Mature CD4 Research Articles

Generation of ex vivo autologous hematopoietic stem cell-derived T lymphocytes for cancer immunotherapy

CD19CAR-T cell therapy demonstrated promising outcomes in relapsed/refractory B-cell malignancies. Nonetheless, the limited T-cell function and ineffective T-cell apheresis for therapeutic purposes are still concern in heavily pretreated patients. We investigated the feasibility of generating hematopoietic stem cell-derived T lymphocytes (HSC-T) for cancer immunotherapy. The patients’ autologous peripheral blood HSCs were enriched for CD34+ and CD3+ cells. The CD34+ cells were then cultured following three steps of lymphoid progenitor differentiation, T-cell differentiation, and T-cell maturation processes. HSC-T cells were successfully generated with robust fold expansion of 3735 times. After lymphoid progenitor differentiation, CD5+ and CD7+ cells remarkably increased (65–84 %) while CD34+ cells consequentially declined. The mature CD3+ cells were detected up to 40 % and 90 % on days 42 and 52, respectively. The majority of HSC-T population was naïve phenotype compared to CD3-T cells (73 % vs 34 %) and CD8:CD4 ratio was 2:1. The higher level of cytokine and cytotoxic granule secretion in HSC-T was observed after activation. HSC-T cells were assessed for clinical application and found that CD19CAR-transduced HSC-T cells demonstrated higher cytokine secretion and a trend of superior cytotoxicity against CD19+ target cells compared to control CAR-T cells. A chronic antigen stimulation assay revealed similar T-cell proliferation, stemness, and exhaustion phenotypes among CAR-T cell types. In conclusions, autologous HSC-T was feasible to generate with preserved T-cell efficacy. The HSC-T cells are potentially utilized as an alternative option for cellular immunotherapy.

Hydrogel based on M1 macrophage lysate and alginate loading with oxaliplatin for effective immunomodulation to inhibit melanoma progression, recurrence and metastasis

Despite the monumental success of immunotherapy in treating melanoma clinically, it still confronts significant challenges, chiefly that singular immunomodulatory tactics are insufficient to suppress the recurrence and metastasis of melanoma. Herein, these challenges are addressed by a hydrogel based on M1 macrophage lysate and alginate (M1LMHA) loaded with oxaliplatin (OXA), named M1LMHA@OXA.The results obtained from scanning electron microscopy and confocal microscopy indicate that the structure and morphology of M1LMHA@OXA remain unchanged. Flow cytometry results reveal that M1LMHA@OXA significantly promotes the maturation of dendritic cells (DCs) and enhances the proliferation of T lymphocytes. In a subcutaneous melanoma transplant model, M1LMHA@OXA effectively suppressed tumor growth in comparison to OXA alone and M1LMHA alone. Flow cytometry demonstrated that M1LMHA@OXA markedly increased the number of mature DCs and CD8+ T cells at the tumor site, while significantly reducing the quantity of M2-like tumor-associated macrophages (TAM) and enhancing the presence of M1 macrophages. Enzyme-linked immunosorbent assay (ELISA) results indicated that following treatment with M1LMHA@OXA, the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the bloodstream of mice were significantly elevated, whereas interleukin-10 (IL-10) exhibited no significant difference. This outcome further corroborates the ability of M1LMHA@OXA to substantially bolster the immune capability of mice. Similar results have also been observed in a melanoma subcutaneous transplantation recurrence model, and optical imaging of the lungs of mice revealed that M1LMHA@OXA inhibited tumor metastasis to the lungs. Notably, M1LMHA@OXA exhibits an exceptional therapeutic effect on the growth, post-surgical recurrence, and metastasis of the B16F10 melanoma. Therefore, this study provides a straightforward strategy that leverages the cooperative regulation of multiple immune cells to thwart the proliferation, recurrence, and spread of melanoma.

Divergent Immediate and Delayed Effects of Juvenile Exposure to Doxorubicin on the Thymus in C57BL/6 Mice.

