Identification of Leptospiral Protein Antigens Recognized by WC1+ γδ T Cell Subsets as Target for Development of Recombinant Vaccines.

Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. γδ T cells play a role in the protective immune response in livestock species against Leptospira, while human γδ T cells also respond to Leptospira. Thus, activation of γδ T cells has emerged as a potential component in the optimization of vaccine strategies. Bovine γδ T cells proliferate and produce gamma interferon (IFN-γ) in response to vaccination with inactivated leptospires, and this response is mediated by a specific subpopulation of the WC1-bearing γδ T cells. WC1 molecules are members of the group B scavenger receptor cysteine-rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and γδ TCR coreceptors, the WC1 system has been proposed as a novel target to engage γδ T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ γδ T cells and identify two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific γδ T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+ γδ T cells respond. Identification of protein antigens will enhance our understanding of the role γδ T cells play in the leptospiral immune response and in recombinant vaccine development.

Defining the caprine γδ T cell WC1 multigenic array and evaluation of its expressed sequences and gene structure conservation among goat breeds and relative to cattle.

Workshop cluster 1 (WC1) molecules are part of the scavenger receptor cysteine-rich (SRCR) superfamily and act as hybrid co-receptors for the γδ T cell receptor and as pattern recognition receptors for binding pathogens. These members of the CD163 gene family are expressed on γδ T cells in the blood of ruminants. While the presence of WC1+ γδ T cells in the blood of goats has been demonstrated using monoclonal antibodies, there was no information available about the goat WC1 gene family. The caprine WC1 multigenic array was characterized here for number, structure and expression of genes, and similarity to WC1 genes of cattle and among goat breeds. We found sequence for 17 complete WC1 genes and evidence for up to 30 SRCR a1 or d1 domains which represent distinct signature domains for individual genes. This suggests substantially more WC1 genes than in cattle. Moreover, goats had seven different WC1 gene structures of which 4 are unique to goats. Caprine WC1 genes also had multiple transcript splice variants of their intracytoplasmic domains that eliminated tyrosines shown previously to be important for signal transduction. The most distal WC1 SRCR a1 domains were highly conserved among goat breeds, but fewer were conserved between goats and cattle. Since goats have a greater number of WC1 genes and unique WC1 gene structures relative to cattle, goat WC1 molecules may have expanded functions. This finding may impact research on next-generation vaccines designed to stimulate γδ T cells.

Characterization of the domestic goat γδ T cell receptor gene loci and gene usage.

Goats and cattle diverged 30 million years ago but retain similarities in immune system genes. Here, the caprine T cell receptor (TCR) gene loci and transcription of its genes were examined and compared to cattle. We annotated the TCR loci using an improved genome assembly (ARS1) of a highly homozygous San Clemente goat. This assembly has already proven useful for describing other immune system genes including antibody and leucocyte receptors. Both the TCRγ (TRG) and TCRδ (TRD) loci were similarly organized in goats as in cattle and the gene sequences were highly conserved. However, the number of genes varied slightly as a result of duplications and differences occurred in mutations resulting in pseudogenes. WC1+ γδ T cells in cattle have been shown to use TCRγ genes from only one of the six available cassettes. The structure of that Cγ gene product is unique and may be necessary to interact with WC1 for signal transduction following antigen ligation. Using RT-PCR and PacBio sequencing, we observed the same restriction for goat WC1+ γδ T cells. In contrast, caprine WC1+ and WC1- γδ T cell populations had a diverse TCRδ gene usage although the propensity for particular gene usage differed between the two cell populations. Noncanonical recombination signal sequences (RSS) largely correlated with restricted expression of TCRγ and δ genes. Finally, caprine γδ T cells were found to incorporate multiple TRD diversity gene sequences in a single transcript, an unusual feature among mammals but also previously observed in cattle.

A Consensus Definitive Classification of Scavenger Receptors and Their Roles in Health and Disease.

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.

Synthetic Protein Mimics for Functional Protein Delivery.

