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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. [ABSTRACT FROM AUTHOR]

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 pentostatinbased 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Troublesome T cells: Many HIV vaccines aim to generate robust T cell responses, but two new studies show that this is not always straightforward. Chamcha et al. analyzed data from several nonhuman primate vaccine studies with SIV/SHIV challenges and discovered that achieving a certain threshold of vaccine-induced IFN-γ+ CD4 T cells lowered vaccine efficacy, possibly by providing target cells for the virus. Kallas et al. vaccinated people with HIV Gag/Pol alone or with Env to see whether antigenic competition could interfere with CD4 T cell responses; lower responses to Gag/Pol were detected when Env was also administered, indicating that including multiple antigens in a vaccine may preclude maximal T cell responses. Together, these studies highlight how we must tread carefully on the path to an effective HIV vaccine. 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 × 1010 particle units (PU) of rAd5 Gag/Pol and EnvA/B/C (3:1:1:1 mixture) or 5 × 109 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]