Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

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.

OMIP-084: 28-color full spectrum flow cytometry panel for the comprehensive analysis of human γδ T cells.

Using full spectrum flow cytometry, we designed a 28-color panel for the analysis of markers known to be associated with the γδ T cell immune response. This panel allows the classification of γδ T cell subsets via specific V gene usage (Vγ9, Vδ1, Vδ2, and Vδ3) of their T cell receptor (TCR) and according to their functional differentiation. Phenotypical surface receptors to distinguish different stages of cell maturation included CD45RA, CD27, CD28, CD127, CD57, and CD16; chemokine receptors CXCR6, CCR5, CCR6, and CX3CR1; NK-associated markers NKG2A, NKG2D, CD56, and CD161, checkpoint-inhibitor PD-1, and activating receptors CD38 and CD25. T cell lineage markers for the analysis of αß T cells (CD4 and CD8) and MAIT cells (Vα7.2) were also included. This optimized multicolor panel allows a comprehensive immune-profiling of all main human γδ T cell subsets and is suitable for longitudinal or exploratory analysis of γδ T cell development and γδ T cell dynamics in clinical cohorts.

Mutual interplay between IL-17-producing γδT cells and microbiota orchestrates oral mucosal homeostasis.

γδT cells are a major component of epithelial tissues and play a role in tissue homeostasis and host defense. γδT cells also reside in the gingiva, an oral tissue covered with specialized epithelium that continuously monitors the challenging dental biofilm. Whereas most research on intraepithelial γδT cells focuses on the skin and intestine epithelia, our knowledge on these cells in the gingiva is still incomplete. In this study, we demonstrate that even though the gingiva develops after birth, the majority of gingival γδT cells are fetal thymus-derived Vγ6+ cells, and to a lesser extent Vγ1+ and Vγ4+ cells. Furthermore, we show that γδT cells are motile and locate preferentially in the epithelium adjacent to the biofilm. Vγ6+ cells represent the major source of IL-17-producing cells in the gingiva. Chimeric mice and parabiosis experiments indicated that the main fraction of gingival γδT cells is radioresistant and tissue-resident, persisting locally independent of circulating γδT cells. Notably, gingival γδT cell homeostasis is regulated by the microbiota as the ratio of Vγ6+ and Vγ4+ cells was reversed in germ-free mice, and their activation state was decreased. As a consequence, conditional ablation of γδT cells results in elevated gingival inflammation and subsequent alterations of oral microbial diversity. Taken together, these findings suggest that oral mucosal homeostasis is shaped by reciprocal interplays between γδT cells and local microbiota.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

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.

Eomes expression reports the progressive differentiation of IFN-γ-producing Th1-like γδ T cells.

The transcription factor Eomesodermin (Eomes) plays a crucial role in regulating cytotoxic function, development, and survival of immune cells. γδ T cells can express Eomes, but its contribution to their differentiation is unknown. Using Eomes-IRES-GFP mice, we show that Eomes+ γδ T cells are unequally distributed among organs, with the highest proportion in spleen. While the majority of Eomes+ γδ T cells expressed Vγ1+ and Vγ4+ TCRs, Eomes was absent in Vγ5+ , Vγ6+ , and Vγ7+ subsets. Moreover, Eomes was co-expressed in γδ T cells with Th1 lineage-related factors such as CD27, T-bet, and Ly6C, but not with Th17 lineage-related genes. Eomes+ and Eomes- γδ T-cell populations showed distinct gene expression profiles, with an increase of cytotoxic-related genes in Eomes+ γδ T cells. Furthermore, Eomes could be induced in peripheral γδ T cells by IL-12 and IL-4, and Eomes+ γδ T cells presented a higher proliferation rate and IFN-γ production when stimulated in vitro with IL-12 and IL-18. However, γδ T cells with very high Eomes levels displayed an exhausted phenotype with high levels of PD-1, and were less capable of IFN-γ production. Together, this study highlights Eomes as a marker for the differentiation of Th1-like effector γδ T cells.

Effector γδ T Cell Differentiation Relies on Master but Not Auxiliary Th Cell Transcription Factors.

