodigy. Fresh apheresis products from rhG-CSF-mobilized related donors were processed using either device. Cryopreserved apheresis products intended for destruction were thawed (Viathaw, Cytiva), washed on Sepax-II and resuspended in MACS GMP PBS/MgCl₂ with HSA and rh-DNase prior to CD34+ selection on Prodigy. Flow cytometry (Stem Kit-Beckman Coulter) was used for CD34+ and CD45+ cell quantification. From fresh products, immunoselections yielded similar CD34+ purities (95% versus 97%) and viabilities (95% versus 99%) for CliniMACS Plus (n = 10) and Prodigy (n = 10), respectively. T-cell depletion was slightly better with the CliniMACS Plus (0.1 × 10⁴ versus 0.4 × 10⁴ CD3⁺/kg), while CD34+ recovery was more efficient with Prodigy (66% versus 53%). Processing thawed apheresis with the Prodigy archieved 98% CD34+ purity, 51% recovery, and 0.3 × 10⁴ CD3⁺/kg, all within our center's acceptance criteria. The fully automated workflow significantly reduced hands-on time (2 h versus 5 h) and operator intervention. Prodigy platform enables reproducible CD34+ immunoselection from both fresh and thawed products, improving standardization, efficiency, and flexibility in graft engineering and ATMP manufacturing. The ability to process thawed products also broadens clinical options whenever cryopreserved products are available.

Comparison of Viral Infection Rates after T-cell Depletion in Pediatric HCT: Alemtuzumab Vs Post-Transplant Cyclophosphamide