The understanding of alterations within the immune system following doxorubicin (DOX) chemotherapy, and subsequent restoration, in childhood cancer survivors remains limited. This investigation endeavors to elucidate the immediate and delayed changes in thymic immune cell populations and their phenotypes in response to clinically relevant low doses of DOX in a juvenile mouse model. Male mice underwent a regimen of repeated low-dose DOX intraperitoneal injections at 4 mg/kg/week for three consecutive weeks. One week after the last dose of DOX, a subset of mice was euthanized to assess the immediate effects of DOX administration. A second subset of mice was euthanized five weeks after the last DOX dose to evaluate the delayed effects. Thymic samples were collected for multiparameter flow cytometry analysis to evaluate alterations in immune cell composition and phenotype. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure gene expression of- cytokines and senescence markers. One week following DOX administration, DOX treatment resulted in significant decline in thymus weight, with notable alterations in immune cell subpopulations. Reduced frequencies of mature CD3+CD4+ and CD3+CD8+ T cells were observed, along with changes in proliferation and exhaustion markers. Gene expression analysis revealed upregulation of Foxn, Pax1, Ifnγ, and Il7 alongside decreased Il6 and Il17 expression. Furthermore, Cdkn1a (p21Cip1) expression was elevated, suggesting immunosenescence. Five weeks following DOX administration, delayed effects of DOX treatment manifested in rebound increase in thymus weight and altered frequencies of CD4+ and CD8+ T cell subsets, with distinct patterns of proliferation and exhaustion observed. Notably, central memory CD4+ T cells exhibited significant decrease in frequency, while naive and effector memory CD4+ T cells showed reduced proliferation (Ki67+) and PD1 expression. Similar trends were observed in CD8+ T cell subsets, indicating selective effects of DOX on T cell differentiation and function. Although expression of thymus-related genes was normalized, p21Cip1 gene expression remained elevated. DOX treatment elicits a multifaceted influence on immune cell subsets and thymic weight. Immediate effects included thymic atrophy and reductions in mature T cell populations, while delayed effects showed rebound thymic hyperplasia and selective changes in CD4+ and CD8+ T cell subsets. Notably, both central memory and effector memory T cells exhibited reduced proliferation and exhaustion, suggesting unique impacts of DOX on immune cell function. The enduring elevation in p21Cip1 gene expression 5 weeks after DOX treatment suggests an immunosenescent phenotype. These observations collectively illuminate the formidable task of preserving immune competence and overall well-being in childhood cancer survivors subjected to DOX therapy.

Lenalidomide and pomalidomide modulate hematopoietic cell expansion and differentiation in the presence of MSC

Cytopenia is a well-documented complication in the treatment of hematological malignancies with lenalidomide and pomalidomide. Although prior studies have highlighted direct effects on hematopoietic cells to explain this adverse effect, the involvement of hematopoietic-supportive stroma remains less understood. This study examined the effects of lenalidomide/pomalidomide on the expansion and differentiation of human CD34+ hematopoietic stem/progenitor cells (HSPCs) in vitro, in co-culture with human bone-marrow mesenchymal stromal/stem cells (MSCs). Our findings indicate that lenalidomide/pomalidomide increases the population of immature CD34+CD38− cells while decreasing the number of mature CD34+CD38+ cells, suggesting a mechanism that inhibits early HSPC maturation. This effect persisted across myeloid, megakaryocytic, and erythroid lineages, with MSCs playing a key role in preserving immature progenitors and inhibiting their differentiation. Furthermore, in myeloid differentiation assays augmented by granulocyte-colony stimulating factor, lenalidomide/pomalidomide not only enhanced the presence of CD34+ cells with mature myeloid markers such as CD11b but also reduced the populations lacking CD34 yet positive for these markers, irrespective of MSC presence. Thus, while MSCs support the presence of these immature cell populations, they simultaneously inhibit their maturation. This finding provides novel mechanistic insights into lenalidomide- and pomalidomide-induced cytopenia, and could guide therapeutic strategies for its mitigation.

Carotid artery transplantation of brain endothelial cells enhances neuroprotection and neurorepair in ischaemic stroke rats.

Brain microvascular endothelial cells (BMECs), an important component of the neurovascular unit, can promote angiogenesis and synaptic formation in ischaemic mice after brain parenchyma transplantation. Since the therapeutic efficacy of cell-based therapies depends on the extent of transplanted cell residence in the target tissue and cell migration ability, the delivery route has become a hot research topic. In this study, we investigated the effects of carotid artery transplantation of BMECs on neuronal injury, neurorepair, and neurological dysfunction in rats after cerebral ischaemic attack. Purified passage 1 endothelial cells (P1-BMECs) were prepared from mouse brain tissue. Adult rats were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Then, the rats were treated with 5 × 105 P1-BMECs through carotid artery infusion or tail vein injection. We observed that carotid artery transplantation of BMECs produced more potent neuroprotective effects than caudal injection in MCAO rats, including reducing infarct size and alleviating neurological deficits in behavioural tests. Carotid artery-transplanted BMECs displayed a wider distribution in the ischaemic rat brain. Immunostaining for endothelial progenitor cells and the mature endothelial cell markers CD34 and RECA-1 showed that carotid artery transplantation of BMECs significantly increased angiogenesis. Carotid artery transplantation of BMECs significantly increased the number of surviving neurons, decreased the cerebral infarction volume, and alleviated neurological deficits. In addition, we found that carotid artery transplantation of BMECs significantly enhanced ischaemia-induced hippocampal neurogenesis, as measured by doublecortin (DCX) and Ki67 double staining within 2 weeks after ischaemic injury. We conclude that carotid artery transplantation of BMECs can promote cerebral angiogenesis, neurogenesis, and neurological function recovery in adult rats after ischaemic stroke. Our results suggest that carotid injection of BMECs may be a promising new approach for treating acute brain injuries.

