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Helicobacter pylori infects roughly half the world's population, causing gastritis, peptic ulcers, and gastric cancer in a subset. These pathologies occur in response to a chronic inflammatory state, but it is not fully understood how H. pylori controls this process. We characterized the inflammatory response of H. pylori mutants that cannot produce the quorum sensing molecule autoinducer 2 (AI-2) by deleting the gene for the AI-2 synthase, luxS . Our work shows that H. pylori luxS mutants colonize the stomach normally but recruit high numbers of CD4 + T cells to the stomach during chronic infection. This increase in the number of CD4 + T cells correlated with elevated expression of CXCL9 , a chemokine important for T cell recruitment. Together, our results suggest that H. pylori may utilize AI-2 signaling to modulate the inflammatory response during chronic infection., Importance: Many bacteria signal to each other using quorum sensing signals. One type of signal is called autoinducer 2 (AI-2), which is produced and sensed by the LuxS enzyme found in many bacteria, including the gastric pathogen Helicobacter pylori . H. pylori establishes chronic infections that last for decades and lead to serious disease outcomes. How AI-2 signaling and LuxS contribute to chronic H. pylori infection has not been studied. In this work, we analyzed how loss of H. pylori -created AI-2, via mutation of luxS , affects H. pylori chronic infection. luxS mutants did not have significant colonization defects, similar to their reported phenotype during early infection, but they did have high stomach levels of effector and regulatory T cells and T-cell-recruiting chemokines. These results suggest that H. pylori LuxS may play more of a role in modulating the immune response versus colonization., Competing Interests: The authors declare no conflict of interest.

Distinct routes of cellular production from hematopoietic stem cells (HSCs) have defined our current view of hematopoiesis. Recently, we challenged classical views of platelet generation, demonstrating that megakaryocyte progenitors (MkPs), and ultimately platelets, can be specified via an alternate and additive route of HSC-direct specification specifically during aging. This "shortcut" pathway generates hyperactive platelets likely to contribute to age-related platelet-mediated morbidities. Here, we used single-cell RNA/CITEseq to demonstrate that these age-unique, non-canonical (nc)MkPs can be prospectively defined and experimentally isolated from wild type mice. Surprisingly, this revealed that a rare population of ncMkPs also exist in young mice. Young and aged ncMkPs are functionally distinct from their canonical (c)MkP counterparts, with aged ncMkPs paradoxically and uniquely exhibiting enhanced survival and platelet generation capacity. We further demonstrate that aged HSCs generate significantly more ncMkPs than their younger counterparts, yet this is accomplished without strict clonal restriction. Together, these findings reveal significant phenotypic, functional, and aging-dependent heterogeneity among the MkP pool and uncover unique features of megakaryopoiesis throughout life, potentially offering cellular and molecular targets for mitigation of age-related adverse thrombotic events., Competing Interests: Declaration of interests The authors declare no competing interests.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.

