Peripheral Blood CD4 Research Articles

Efficacy of ofatumumab combined with corticosteroids in the treatment of autoimmune encephalitis: a 6-month cohort study.

This study investigated the efficacy of ofatumumab (OFA) combined with corticosteroids in autoimmune encephalitis (AE) patients refractory to conventional treatment. Eighteen AE patients admitted to Ruijin Hospital between June 2022 and September 2023 received four subcutaneous 20-mg OFA injections at 0, 1, 6, and 12 weeks combined with standard corticosteroid therapy. Clinical symptoms, modified Rankin scale (mRS) scores, Clinical Assessment Scale for Autoimmune Encephalitis (CASE) scores, serum immunoglobulin (Ig) levels (IgG and IgM), and peripheral blood CD20 + B cell levels were documented before OFA administration and at 1, 2, 6, 12, and 24 weeks post-treatment. OFA treatment significantly improved psychiatric symptoms (P = 0.025), cognitive dysfunction (P = 0.008), and seizure frequency (P = 0.014). The mRS and CASE scores improved after two injections in patients with anti-NMDAR encephalitis (P = 0.041), but not in patients with anti-LGI1 encephalitis (P > 0.05). The mRS scores significantly improved at 12 weeks post-treatment in antibody-negative encephalitis. Blood CD20 + B cell levels dropped to zero after an average of 2.5 injections. No significant changes could be observed in serum IgG and IgM levels (P > 0.05). Seven patients had mild fever or pulmonary infections post-treatment. This study suggests that OFA is a safe and effective treatment for a subset of AE patients, particularly in cases of anti-NMDAR encephalitis, though further research is needed on long-term outcomes and recurrence.

Characteristics of cytokines/chemokines associated with disease severity and adverse prognosis in COVID-19 patients

BackgroundCytokines and chemokines as crucial participants in innate immune response play significant roles during SARS-CoV-2 infection, yet excessive immune response exacerbates the severity of COVID-19.PurposeThis study aims to investigate the involvement of which cytokines/chemokines in the cytokine storm of COVID-19, as well as the changes in cytokine/chemokine levels during the course of COVID-19, simultaneously exploring the diagnostic and prognostic value of the relevant cytokines/chemokines for COVID-19.MethodsFlow cytometry was employed to detect the levels of cytokines and chemokines in the serum of 50 COVID-19 patients.ResultsCompared with severe COVID-19 patients, the levels of cytokines IL-6, IL-8, IL-10, sCD25, and chemokines IP-10 and MIG in the peripheral blood of non-severe patients were significantly reduced, while only IL-6, IL-10, and IP-10 levels were significantly decreased compared to non-survivors of COVID-19. Meanwhile, serum concentrations of IP-10, MCP-1, sTREM-1, IL-10, and the neutrophil-to-lymphocyte ratio (NLR) in peripheral blood could distinguish between COVID-19 survivors and non-survivors and were significantly associated with mortality. Among them, the concentration of IP-10 was shown to be the most powerful indicator for predicting adverse outcomes in COVID-19 patients (AUC: 0.715); however, its combined detection with the conventional inflammatory marker NLR did not improve the predictive value for adverse outcomes in COVID-19 patients. Additionally, serum IP-10 levels were negatively correlated with peripheral blood NK cell count and total lymphocyte count, while sTREM-1 levels were positively correlated with peripheral blood CD4+ T cell count and CD3+ T cell count. Meanwhile, IL-8 levels were positively correlated with total lymphocyte count in peripheral blood. Finally, the serum levels of cytokines/chemokines in non-survivors of COVID-19 increased significantly before death, while in survivors, they returned to normal levels before discharge.ConclusionsSeverely ill and non-surviving COVID-19 patients exhibit compromised immune function, with significantly higher levels of inflammation, cytokine/chemokine storms, and immune dysregulation compared to non-severe patients. Serum concentrations of IP-10, MCP-1, sTREM-1, and IL-10 levels can serve as biomarkers to predict adverse outcomes in COVID-19.

Study on the correlation between viral load and activation and exhaustion levels of CD8+T cells in HIV/AIDS patients