Tp53 Genetic Mutations in Chronic Lymphocytic Leukemia Patients in Iraq

Abstract BACKGROUND: Chronic lymphocytic leukemia (CLL) is a malignant clonal proliferative condition of mature CD5+ B cells, with a 4.2/100,000 annual incidence rate and the median age at diagnosis is 72 years. CLL impacts mainly the elderly in Western nations. About 4%–10% of patients have 17p deleted at diagnosis, which includes tumor-suppressor protein (TP53) gene deletions and/or mutations. However, TP53 can also be acquired during the course of the disease, with a predictable incidence of 42%–45% in refractory CLL. Even when this mutation develops in a small percentage of neoplastic cells, the attending of subclonal TP53 changes has been linked to a bad prognosis. TP53 mutations are correlated with significantly lower lifespan and indicate impaired tolerance to chemoimmunotherapy, making them among the most powerful prognostic markers-guiding treatment decisions in CLL. MATERIALS AND METHODS: This cross-sectional study was performed at different centers in Iraq, including Euphrates Center for Cancerous Tumors, Najaf, and the Hematology center-medical City, Baghdad, Imam al Hussien Medical City, Karbala, from October 2023 to June 2024, with patients diagnosed by blood film and immunophenotyping CLL, included 63 patients. RESULTS: DNA Sequencing of TP53 genes investigated by Sanger technique to detect TP53 variations. All potential genotype variations of the TP53 gene were detected across several single-nucleotide polymorphisms (SNPs). CONCLUSIONS: The vast majority, 93% of our patients, carry variant-type mutations of TP53. The genotype of SNP rs1597359353 was found P < 0.05, which is significant according to the White blood count (WBCs) count, although all patients on treatment or follow-up had TP53 mutations.

DIPG-32. NEOANTIGEN HEAT SHOCK PROTEIN VACCINE, RHSC-DIPGVAX, IS ASSOCIATED WITH INCREASED VACCINE-INDUCED B CELLS AND BCR/TCR REPERTOIRE DIVERSITY IN PEDIATRIC PATIENTS WITH DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND Diffuse midline gliomas (DMG/DIPG) remain a devastating class of tumors with limited treatment options. Cancer vaccines educate the immune system to tumor neoantigens to facilitate immune-mediated tumor destruction. DMGs have high-frequency, co-occurring neoantigens and are therefore a relevant target for activation of anti-tumor immunity via vaccine. To this end, we have initiated a clinical trial (NCT04943848) to determine if a neoantigen peptide-based vaccine with recombinant human heat-shock constitutive protein 70 and QS-21 adjuvant can promote anti-tumor immunity against DMG/DIPG. NCT04943848 is ongoing and now enrolling subsequent arms with rHSC-DIPGVax plus checkpoint blockade. METHODS Whole blood was collected before the first vaccine cycle (C1D1). Vaccine doses were administered every 2 weeks and blood was collected prior to the third or fifth vaccine cycle (C3D1 & C5D1). Peripheral blood mononuclear cells (PBMCs) were isolated and stored at -160°C. PBMCs from each patient, pre- and post-vaccine, were stained with human T-cell, myeloid, and B-cell antibody panels before spectral flow cytometry, single-cell RNA sequencing, and genomic DNA isolation for T- and B-cell receptor (TCR/BCR) sequencing. Spectral flow cytometry data was analyzed via FlowJo v10. TCR/BCR sequencing was processed via MiXCR and Immunarch. RESULTS Following vaccination, there were higher percentages of two mature CD20+CD19+IgM+ B cell populations, including a 4-1BBL+CD86+ population previously shown to induce robust CD8+ T-cell activation in adults with glioblastoma. Interestingly, given the recent discovery of TIM3’s importance in DIPG, we also identified TIM3-expressing CD4+ and CD8+ T cell populations. Finally, TCR/BCR sequencing demonstrated increased receptor repertoire diversity after vaccination, which is indicative of the vaccine shaping adaptive immunity. CONCLUSIONS Peripheral immune populations and TCR/BCR sequences change during vaccination with rHSC-DIPGVax. Ongoing analyses will determine the specificity of the patient immune response to vaccine antigens and correlate changes in immune populations to clinical course.

