The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1 + stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.

O ne major obstacle toward an effective human immunodeficiency virus type 1 (HIV-1) cure is the establishment of a pool of long-lived latently infected cells early after infection (1). Using the rhesus macaque model of simian immunodeficiency virus (SIV) infection, it was recently shown that latent reservoirs could be seeded as early as 3 days after SIV exposure and before the detection of viremia in blood (2). The vast majority of cells infected with HIV-1 will die as a consequence of the infection or will be eliminated by the immune system. A minority of infected cells, however, turns into latently infected resting cells. These cells can either be reactivated and release de novo HIV-1 particles (3) or persist and homeostatically proliferate as long-lived memory T cells (4, 5). While current antiretroviral treatments (ARTs) can efficiently suppress HIV-1 replication and have dramatically improved the life expectancy and life quality of infected individuals, ART cannot eradicate the latent viral reservoir.Several different biological processes have been described to maintain latency in HIV-1-infected cells. Host transcription factors (TFs) such as nuclear factor kappa light-chain enhancer of activated B cells (NF-B) have multiple binding sites in the 5= long terminal repeat (LTR) of the HIV-1 genome, and their binding has been demonstrated to be necessary to initiate HIV-1 transcription (6). Sequestration of these TFs in the cytoplasm is one of the mechanisms enabling viral latency (7). Another described HIV-1 latency mechanism involves histone deacetylase (HDAC)-mediated epigenetic silencing (8). During latency establishment, HDAC molecules are recruited toward the 5= LTR of HIV-1 (9, 10) and therefore maintain the LTR in a repressed state (11). Several HDAC inhibitors (HDACis) targeting HDAC molecules have been tested for their ability to reactivate latently HIV-1-infected cells, including vorinostat, panobinostat, entinostat, and romidepsin (RMD). These HDACis proved to efficiently induce HIV-1 expression in latently infected resting CD4 ϩ T cells from HIV-1-infected individuals (8,12,13). RMD, a drug that has been used for the treatment of peripheral T-cell lymphoma,

Summary The persistence of long-lived memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, which are provided in niches organized by stromal cells. Using an ex vivo system in which we supply the known survival signals, direct cell contact to stromal cells, and the soluble cytokine a proliferation-inducing ligand (APRIL), we have elucidated the critical signaling pathways required for the survival of long-lived plasma cells. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to critical inactivation of Forkhead-Box-Protein O1/3 (FoxO1/3) and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Accordingly, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, by the nuclear factor κB (NF-κB) pathway, blocks activation of the endoplasmic-reticulum-stress-associated initiator caspase 12. Thus, stromal-cell-contact-induced PI3K and APRIL-induced NF-κB signaling provide the necessary and complementary signals to maintain bone marrow memory plasma cells.