Objective: To investigate and analyze the correlation between the expression levels of CD38, HLA-DR and programmed cell death-1 (PD-1) on peripheral blood CD8+T cells and HIV-1 RNA viral load, immune activation and exhaustion in patients with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Methods: A total of 81 HIV/AIDS patients (64 without antiretroviral therapy and 17 with therapy) and 40 healthy donors in the same period were enrolled as the control group. Flow cytometry was used to analyze the CD4+T lymphocyte count and the expression levels of activation markers CD38 and HLA-DR and apoptosis marker PD-1 on CD8+T cells. HIV-1 RNA in the plasma of HIV-1 infected patients was quantitatively detected by real-time fluorescence quantitative polymerase chain reaction. Variance analysis was used to compare the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells between HIV/AIDS patients and healthy controls. Spearman correlation analysis was used to analyze the correlation between different T lymphocyte counts and HIV RNA viral load, and the correlation between HIV RNA viral load and peripheral blood CD8+T cell CD38, HLA-DR and PD-1. Results: Among the 81 HIV/AIDS patients, 69 (85.19%) were males and 12 (14.81%) were females, with an age M (Q1, Q3) of 58 (36.5, 65.0) years. There were 60 HIV/AIDS patients over 55 years old (74.07%) and 21 HIV/AIDS patients between 18 and 55 years old (25.93%). The results of variance analysis showed that compared with the healthy control group, the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells in HIV/AIDS patients increased, and the differences were statistically significant (all P<0.05). In addition, the expression of CD38, HLA-DR and PD-1 increased significantly in patients with CD4+T cell count less than 350 cells/μl, and the differences were statistically significant (all P<0.05). Spearman correlation analysis showed that CD4+and CD4+/CD8+were negatively correlated with viral load in HIV/AIDS patients (r=-0.407 and -0.378, respectively, both P<0.05), and CD8+was positively correlated with viral load (r=0.356, P<0.05). When the HIV RNA level was≤105 CPs/ml, there was no correlation between the HIV RNA level and the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells (all P>0.05). However, when the level of HIV RNA was>105 CPs/ml, the level of HIV RNA was positively correlated with the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells (r=0.412, 0.387, 0.395, respectively, all P<0.05). Conclusions: The activation levels of CD38 and HLA-DR and the expression of PD-1 on CD8+T cells in the peripheral blood of HIV/AIDS patients are increased. When the viral load is high, the HIV RNA viral load is positively correlated with the activation and exhaustion levels of CD8+T cells.

Early Mixed Donor Chimerism is a Strong Negative Prognostic Indicator in Allogeneic Stem Cell Transplant for AML and MDS

Allogeneic bone marrow transplantation remains the most potent curative therapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) due to the graft-versus-tumor effect provided by donor cells. Donor chimerism is utilized early after transplantation to evaluate engraftment and to monitor the persistence of donor hematopoiesis. Literature is conflicting regarding to the prognostic utility of early mixed donor chimerism, chimerism kinetic patterns as well as factors associated with it and we sought to clarify this uncertainty. In this single-centre retrospective analysis, 141 adults aged 18 years of age or older with AML (n = 104) and MDS (n = 37) who received their first transplant from HLA matched related, matched unrelated or mismatched related (haploidentical) donors between 2016 and 2022 and had at least day 30 chimerism measured were included. Approximately 30% received post-transplant cyclophosphamide for graft-versus-host disease (GVHD) prophylaxis and 67% of subjects received reduced-intensity conditioning. Chimerism was measured using STR-PCR from unfractionated peripheral blood mononuclear cells (whole blood; WB) and CD3+ (T cell; TC) compartment at each time point. Complete donor chimerism was defined as ≥95% whereas <95% defined as mixed. Competing risk analysis was used to estimate cumulative incidence of relapse with kinetic calculations completed using an increment factor. Kaplan-Meier was used for overall survival (OS) and relapse-free survival (RFS). Cox proportional hazards regression was used to explore prognostic factors for OS and RFS. Both day 30 mixed WB and TC donor chimerism were individually associated with an increased risk of relapse and worse overall and relapse-free survival at days 30, 60 and 90 post-transplant. Day 30 mixed WB was more specific for relapse (86%), while mixed TC was more sensitive (67%). Complete day 30 chimerism had a negative predictive value of 63% and 70% and positive predictive value of 57% and 67% for WB and TC, respectively. Day 30 WB and TC donor chimerism of <88.92% and 89.29% had specificities of 79.17% and 82.19% although sensitivities only approximated 50%. Evaluating the kinetics of chimerism over the first 90 days provided additional information for prognosticating relapse than absolute chimerism values at individual time points in both WB day 30 to 90 [HR, 1.75 (95% CI, 1.04 to 2.94); P < .035] and TC day 60 to 90 [HR, 1.32 (95% CI, 1.03, 1.69); P < .29]. Twice as many patients with complete chimerism developed acute GVHD compared to those with mixed chimerism. Factors that were found to be associated with day 30 mixed TC chimerism were donor source, ATG GVHD prophylaxis, myeloablative conditioning and female sex, while only donor source was associated with mixed WB. Both TC and WB day 30 mixed chimerism were associated with an increased risk of relapse. Early mixed WB and TC chimerism is strongly associated with a worse overall and relapse-free survival. The serial measurement of chimerism early post-transplant for monitoring chimerism kinetics provides additional prognostic information beyond the absolute donor chimerism value at a single time point. Haploidentical stem cell transplants were associated with a lower likelihood of mixed chimerism than other donor sources.