The use of proteins as biological tools and therapeutic agents is limited due to the fact that proteins do not effectively cross the plasma membrane of cells. Here, we report a novel class of protein transporter molecules based on protein transduction domain mimics (PTDMs) synthesized via ring opening metathesis polymerization (ROMP). The PTDMs reported here were specifically inspired by amphiphilic peptides known to deliver functional proteins into cells via noncovalent interactions between the peptide and the cargo. This contrasts with peptides like TAT, penetratin, and R9, which often require covalent fusion to their cargoes. Using the easily tunable synthetic ROMP platform, the importance of a longer hydrophobic segment with cationic guanidinium groups was established through the delivery of EGFP into Jurkat T cells. The most efficient of these protein transporters was used to deliver functional Cre Recombinase with ∼80% knockdown efficiency into hard to transfect human T cells. Additionally, a C-terminally deleted form of the transcription factor Runx1 (Runx1.d190) was delivered into primary murine splenocytes, producing a 2-fold increase in c-Myc mRNA production, showcasing the versatility of this platform to deliver biologically active proteins into hard to transfect cell types.

The endocytosis and signaling of the γδ T cell coreceptor WC1 are regulated by a dileucine motif.

WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

WC1 is a hybrid γδ TCR coreceptor and pattern recognition receptor for pathogenic bacteria.

WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials.

Signal transduction by different forms of the γδ T cell-specific pattern recognition receptor WC1.

WC1 coreceptors are scavenger receptor cysteine-rich (SRCR) family members, related to T19 in sheep, SCART in mice, and CD163c-α in humans, and form a 13-member subfamily in cattle exclusively expressed on γδ T cells. Subpopulations of γδ T cells are defined by anti-WC1 mAbs and respond to different pathogen species accordingly. In this study, variegated WC1 gene expression within subpopulations and differences in signaling and cell activation due to endodomain sequences are described. The endodomains designated types I to III differ by a 15- or 18-aa insert in type II and an additional 80 aa containing an additional eight tyrosines for type III. Anti-WC1 mAbs enhanced cell proliferation of γδ T cells when cross-linked with the TCR regardless of the endodomain sequences. Chimeric molecules of human CD4 ectodomain with WC1 endodomains transfected into Jurkat cells showed that the tyrosine phosphorylation of the type II was the same as that of the previously reported archetypal sequence (type I) with only Y24EEL phosphorylated, whereas for type III only Y199DDV and Y56TGD were phosphorylated despite conservation of the Y24EEL/Y24QEI and Y199DDV/I tyrosine motifs among the three types. Time to maximal phosphorylation was more rapid with type III endodomains and sustained longer. Differences in tyrosine phosphorylation were associated with differences in function in that cross-linking of type III chimeras with TCR resulted in significantly greater IL-2 production. Identification of differences in the signal transduction through the endodomains of WC1 contributes to understanding the functional role of the WC1 coreceptors in the γδ T cell responses.

Specific recognition of mycobacterial protein and peptide antigens by γδ T cell subsets following infection with virulent Mycobacterium bovis.

Promoting effective immunity to Mycobacterium bovis infection is a challenge that is of interest to the fields of human and animal medicine alike. We report that γδ T cells from virulent M. bovis-infected cattle respond specifically and directly to complex, protein, and nonprotein mycobacterial Ags. Importantly, to our knowledge, we demonstrate for the first time that bovine γδ T cells specifically recognize peptide Ags derived from the mycobacterial protein complex ESAT6:CFP10 and that this recognition requires direct contact with APCs and signaling through the T cell Ag receptor but is independent of MHC class I or II. Furthermore, we show that M. bovis infection in cattle induces robust IL-17A protein responses. Interestingly, in contrast to results from mice, bovine CD4 T cells, and not γδ T cells, are the predominant source of this critical proinflammatory mediator. Bovine γδ T cells are divided into subsets based upon their expression of Workshop Cluster 1 (WC1), and we demonstrate that the M. bovis-specific γδ T cell response is composed of a heterogeneous mix of WC1-expressing populations, with the serologically defined WC1.1(+) and WC1.2(+) subsets responding in vitro to mycobacterial Ags and accumulating in the lesions of M. bovis-infected animals. The results described in this article enhance our understanding of γδ T cell biology and, because virulent M. bovis infection of cattle represents an excellent model of tuberculosis in humans, contribute to our overall understanding of the role of γδ T cells in the mycobacterial-specific immune response.

