These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

T cell help to B cells is a fundamental property of adaptive immunity, yet only recently have many of the cellular and molecular mechanisms of T cell help emerged. T follicular helper (Tfh) cells are the CD4(+) T helper cells that provide cognate help to B cells for high-affinity antibody production in germinal centers (GC). Tfh cells produce interleukin-21 (IL-21), and we show that IL-21 was necessary for GC formation. However, the central role of IL-21 in GC formation reflected its effects on Tfh cell generation rather than on B cells. Expression of the inducible costimulator (ICOS) was necessary for optimal production of IL-21, indicative of interplay between these two Tfh cell-expressed molecules. Finally, we demonstrate that IL-21's costimulatory capacity for T helper cell differentiation operated at the level of the T cell receptor signalosome through Vav1, a signaling molecule that controls T cell helper function. This study reveals a previously unappreciated role for Tfh cells in the formation of the GC and isotype switching through a CD4(+) T cell-intrinsic requirement for IL-21.

Highlights d Activated T cell signatures/populations drive response to anti-PD-1-based therapies d EOMES + CD69 + CD45RO + effector memory T cells are associated with response d EOMES + CD69 + CD45RO + expression is associated with longer PFS and tumor shrinkage d Non-responders with TIL-hot tumors express other immune drug targets

Significantly higher levels of plasma CXCL13 [chemokine (C-X-C motif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans, making the monitoring of GC activity by direct assessment of GC B cells and germinal center CD4 + T follicular helper (GC Tfh) cells problematic. The CXCL13-CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS + (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings.T he germinal center (GC) reaction is a critical immunological process that occurs in draining lymph nodes after immunization (1). The GC response consists of antigen-specific B cells undergoing affinity maturation through a process of somatic hypermutation (SHM) of the B-cell receptor. SHM is necessary for producing high-affinity Ab responses after immunizations and infections. Influenza neutralizing Abs have substantial SHM. Particularly high levels of SHM, 15-30% amino acid mutation (2, 3), are present and necessary for broad Ab neutralization of diverse HIV strains (4, 5). Therefore, as candidate influenza and HIV vaccines are evaluated for the ability to induce broadly neutralizing antibodies (bnAbs), the quantitation and functional characterization of GC responses will be a key parameter for study. Serological analysis of vaccine-specific Ab titers provides important information, but those data are limited. Serological outcomes are measured at time points long after initial immunizations. Neutralizing Ab responses are commonly only measurable after multiple boosts. Those outcomes likely depend on GC activity and affinity maturation at much earlier time points. Several state of the art HIV vaccine strategies rely on long, multistage immunization protocols (6, 7). With bnAb responses as the goal, means of early analysis of the immune response will be essential to understand and improve on vaccination schemes that may end in failure or only partial success. One critical parameter to assess will be the ability of each immunization to generate GC responses.Central to the GC re...

Respiratory viral infections are a significant burden to healthcare worldwide. Many whole genome expression profiles have identified different respiratory viral infection signatures, but these have not translated to clinical practice. Here, we performed two integrated, multi-cohort analyses of publicly available transcriptional data of viral infections. First, we identified a common host signature across different respiratory viral infections that could distinguish (a) individuals with viral infections from healthy controls and from those with bacterial infections, and (b) symptomatic from asymptomatic subjects prior to symptom onset in challenge studies. Second, we identified an influenza-specific host response signature that (a) could distinguish influenza-infected samples from those with bacterial and other respiratory viral infections, (b) was a diagnostic and prognostic marker in influenza-pneumonia patients and influenza challenge studies, and (c) was predictive of response to influenza vaccine. Our results have applications in the diagnosis, prognosis, and identification of drug targets in viral infections.