Alterations in T cell immunity over 6–12 months post-COVID-19 infection in convalescent individuals: a screening study

Acute COVID-19 is a viral infection caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that results in dramatically decreased peripheral blood CD3+T cell count apparently due to alterations of thymic T cell maturation, that can persist long term afterwards. Therefore, we analyzed the levels of peripheral blood TRECs (T-cell receptor excision circles), and investigated the main alterations in peripheral blood T cell subsets in COVID-19 convalescents. We performed molecular quantification of TRECs with “TREC/KREC-AMP PS” kit and flow cytometric analysis of peripheral blood lymphocytes from three groups of patients. The first group contained 109 samples from COVID-19 convalescents (6–12 month post-acute COVID-19) with normal levels of TRECs (TRECn); the second was formed from COVID-19 convalescents (6–12 month post-acute COVID-19) with decreased levels of TRECs (TREClow, n = 29), and healthy control group (HC, n = 18). We noticed no significant differences between all three groups in CD3+T cell relative and absolute numbers. However, CD4+T cell frequencies were decreased in TREClow and TRECn groups compared to HC (40.8% (31.6; 50.1) and 46.4% (40.0; 53.0) vs 53.5% (47.36; 56.9), p 0.001 and р = 0.004, respectively). Furthermore, Th cell levels were decreased in TREClow patients vs HC and TRECn groups (701 cell/1 µL (478; 807) vs 1005 cell/1 µL (700; 1419), р = 0.020, and 876 cell/ 1 µL (661; 1046), р = 0.008, respectively). Finally, both groups of COVID-19 convalescents had increased frequencies of circulating CD8+T cells — 29.4% (20.7; 39.7) in TREClow group, 26.5% (21.1; 32.7) in TRECn group vs 21.3% (17.1; 26.0) in healthy controls (p = 0.024 and р = 0.026, respectively). In TRECn group, CD8+T cell count was elevated vs control range (508 cell/1 µL (372; 622) vs 356 cell/1 µL (247; 531), р = 0.044). Thus, COVID-19 convalescents (6–12 month post-acute COVID-19) showed an imbalance in CD4+and CD8+T cell level even at 6–12 months post-acute SARS-CoV-2 infection, and the observed changes in peripheral blood T cells could be closely related to the alterations in thymic T cell maturation and differentiation. Such a long-term decline in TREC levels in the circulation may have a profound impact on immune system functions and requires immunocorrection therapy.

Mechanism of PD-1/PD-L1 in Regulating cTfr/cTfh Balance in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is frequent, an imbalance between helper cells (Th) and regulatory T cells (Treg) is the fundamental immunological cause of RA. This study investigates how recombinant human programmed cell death 1 (PD-L1) protein affects circulating T follicular helper (cTfh), circulating T follicular regulatory (cTfr), and their equilibrium. Magnetic bead sorting was used to select CD4+CXCR5+T cells from RA patients' and healthy individuals' peripheral blood mononuclear cells for in vitro growth. Recombinant human PD-L1 protein stimulated CD4+CXCR5+T cells. Cell counting kit 8 (CCK-8), flow cytometry surface labeling, ELISA, and RT-PCR were used to measure CD4+CXCR5+T cell proliferation inhibition, cTfh and cTfr frequencies, IL-21 expression, and PI3K, AKT, Bcl-6, and Blimp-1 mRNA levels. The recombinant human PD-L1 protein dose-dependently inhibited the proliferation of CD4+CXCR5+T cells in active RA peripheral blood. However, it has a weaker inhibitory effect on healthy peripheral blood CD4+CXCR5+T cells. PD-L1 protein decreased cTfh in active RA peripheral blood CD4+CXCR5+T overall cultured cells but did not affect cTfr; The cTfr/cTfh ratio increased but did not affect the frequency of cTfh and cTfr in healthy persons' cultured CD4+CXCR5+T cells. PD-L1 protein reduced IL-21 in CD4+CXCR5+T cell culture supernatant from active RA peripheral blood. Recombinant human PD-L1 protein lowered PI3K, AKT, and Bcl-6 mRNA in active RA peripheral blood CD4+CXCR5+T cell culture, including significant differences. But Blinmp-1 mRNA variations were neither substantial nor statistically different. PD-1/PD-L1 limits cTfh proliferation, differentiation, and activation via the PI3K/AKT signaling pathway regulates its immunological balance with cTfr, and corrects the cTfr/cTfh imbalance by controlling their interaction.