Ferroptosis-induced immunomodulation with biometabolic MOF@COF nanovaccine for self-boosting anti-tumor immunotherapy

Ferroptosis, a regulated cell death pathway, presents a compelling approach for augmenting anti-tumor immunity, addressing challenges associated with immune escape and inadequate immunogenicity linked to apoptosis resistance. In this study, we introduce a biometabolic MOF@COF nanovaccine (NMCAH) designed to evoke self-amplifying anti-tumor responses through ferroptosis-mediated immune stimulation. The nanovaccine demonstrates the capacity to induce the generation of reactive oxygen species and deplete glutathione within tumors, initiating immunogenic ferroptosis. Simultaneously, it facilitates the polarization of macrophages toward the M1 phenotype and the maturation of CD8+ T cells. Upon priming, these immune cells autonomously amplify the response in a self-reinforcing manner. Notably, IFN-γ secretion by CD8+ T cells reinforces macrophage polarization and induces secondary ferroptosis via ACSL4 up-regulation. This intricately coordinated interplay between ferroptosis and adaptive immunity holds significant promise in overcoming the immunosuppressive tumor microenvironment. Our study establishes a versatile nanotherapeutic platform for advancing cancer immunotherapy by leveraging the immunogenic characteristics of regulated cell death.

Single-cell transcriptomics reveals antigen-presenting capacity and therapeutic resistance potential of immunomodulatory endothelial cells in colorectal cancer.

The heterogeneity of tumor endothelial cells (TECs) hinders the efficacy of antiangiogenic therapies (AATs). Only a small percentage of angiogenic TECs are considered effective targets for AATs. Immunomodulatory ECs (IMECs), as a newly focused functional subgroup of endothelial cells (ECs), are being evaluated for their ability to regulate tumor immune balance and influence existing AATs. Based on single-cell transcriptome data from colorectal cancer in a publicly available database, we conducted a wide array of bioinformatic approaches to study EC subsets that meet the IMECs definition. Our investigation encompassed the gene expression signatures of these subsets, cellular composition differences, cell-cell interactions. Two subsets that meet the IMECs definition were found in tumors and para-cancerous tissues. Combined with the results of gene ontological analysis and interaction with CD4+ T cells, we found that IMECs can present MHC-II antigens to mature CD4+ T cells. There were differences in the level of interaction between IMECs and different types of mature CD4+ T cell subsets. In addition, IMEC subsets had different expression levels of angiogenesis related genes. The angiogenesis score of IMECs decreased after patients received immunotherapy. IMEC subsets do not depend on a single proangiogenic receptor and are involved in regulating angiogenesis, which may reduce the efficacy of AATs. The adverse effects of specific IMEC subsets on AATs were validated in the RNA-seq dataset of the bevacizumab treatment group. Our study suggests the potential MHC-II antigen presentation capacity of IMECs and the enhanced angiogenesis characteristics within tumors. The function of IMECs in the vascular network may have a potentially adverse effect on AATs. Controlling the functional properties of IMECs may be a new angle for tumor therapy.

SKI Regulates Medullary Thymic Epithelial Cell Differentiation to Control Peripheral T Cell Responses in Mice.

The thymus is an important site for the establishment of an appropriate immune response through positive and negative selection of developing T cells. During selection, developing T cells interact with cortical and medullary thymic epithelial cells (TECs), termed cTECs and mTECs, respectively. Using a Foxn1Cre+/-SKIfl/fl mouse model, we found that TEC-specific deletion of SKI reduced the mTEC compartment in the thymus and that tissue-restricted Ag expression in mTECs was altered. This decrease in the medullary area led to a decrease in CD4 thymocyte cellularity; however, mature CD4 cellularity in the spleen remained normal. Interestingly, naive CD4 T cells purified from SKI-deleted mice showed a defect in proliferation invitro after global TCR stimulation, and these mice were significantly protected from developing experimental autoimmune encephalomyelitis compared with the control mice. Overall, our findings suggest that SKI signaling in the thymus regulates mTEC differentiation and function as well as downstream peripheral T cell responses and provide evidence for targeting SKI in T cell-driven autoimmune diseases such as multiple sclerosis.

