The γδ T Cell Club: bringing a global scientific community together in the age of Zoom.

Immunologists are very social people-they love to meet other immunologists and talk about immunology (and immunologists). Constantly! γδ T-cell researchers are no exception. On the contrary, as there are not so many of them compared to, say, researchers working on dendritic cells, they especially crave frequent interactions with like-minded scientists. This is where the technological solutions being developed during the coronavirus disease 2019 (COVID-19) pandemic come into play that have, almost overnight, allowed researchers to hold meetings and lectures online. We here describe how we set up the virtual 'γδ T Cell Club', a monthly webinar series that aims to bring the field closer together, and present our musings about what we have learned from this experience, which we hope is useful for other researchers interested in connecting online.

Mouse genome rewriting and tailoring of three important disease loci.

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.

MAVS signaling is required for preventing persistent chikungunya heart infection and chronic vascular tissue inflammation.

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.

Helminth resistance is mediated by differential activation of recruited monocyte-derived alveolar macrophages and arginine depletion.

Macrophages are known to mediate anti-helminth responses, but it remains uncertain which subsets are involved or how macrophages actually kill helminths. Here, we show rapid monocyte recruitment to the lung after infection with the nematode parasite Nippostrongylus brasiliensis. In this inflamed tissue microenvironment, these monocytes differentiate into an alveolar macrophage (AM)-like phenotype, expressing both SiglecF and CD11c, surround invading parasitic larvae, and preferentially kill parasites in vitro. Monocyte-derived AMs (Mo-AMs) express type 2-associated markers and show a distinct remodeling of the chromatin landscape relative to tissue-derived AMs (TD-AMs). In particular, they express high amounts of arginase-1 (Arg1), which we demonstrate mediates helminth killing through L-arginine depletion. These studies indicate that recruited monocytes are selectively programmed in the pulmonary environment to express AM markers and an anti-helminth phenotype.

Transcriptional programming and gene regulation in WC1+ γδ T cell subpopulations.

γδ T cells represent a high proportion of lymphocytes in the blood of ruminants with the majority expressing lineage-specific glycoproteins from the WC1 family. WC1 receptors are coded for by a multigenic array whose genes have variegated but stable expression among cells in the γδ T cell population. WC1 molecules function as hybrid pattern recognition receptors as well as co-receptors for the TCR and are required for responses by the cells. Because of the variegated gene expression, WC1+ γδ T cells can be divided into two main populations known as WC1.1+ and WC1.2+ based on monoclonal antibody reactivity with the expressed WC1 molecules. These subpopulations differ in their ability to respond to specific pathogens. Here, we showed these populations are established in the thymus and that WC1.1+ and WC1.2+ subpopulations have transcriptional programming that is consistent with stratification towards Tγδ1 or Tγδ17. WC1.1+ cells exhibited the Tγδ1 phenotype with greater transcription of Tbx21 and production of more IFNγ while the WC1.2+ subpopulation tended towards Tγδ17 programming producing higher levels of IL-17 and had greater transcription of Rorc. However, when activated both WC1+ subpopulations' cells transcribed Tbx21 and secreted IFNγ and IL-17 reflecting the complexity of these subpopulations defined by WC1 gene expression. The gene networks involved in development of these two subpopulations including expression of their archetypal genes wc1-3 (WC1.1+) and wc1-4 (WC1.2+) were unknown but we report that SOX-13, a γδ T cell fate-determining transcription factor, has differential occupancy on these WC1 gene loci and suggest a model for development of these subpopulations.

Beyond neutralization for BNT162b2 mRNA vaccination.

Mounting a robust immune response against SARS-CoV-2 requires neutralization as well as effector T cell functions. In this issue of Cell Host Microbe, Tauzin et al. characterize the humoral and T cell responses after a single dose of BNT162b2 mRNA vaccine in individuals with or without previous exposure to SARS-CoV-2.

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

Special features of γδ T cells in ruminants.

