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Journal of Experimental & Clinical... Jun 2024Phosphoinositide-3-kinase γ (PI3Kγ) plays a critical role in pancreatic ductal adenocarcinoma (PDA) by driving the recruitment of myeloid-derived suppressor cells...
Phosphoinositide-3-kinase γ (PI3Kγ) plays a critical role in pancreatic ductal adenocarcinoma (PDA) by driving the recruitment of myeloid-derived suppressor cells (MDSC) into tumor tissues, leading to tumor growth and metastasis. MDSC also impair the efficacy of immunotherapy. In this study we verify the hypothesis that MDSC targeting, via PI3Kγ inhibition, synergizes with α-enolase (ENO1) DNA vaccination in counteracting tumor growth.Mice that received ENO1 vaccination followed by PI3Kγ inhibition had significantly smaller tumors compared to those treated with ENO1 alone or the control group, and correlated with i) increased circulating anti-ENO1 specific IgG and IFNγ secretion by T cells, ii) increased tumor infiltration of CD8 T cells and M1-like macrophages, as well as up-modulation of T cell activation and M1-like related transcripts, iii) decreased infiltration of Treg FoxP3 T cells, endothelial cells and pericytes, and down-modulation of the stromal compartment and T cell exhaustion gene transcription, iv) reduction of mature and neo-formed vessels, v) increased follicular helper T cell activation and vi) increased "antigen spreading", as many other tumor-associated antigens were recognized by IgG2c "cytotoxic" antibodies. PDA mouse models genetically devoid of PI3Kγ showed an increased survival and a pattern of transcripts in the tumor area similar to that of pharmacologically-inhibited PI3Kγ-proficient mice. Notably, tumor reduction was abrogated in ENO1 + PI3Kγ inhibition-treated mice in which B cells were depleted.These data highlight a novel role of PI3Kγ in B cell-dependent immunity, suggesting that PI3Kγ depletion strengthens the anti-tumor response elicited by the ENO1 DNA vaccine.
Topics: Animals; Mice; Vaccines, DNA; Pancreatic Neoplasms; B-Lymphocytes; Class Ib Phosphatidylinositol 3-Kinase; Humans; Cell Line, Tumor; Cancer Vaccines; Disease Models, Animal; Myeloid-Derived Suppressor Cells
PubMed: 38824552
DOI: 10.1186/s13046-024-03080-1 -
Genome Biology May 2024Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of...
BACKGROUND
Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence.
RESULTS
Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers.
CONCLUSIONS
Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.
Topics: Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Histone-Lysine N-Methyltransferase; Cell Cycle Proteins; Cell Line, Tumor; Pyrimidines; Pyrimidinones; Myeloid-Lymphoid Leukemia Protein; Pyrazoles; Protein-Tyrosine Kinases; Antineoplastic Agents; Cell Cycle; Core Binding Factor Alpha 2 Subunit
PubMed: 38822412
DOI: 10.1186/s13059-024-03260-4 -
Blood Cancer Journal May 2024B-cell-maturation-antigen (BCMA)-directed therapies are highly active for multiple myeloma, but infections are emerging as a major challenge. In this retrospective,... (Comparative Study)
Comparative Study
B-cell-maturation-antigen (BCMA)-directed therapies are highly active for multiple myeloma, but infections are emerging as a major challenge. In this retrospective, single-center analysis we evaluated infectious complications after BCMA-targeted chimeric-antigen-receptor T-cell therapy (CAR-T), bispecific-antibodies (BsAb) and antibody-drug-conjugates (ADC). The primary endpoint was severe (grade ≥3) infection incidence. Amongst 256 patients, 92 received CAR-T, 55 BsAb and 109 ADC. The incidence of severe infections was higher with BsAb (40%) than CAR-T (26%) or ADC (8%), including grade 5 infections (7% vs 0% vs 0%, respectively). Comparing T-cell redirecting therapies, the incidence rate of severe infections was significantly lower with CAR-T compared to BsAb at 1-year (incidence-rate-ratio [IRR] = 0.43, 95%CI 0.25-0.76, P = 0.004). During periods of treatment-emergent hypogammaglobulinemia, BsAb recipients had higher infection rates (IRR:2.27, 1.31-3.98, P = 0.004) and time to severe infection (HR 2.04, 1.05-3.96, P = 0.036) than their CAR-T counterparts. During periods of non-neutropenia, CAR-T recipients had a lower risk (HR 0.44, 95%CI 0.21-0.93, P = 0.032) and incidence rate (IRR:0.32, 95% 0.17-0.59, P < 0.001) of severe infections than BsAb. In conclusion, we observed an overall higher and more persistent risk of severe infections with BsAb. Our results also suggest a higher infection risk during periods of hypogammaglobulinemia with BsAb, and with neutropenia in CAR-T recipients.