Zinc deficiency impairs the development of human regulatory B cells from purified B cells

Zinc is a vital trace element, important for many different immune processes and adequate functionality. B cell development is known to be dependent on sufficient zinc supply. Recently a regulatory B cell (Breg) population has been identified, as CD19+IL-10+ B cells, able to regulate immune responses by secretion of anti-inflammatory cytokines, such as IL-10. Due to their promotion of an anti-inflammatory milieu, Bregs could reduce or might even prevent excessive pro-inflammatory responses. Hence, having and maintaining Bregs could be interesting for patients suffering from allergies, asthma, and autoimmune diseases. Therefore, understanding Breg generation, required signaling, and their developmental requirements are important. Since our group could previously show that zinc is important for regulatory T cells, we aimed to determine the effect of zinc deficiency on Breg development from human peripheral blood CD19+ B cells. We observed highest Breg generation with a combined stimulus of CD40L and the toll like receptor (TLR) ligand, CpG-ODN2006. Using this stimulus, we observed that zinc deficient medium significantly decreased Breg generation from purified B cells. This was not seen in Bregs generated from peripheral blood mononuclear cells (PBMCs) without B cell enrichment suggesting a compensatory mechanism. In line with literature, our data also confirms Bregs develop from CD19+ B cells, since total CD19+ frequencies remained unchanged, while Breg frequencies varied between stimuli and zinc media conditions. Our study shows for the first time that zinc deficiency significantly impairs Breg development, which provides an important new perspective for clinical applications and therapeutic strategies.

Comparative analysis of non-tuberculous mycobacterial lung disease and lung colonization: a case-control study

BackgroundNon-tuberculous mycobacteria (NTM) are common opportunistic pathogens, and the most common infection site is lung. NTM are found commonly in the environment. Many patients have NTM lung colonization (NTM-Col). NTM lung disease (NTM-LD) have no specific sympotms, though it is hard to differentiate NTM-LD and NTM-Col under this circumstance. The aim of this study is to explore the differences between NTM-LD and NTM-Col for future clinical diagnosis and treatment.MethodsWe retrospectively enrolled patients who had a history of NTM isolated from respiratory specimens in Peking Union Medical College Hospital (PUMCH) from January 1st, 2013 to December 31st, 2022. Patients were classified into NTM-LD group and NTM-Col group. Demographic characteristics, clinical manifestations, laboratory tests and imaging findings of the two groups were compared. Comparative analysis was also performed in peripheral blood lymphocyte subsets among three groups.ResultsA total of 127 NTM-LD patients and 37 NTM-Col patients were enrolled. Proportion of patients with bronchiectasis was higher in NTM-LD group than in NTM-Col group (P = 0.026). Predominant NTM isolates were Mycobacterium avium complex (MAC). NTM-LD group had a higher proportion of Mycobacterium intracellulare (P = 0.004). CD4+ T cells counts was lower in NTM-LD group (P = 0.041) than in NTM-Col group. Imaging finding of bronchiectasis (P = 0.006) was higher in NTM-LD group than in NTM-Col group. Imaging findings of bronchiectasis (OR = 6.282, P = 0.016), and CD4+ T cell count (OR = 0.997, P = 0.012) were independent associated factors for differential diagnosis between NTM-LD and NTM-Col.ConclusionNTM isolates from both NTM-LD and NTM-Col patients were predominantly MAC, with a higher Mycobacterium intracellulare isolation rate in NTM-LD group. Imaging findings of bronchiectasis and lower peripheral blood CD4+ T cell count may be helpful to separate the diagnosis of NTM-LD from NTM-Col.

LncRNA SNHG16 Inhibits Intracellular M. tuberculosis Growth Involving Cathelicidin Pathway, Autophagy, and Effector Cytokines Production.

Long noncoding small nucleolar RNA (LncRNA) host gene 16 (SNHG16) is associated with certain diseases, including cancers. However, its role and mechanism in Mycobacterium tuberculosis (Mtb) infection remain unclear. Here, we demonstrated that SNHG16 expression levels were suppressed in peripheral blood mononuclear cells (PBMCs) and CD14+ monocytes of tuberculosis (TB) patients. SNHG16 was up-regulated by acute Mtb infection of PBMCs from healthy control (HC) subjects. Such TB suppression of SNHG16 was consistent with an immunosuppressive-like state driven by IL-10 signaling as seen in TB patients. Notably, SNHG16 limited Mtb growth in macrophages/monocytes through autophagy and vitamin D receptor (VDR)-dependent cathelicidin (CAMP) antimicrobial pathways. Concurrently, SNHG16 was highly expressed in lymphocytes, including CD8+ and Vγ2 Vδ2 T-cell subsets in HCs. SNHG16 overexpression in lymphocytes allowed them to control Mtb infection in macrophages, and SNHG16 epigenetically increased the expression of anti-Mtb effector cytokines in lymphocytes by developing more accessible chromatin states in gene loci encoding IFN-γ, TNF-α, and Granzyme B. Furthermore, the adoptive transfer of SNHG16-overexpressing human PBMCs into Mtb-infected SCID mice conferred protective immunity against Mtb infection. Thus, SNHG16 drove the induction of pleiotropic effector functions that inhibited intracellular Mtb growth in vitro and in vivo, serving as an immunotherapy target in TB.