Background: Elicitation of broad immune responses is understood to be required for an efficacious preventative HIV vaccine. This Phase 1 randomized controlled trial evaluated whether administration of vaccine antigens separated at multiple injection sites vs combined, fractional delivery at multiple sites affected T-cell breadth compared to standard, single site vaccination., Methods: We randomized 90 participants to receive recombinant adenovirus 5 (rAd5) vector with HIV inserts gag, pol and env via three different strategies. The Standard group received vaccine at a single anatomic site (n = 30) compared to two polytopic (multisite) vaccination groups: Separated (n = 30), where antigens were separately administered to four anatomical sites, and Fractioned (n = 30), where fractions of each vaccine component were combined and administered at four sites. All groups received the same total dose of vaccine., Findings: CD8 T-cell response rates and magnitudes were significantly higher in the Fractioned group than Standard for several antigen pools tested. CD4 T-cell response magnitudes to Pol were higher in the Separated than Standard group. T-cell epitope mapping demonstrated greatest breadth in the Fractioned group (median 8.0 vs 2.5 for Standard, Wilcoxon p = 0.03; not significant after multiplicity adjustment for co-primary endpoints). IgG binding antibody response rates to Env were higher in the Standard and Fractioned groups vs Separated group., Interpretation: This study shows that the number of anatomic sites for which a vaccine is delivered and distribution of its antigenic components influences immune responses in humans., Funding: National Institute of Allergy and Infectious Diseases, NIH., Competing Interests: Declaration of interests MAA is employed by NIAID, the funder of the study. HVT, KJB, SAK, AFG, LC, GDT, NF, NG, SCDR reports receiving institutional grants from the NIH. IF reports institutional grants and participation on Boards of Gilead and Viiv; LB reports institutional grants and participation on Boards of the FDA and NIAID; MJM reports institutional grants, participation on Boards of Ragon Institute, Keystone Symposia and NIH VRC, and a patent on HIV immunogens. The rest of the authors as well as HVTN 085 Study Team declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19 + CD5 + clonal B lymphocytes in the blood, bone marrow, and peripheral lymphoid organs. Treatment options for patients range from historical chemoimmunotherapy (CIT) to small molecule inhibitors targeting pro-survival pathways in leukemic B cells, such as the Bruton's tyrosine kinase inhibitor ibrutinib (IBR). Using biobanked blood samples obtained pre-therapy and at standard response evaluation timepoints, we performed an in-depth evaluation of the blood innate and adaptive immune compartments between pentostatin-based CIT and IBR and looked for correlations with clinical sequelae. CD4 + conventional T cells and CD8 + cytotoxic T cells responded similarly to CIT and IBR, although exhaustion status differed. Both treatments dramatically increased the prevalence and functional status of monocyte, dendritic cell, and natural killer cell subsets. As expected, both regimens reduced clonal B cell levels however, we observed no substantial recovery of normal B cells. Although improvements in most immune subsets were observed with CIT and IBR at response evaluation, both patient groups remained susceptible to infections and secondary malignancies during the study., Competing Interests: Research funding has been provided to the institution from Janssen, AstraZeneca, Merck, and Genentech for clinical studies in which SP is a principal investigator. Honoraria has been provided to the institution from Pharmacyclics, Merck, AstraZeneca, Janssen, Genentech, Amgen, TG Therapeutics, Novalgen Limited, Kite Pharma, and AbbVie for SP’s participation in consulting activities/advisory board meetings. Research funding to NK has been provided from Acerta Pharma BV, Celgene, Genentech, Pharmacyclics, and Tolero Pharmaceutical. NK also participates in the data safety monitoring committees of Agios Pharm, Astra Zeneca, Celgene, Cytomx Therapeutics, Genentech, Infinity Pharm, Morpho-Sys, and Pharmacyclics he is not personally compensated for his participation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Welch, Manso, Gwin, Lothert, Parikh, Kay and Medina.)

The HIV Vaccine Trials Network (HVTN) conducts clinical trials on 4 continents in pursuit of a safe and effective HIV vaccine. Cellular immune responses to vaccination that define vaccine immunogenicity and/or immune correlates of protection can be measured using multiparameter intracellular cytokine staining (ICS) assays. The HVTN cellular immunology laboratory, located in Seattle, WA, conducts ICS assays for vaccine trials according to Good Clinical Laboratory Practices (GCLP). In 2013, the HVTN established a second GCLP compliant cellular immunology laboratory in Cape Town, South Africa to assess vaccine immunogenicity for HVTN trials conducted on the African continent. To ensure ICS readouts in the 2 laboratories were directly comparable, we conducted concordance testing using PBMC from healthy controls and vaccine trial participants. Despite standardized procedures and instrumentation, shared quality control measures and quality assurance oversight, several factors impacted our ability to obtain close agreement in T-cell responses measured in the 2 laboratories. One of these was the type of fetal bovine serum (FBS) used in the assay, which impacted lymphocyte cell viability and background responses. In addition, the differences in supernatant removal technique also significantly affected our ability to detect positive responses to vaccine antigens. Standardization of these factors allowed us to achieve and maintain ICS assay concordance across the 2 laboratories over multiple years, accelerating our efforts to evaluate HIV vaccines. The insights gained in this process are valuable for assay transfer efforts by groups of investigators that need to directly compare data generated in different laboratories around the globe., (©2022 Society for Leukocyte Biology.)