Bovine gamma delta T cells and the function of gamma delta T cell specific WC1 co-receptors.

The study of γδ T cells in ruminants dates to the discovery of the γδ TCR in humans and mice. It is important since cattle offer an alternative model to the mouse for evaluating the role of γδ T cells in zoonotic disease research and for control of disease reservoirs in non-human animals. In addition, maintaining the health of cattle and other members of the order Artiodactyla is critical to meet the global human need for animal-source protein. In this review, we examine the bovine γδ T cell responses to Mycobacteria, which infects a third of the human population, and bovine γ and δ TCR diversity and the relationship to the TCR of human mycobacteria-responsive γδ T cells. We review the utilization of the γδ T cell specific scavenger receptor cysteine-rich (SRCR) glycoproteins known as WC1, and that are part of the CD163 family, which function as both γδ T cell activating co-receptors and pattern recognition receptors (PRR) for bovine γδ T cells and highlight the presence and evolution of this multigenic array, with potential for the same function, in birds, reptiles, jawless and bony fishes, and prototherian and eutherian mammals.

Novel protein transduction domain mimics as nonviral delivery vectors for siRNA targeting NOTCH1 in primary human T cells.

RNA interference technology has recently been highlighted as a powerful research method as well as a potential therapeutic treatment for several diseases. However, the delivery of small interfering RNA (siRNA) into T cell lines and primary blood cells is exceedingly challenging, as they are resistant to transfection by conventional reagents. As a result, there is an unmet need for nonviral, efficient, and easily prepared carriers for siRNA delivery into hard-to-transfect cell types. Here, we report a novel system based on protein transduction domain mimics (PTDMs), generated by ring opening metathesis polymerization, for intracellular delivery of siRNA molecules. PTDM-based siRNA delivery induced efficient NOTCH1 knockdown in Jurkat T cells and human peripheral blood mononuclear cells without any measured toxicity. Furthermore, delivering siRNA to NOTCH1 in human peripheral blood cells modulated cell proliferation and differentiation of T cells into T(H)1 cells.

T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis.

African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG) coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB) and Follicular B (FoB) cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves.

Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes.

BACKGROUND: WC1 co-receptors belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1(+) γδ T cells. We have previously identified partial or complete genomic sequences for thirteen different WC1 genes through annotation of the bovine genome Btau_3.1 build. We also identified two WC1 cDNA sequences from other cattle that did not correspond to sequences in the Btau_3.1 build. Their absence in the Btau_3.1 build may have reflected gaps in the genome assembly or polymorphisms among animals. Since the response of γδ T cells to bacterial challenge is determined by WC1 gene expression, it was critical to understand whether individual cattle or breeds differ in the number of WC1 genes or display polymorphisms. RESULTS: Real-time quantitative PCR using DNA from the animal whose genome was sequenced ("Dominette") and sixteen other animals representing ten breeds of cattle, showed that the number of genes coding for WC1 co-receptors is thirteen. The complete coding sequences of those thirteen WC1 genes is presented, including the correction of an error in the WC1-2 gene due to mis-assembly in the Btau_3.1 build. All other cDNA sequences were found to agree with the previous annotation of complete or partial WC1 genes. PCR amplification and sequencing of the most variable N-terminal SRCR domain (domain 1 which has the SRCR "a" pattern) of each of the thirteen WC1 genes showed that the sequences are highly conserved among individuals and breeds. Of 160 sequences of domain 1 from three breeds of cattle, no additional sequences beyond the thirteen described WC1 genes were found. Analysis of the complete WC1 cDNA sequences indicated that the thirteen WC1 genes code for three distinct WC1 molecular forms. CONCLUSION: The bovine WC1 multi-gene family is composed of thirteen genes coding for three structural forms whose sequences are highly conserved among individual cattle and breeds. The sequence diversity necessary for WC1 genes to function as a multi-genic pattern recognition receptor array is encoded in the genome, rather than generated by recombinatorial diversity or hypermutation.