Immunotoxicity of 2-Acetyl-4-tetrahydroxybutylimidazole in BALB/c mice with different vitamin B6 nutritional statuses

Caramel color is a widely used food pigment, and 2-Acetyl-4-tetrahydroxybutylimidazole (THI) is a by-products of Class III caramel color. Some studies have shown that THI can reduce the number of peripheral blood lymphocytes. However, the comprehensive mechanism of THI immunotoxicity requires further study. In this study, the effects of THI on lymphocyte count, humoral immunity, cellular immunity and nonspecific immunity were determined and the effect of the nutritional status of VB6 on THI immunotoxicity was evaluated. Female BALB/c mice were divided into 3 groups and fed chow containing different doses of VB6: VB6-normal (6 mg/kg VB6), VB6-deprived (0.5 mg/kg VB6) or VB6-enhanced (12 mg/kg VB6) feed. Each group was further divided into 4 subgroups and treated with THI (0.5, 2.5 or 12.5 mg/kg bw) or the solvent control by gavage for 30 days. The thymic cortical thickness was measured with ViewPoint; the proportions of major immune cells and T cells in peripheral blood and tissues were detected via flow cytometry; the transformation and proliferation abilities of T and B cells were detected via T and B lymphocyte proliferation assays; NK cell activity was assessed via lactate dehydrogenase assays; humoral immune function was assessed via plaque-forming cell assays; and the immune function of T lymphocytes was assessed via delayed type hypersensitivity assays. The results showed that compared with those in the corresponding control group, the white blood cell count and lymphocyte count decreased significantly in all the VB6-deprived groups, in the 2.5 and 12.5 mg/kg VB6 groups, and in the 12.5 mg/kg VB6-enhanced group. With increasing THI dose, the thymic cortical layer became thinner. In the thymus, THI increased the proportions of CD3+ T cells and mature CD8+ T cells and decreased the proportions of immature double-positive, double-negative T cells and CD69-expressing lymphocytes. The proportions of naïve T cells and Tcm (central memory T) cells related to homing decreased. The proportion of mature T cells in the spleen decreased significantly. The proliferation of T cells stimulated by ConA decreased after THI exposure. VB6-deficient mice were more sensitive to THI immunotoxicity, and supplementation with VB6 had a certain protective effect on these mice. The results of the PFC and NK cell activity assays indicated that THI exposure might not affect humoral immune or innate immune function.

CCR6+ T helper cells and regulatory T cells in the blood and gastric mucosa during Helicobacter pylori infection.

Helicobacter pylori (H. pylori) can evade the host's immune response and persist for a long time on the gastric mucosa. T helper (Th) cells appear to be involved in the control of H. pylori bacteria but promote mucosal inflammation. In contrast, regulatory T cells (Tregs) may reduce inflammation but promote H. pylori persistence. CC motif chemokine receptor 6 (CCR6) is involved in the migration of various cells into inflamed gastric mucosa. In this study, we examined CCR6+ Th cells and CCR6+ Tregs during H. pylori infection in humans. Isolation of cells from blood and mucosal biopsies, magnetic separation of В cells, CD4+ and CD4+CCR6+CD45RO+ T cells, antigen-specific activation, B cell response in vitro, flow cytometry, determination of CD4+CD25hiFoxP3+ Tregs and various groups of Th cells. CD4+CCR6+ blood lymphocytes from healthy donors included Th cells and Tregs. These CCR6+ Th cells produced proinflammatory cytokines and also stimulated plasma cell maturation and antibody production in vitro. H. pylori gastritis and peptic ulcer disease were associated with an increase in the number of circulate CD4+CCR6+CD45RO+ cells and the percentage of Th1, Th17 and Th1/17 cells in this lymphocyte subgroup. In H. pylori-positive patients, circulating CD4+CCR6+ cells contained a higher proportion of H. pylori-specific cells compared with their CD4+CCR6- counterparts. H. pylori infection strongly increased the content of CD4+ lymphocytes in the inflamed gastric mucosa, with the majority of these CD4+ lymphocytes expressing CCR6. CD4+CCR6+ lymphocytes from H. pylori-infected stomach included Tregs and in vivo activated T cells, some of which produced interferon-γ without ex vivo stimulation. H. pylori infection causes an increase in the number of mature CD4+CCR6+ lymphocytes in the blood, with a pro-inflammatory shift in their composition and enrichment of the gastric mucosa with CD4+CCR6+ lymphocytes, including CCR6+ Th1 cells and Tregs.

The Impact of Phenotype of Inflammatory Bowel Diseases, Inflammation Activity and Therapy on Mucosal Mature Cd83+ Dendritic Cell.