γδ T lymphocytes are programmed into distinct IFN-γ-producing CD27(+) (γδ27(+)) and IL-17-producing CD27(-) (γδ27(-)) subsets that play key roles in protective or pathogenic immune responses. Although the signature cytokines are shared with their αß Th1 (for γδ27(+)) and Th17 (for γδ27(-)) cell counterparts, we dissect in this study similarities and differences in the transcriptional requirements of murine effector γδ27(+), γδ27(-)CCR6(-), and γδ27(-)CCR6(+) γδ T cell subsets and αß T cells. We found they share dependence on the master transcription factors T-bet and RORγt for IFN-γ and IL-17 production, respectively. However, Eomes is fully dispensable for IFN-γ production by γδ T cells. Furthermore, the Th17 cell auxiliary transcription factors RORα and BATF are not required for IL-17 production by γδ27(-) cell subsets. We also show that γδ27(-) (but not γδ27(+)) cells become polyfunctional upon IL-1ß plus IL-23 stimulation, cosecreting IL-17A, IL-17F, IL-22, GM-CSF, and IFN-γ. Collectively, our in vitro and in vivo data firmly establish the molecular segregation between γδ27(+) and γδ27(-) T cell subsets and provide novel insight on the nonoverlapping transcriptional networks that control the differentiation of effector γδ versus αß T cell subsets.

Opi1p translocation to the nucleus is regulated by hydrogen peroxide in Saccharomyces cerevisiae.

During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.

Systems biology analysis reveals distinct molecular signatures associated with immune responsiveness to the BNT162b COVID-19 vaccine.

BACKGROUND: Human immune responses to COVID-19 vaccines display a large heterogeneity of induced immunity and the underlying immune mechanisms for this remain largely unknown. METHODS: Using a systems biology approach, we longitudinally profiled a unique cohort of female high and low responders to the BNT162b vaccine, who were known from previous COVID-19 vaccinations to develop maximum and minimum immune responses to the vaccine. We utilized high dimensional flow cytometry, bulk and single cell mRNA sequencing and 48-plex serum cytokine analyses. FINDINGS: We revealed early, transient immunological and molecular signatures that distinguished high from low responders and correlated with B and T cell responses measured 14 days later. High responders featured a distinct transcriptional activity of interferon-driven genes and genes connected to enhanced antigen presentation. This was accompanied by a robust cytokine response related to Th1 differentiation. Both transcriptome and serum cytokine signatures were confirmed in two independent confirmatory cohorts. INTERPRETATION: Collectively, our data contribute to a better understanding of the immunogenicity of mRNA-based COVID-19 vaccines, which might lead to the optimization of vaccine designs for individuals with poor vaccine responses. FUNDING: German Center for Infection Research, German Center for Lung Research, German Research Foundation, Excellence Strategy EXC 2155 "RESIST" and European Regional Development Fund.

RORγt+ c-Maf+ Vγ4+ γδ T cells are generated in the adult thymus but do not reach the periphery.

T cell receptor (TCR) Vγ4-expressing γδ T cells comprise interferon γ (IFNγ)- and interleukin-17 (IL-17)-producing effector subsets, with a preference for IL-17 effector fate decisions during early ontogeny. The existence of adult-thymus-derived IL-17+ T cells (γδ17) remains controversial. Here, we use a mouse model in which T cells are generated exclusively in the adult thymus and employ single-cell chromatin state analysis to study their development. We identify adult-thymus-derived Vγ4 T cells that have all the molecular programs to become IL-17 producers. However, they have reduced IL-17 production capabilities and rarely reach the periphery. Moreover, this study provides high-resolution profiles of Vγ4 T cells in the adult thymus and lymph nodes and identifies Zeb1 as a potential γδ17 cell regulator. Together, this study provides valuable insights into the developmental traits of Vγ4 T cells during adulthood and supports the idea of age-specific signals required for thymic export and/or peripheral maturation of γδ17 cells.

Omicron infection-associated T- and B-cell immunity in antigen-naive and triple-COVID-19-vaccinated individuals.

Since early 2022, various Omicron variants have dominated the SARS-CoV-2 pandemic in most countries. All Omicron variants are B-cell immune escape variants, and antibodies induced by first-generation COVID-19 vaccines or by infection with earlier SARS-CoV-2 variants largely fail to protect individuals from Omicron infection. In the present study, we investigated the effect of Omicron infections in triple-vaccinated and in antigen-naive individuals. We show that Omicron breakthrough infections occurring 2-3.5 months after the third vaccination restore B-cell and T-cell immune responses to levels similar to or higher than those measured 14 days after the third vaccination, including the induction of Omicron-neutralizing antibodies. Antibody responses in breakthrough infection derived mostly from cross-reacting B cells, initially induced by vaccination, whereas Omicron infections in antigen-naive individuals primarily generated B cells binding to the Omicron but not the Wuhan spike protein. Although antigen-naive individuals mounted considerable T-cell responses after infection, B-cell responses were low, and neutralizing antibodies were frequently below the limit of detection. In summary, the detection of Omicron-associated B-cell responses in primed and in antigen-naive individuals supports the application of Omicron-adapted COVID-19 vaccines, but calls into question their suitability if they also contain/encode antigens of the original Wuhan virus.