Topics: Humans; Multiple Myeloma; B-Cell Maturation Antigen; Male; Female; Middle Aged; Aged; Retrospective Studies; Immunotherapy, Adoptive; Adult; Infections; Antibodies, Bispecific; Aged, 80 and over; Incidence; Immunoconjugates
PubMed: 38821925
DOI: 10.1038/s41408-024-01043-5 -
AIDS (London, England) May 2024People living with HIV (PLWH) are at greater risk of developing lung diseases even when they are antiretroviral therapy (ART)-adherent and virally suppressed. The most...
People living with HIV (PLWH) are at greater risk of developing lung diseases even when they are antiretroviral therapy (ART)-adherent and virally suppressed. The most common pulmonary function abnormality in PLWH is that of impaired diffusing capacity of the lungs for carbon monoxide (DLCO), which is an independent risk factor for increased mortality in PLWH. Earlier work has identified several plasma biomarkers of inflammation and immune activation to be associated with decreased DLCO. However, the underpinning molecular mechanisms of HIV-associated impaired DLCO are largely unknown. We conducted a pilot study of PLWH with normal DLCO (values greater than or equal to the lower limit of normal, DLCO≥LLN) or abnormal DLCO (DLCO
belong to 4 categories: 1. Nine genes in inflammation and immune activation pathways, 2. seven upregulated genes that are direct targets of the interferon signaling pathway, 3. seven B-cell specific genes that are downregulated, and 4. three miscellaneous genes. These results were corroborated using the bioinformatics tools DAVID (Database for Annotation, Visualization and Integrated Discovery) and GSEA (Gene Sets Enrichment Analysis). Together, the data provides preliminary evidence for the involvement of sustained interferon signaling as a molecular mechanism for impaired DLCO in PLWH. We discuss the implication of these findings in the context of inflammation and interferon as potential therapeutic targets to treat HIV-associated lung dysfunction. PubMed: 38819840
DOI: 10.1097/QAD.0000000000003946 -
Cytotherapy May 2024The success of chimeric antigen receptor (CAR) T-cell therapy in treating B-cell malignancies has led to the evaluation of CAR T-cells targeting a variety of other...
BACKGROUND AIMS
The success of chimeric antigen receptor (CAR) T-cell therapy in treating B-cell malignancies has led to the evaluation of CAR T-cells targeting a variety of other malignancies. Although the efficacy of CAR T-cells is enhanced when administered post-lymphodepleting chemotherapy, this can trigger bone marrow suppression and sustained cytopenia after CD19.CAR T-cell therapy. Additionally, systemic inflammation associated with CAR T-cell activity may contribute to myelosuppression. Cytopenias, such as neutropenia and thrombocytopenia, elevate the risk of severe infections and bleeding, respectively. However, data on the incidence of prolonged cytopenias after immune effector therapy in the solid tumor context remain limited.
OBJECTIVE
We compared the incidence of prolonged cytopenias after immune effector therapy including genetically modified T-cells, virus-specific T-cells (VSTs) and NKT-cells, as well non-gene-modified VSTs for leukemia, lymphoma, and solid tumors (ST) to identify associated risk factors.
METHODS
A retrospective analysis was conducted of 112 pediatric and adult patients with relapsed and/or refractory cancers who received lymphodepleting chemotherapy followed by immune effector therapy. Patients treated with 13 distinct immune effector cell therapies through 11 single-center clinical trials and 2 commercial products over a 6-year period were categorized into 3 types of malignancies: leukemia, lymphoma and ST. We obtained baseline patient characteristics and adverse events data for each participant, and tracked neutrophil and platelet counts following lymphodepletion.