Hematopoietic stem cell (HSC) multipotency and self-renewal are typically defined through serial transplantation experiments. Host conditioning is necessary for robust HSC engraftment, likely by reducing immune-mediated rejection and by clearing limited HSC niche space. Because irradiation of the recipient mouse is non-specific and broadly damaging, there is a need to develop alternative models to study HSC performance at steady-state and in the absence of radiation-induced stress. We have generated and characterized two new mouse models where either all hematopoietic cells or only HSCs can be specifically induced to die in vivo or in vitro. Hematopoietic-specific Vav1-mediated expression of a loxP-flanked diphtheria-toxin receptor (DTR) renders all hematopoietic cells sensitive to diphtheria toxin (DT) in "Vav-DTR" mice. Crossing these mice to Flk2-Cre mice results in "HSC-DTR" mice which exhibit HSC-selective DT sensitivity. We demonstrate robust, rapid, and highly selective cell ablation in these models. These new mouse models provide a platform to test whether HSCs are required for long-term hematopoiesis in vivo, for understanding the mechanisms regulating HSC engraftment, and interrogating in vivo hematopoietic differentiation pathways and mechanisms regulating hematopoietic homeostasis., (© 2022. The Author(s).)

Hematopoietic stem cells (HSCs) have been studied extensively since their initial functional description in 1961 when Dr. James Till and Dr. Ernest McCulloch developed the first in vivo clonal strategy, termed the spleen colony-forming unit (CFU-S) assay, to assess the functional capacity of bone marrow-derived hematopoietic progenitors at the single-cell level. Through transplantation of bone marrow cells and analysis of the resulting cellular nodules in the spleen, the CFU-S assay revealed both the self-renewal and clonal differentiation capacity of hematopoietic progenitors. Further development and use of this assay have identified highly proliferative, self-renewing, and differentiating HSCs that possess clonal, multilineage differentiation. The CFU-S strategy has also been adapted to interrogating single purified hematopoietic stem and progenitor cell populations, advancing our knowledge of the hematopoietic hierarchy. In this review, we explore the major discoveries made with the CFU-S assay, consider its modern use and recent improvements, and compare it with commonly used long-term transplantation assays to determine the continued value of the CFU-S assay for understanding HSC biology and hematopoiesis., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Transcription factor (TF) expression levels drive developmental programs, including cell fate and function, and their measurement by flow cytometry allows for robust downstream analysis. However, significant batch-to-batch variability between replicative experiments precludes direct comparison of absolute values across experimental conditions. Here, we present a flow cytometry protocol to measure the relative abundance of multiple TFs simultaneously in single cells, allowing for direct comparison across experimental conditions/time points. This protocol uses bone marrow cells but can be adapted for other cell types. For complete details on the use and execution of this protocol, please refer to Manso et al. (2021) and Manso et al. (2019)., Competing Interests: B.A.M. is currently affiliated with the University of California Santa Cruz, Santa Cruz, CA, USA. This protocol is submitted in support of work done while affiliated with the Mayo Clinic, Rochester, MN, USA., (© 2021 The Author(s).)

TNFα is implicated in chronic lymphocytic leukemia (CLL) immunosuppression and disease progression. TNFα is constitutively produced by CLL B cells and is a negative regulator of bone marrow (BM) myelopoiesis. Here, we show that co-culture of CLL B cells with purified normal human hematopoietic stem and progenitor cells (HSPCs) directly altered protein levels of the myeloid and erythroid cell fate determinants PU.1 and GATA-2 at the single-cell level within transitional HSPC subsets, mimicking ex vivo expression patterns. Physical separation of CLL cells from control HSPCs or neutralizing TNFα abrogated upregulation of PU.1, yet restoration of GATA-2 required TNFα neutralization, suggesting both cell contact and soluble-factor-mediated regulation. We further show that CLL patient BM myeloid progenitors are diminished in frequency and function, an effect recapitulated by chronic exposure of control HSPCs to low-dose TNFα. These findings implicate CLL B-cell-derived TNFα in impaired BM myelopoiesis., Competing Interests: Research funding to S.A.P. has been provided from 10.13039/100014491Pharmacyclics, 10.13039/501100013650MorphoSys, Janssen, 10.13039/100004325AstraZeneca, 10.13039/100013252TG Therapeutics, 10.13039/100006436Celgene, 10.13039/100006483AbbVie, and Ascentage Pharma for clinical studies in which S.A.P. is a principal investigator. S.A.P. also participated in Advisory Board meetings of Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie (he was not personally compensated for his participation). Research funding to N.E.K. has been provided from Acerta Pharma BV, 10.13039/100006436Celgene, 10.13039/100004328Genentech, 10.13039/100014491Pharmacyclics, and Tolero Pharmaceutical. N.E.K. also participates in the data safety monitoring committees of Agios Pharm, Astra Zeneca, Celgene, Cytomx Therapeutics, Genentech, Infinity Pharm, Morpho-Sys, and Pharmacyclics (he was not personally compensated for his participation). All other authors declare no competing interests., (© 2020 The Author(s).)