BNT162b2-boosted immune responses six months after heterologous or homologous ChAdOx1nCoV-19/BNT162b2 vaccination against COVID-19.

Heterologous prime/boost vaccination with a vector-based approach (ChAdOx-1nCov-19, ChAd) followed by an mRNA vaccine (e.g. BNT162b2, BNT) has been reported to be superior in inducing protective immunity compared to repeated application of the same vaccine. However, data comparing immunity decline after homologous and heterologous vaccination as well as effects of a third vaccine application after heterologous ChAd/BNT vaccination are lacking. Here we show longitudinal monitoring of ChAd/ChAd (n = 41) and ChAd/BNT (n = 88) vaccinated individuals and the impact of a third vaccination with BNT. The third vaccination greatly augments waning anti-spike IgG but results in only moderate increase in spike-specific CD4 + and CD8 + T cell numbers in both groups, compared to cell frequencies already present after the second vaccination in the ChAd/BNT group. More importantly, the third vaccination efficiently restores neutralizing antibody responses against the Alpha, Beta, Gamma, and Delta variants of the virus, but neutralizing activity against the B.1.1.529 (Omicron) variant remains severely impaired. In summary, inferior SARS-CoV-2 specific immune responses following homologous ChAd/ChAd vaccination can be compensated by heterologous BNT vaccination, which might influence the choice of vaccine type for subsequent vaccination boosts.

IL-4-Producing Vγ1+/Vδ6+ γδ T Cells Sustain Germinal Center Reactions in Peyer's Patches of Mice.

The mucosal immune system is the first line of defense against pathogens. Germinal centers (GCs) in the Peyer's patches (PPs) of the small intestine are constantly generated through stimulation of the microbiota. In this study, we investigated the role of γδ T cells in the GC reactions in PPs. Most γδ T cells in PPs localized in the GCs and expressed a TCR composed of Vγ1 and Vδ6 chains. By using mice with partial and total γδ T cell deficiencies, we found that Vγ1+/Vδ6+ T cells can produce high amounts of IL-4, which drives the proliferation of GC B cells as well as the switch of GC B cells towards IgA. Therefore, we conclude that γδ T cells play a role in sustaining gut homeostasis and symbiosis via supporting the GC reactions in PPs.

Clonal expansion of CD8+ T cells reflects graft-versus-leukemia activity and precedes durable remission following DLI.

Donor lymphocyte infusion (DLI) is a standard of care for relapse of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Currently it is poorly understood how and when CD8+ αß T cells exert graft-versus-leukemia (GVL) activity after DLI. Also, there is no reliable biomarker to monitor GVL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αß T-cell clones in patients with DLI. In this prospective clinical study of 29 patients, we performed deep T-cell receptor ß (TRB ) sequencing of sorted CD8+ αß T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GVL, longitudinal analyses revealed a preferential expansion of distinct CD8+TRB clones (n = 14). This did not occur in samples of patients without signs of GVL (n = 11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36-month study follow-up. Furthermore, absence of clonal outgrowth of the CD8+TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+TRB diversity at day 15 after DLI (n = 13) had a lower relapse incidence (P = .0040) compared with patients without clonal expansion (n = 6). In conclusion, CD8+TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.

Immune responses against SARS-CoV-2 variants after heterologous and homologous ChAdOx1 nCoV-19/BNT162b2 vaccination.

Currently approved viral vector-based and mRNA-based vaccine approaches against coronavirus disease 2019 (COVID-19) consider only homologous prime-boost vaccination. After reports of thromboembolic events, several European governments recommended using AstraZeneca's ChAdOx1-nCov-19 (ChAd) only in individuals older than 60 years, leaving millions of already ChAd-primed individuals with the decision to receive either a second shot of ChAd or a heterologous boost with mRNA-based vaccines. However, such combinations have not been tested so far. We used Hannover Medical School's COVID-19 Contact Study cohort of healthcare professionals to monitor ChAd-primed immune responses before and 3 weeks after booster with ChAd (n = 32) or BioNTech/Pfizer's BNT162b2 (n = 55). Although both vaccines boosted prime-induced immunity, BNT162b2 induced significantly higher frequencies of spike-specific CD4+ and CD8+ T cells and, in particular, high titers of neutralizing antibodies against the B.1.1.7, B.1.351 and P.1 variants of concern of severe acute respiratory syndrome coronavirus 2.