Background: Crohn's disease (CD) and ulcerative colitis (UC) are well-defined phenotypes of chronic inflammatory bowel diseases (IBDs). A mechanism of inflammation in these diseases is partially controlled by the intestinal dendritic cell (DC). In this study, we observed a mature CD83+ DC in colonic bioptic samples, and its correlation with disease phenotype and activity. Methods: The study included 219 subjects: 100 with UC, 44 with CD and 75 healthy subjects. Colonic biopsy specimens were incubated with the primary antibody Anti-CD83. Intraepithelial CD83+ DCs were counted per 100 enterocytes. The presence of CD83+ DC was analysed according to the type of IBD, histopathologic inflammation activity and treatment outcome. Results: The presence of mature CD83+ DCs (0, ≥1) differed according to disease types of IBD (p = 0.001), histologic inflammation activity (p = 0.049) and applied therapy (p = 0.001). The odds for CD83+ DC presence were 5.2 times higher in the CD group than in the control/UC group. The odds for CD83+ DC presence were 2.6 times higher in subjects without inflammation or chronic inflammation than with acute inflammation. They were also 3.7 times higher in subjects without therapy. The cut-off value 0.5 CD83+ DC (Rock analysis area = 0.699; SE 0.046; p < 0.001; 95% CI: 0.609-0.788) had been assessed as a differentiation marker between UC and CD. Conclusion: Presence of CD83+ DC could be used as a possible parameter in distinction between UC and CD, as well as a predictor of inflammation activity and treatment outcome.

Pathological proliferation: a potential mechanism for poor CD4+ T cell recovery in people living with HIV.

People living with HIV (PLWH) fail to achieve normalization of CD4+ T cell counts and function, especially in immunological non-responders (INRs). The frequencies of Ki67+CD4+ T cells were inversely associated with CD4+ T cell counts in HIV infected patients. Early ART did not normalize CD4+ T cell proliferation. However, the features of the abnormal proliferation CD4+ T cell in INRs are far from known. PLWH were divided into INRs (n= 16) and immunological responders (IRs, n= 53) groups. Mass cytometry was applied to peripheral blood T cells to profile the immune cells and liquid chip technique was used to measure plasma levels of cytokines and chemokines. Correlation analyses were conducted to evaluate associations between the degree of CD4+ T cell proliferation and immune function. The percentage of Ki67+ CD4+ T cells were significant higher in INRs, and we defined these cells with significant higher level of Ki67, as over-proliferating cells. No significant difference of markers' expression (HLA-DR, CD38, CD57, PD-1, PD-L1, CD107a, perforin) was found between INRs and IRs. Compared with naïve CD4+ T cells in INRs, Ki67+ CD4+ T cells exhibited lower levels of CD57 and CD38. Whereas Ki67+ T cells exhibited higher levels of CD38 and CD57 and activation compared with differentiated mature central memory CD4+ T cells and effector memory CD4+ T cells. Ki67+ cells did not show higher levels of senescence and activation compared to certain Ki67- CD4+ central memory T cells in IRs. Furthermore, Ki67+ CD4+ Tcm cells exhibited positive correlations with pro-inflammatory cytokines. We proposed and validated the hypothesis of "pathological proliferation" in INRs: excessive proliferation of CD4+ T cells in INRs may be accompanied by aberrant activation, senescence and loss of immune function. Eventually, such over-proliferating but poor-quality cells in INRs result in incomplete recovery of both CD4+ T cell counts and function. An intervention that enhancing the proliferative capacity or functional ability or both of CD4+ T cell in INRs might therefore be beneficial.

Abstract 629: Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem cells

Abstract Introduction: In patients with acute myeloid leukemia (AML), CD123 has been shown to have high expression on blast cells and leukemic stem cells (LSC) compared with normal hematopoietic stem cells (HSCs) and other more mature CD34+ subsets (Zhou J, World J Stem Cells 2014). LSCs are inherently resistant to standard of care chemotherapeutics and LSC persistence after chemotherapy is associated with disease relapse. VIP943 is an ADC consisting of an anti-CD123 antibody, a unique linker cleaved intracellularly by legumain which is required for release, and activation of a novel kinesin spindle protein inhibitor (KSPi) payload that accumulates inside the cell. Herein, we evaluated the potential of VIP943 to target LSC and progenitor cells. Methods: Cytotoxicity assay: Bone marrow aspirates (BMA) and peripheral blood from previously untreated patients with primary or transformed from myelodysplastic syndrome AML were used in this experiment. Cells were treated with VIP943 (66 nM or 330 nM). After the end of the 48 h or 72 h incubation, the CD123+ cells and the CD34+CD38- LSC were detected by flow cytometry. Characterization of progenitor cell population: Fresh BM samples from healthy volunteers (HVBM) were stained with specific monoclonal antibodies to identify progenitor cell populations. Depletion assay: The effect of VIP943 on HVBM was evaluated by measuring the depletion of CD34+ progenitor cell populations in comparison to the anti-CD33-ADC, gemtuzumab ozogamicin (gem-oz). At the end of the incubation time, red blood cells were lysed; the remaining cells were stained with a cocktail of antibodies to discriminate between progenitor cells and analyzed by flow cytometry. Results: Treatment of patient-derived CD123+ leukemic blasts with VIP943 resulted in a 50% reduction after 48 h and up to 80% at 72 h. A reduction of &amp;gt;70% of CD123+CD34+CD38- LSCs derived from BM aspirates of untreated patients with AML was achieved after incubation with VIP943 up to 72h. In addition, HVBM samples derived from five healthy volunteers were treated with VIP943 or gem-oz in a depletion assay, where gem-oz was toxic to CD34+ cells with an EC50 of 0.16 µM in contrast to VIP943 with an EC50 value of 8.83 µM. Conclusions: In this in vitro analysis of primary samples from patients with AML, activity of VIP943 is observed in AML patient-derived CD123+ blasts as well as chemoresistant LSCs. At anticipated pharmacologic levels, VIP943 shows no adverse effects on HSCs unlike gem-oz suggesting an improved therapeutic index. In the ongoing first-in-human dose-escalation study in subjects with advanced CD123+ hematologic malignancies, VIP943 demonstrates a promising safety profile (NCT06034275). Citation Format: Beatrix Stelte-Ludwig, Tibor Schomber, Melanie M. Frigault, Joseph Birkett, Amy J. Johnson, Anne-Sophie Rebstock, Sebastian Ludwig, Raquel Izumi, Ahmed Hamdy. Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 629.

CD4 T Cell-Derived IL-21 Is Critical for Sustaining Plasmodium Infection-Induced Germinal Center Responses and Promoting the Selection of Memory B Cells with Recall Potential.

Development of Plasmodium-specific humoral immunity is critically dependent on CD4 Th cell responses and germinal center (GC) reactions during blood-stage Plasmodium infection. IL-21, a cytokine primarily produced by CD4 T cells, is an essential regulator of affinity maturation, isotype class-switching, B cell differentiation, and maintenance of GC reactions in response to many infection and immunization models. In models of experimental malaria, mice deficient in IL-21 or its receptor IL-21R fail to develop memory B cell populations and are not protected against secondary infection. However, whether sustained IL-21 signaling in ongoing GCs is required for maintaining GC magnitude, organization, and output is unclear. In this study, we report that CD4+ Th cells maintain IL-21 expression after resolution of primary Plasmodium yoelii infection. We generated an inducible knockout mouse model that enabled cell type-specific and timed deletion of IL-21 in peripheral, mature CD4 T cells. We found that persistence of IL-21 signaling in active GCs had no impact on the magnitude of GC reactions or their capacity to produce memory B cell populations. However, the memory B cells generated in the absence of IL-21 exhibited reduced recall function upon challenge. Our data support that IL-21 prevents premature cellular dissolution within the GC and promotes stringency of selective pressures during B cell fate determination required to produce high-quality Plasmodium-specific memory B cells. These data are additionally consistent with a temporal requirement for IL-21 in fine-tuning humoral immune memory responses during experimental malaria.

Intra-bone marrow injection with engineered Lactococcus lactis for the treatment of metastatic tumors: Primary report

Bone marrow has the capacity to produce different types of immune cells, such as natural killer cells, macrophages, dendritic cells (DCs) and T cells. Improving the activation of immune cells in the bone marrow can enhance the therapy of bone metastases. Previously, we designed an engineered probiotic Lactococcus lactis, capable of expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and co-stimulator OX40 ligand (FOLactis), and proved that it can induce the activation and differentiation of several immune cells. In this research, we successfully establish mouse models of bone metastasis, lung metastasis and intraperitoneal dissemination, and we are the first to directly inject the probiotics into the bone marrow to inhibit tumor growth. We observe that injecting FOLactis into the bone marrow of mice can better regulate the immune microenvironment of tumor-bearing mice, resulting in a tumor-suppressive effect. Compared to subcutaneous (s.c.) injection, intra-bone marrow (IBM) injection is more effective in increasing mature DCs and CD8+ T cells and prolonging the survival of tumor-bearing mice. Our results confirm that IBM injection of FOLactis reprograms the immune microenvironment of bone marrow and has remarkable effectiveness in various metastatic tumor models.