RESULTS
Of 112 patients, 104 (92.9%) experienced cytopenias and 88 (79%) experienced severe cytopenias. Patients with leukemia experienced significantly longer durations of severe neutropenia (median duration of 14 days) compared with patients with lymphoma (7 days) or ST (11 days) (P = 0.002). Patients with leukemia also had a higher incidence of severe thrombocytopenia (74.1%), compared with lymphoma (46%, P = 0.03) and ST (14.3%, P < 0.0001). Prolonged cytopenias were significantly associated with disease type (63% of patients with leukemia, 44% of patients with lymphoma, and 22.9% of patients with ST, P = 0.006), prior hematopoietic stem cell transplant (HSCT) (66.7% with prior HSCT versus 38.3% without prior HSCT, P = 0.039), and development of immune effector cell-associated neurotoxicity syndrome (ICANS) (75% with ICANS versus 38% without ICANS, P = 0.027). There was no significant association between prolonged cytopenias and cytokine release syndrome.
CONCLUSIONS
Immune effector recipients often experience significant cytopenias due to marrow suppression following lymphodepletion regardless of disease, but prolonged severe cytopenias are significantly less common after treatment of patients with lymphoma and solid tumors.
PubMed: 38819365
DOI: 10.1016/j.jcyt.2024.04.075 -
Helicobacter 2024Helicobacter 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...
BACKGROUND
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.
MATERIALS AND METHODS
Isolation of cells from blood and mucosal biopsies, magnetic separation of В cells, CD4 and CD4CCR6CD45RO T cells, antigen-specific activation, B cell response in vitro, flow cytometry, determination of CD4CD25FoxP3 Tregs and various groups of Th cells.
RESULTS
CD4CCR6 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 CD4CCR6CD45RO cells and the percentage of Th1, Th17 and Th1/17 cells in this lymphocyte subgroup. In H. pylori-positive patients, circulating CD4CCR6 cells contained a higher proportion of H. pylori-specific cells compared with their CD4CCR6 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. CD4CCR6 lymphocytes from H. pylori-infected stomach included Tregs and in vivo activated T cells, some of which produced interferon-γ without ex vivo stimulation.
CONCLUSION
H. pylori infection causes an increase in the number of mature CD4CCR6 lymphocytes in the blood, with a pro-inflammatory shift in their composition and enrichment of the gastric mucosa with CD4CCR6 lymphocytes, including CCR6 Th1 cells and Tregs.
Topics: Humans; Receptors, CCR6; Helicobacter Infections; T-Lymphocytes, Regulatory; Gastric Mucosa; Helicobacter pylori; T-Lymphocytes, Helper-Inducer; Male; Female; Adult; Middle Aged; Flow Cytometry; Aged
PubMed: 38819071
DOI: 10.1111/hel.13097 -
Antioxidants & Redox Signaling May 2024Down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) contributes to doxorubicin (DOX)-induced myocardial oxidative stress, and inhibition of...
AIMS
Down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) contributes to doxorubicin (DOX)-induced myocardial oxidative stress, and inhibition of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) increased Nrf2 protein level in rat heart suffered ischemia/reperfusion, indicating a connection between MALT1 and Nrf2. This study aims to explore the role of MALT1 in DOX-induced myocardial oxidative stress and the underlying mechanisms.
RESULTS
The mice received a single injection of DOX (15 mg/kg, i.p.) to induce myocardial oxidative stress, evidenced by increases in the levels of reactive oxidative species while decreases in the activities of anti-oxidative enzymes, concomitant with a down-regulation of Nrf2; these phenomena were reversed by MALT1 inhibitor. Similar phenomena were observed in DOX-induced oxidative stress in cardiomyocytes. Mechanistically, knockdown or inhibition of MALT1 notably attenuated the interaction between Nrf2 and MALT1, and decreased the k48-linked ubiquitination of Nrf2. Furthermore, inhibition or knockdown of calcium/calmodulin-dependent protein kinase II (CaMKII-δ) reduced the phosphorylation of caspase recruitment domain-containing protein 11 (CARD11), and subsequently disrupted the assembly of CARD11, B-cell lymphoma 10 (BCL10) and MALT1 (CBM) complex, and reduced the MALT1-dependent k48-linked ubiquitination of Nrf2 in DOX-treated mice or cardiomyocytes.
INNOVATION AND CONCLUSION
The E3 ubiquitin ligase function of MALT1 accounts for the down-regulation of Nrf2 and aggravation of myocardial oxidative stress in DOX-treated mice, and CaMKII-δ-dependent phosphorylation of CARD11 triggered the assembly of CBM complex and subsequent activation of MALT1.
PubMed: 38814831
DOI: 10.1089/ars.2023.0543 -
Methods in Molecular Biology (Clifton,... 2024Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have...
Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than two decades to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs) and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers (i.e. caIKK), and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs and mRNA-electroporated T cells for therapeutic applications in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types; (2) scalability from 10 to approximately 10 cells per shot; (3) high transfection efficiency (80-99%); (4) homogenous protein expression; (5) GMP compliance if the EP is performed in a class A clean room; and (6) no transgene integration into the genome. The provided protocol involves: OptiMEM as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time has to be altered. Thus, we share an overview of proven electroporation settings (including recovery media), which we have established for various cell types. Next to the basic protocol, we also provide an extensive list of hints and tricks, which, in our opinion, are of great value for everyone who intends to use this transfection technique.
Topics: Electroporation; Humans; RNA, Messenger; Transfection; Dendritic Cells; T-Lymphocytes; Antigens; B-Lymphocytes
PubMed: 38814397
DOI: 10.1007/978-1-0716-3770-8_10 -
Cellular and Molecular Biology... May 2024Non-Hodgkin lymphoma (NHL) is a lymphoproliferative disorder derived from either B or T lymphocytes. Among NHL, activated B-cell-like (ABC) diffuse large B-cell lymphoma...
Non-Hodgkin lymphoma (NHL) is a lymphoproliferative disorder derived from either B or T lymphocytes. Among NHL, activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) and T cell non-Hodgkin lymphomas (T-NHL) are poor prognosis and aggressive subtypes. Macrophages are professional phagocytic cells and dendritic cells (DCs) are professional antigen-presenting cells in immune system. Doxorubicin (Dox) and Etoposide (ET) are the most effective anti-cancer drugs. A20 and CYLD are negative regulators of NF-κB-dependent functions in many cell types. Little is known about the roles of A20 and CYLD in regulating functions of DCs and macrophages from NHL. The present study, therefore, explored whether A20/CYLD expression contributes to functions of DCs and macrophages from NHL. To this end, blood samples of seventy-nine patients with ABC DLBCL and T-NHL were examined. Gene expression profile was determined by quantitative RT-PCR and immunophenotype, cell apoptosis and phagocytosis by flow cytometry. As a result, immunophenotypic analysis showed that the numbers of CD13+CD117-, CD56+CD40+ and CD23+CD40+ expressing cells were significantly elevated in ABC DLBCL cases compared to healthy individuals and T-NHL patients. Interestingly, upon treatment of Dox and ET, the phagocytosis of lymphoma cells was significantly reduced by CD11c+CD123- DCs and the percentage of CD56+ mature DCs was significantly enhanced in ABC DLBCL patients only in the presence of A20 siRNA, but not CYLD siRNA. In conclusion, ABC DLBCL patients with low A20 expression were defective in elimination of lymphoma cells by DCs and linked to killer DC expansion in circulation.
Topics: Humans; Dendritic Cells; Phagocytosis; Tumor Necrosis Factor alpha-Induced Protein 3; Female; Lymphoma, Large B-Cell, Diffuse; Middle Aged; Male; Lymphoma, Non-Hodgkin; Apoptosis; Aged; Adult; Macrophages; Doxorubicin; B-Lymphocytes; Immunophenotyping
PubMed: 38814241
DOI: 10.14715/cmb/2024.70.5.2 -
European Journal of Immunology May 2024Studies have traditionally focused on the role of T cells in chronic hepatitis B (CHB), but recent evidence supports a role for B cells. The enrichment of so-called...
Studies have traditionally focused on the role of T cells in chronic hepatitis B (CHB), but recent evidence supports a role for B cells. The enrichment of so-called atypical memory (AtM) B cells, which show reduced signaling and impaired differentiation, is believed to be a characteristic feature of CHB, potentially contributing to the observed dysfunctional anti-HBsAg B-cell responses. Our study, involving 62 CHB patients across clinical phases, identified AtM B cells expressing IFNLR1 and interferon-stimulated genes. Contrary to previous reports, we found relatively low frequencies of AtM B cells in the liver, comparable to peripheral blood. However, liver plasma cell frequencies were significantly higher, particularly during phases with elevated viral loads and liver enzyme levels. Liver plasma cells exhibited signs of active proliferation, especially in the immune active phase. Our findings suggest a potential role for plasma cells, alongside potential implications and consequences of local proliferation, within the livers of CHB patients. While the significance of AtM B cells remains uncertain, further investigation is warranted to determine their responsiveness to interferons and their role in CHB.
PubMed: 38813721
DOI: 10.1002/eji.202451085