Intranasal Delivery of MVA Vector Vaccine Induces Effective Pulmonary Immunity Against SARS-CoV-2 in Rodents.

Antigen-specific tissue-resident memory T cells (Trms) and neutralizing IgA antibodies provide the most effective protection of the lungs from viral infections. To induce those essential components of lung immunity against SARS-CoV-2, we tested various immunization protocols involving intranasal delivery of a novel Modified Vaccinia virus Ankara (MVA)-SARS-2-spike vaccine candidate. We show that a single intranasal MVA-SARS-CoV-2-S application in mice strongly induced pulmonary spike-specific CD8+ T cells, albeit restricted production of neutralizing antibodies. In prime-boost protocols, intranasal booster vaccine delivery proved to be crucial for a massive expansion of systemic and lung tissue-resident spike-specific CD8+ T cells and the development of Th1 - but not Th2 - CD4+ T cells. Likewise, very high titers of IgG and IgA anti-spike antibodies were present in serum and broncho-alveolar lavages that possessed high virus neutralization capacities to all current SARS-CoV-2 variants of concern. Importantly, the MVA-SARS-2-spike vaccine applied in intramuscular priming and intranasal boosting treatment regimen completely protected hamsters from developing SARS-CoV-2 lung infection and pathology. Together, these results identify intramuscular priming followed by respiratory tract boosting with MVA-SARS-2-S as a promising approach for the induction of local, respiratory as well as systemic immune responses suited to protect from SARS-CoV-2 infections.

Reappearance of effector T cells is associated with recovery from COVID-19.

BACKGROUND: Elucidating the role of T cell responses in COVID-19 is of utmost importance to understand the clearance of SARS-CoV-2 infection. METHODS: 30 hospitalized COVID-19 patients and 60 age- and gender-matched healthy controls (HC) participated in this study. We used two comprehensive 11-colour flow cytometric panels conforming to Good Laboratory Practice and approved for clinical diagnostics. FINDINGS: Absolute numbers of lymphocyte subsets were differentially decreased in COVID-19 patients according to clinical severity. In severe disease (SD) patients, all lymphocyte subsets were reduced, whilst in mild disease (MD) NK, NKT and γδ T cells were at the level of HC. Additionally, we provide evidence of T cell activation in MD but not SD, when compared to HC. Follow up samples revealed a marked increase in effector T cells and memory subsets in convalescing but not in non-convalescing patients. INTERPRETATION: Our data suggest that activation and expansion of innate and adaptive lymphocytes play a major role in COVID-19. Additionally, recovery is associated with formation of T cell memory as suggested by the missing formation of effector and central memory T cells in SD but not in MD. Understanding T cell-responses in the context of clinical severity might serve as foundation to overcome the lack of effective anti-viral immune response in severely affected COVID-19 patients and can offer prognostic value as biomarker for disease outcome and control. FUNDING: Funded by State of Lower Saxony grant 14-76,103-184CORONA-11/20 and German Research Foundation, Excellence Strategy - EXC2155"RESIST"-Project ID39087428, and DFG-SFB900/3-Project ID158989968, grants SFB900-B3, SFB900-B8.

Single-Cell Transcriptomics Identifies the Adaptation of Scart1+ Vγ6+ T Cells to Skin Residency as Activated Effector Cells.

IL-17-producing γδ T cells express oligoclonal Vγ4+ and Vγ6+ TCRs, mainly develop in the prenatal thymus, and later persist as long-lived self-renewing cells in all kinds of tissues. However, their exchange between tissues and the mechanisms of their tissue-specific adaptation remain poorly understood. Here, single-cell RNA-seq profiling identifies IL-17-producing Vγ6+ T cells as a highly homogeneous Scart1+ population in contrast to their Scart2+ IL-17-producing Vγ4+ T cell counterparts. Parabiosis demonstrates that Vγ6+ T cells are fairly tissue resident in the thymus, peripheral lymph nodes, and skin. There, Scart1+ Vγ6+ T cells display tissue-specific gene expression signatures in the skin, characterized by steady-state production of the cytokines IL-17A and amphiregulin as well as by high expression of the anti-apoptotic Bcl2a1 protein family. Together, this study demonstrates how Scart1+ Vγ6+ T cells undergo tissue-specific functional adaptation to persist as effector cells in their skin habitat.