Peripheral blood lymphocyte subpopulations as predictive biomarkers for first-line programmed death 1 inhibitors efficacy in esophageal squamous cell carcinoma: A retrospective study.

Esophageal cancer (EC) poses a significant global health burden, necessitating effective treatment strategies. Immune checkpoint inhibitors have emerged as a promising therapeutic option for EC, but the identification of predictive biomarkers remains crucial for optimizing patient outcomes. We conducted a retrospective analysis of medical records from advanced esophageal squamous cell carcinoma patients treated with first-line programmed death 1 inhibitors. Peripheral blood lymphocyte subpopulations were evaluated using flow cytometry, while hematological tests provided data on neutrophil, lymphocyte, and monocyte counts. Cox regression and logistic regression analyses were employed to explore the association between lymphocyte subpopulations, baseline characteristics, and progression-free survival (PFS). Among the 100 initially included patients, 70 met eligibility criteria. Multivariate Cox regression analysis revealed a significant association between high CD16+CD56+ lymphocyte proportions and longer PFS, independent of other clinical variables. Similarly, a high CD4+/CD8+ ratio was correlated with prolonged PFS. Kaplan-Meier survival curves supported these findings. Logistic regression analysis indicated no significant differences in the CD4+/CD8+ ratio and CD16+CD56+ lymphocytes concerning baseline characteristics, suggesting their potential as independent prognostic markers. Our study highlights the predictive value of peripheral blood CD16+CD56+ lymphocytes and the CD4+/CD8+ ratio for the efficacy of programmed death 1 inhibitors in advanced esophageal squamous cell carcinoma patients. These findings underscore the importance of peripheral blood biomarkers in guiding personalized immunotherapy strategies and improving outcomes for EC patients.

Downregulating LKB1 in bone marrow mesenchymal stem cells could inhibit CD4+ T cell proliferation via the PD-1/PD-L1 signaling pathway

BackgroundOur previous research has shown that LKB1 in amniotic mesenchymal stem cells (MSCs) serves as a vital regulator of regulatory T cell differentiation and T cell proliferation, which may have a similar role in bone marrow MSCs (BMMSCs). Therefore, we investigated the role of LKB1 in BMMSCs for regulating CD4+ T cell proliferation in the bone micro-environment of AML. MethodsRT-PCR was used to assessed LKB1 expression in BMMSCs derived from AML patients and healthy controls. Subsequently, LKB1 was knocked down in the BMMSCs line HS-5 (HS-5-LKB1KD). Co-cultures in vitro were established to analyze the effect of HS-5-LKB1KD on CD4+ T cell. Flow cytometry was employed to measure PD-L1 and CD4+ T cell proliferation levels. Western blot was utilized to detect related proteins. ResultsThe expression of LKB1 in BMMSCs derived from AML patients was decreased. Knockdown of LKB1 in HS-5 resulted in upregulation of PD-L1 expression. Co-culture of peripheral blood CD4+ T cell with HS-5-LKB1KD exhibited reduced CD4+ T cell proliferation compared to co-culture with HS-5-LKB1con. Furthermore, blocking PD-L1 in the co-culture conditions could restore the reduced CD4+ T cell proliferation. Additionally, it was found that upregulation of the Wnt signaling pathway-related proteins following LKB1 knockdown in HS-5, indicating that downregulating LKB1 could promote PD-L1 expression through activation of the Wnt signaling pathway. ConclusionsThe decreased expression of LKB1 in BMMSCs may activate the Wnt signaling pathway, leading to increased PD-L1 expression. This inhibited CD4+ T cell proliferation, which might lead to impaired anti-tumor immunity in AML patients and promote AML progression.

Effect and Influencing Factors of Peripheral Blood Hematopoietic Stem Cells Collection from Unrelated Donors

To analyze the effect of peripheral blood hematopoietic stem cells (PBSC) collection from unrelated donors and its influencing factors. A retrospective analysis was conducted on the mobilization and collection of PBSC from 113 unrelated donors at Yueyang Central Hospital from January 2021 to December 2023. 113 donors were successfully mobilized. The average count of PBSC mononuclear cells (MNC) and CD34+ cells were (12.40±7.41)×108/kg and (10.64±8.07)×106/kg, respectively. Univariate analysis showed that the PBSC CD34+ cells ratio of male donors was significantly higher than that in female donors (P =0.015). The peripheral blood (PB) white blood cell (WBC) count before collection was positively correlated with the PBSC nucleated cells count (r =0.388), and the donor's body weight, the PB CD34+ cell ratio before collection were positively correlated with the PBSC CD34+ cell ratio (r =0.259, r =0.780). The daily dose of rhG-CSF was negatively correlated with the PBSC CD34+ cell ratio (r =-0.285). Both rhG-CSF agents achieved successful mobilization. Multivariate analysis showed that PB WBC count before collection was a factor affecting the count of PBSC nucleated cells (P <0.001), while the PB CD34+cell ratio before collection was a factor affecting the PBSC CD34+ cell ratio (P <0.001). The mobilization and collection of PBSC from unrelated donors are good, and the PB WBC count and CD34+ cell ratio before collection are reliable indicators for predicting the collection effect.