Distinct transcriptomes and autocrine cytokines underpin maturation and survival of antibody-secreting cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibody types, some of which are produced by long-lived plasma cells (LLPC). Active SLE generates increased circulating antibody-secreting cells (ASC). Here, we examine the phenotypic, molecular, structural, and functional features of ASC in SLE. Relative to post-vaccination ASC in healthy controls, circulating blood ASC from patients with active SLE are enriched with newly generated mature CD19−CD138+ ASC, similar to bone marrow LLPC. ASC from patients with SLE displayed morphological features of premature maturation and a transcriptome epigenetically initiated in SLE B cells. ASC from patients with SLE exhibited elevated protein levels of CXCR4, CXCR3 and CD138, along with molecular programs that promote survival. Furthermore, they demonstrate autocrine production of APRIL and IL-10, which contributed to their prolonged in vitro survival. Our work provides insight into the mechanisms of generation, expansion, maturation and survival of SLE ASC.

Integrative single-cell analysis: dissecting CD8 + memory cell roles in LUAD and COVID-19 via eQTLs and Mendelian Randomization

Lung adenocarcinoma exhibits high incidence and mortality rates, presenting a significant health concern. Concurrently, the COVID-19 pandemic has emerged as a grave global public health challenge. Existing literature suggests that T cells, pivotal components of cellular immunity, are integral to both antiviral and antitumor responses. Yet, the nuanced alterations and consequent functions of T cells across diverse disease states have not been comprehensively elucidated. We gathered transcriptomic data of peripheral blood mononuclear cells from lung adenocarcinoma patients, COVID-19 patients, and healthy controls. We followed a standardized analytical approach for quality assurance, batch effect adjustments, and preliminary data processing. We discerned distinct T cell subsets and conducted differential gene expression analysis. Potential key genes and pathways were inferred from GO and Pathway enrichment analyses. Additionally, we implemented Mendelian randomization to probe the potential links between pivotal genes and lung adenocarcinoma susceptibility. Our findings underscored a notable reduction in mature CD8 + central memory T cells in both lung adenocarcinoma and COVID-19 cohorts relative to the control group. Notably, the downregulation of specific genes, such as TRGV9, could impede the immunological efficacy of CD8 + T cells. Comprehensive multi-omics assessment highlighted genetic aberrations in genes, including TRGV9, correlating with heightened lung adenocarcinoma risk. Through rigorous single-cell transcriptomic analyses, this investigation meticulously delineated variations in T cell subsets across different pathological states and extrapolated key regulatory genes via an integrated multi-omics approach, establishing a robust groundwork for future functional inquiries. This study furnishes valuable perspectives into the etiology of multifaceted diseases and augments the progression of precision medicine.

Immunohistochemical profile of gingiva biopsy samples from the edentulous alveolar ridge in the jaw

Relevance. Despite the high success rate of dental implantation in restoring anatomical occlusion and achieving desired aesthetic effects, complications, often mediated by peri-implantitis, remain an area requiring further study. This study aimed to morphologically assess gingiva biopsy samples from the edentulous alveolar ridge of the jaw, using immunohistochemical markers for immunophenotyping different subpopulations of inflammatory cells, inter alia.Material and methods. Gingiva biopsy specimens from 25 patients underwent descriptive histological and immunohistochemical analyses, employing markers such as CD3, CD20, TBX21, GATA3, Foxp3, CD68, CD80, CD163, CD1a, and CD83. The study includes clinical-anamnestic data analysis, descriptive histological and immunohistochemical observations, and a quantitative analysis of the abundance of various immunopositive cell subpopulations.Results. The data indicated that approximately one third of the gum tissues exhibited signs of a chronic inflammatory response, predominantly characterized by the primary T-cell subpopulation of T-helper 1 cells, a minimal presence of regulatory T-cells, and an almost complete absence of T-helper 2 cells. Additionally, an inflammatory cell mix was observed, comprising a significant proportion of B-cells, monocyte-macrophage cells (mainly M2 macrophage subpopulation), and mature CD83+ dendritic cells. These changes reflect a pro-inflammatory local immune response, crucial to consider in future implantation planning.Conclusion. The analysis of this pilot clinical case series enhances our understanding of the gingiva condition in the edentulous alveolar ridge before dental implantation and opens avenues for further research in this field.