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.

Background: Coronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Anti-viral immune response is crucial to achieve pathogen clearance, however in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options. Methods: We performed a flow cytometric characterization of immune cells subsets from 30 COVID-19 patients and correlated these data with clinical outcomes. Results: COVID-19 patients showed decreased numbers of circulating T, B and NK cells, and exhibited a skewing of CD8+ T cells towards a terminally differentiated/senescent phenotype. In agreement, T CD4+, T CD8+ but also NK cells displayed reduced anti-viral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in COVID-19 patients, particularly in those that required intensive care. The latter group of patients showed also increased serum IL-6 levels, that correlated to the frequency of granzyme-expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells. Conclusion: In conclusion, the association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore anti-viral mechanisms.

The novel coronavirus SARS-CoV-2 enters into the human body mainly through the ACE2 + TMPRSS2+ nasal epithelial cells. The initial host response to this pathogen occurs in a peculiar immune microenvironment that, starting from the Nasopharynx-Associated Lymphoid Tissue (NALT) system, is the product of a long evolutionary process that is aimed to first recognize exogenous airborne agents. In the present work, we want to critically review the latest molecular and cellular findings on the mucosal response to SARS-CoV-2 in the nasal cavity and in NALT, and to analyze its impact in the subsequent course of COVID-19. Finally, we want to explore the possibility that the regulation of the systemic inflammatory network against the virus can be modulated starting from the initial phases of the nasal and nasopharyngeal response and this may have several clinical and epidemiological implications starting from a mucosal vaccine development.

The characterization of the adaptive immune response to COVID-19 vaccination in individuals who recovered from SARS-CoV-2 infection may define current and future clinical practice. To determine the effect of two doses BNT162b2 mRNA COVID-19 vaccination schedule in individuals who recovered from COVID-19 (COVID-19 recovered) compared to naïve subjects, we evaluated SARS-CoV-2 Spike-specific T and B cell responses, as well as specific IgA, IgG, IgM and neutralizing antibodies titers in 22 individuals who received BNT162b2 mRNA COVID-19 vaccine, 11 of which had a previous history of SARS-CoV-2 infection.Evaluations were performed before vaccination and then weekly until 7 days post second injection. Data obtained clearly showed that one vaccine dose is sufficient to increase both cellular and humoral immune response in COVID-19 recovered subjects without any additional improvement after the second dose. On the contrary, the second dose is proved mandatory in naïve ones to further enhance the immune response. These findings were further confirmed at serological level in a larger cohort of naïve (68) and COVID-19 recovered (29) subjects, tested up to 50 days post vaccination. These results question whether a second vaccine injection in COVID-19 recovered subjects is required and indicate that millions of vaccine doses may be redirected to naïve individuals, thus shortening the time to reach herd immunity.

T helper17 (Th17) lymphocytes represent a third arm of the CD4+ T-cell effector responses, in addition to Th1 and Th2 cells. Th17 cells have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in inflammatory sites. In humans, Keywords: CD161 r IL4I1 r RORC r Th17 Th1 cells derived from Th17 cells express CD161, whereas classic Th1 cells do not; these Th17-derived Th1 cells have been termed nonclassic Th1 cells. In this study, we examined similarities and differences between classic and nonclassic human Th1 cells by assessing a panel of T-cell clones, as well as CD161Introduction CD4 + T helper (Th) cells can be classified into lineages on the basis of cytokine production, the expression of specific transcription factors, and the immunological function they mediate: Th1 cells, which express the transcription factor T-box expressed in T cells (T-bet) and secrete IFN-γ, protect the host against intracellular infections; Th2 cells, which express GATA-3 and secrete IL-4, IL-5, and IL-13, mediate host defense against helminths [1,2]. Recently, additional subsets that preferentially produce distinct cytokines Correspondence: Prof. Francesco Annunziato e-mail: f.annunziato@dmi.unifi.it have been described. The most studied subset includes cells that selectively produce IL-17A (Th17 cells), express the transcription factor RAR-related orphan receptor (ROR)γt, and protect the host against infection with extracellular pathogens [3,4]. Human Th17 cells are, at least partially, different from murine Th17 cells [5,6]. Indeed, human Th17 cells express not only distinctive Th17 molecules, such as IL-23 receptor (IL-23R) and RORC, but also those typical of the Th1 phenotype such as IL-12Rβ2 and T-bet [6]. Moreover, we have previously discovered a subset of human Th17 cells that are also able to produce IFN-γ, named Th17/Th1 * These authors contributed equally to this work. To investigate the main features of these two different Th1 subsets, CD4+ CD161 + and CD4 + CD161 − T-cell populations were purified from peripheral blood (PB) of healthy human subjects and then cloned under limiting dilution conditions. As expected, clones derived from the CD4 + CD161 + T-cell fraction exhibited a Th17, a Th17/Th1, or a Th1 phenotype, whereas virtually all clones generated from the CD4 + CD161 − T-cell subset had a Th1 phenotype and none of them was able to produce IL-17A ( Fig. 1A and B). CD161 − Th1 clones were considered as classic Th1 cells, whereas CD161 + Th1 cells were considered as nonclassic (Th17-derived) cells. Transcription factorsThe expression of the transcription factors T-bet and RORC by classic and nonclassic Th1, as well as by Th17 or Th17/Th1, cells were compared by assessing 20 randomly selected T-cell clones from each phenotype with quantitative RT-PCR. In agreement with previously published data [15,16], the Th1-related transcription factor T-bet was found to be expressed by all four types of T-cell clones analyzed, reaching the highest mRNA levels in both classic and nonclassic-Th1 c...