A Tissue-Engineered Model of T-Cell–Mediated Oral Mucosal Inflammatory Disease

T-cell-mediated oral mucocutaneous inflammatory conditions including oral lichen planus (OLP) are common but development of new treatments aimed at relieving symptoms and controlling OLP progression are hampered by the lack of experimental models. Here, we developed a tissue-engineered oral mucosal equivalent (OME) containing polarised T-cells to replicate OLP pathogenesis. Peripheral blood CD4+ and CD8+ T-cells were isolated, activated and polarised into Th1 and cytotoxic T-cells (Tc). OME were constructed by culturing oral keratinocytes on an oral fibroblast-populated hydrogel to produce a stratified squamous epithelium. OME stimulated with IFN-γ and TNF-α or medium from Th1 cells caused increased secretion of inflammatory cytokines/chemokines. A model of T-cell-mediated inflammatory disease was developed by combining OME on top of a Th1/Tc-containing hydrogel, followed by epithelial stimulation with IFN-γ/TNF-α. T-cell recruitment towards the epithelium was associated with increased secretion of T-cell chemoattractants CCL5, CXCL9 and CXCL10. Histological assessment showed tissue damage associated with cleaved-caspase-3 and altered laminin-5 expression. Treatment with inhibitors directed against JAK, KCa3.1 channels or clobetasol in solution and/or via a mucoadhesive patch prevented cytokine/chemokine release and tissue damage. This disease model has potential to probe for mechanisms of pathogenesis or as a test platform for novel therapeutics or treatment modalities.

Expression of immune checkpoint markers in peripheral blood lymphocytes of women with fibroadenoma of the breast

Changes of the immune state in patients with fibroadenoma (FA) or other benign lesions of the breast, as well as the involvement of immune checkpoints in pathogenesis of these lesions, remain underexplored. The aim of this study was to compare the expression level of the key immune checkpoint markers CD279/PD-1, CD274/PD-L1, CD366/TIM3, and CD223/LAG3 in total circulating lymphocytes, T cell population as well as in its CD4 and CD8 subsets in peripheral blood from women with breast FA and healthy controls. Blood samples were taken from 12 women diagnosed with FA of the breast (aged 23-54 years, FA group) and 15 healthy women (aged 22-52 years, control group). Sample uptake was performed immediately before surgery, and samples were further analyzed by multicolor flow cytometry using monoclonal antibodies CD3-VioBlue, CD4/CD8-FITC, PD1-PE, PD-L1-PerCP-Cy.5.5, and TIM3/LAG3-APC. Each sample was incubated with 4 antibody combinations: CD3/CD4/PD1/PD-L1/TIM3, CD3/CD4/PD1/PD-L1/LAG3, CD3/CD8/PD1/PD-L1/TIM3, and CD3/CD8/PD1/PD-L1/LAG3. First, mono-expression of each of the 4 immune checkpoint markers was evaluated in the lymphocyte gate from both investigated groups. In FA samples, a significant increase in PD-L1 expression (assessed as percent of positive cells and fluorescence intensity change) was observed. Regarding expression of immune checkpoints in CD3+ T cells, along with significantly increased %PD-L1+, elevated numbers of PD1+T cells were detected. As for the differences in immune checkpoint expression changes between CD4+ and CD8+ T cell subsets, FA patient group demonstrated a more prominent increase in the amount of CD8+PD1+T cells relative to CD4 subset. The profiles of PD-L1 changes in CD4 and CD8 subpopulations were comparable showing, in both cases, a significant increase in FA sample group. We also analyzed changes in co-expression of any two immune checkpoint markers in CD4+ and CD8+ T cell subsets. The most noticeable was as increase in the prevalence of PD1+PD-L1+ phenotype in both T cell subpopulations from FA patients compared to the controls. With respect to co-expression of 3 checkpoint markers in CD4+ and CD8+ T cells, a significant increase in the percentage of PD1+PD-L1+TIM3+ cells among CD4 T helpers was found in FA. Thus, specific changes of T cell phenotype related to (co-)expression of immune checkpoint regulators may occur in systemic circulation of women with breast FA.

The Role of NK and T Cells in Endometriosis.