γδ T cells in artiodactyls: Focus on swine.

Vaccination is the most effective medical strategy for disease prevention but there is a need to improve livestock vaccine efficacy. Understanding the structure of the immune system of swine, which are considered a γδ T cell "high" species, and thus, particularly how to engage their γδ T cells for immune responses, may allow for development of vaccine optimization strategies. The propensity of γδ T cells to home to specific tissues, secrete pro-inflammatory and regulatory cytokines, exhibit memory or recall responses and even function as antigen-presenting cells for αß T cells supports the concept that they have enormous potential for priming by next generation vaccine constructs to contribute to protective immunity. γδ T cells exhibit several innate-like antigen recognition properties including the ability to recognize antigen in the absence of presentation via major histocompatibility complex (MHC) molecules enabling γδ T cells to recognize an array of peptides but also non-peptide antigens in a T cell receptor-dependent manner. γδ T cell subpopulations in ruminants and swine can be distinguished based on differential expression of the hybrid co-receptor and pattern recognition receptors (PRR) known as workshop cluster 1 (WC1). Expression of various PRR and other innate-like immune receptors diversifies the antigen recognition potential of γδ T cells. Finally, γδ T cells in livestock are potent producers of critical master regulator cytokines such as interferon (IFN)-γ and interleukin (IL)-17, whose production orchestrates downstream cytokine and chemokine production by other cells, thereby shaping the immune response as a whole. Our knowledge of the biology, receptor expression and response to infectious diseases by swine γδ T cells is reviewed here.

Assessment of Scavenger Receptor Cysteine-Rich Domain Binding to Bacteria.