T cell responses have been implicated in reduced risk of HIV acquisition in uninfected persons and control of viral replication in HIV-infected individuals. HIV Gag-specific T cells have been predominantly associated with post-infection control, whereas Env antigens are the target for protective antibodies; therefore, inclusion of both antigens is common in HIV vaccine design. However, inclusion of multiple antigens may provoke antigenic competition, reducing the potential effectiveness of the vaccine. HVTN 084 was a randomized, multicenter, double-blind phase 1 trial to investigate whether adding Env to a Gag/Pol vaccine decreases the magnitude or breadth of Gag/Pol-specific T cell responses. Fifty volunteers each received one intramuscular injection of 1 × 10 10 particle units (PU) of rAd5 Gag/Pol and EnvA/B/C (3:1:1:1 mixture) or 5 × 10 9 PU of rAd5 Gag/Pol. CD4 + T cell responses to Gag/Pol measured 4 weeks after vaccination by cytokine expression were significantly higher in the group vaccinated without Env, whereas CD8 + T cell responses did not differ significantly between the two groups. Mapping of individual epitopes revealed greater breadth of the Gag/Pol-specific T cell response in the absence of Env compared to Env coimmunization. Addition of an Env component to a Gag/Pol vaccine led to reduced Gag/Pol CD4 + T cell response rate and magnitude as well as reduced epitope breadth, confirming the presence of antigenic competition. Therefore, T cell-based vaccine strategies should aim at choosing a minimalist set of antigens to reduce interference of individual vaccine components with the induction of the maximally achievable immune response., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The consequences of immune dysfunction in B-chronic lymphocytic leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The well-described immune abnormalities are not able to consistently explain these complications. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. Numbers of CD34 + BM hematopoietic progenitors responsive in standard colony-forming unit (CFU) assays, including CFU-GM/GEMM and CFU-E, were significantly reduced. Flow cytometry revealed corresponding reductions in frequencies of all hematopoietic stem and progenitor cell (HSPC) subsets assessed in CLL patient marrow. Consistent with the reduction in HSPCs, BM resident monocytes and natural killer cells were reduced, a deficiency recapitulated in blood. Finally, we report increases in protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients that provides new insight into the etiology of the complex immunodeficiency state in CLL.

iNKT cells are a unique lineage of T cells that recognize glycolipid presented by CD1d. In the thymus, they differentiate into iNKT1, iNKT2 and iNKT17 effector subsets, characterized by preferential expression of Tbet, Gata3 and ROR-γt and production of IFN-γ, IL-4 and IL-17, respectively. We demonstrate that the transcriptional regulator Runx1 is essential for the generation of ROR-γt expressing iNKT17 cells. PLZF-cre Runx1 cKO mice lack iNKT17 cells in the thymus, spleen and liver. Runx1-deficient iNKT cells have altered expression of several genes important for iNKT17 differentiation, including decreased expression of IL-7Rα, BATF and c-Maf and increased expression of Bcl11b and Lef1. However, reduction of Lef1 expression or introduction of an IL-7Rα transgene is not sufficient to correct the defect in iNKT17 differentiation, demonstrating that Runx1 is a key regulator of several genes required for iNKT17 differentiation. Loss of Runx1 leads to a severe decrease in iNKT cell numbers in the thymus, spleen and liver. The decrease in cell number is due to a combined decrease in proliferation at Stage 1 during thymic development and increased apoptosis. Thus, we describe a novel role of Runx1 in iNKT cell development and differentiation, particularly in orchestrating iNKT17 differentiation.

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7-30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.