Endometriosis, a debilitating condition, affects one in ten women of reproductive age. Its pathophysiology remains unclear, though deficiencies in immune surveillance are thought to create an environment conducive to the evasion of ectopic endometrial cells from the immune system. Our research explores the immunological impact of endometriosis both locally and systemically, emphasizing natural killer (NK) and T cell subpopulations. We incorporated 62 female patients who underwent laparoscopic surgery; of those, 47 had endometriosis, and 15 were controls. We collected peritoneal fluid (PF) and peripheral blood (PB) samples which were tagged with monoclonal antibodies and subsequently scrutinized using flow cytometry. Our findings revealed significant differences in immunological profiles based on demographic factors and symptomatology. In the endometriosis cohort, there was an increase in PB CD56HiCD16dim and PF CD8+ CD56dimCD16Hi NK cells. CD16+ CD4 T cell levels were significantly lower in the PB of endometriosis patients who smoke. Individuals with more severe disease displayed significantly higher levels of PB CD16+ CD8 T cells, which also increased in those with non-menstrual pelvic pain. Dysmenorrhea severity correlated with a progressive increase in PF CD8+ CD56dimCD16Hi NK cells. These variations in specific lymphocyte subsets, namely, within NK and T cells, suggest potential immunological mechanisms in the evolution and clinical presentation of endometriosis.

UM171 enhances fitness and engraftment of gene-modified hematopoietic stem cells from patients with sickle cell disease

AbstractHematopoietic stem cell (HSC) transplantation with lentiviral vector (LVV)-transduced autologous cells has proven an effective therapeutic strategy for sickle cell disease (SCD). However, ex vivo culture or proliferative stress associated with in vivo reconstitution may amplify any underlying genetic risk of leukemia. We aimed to minimize culture-induced stress and reduce genomic damage during ex vivo culture and enhance stem cell fitness and reconstitution of SCD CD34+ cells transduced with BCL11A shmiR-encoding LVV. UM171, a pyrimidoindole derivative, can expand normal HSCs during in vitro culture and has been shown to be safe and effective using umbilical cord blood. We examined the effect of UM171 during ex vivo LVV transduction of SCD HSCs. Culture of SCD CD34+ HSCs with UM171 during transduction reduced DNA damage and reactive oxygen species, decreased apoptosis, and was associated with increased numbers of immunophenotypically defined long-term HSCs. UM171 increased the engraftment of LVV-transduced human HSCs in immunodeficient mice and barcode tracing revealed increased clonal diversity of engrafting cells. In competitive transplantation assays, analysis of bone marrow showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions. In summary, exposure of SCD peripheral blood CD34+ cells to UM171 during LVV transduction enhances stem cell fitness. These findings suggest manufacturing of genetically modified HSCs in the presence of UM171 may improve efficacy, safety, and sustainability of gene therapy using ex vivo approaches. BCL11A shmiR-encoding LVV is in clinical trials to treat SCD (NCT03282656), UM171 is in clinical trials to culture umbilical cord blood (NCT02668315).

Preemptive Approach to Plerixafor Use Is Optimal in Patients With Relapsed/Refractory Germ Cell Tumors Undergoing Peripheral Blood Hematopoietic Stem Cell Collection: Effect on Collection Days, Yields, and Cost.

Evidence describing the use of plerixafor in the off-label population of relapsed/refractory germ cell tumors (GCT) is limited. Weaim to describe the effect of rescue versus preemptive plerixafor use on apheresis collection days, collection yields, and cost. We retrospectively collected data on 77 consecutive patients (at least 15 years of age) with GCT who underwent peripheral blood stem cell (PBSC) collection for autologous stem cell transplant between January 1, 2020 and May 1, 2022. Depending on insurance approval, plerixafor was given either as "rescue" (after a first apheresis collection of < 5 × 106 CD34+ cells/kg) or as "preemptive" on Day 4 of granulocyte-colony stimulating factor (G-CSF) prior to the first apheresis collection, if the Day 4 peripheral blood CD34+ count was < 40 cells/μL. A total of 66% of patients who received preemptive plerixafor completed collection in 1 day, similar to good mobilizers who only needed G-CSF (71%, p = 0.366). In contrast, all poor mobilizers in the rescue group required at least 2 days of collection and had lower CD34+ cell yields than the preemptive group (7.15 vs. 9.81 × 106/kg, p = 0.0055). A cost analysis revealed that preemptive plerixafor may save approximately $7000 per patient compared with a rescue approach. Preemptive plerixafor in GCT patients undergoing PBSC collection allows relatively poor mobilizers to collect in fewer days and with lower overall cost. Fewer apheresis procedures result in less risk to the patient, increased patient satisfaction, and the ability to schedule more patients within the constraints of staffing.

RPL26 variants: A rare cause of Diamond-Blackfan anemia syndrome with multiple congenital anomalies at the forefront

PurposeDiamond-Blackfan anemia syndrome (DBS) is a rare congenital disorder originally characterized by bone marrow failure with or without various congenital anomalies. At least 24 genes are implicated, the vast majority encoding for ribosomal proteins. RPL26 (ribosomal protein L26) is an emerging candidate (DBA11, MIM#614900). We aim to further delineate this rare condition. MethodsPatients carrying heterozygous RPL26 variants were recruited. In one of them, erythroid proliferation and differentiation from peripheral blood CD34+ cells were studied by flow cytometry, and RPL26 expression by quantitative reverse transcription polymerase chain reaction and immunoblotting. ResultsWe report on 8 affected patients from 4 families. Detailed phenotyping reveals that RPL26 is mainly associated with multiple congenital anomalies (particularly radial ray anomalies), albeit with variable expression. Mandibulofacial dysostosis and neural tube defects are potential features in DBA11, expanding the growing list of DBS abnormalities. In 1 individual, we showed that RPL26 haploinsufficiency was responsible for subclinical impairment in erythroid proliferation and enucleation. The absence of hematological involvement in 4 adults from this series contributes to the mounting evidence that bone marrow failure is not universally central to all DBS genes. ConclusionWe confirm RPL26 as a DBS gene and expand the phenotypic spectrum of the gene and the disease.

Efficacy and Safety of Rituximab Treatment for Anti-N-Methyl-d-Aspartate Receptor Encephalitis Without Tumor in Children

BackgroundTo evaluate the efficacy and safety of rituximab treatment for anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis without tumor in children. MethodsEighteen pediatric patients with NMDAR encephalitis treated with rituximab after failure of intravenous immunoglobulin (IVIG) and methylprednisolone treatment were analyzed retrospectively in terms of their medical history, clinical features, laboratory examination results, and treatments. The modified Rankin scale (mRS) score, peripheral blood CD19+ B cells, recurrence, and adverse events were used to evaluate the efficacy and safety of rituximab. ResultsThe patients were treated with rituximab 3.2 ± 1.0 days after the end of IVIG and methylprednisolone treatment. After initial rituximab treatment for four weeks, the mRS score and number of CD19+ B cells in all patients were significantly lower than those before treatment (P < 0.05). At the last follow-up (44.1 months, 17.7 S.D.), all patients had recovered well (mRS ≤2), 14 patients (77.8%) recovered completely (mRS = 0), three patients had recurrent seizures, and one patient had mental and language impairment. Two patients had transient mild adverse events during infusion, and none of the other patients experienced severe adverse events during hospitalization or follow-up. ConclusionsRituximab appears safe and may be effective for the treatment of anti-NMDAR encephalitis without tumor in children refractory to first-line agents.

Expression of γδ T cells and lymphocyte subsets in newly diagnosed multiple myeloma patients

ObjectiveTo retrospectively analyze the expression and significance of γδ T cells and lymphocyte subsets in peripheral blood on patients with multiple myeloma (MM). MethodsFifty-five patients with multiple myeloma admitted to Yangpu Hospital Affiliated to Tongji University from January 2022 to August 2023 were selected as the disease group, and were categorized into stage I group, stage II group and stage III group according to the International Staging System (ISS) of multiple myeloma. All included patients were consecutive visits. Another 30 cases of healthy people with physical examination were selected as the control group. Flow cytometry was used to detect the γδ T cells and lymphocyte subsets in the peripheral blood of the subjects. After clinical indicators of patients and healthy controls were collected, the statistical analysis was conducted. ResultsCompared with the control group, the percentage of γδ T cells and CD8+ T cells in the disease group were higher, while, the absolute numbers of total peripheral blood lymphocytes (LYMT) and CD19+ B cells, percentage of CD19+ B cells, CD3+ T cells, CD4+ T cells were lower (P​<​0.05). The percentage of γδ T cells (P​<​0.001), the percentage of CD19+ B cells (P​=​0.003), the absolute number of CD3+ T cells (P​=​0.010), the absolute number of CD8+ T cells (P​=​0.002) were the significant influential factors of MM. γδ T cells (P​=​0.004), neutrophils (P​<​0.001), neutrophil to lymphocyte ratio (P​=​0.003), and white blood cell count (P​<​0.001) were statistically different in different stages. ROC curve suggested that the area under the curve of the percentage of γδ T cells in predicting the MM were 0.973 (P​<​0.001) which had certain diagnostic value. Conclusionsγδ T cells and lymphocyte subsets (CD8+ T cells, CD19+ B cells, CD3+ T cells, etc.) in patients with newly diagnosis of MM can reflect the immune function of the patient’s organism, which is a key factor of MM. γδ T cells influence the staging of MM patients, and also have certain diagnostic value.