Ruminant γδ T cells were discovered in the mid-1980's shortly after a novel T cell receptor (TCR) gene from murine cells was described in 1984 and the murine TCRγ gene locus in 1985. It was possible to identify γδ T cell populations early in ruminants because they represent a large proportion of the peripheral blood mononuclear cells (PBMC). This null cell population, γδ T cells, was designated as such by its non-reactivity with monoclonal antibodies (mAb) against ovine and bovine CD4, CD8 and surface immunoglobulin (Ig). γδ T cells are non-conventional T cells known as innate-like cells capable of using both TCR as well as other types of receptor systems including pattern recognition receptors (PRR) and natural killer receptors (NKR). Bovine γδ T cells have been shown to respond to stimulation through toll-like receptors, NOD, and NKG2D as well as to cytokines alone, protein and non-protein antigens through their TCR, and to pathogen-infected host cells. The two main populations of γδ T cells are distinguished by the presence or absence of the hybrid co-receptor/PRR known as WC1 or T19. These two populations not only differ by their proportional representation in various tissues and organs but also by their migration into inflamed tissues. The WC1+ cells are found in the blood, skin and spleen while the WC1- γδ T cells predominate in the gut, mammary gland and uterus. In ruminants, γδ T cells may produce IFNγ, IL-17, IL-10 and TGFß, have cytotoxic activity and memory responses. The expression of particular WC1 family members controls the response to particular pathogens and correlates with differences in cytokine responses. The comparison of the WC1 gene families in cattle, sheep and goats is discussed relative to other multigenic arrays that differentiate γδ T cells by function in humans and mice.

Gene characterization and expression of the γδ T cell co-receptor WC1 in sheep.

Sheep are known to express the hybrid co-receptor/pattern recognition receptor WC1 on their γδ T cells but details of the ovine WC1 multigenic array and gene expression were unknown. Annotation of the sheep genome assembly (Oar_rambouillet_v1.0) yielded 15 complete and 42 partial WC1 genes predicted to code for six different protein structures. RT-PCR amplification of the most distal scavenger receptor cysteine rich (SRCR) domain known as a1, which serves as the gene signature, from genomic and cDNA templates verified the majority of annotated genes. As for cattle and goats, sheep a1 domain sequences included WC1.1 and WC1.2 types. A unique ovine gene, WC1-16, had multiple SRCR a-pattern domains in tandem similar to one found in goats. Intracytoplasmic domains of WC1 transcripts had splice variants that may affect signal transduction. The larger number of WC1 genes in sheep and differences in structures and splice variants relative to cattle could have implications in expression patterns and engagement of γδ T cells by pathogens or vaccine constructs.

Special features of γδ T cells in ruminants

Ruminant γδ T cells were discovered in the mid-1980′s shortly after a novel T cell receptor (TCR) gene from murine cells was described in 1984 and the murine TCRγ gene locus in 1985. It was possible to identify γδ T cell populations early in ruminants because they represent a large proportion of the peripheral blood mononuclear cells (PBMC). This null cell population, γδ T cells, was designated as such by its non-reactivity with monoclonal antibodies (mAb) against ovine and bovine CD4, CD8 and surface immunoglobulin (Ig). γδ T cells are non-conventional T cells known as innate-like cells capable of using both TCR as well as other types of receptor systems including pattern recognition receptors (PRR) and natural killer receptors (NKR). Bovine γδ T cells have been shown to respond to stimulation through toll-like receptors, NOD, and NKG2D as well as to cytokines alone, protein and non-protein antigens through their TCR, and to pathogen-infected host cells. The two main populations of γδ T cells are distinguished by the presence or absence of the hybrid co-receptor/PRR known as WC1 or T19. These two populations not only differ by their proportional representation in various tissues and organs but also by their migration into inflamed tissues. The WC1+ cells are found in the blood, skin and spleen while the WC1− γδ T cells predominate in the gut, mammary gland and uterus. In ruminants, γδ T cells may produce IFNγ, IL-17, IL-10 and TGFβ, have cytotoxic activity and memory responses. The expression of particular WC1 family members controls the response to particular pathogens and correlates with differences in cytokine responses. The comparison of the WC1 gene families in cattle, sheep and goats is discussed relative to other multigenic arrays that differentiate γδ T cells by function in humans and mice.

Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties.

Tissue-resident macrophages are a diverse population of cells that perform specialized functions including sustaining tissue homeostasis and tissue surveillance. Here, we report an interstitial subset of CD169+ lung-resident macrophages that are transcriptionally and developmentally distinct from alveolar macrophages (AMs). They are primarily localized around the airways and are found in close proximity to the sympathetic nerves in the bronchovascular bundle. These nerve- and airway-associated macrophages (NAMs) are tissue resident, yolk sac derived, self-renewing, and do not require CCR2+ monocytes for development or maintenance. Unlike AMs, the development of NAMs requires CSF1 but not GM-CSF. Bulk population and single-cell transcriptome analysis indicated that NAMs are distinct from other lung-resident macrophage subsets and highly express immunoregulatory genes under steady-state and inflammatory conditions. NAMs proliferated robustly after influenza infection and activation with the TLR3 ligand poly(I:C), and in their absence, the inflammatory response was augmented, resulting in excessive production of inflammatory cytokines and innate immune cell infiltration. Overall, our study provides insights into a distinct subset of airway-associated pulmonary macrophages that function to maintain immune and tissue homeostasis.

γδ T cells in livestock: Responses to pathogens and vaccine potential.

The immediate objective of our research is to understand the molecular mechanisms underlying activation and potentiation of the protective functional response of WC1+ γδ T cells to pathogens afflicting livestock species. The long-term goal is to incorporate stimulation of these cells into the next generation of vaccine constructs. γδ T cells have roles in the immune response to many infectious diseases including viral, bacterial, protozoan and worm infections, and their functional responses overlap with those of canonical αß T cells, for example they produce cytokines including interferon-γ and IL-17. Stimulation of non-conventional lymphocytes including γδ T cells and αß natural killer T (NKT) cells has been shown to contribute to protective immunity in mammals, bridging the gap between the innate and adaptive immune responses. Because of their innate-like early response, understanding how to engage γδ T-cell responses has the potential to optimize strategies of those that aim to induce pro-inflammatory responses as discussed here.

Bovine T cell receptors and γδ WC1 co-receptor transcriptome analysis during the first month of life.

Here we evaluated neonatal transcription of α, ß, γ and δ TCR and the γδ T cell co-receptor family WC1 in peripheral blood mononuclear cells. A previous report showed a rapid and global shift in transcription of immunoglobulin genes in neonatal calves during the first month after birth but this was not found here for the T cell genes. Transcription frequency of genes within TRAV subgroups correlated with the number of members, indicating a stochastic choice. In contrast, of the approximately 60 TRDV genes those in two of eleven TRDV1 clades and TRDVb3 were transcribed significantly more than the others while those in only one TRBV subgroup were. Transcription of genes in the TRGV5-containing cassette predominated among TRGV genes as a result of their exclusive usage by the WC1+ γδ T cells with a preference for transcription of two of four TRGV genes in that cassette. Finally, we report no large differences in transcription frequencies among the 13 WC1 genes.

Variegated Transcription of the WC1 Hybrid PRR/Co-Receptor Genes by Individual γδ T Cells and Correlation With Pathogen Responsiveness.

γδ T cells have broad reactivity and actively participate in protective immunity against tumors and infectious disease-causing organisms. In γδ-high species such as ruminants and other artiodactyls many γδ T cells bear the lineage-specific markers known as WC1. WC1 molecules are scavenger receptors coded for by a multigenic array and are closely related to SCART found on murine γδ T cells and CD163 found on a variety of cells. We have previously shown that WC1 molecules are hybrid pattern recognition receptors thereby binding pathogens as well as signaling co-receptors for the γδ T cell receptor. WC1+ γδ T cells can be divided into two major subpopulations differentiated by the WC1 genes they express and the pathogens to which they respond. Therefore, we hypothesize that optimal γδ T cell responses are contingent on pathogen binding to WC1 molecules, especially since we have shown that silencing WC1 results in an inability of γδ T cells from primed animals to respond to the pathogen Leptospira, a model system we have employed extensively. Despite this knowledge about the crucial role WC1 plays in γδ T cell biology, the pattern of WC1 gene expression by individual γδ T cells was not known but is critical to devise methods to engage γδ T cells for responses to specific pathogens. To address this gap, we generated 78 γδ T cell clones. qRT-PCR evaluation showed that approximately 75% of the clones had one to three WC1 genes transcribed but up to six per cell occurred. The co-transcription of WC1 genes by clones showed many combinations and some WC1 genes were transcribed by both subpopulations although there were differences in the overall pattern of WC1 genes transcription. Despite this overlap, Leptospira-responsive WC1+ memory γδ T cell clones were shown to have a significantly higher propensity to express WC1 molecules that are known to bind to the pathogen.