The scavenger receptor cysteine-rich SRCR domain is an ancient protein domain found in SR-A and SR-I scavenger receptors, which is characterized by a conserved arrangement of cysteines (Martinez et al., Pharmacol Rev 63(4):967-1000, 2011; Sarrias et al., Crit Rev Immunol 24(1):1-37, 2004; Telfer and Baldwin, Cell Immunol 296(1):76-86, 2015; PrabhuDas et al., J Immunol, 2017. 198(10):3775-3789). SRCR domains are divided into group A and group B SRCR domains by virtue of how many cysteines they contain and the resulting disulfide bonding pattern. Group B SRCR domains, found in WC1, CD163, CD5, CD6, Spα and DMBT1, are approximately 100-110 amino acids long and contain 6-8 cysteines predicted to form 3-4 disulfide bonds. The crystal structure of a CD5 group B SRCR domain predicts a fold of two beta-sheets and an alpha helix (Rodamilans et al., J Biol Chem 282(17):12669-12677, 2007; Wang et al., Mol Immunol 48:801-809, 2011). SRCR domains bind to many different types of chemical compounds found on cells, viruses, and microbes and are usually found in multiples in the extracellular domains of transmembrane proteins or in secreted proteins. Small amino acid differences between these SRCR domains lead to significant differences in binding affinity. In addition, SRCR domain genes contain allelic polymorphisms and can be extensively duplicated. Thus, single and duplicated SRCR domain protein gene loci encode a large tunable binding potential. Binding to pathogen-associated molecular patterns (PAMPs) combined with signaling potential predicts an important role for these molecules in the immune response. WC1 SRCR domains bind to the spirochetes Leptospira and Borrelia (Hsu et al., J Immunol 194(5):2280-2288, 2015). CD6 (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007), Spα (Sarrias et al., J Biol Chem 280(42):35391-35398, 2005), CD163A (Fabriek et al., Blood 113(4):887-892, 2009) and DMBT1 (Madsen et al., Eur J Immunol 33(8):2327-2336, 2003) bind to Gram-positive and Gram-negative bacteria; CD5 binds to yeast (Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009). Identified ligands include lipoteichoic acid, lipopolysaccharide, poly-phosphorylated, and -sulfated compounds such as dextran sulfate sodium, leucine-rich repeat proteins, and fungal mannose (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007; Sarrias et al., J Biol Chem 280(42):35391-35398, 2005; Fabriek et al., Blood 113(4):887-892, 2009; Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009; End et al., Eur J Immunol 39(3):833-842, 2009; Loimaranta et al., J Biol Chem 284(28):18614-18623, 2009). A conserved linear binding motif (VEVLXXXXW) in an external loop in the SRCR domain has been identified in CD163A and DMBT1 and can be used as a peptide that aggregates bacteria (Fabriek et al., Blood 113(4):887-892, 2009; Bikker et al., J Biol Chem 279(46):47699-47703, 2004; Leito et al., Biol Chem 389(9):1193-1200, 2008). In contrast, WC1 binding to bacteria is mediated by a noncontinuous motif in the native protein, and mutation of the VEVLXXXXW motif has no effect upon bacterial binding (Hsu et al., J Immunol 194(5):2280-2288, 2015). Thus, bacterial binding studies with WC1 SRCR domains must be done with native, correctly disulfide bonded, protein, ideally posttranslationally modified in mammalian cells.WC1 is found in the genomes of most mammals, reptiles, and birds and is expressed exclusively on γδ T cells in ruminants. The 13 bovine WC1 genes encode up to 11 extracellular SRCR domains, organized in the SRCR domain pattern of a1-[b2-c3-d4-e5-d6]-[b7-c8-d9-e10-d'11], where the alphabet designations indicate homology between genes and across species (Chen et al., BMC Genet 13:86, 2012; Herzig et al., BMC Evol Biol 10:181, 2010; Herzig and Baldwin, BMC Genomics 10:191, 2009). Some of the signaling co-receptor WC1 molecules are required for the γδ T cell response to Leptospira (Wang et al., Mol Immunol 48:801-809, 2011; Rogers et al., J Immunol 174(6):3386-3393, 2005; Wang et al., Eur J Immunol 39(1):254-266, 2009). The WC1 expressed on responsive γδ T cells is correlated with its direct binding to Leptospira via some of its SRCR domains (Hsu et al., J Immunol 194(5):2280-2288, 2015). Because WC1+ γδ T cells share a restriction in their γδ TCRs and WC1 has TCR co-receptor activity, we hypothesize that WC1 co-ligation with the TCR plays the determining role in the activation of WC1+ γδ T cells by pathogens. Classification of the binding of WC1 SRCR domains, their ligands, and their role in the interaction of 𝛾δ T cells with pathogens relevant to the host will allow these cells to be recruited in next-generation vaccines to pathogens that have significant negative economic and health impact.

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.

Durable antibody and effector memory T cell responses in breastmilk from women with SARS-CoV-2.

Background: Given that only 25% of pregnant women elect to receive a COVID-19 vaccine, maternal SARS-CoV-2 infection remains an important route of conferring protective passive immunity to breastfed infants of mothers who are not vaccinated. Methods: We enrolled 30 lactating participants between December 2020 and March 2021 who had a positive PCR-test and their first COVID-19 symptoms within the previous 21 days. Participants were asked to provide serial bilateral milk samples at 12 timepoints (~ every 3 days) over a period of 35 days. A second set of samples was collected at least four months after the beginning of the first set. Participants also were asked to provide their dried blood spots and infant stool samples. All samples were tested for receptor-binding domain (RBD)-specific immunoglobulin (Ig)A, IgG, and IgM. Milk samples were assessed for neutralizing ability against the spike protein and four SARS-CoV-2 variants: D614G, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Permeability of the breast epithelium was assessed by measuring the sodium to potassium ions (Na:K) in milk. Using flow cytometry, memory CD4 and CD8 T cells (CD45RO+ and CCR7+/-) and mucosal-homing CD4 and CD8 T cells (CD103+) were determined in cells from milk expressed at 35 days and at least 4 months after their first milk donation. Results: Milk antibodies from SARS-CoV-2 positive participants neutralized the spike complex. Milk from 73, 90, and 53% of participants had binding reactivities to RBD-specific IgA, IgG, and IgM, respectively. In contrast to blood spots, which showed increased levels of IgG, but not IgA or IgM, the COVID-19 response in milk was associated with a robust IgA response. Twenty-seven percent of participants had increased breast-epithelium permeability, as indicated by Na:K ≥ 0.6. The percentage of CD45RO+CCR7- effector-memory T cells in the day ≥120 milk samples was significantly higher than day 35 samples (P< 0.05). Conclusions: Antibodies in milk from participants with recent SARS-CoV-2 infection and those who recovered can neutralize the spike complex. For the first time we show that breastmilk T cells are enriched for mucosal memory T cells, further emphasizing the passive protection against SARS-CoV-2 conferred to infants via breastmilk.

De novo designed protein transduction domain mimics from simple synthetic polymers.

Protein transduction domains (PTDs) that readily transverse cellular membranes are of great interest and are attractive tools for the intracellular delivery of bioactive molecules. Learning to program synthetic polymers and oligomers with the appropriate chemical information to capture adequately the biological activity of proteins is critical to our improved understanding of how these natural molecules work. In addition, the versatility of these synthetic mimics provides the opportunity to discover analogs with superior properties compared with their native sequences. Here we report the first detailed structure-activity relationship of a new PTD family of polymers based on a completely abiotic backbone. The synthetic approach easily allows doubling the density of guanidine functional groups, which increases the transduction efficiency of the sequences. Cellular uptake studies on three different cell lines (HEK 293T, CHO, and Jurkat T cells) confirm that these synthetic analogs are highly efficient novel protein transduction domain mimics (PTDMs), which are more effective than TAT(49-57) and nonaarginine (R9) and also highlight the usefulness of polymer chemistry at the chemistry-biology interface.

Gamma Delta TCR and the WC1 Co-Receptor Interactions in Response to Leptospira Using Imaging Flow Cytometry and STORM.

The WC1 cell surface family of molecules function as hybrid gamma delta (γδ) TCR co-receptors, augmenting cellular responses when cross-linked with the TCR, and as pattern recognition receptors, binding pathogens. It is known that following activation, key tyrosines are phosphorylated in the intracytoplasmic domains of WC1 molecules and that the cells fail to respond when WC1 is knocked down or, as shown here, when physically separated from the TCR. Based on these results we hypothesized that the colocalization of WC1 and TCR will occur following cellular activation thereby allowing signaling to ensue. We evaluated the spatio-temporal dynamics of their interaction using imaging flow cytometry and stochastic optical reconstruction microscopy. We found that in quiescent γδ T cells both WC1 and TCR existed in separate and spatially stable protein domains (protein islands) but after activation using Leptospira, our model system, that they concatenated. The association between WC1 and TCR was close enough for fluorescence resonance energy transfer. Prior to concatenating with the WC1 co-receptor, γδ T cells had clustering of TCR-CD3 complexes and exclusion of CD45. γδ T cells may individually express more than one variant of the WC1 family of molecules and we found that individual WC1 variants are clustered in separate protein islands in quiescent cells. However, the islands containing different variants merged following cell activation and before merging with the TCR islands. While WC1 was previously shown to bind Leptospira in solution, here we showed that Leptospira bound WC1 proteins on the surface of γδ T cells and that this could be blocked by anti-WC1 antibodies. In conclusion, γδ TCR, WC1 and Leptospira interact directly on the γδ T cell surface, further supporting the role of WC1 in γδ T cell pathogen recognition and cellular activation.

Subpopulations of swine γδ T cells defined by TCRγ and WC1 gene expression.

γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2+/SWC5-, CD2-/SWC5+, and CD2-/SWC5-). Both CD2- subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2+ cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2+ and CD2- γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors.