Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

Much of the research on the humoral response to allografts has focused on circulating serum antibodies and the long-lived plasma cells that produce these antibodies. In contrast, the interrogation of the quiescent memory B cell compartment is technically more challenging and thus has not been incorporated into the clinical diagnostic or prognostic toolkit. In this review, we discuss new technologies that have allowed this heretofore enigmatic subset of B cells to be identified at quiescence and during a recall response. These technologies in experimental models are providing new insights into memory B cell heterogeneity with respect to their phenotype, cellular function, and the antibodies they produce. Similar technologies are also allowing for the identification of comparable memory alloreactive B cells in transplant recipients. Although much of the focus in transplant immunology has been on controlling the alloreactive B cell population, long-term transplant patient survival is also critically dependent on protection by pathogen-specific memory B cells. Techniques are available that allow the interrogation of memory B cell response to pathogen re-encounter. Thus, we are poised in our ability to investigate how immunosuppression affects allospecific and pathogen-specific memory B cells, and reason that these investigations can yield new insights that will be beneficial for graft and patient survival.

Short peptides are poorly immunogenic when delivered sublingually - under the tongue. Nanomaterial delivery of peptides could be utilized to improve immunogenicity towards designed sublingual vaccines, but nanomaterials have not been widely successful in sublingual vaccines owing to the challenges of transport through the sublingual mucosa. Here, we report that the sublingual immunogenicity of peptides is negligible, even in the presence of sublingual adjuvants or when PEGylated, but can be dramatically enhanced by assembly into supramolecular polymer-peptide nanofibers bearing low-molecular weight PEG, optimally between 2000 and 3000 Da. Neither PEGylation nor a sublingual adjuvant were capable of rendering peptides immunogenic without assembly into nanofibers. We found that PEG decreased nanofiber interactions with mucin and promoted longer residence time at the sublingual immunization site. Parallel investigations with shortened nanofibers indicated that the size of the assemblies had a surprisingly negligible influence over sublingual immunogenicity. In mice, optimized formulations were capable of raising strong and highly durable systemic antibody responses, antibodies in the upper respiratory and reproductive tracts, and systemic antigen-specific T-cell responses. These nanofiber-based sublingual vaccines were effective with both protein and nucleotide adjuvants and raised responses against both a model peptide epitope and a peptide epitope from M. tuberculosis. Further, PASylation (modification of nanofibers with peptide sequences rich in Pro, Ala, and Ser) could be substituted for PEGylation to also achieve sublingual immunogenicity. These findings indicated that surface properties supersede nanomaterial size in modulating sublingual nanomaterial immunogenicity, having important implications for the design of synthetic sublingual vaccines.

Oral antigen exposure is a powerful, non-invasive route to induce immune tolerance to dietary antigens, and has been modestly successful at prolonging graft survival in rodent models of transplantation. To harness the mechanisms of oral tolerance for promoting long-term graft acceptance, we developed a mouse model where the antigen ovalbumin (OVA) was introduced orally prior to transplantation with skin grafts expressing OVA. Oral OVA treatment pre-transplantation promoted permanent graft acceptance and linked tolerance to skin grafts expressing OVA fused to the additional antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party allografts were spontaneously accepted. Oral OVA treatment promoted an anergic phenotype in OVA-reactive CD4+ and CD8+ conventional T cells (Tconvs) and expanded OVA-reactive Tregs pre-transplantation. However, skin graft acceptance following oral OVA resisted partial depletion of Tregs and blockade of PD-L1. Mechanistically, we revealed a role for the proximal gut draining lymph nodes (gdLNs) in mediating this effect, as an intestinal infection that drains to the proximal gdLNs prevented tolerance induction. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance.

B cells are unique antigen-presenting cells because their antigen presentation machinery is closely tied to the B cell receptor. Autoreactive thymic B cells can efficiently present cognate self-antigens to mediate CD4+ T cell-negative selection. However, the nature of thymocyte-thymic B cell interaction and how this interaction affects the selection of thymic B cell repertoire and, in turn, the T cell repertoire are not well understood. Here we demonstrate that a large percentage of thymic B cells have undergone class switching intrathymically. Thymic B cell class switching requires cognate interaction with specific T cells. Class-switched thymic B cells have a distinct repertoire compared with unswitched thymic B cells or splenic B cells. Particularly, autoreactive B cell specificities preferentially expand in the thymus by undergoing class switching, and these enriched, class-switched autoreactive thymic B cells play an important role in CD4 T cell tolerance.

Humoral alloreactivity has been recognized as a common cause of kidney transplant dysfunction. B-cell activation, differentiation, and antibody production are dependent on IL-21+CXCR5+follicular T-helper (Tfh) cells. Here, we studied whether belatacept, an inhibitor of the costimulatory CD28-CD80/86-pathway, interrupts the crosstalk between Tfh- and B-cells more efficiently than the calcineurin inhibitor tacrolimus. The suppressive effects of belatacept and tacrolimus on donor antigen-driven Tfh-B-cell interaction were functionally studied in peripheral blood mononuclear cells from 40 kidney transplant patients randomized to a belatacept- or tacrolimus-based immunosuppressive regimen. No significant differences in uncultured cells or donor antigen-stimulated cells were found between belatacept- and tacrolimus-treated patients in the CXCR5+Tfh cell generation and activation (upregulation of PD-1). Belatacept and tacrolimus in vitro minimally inhibited Tfh-cell generation (by ~6-7%) and partially prevented Tfh-cell activation (by ~30-50%). The proportion of IL-21+-activated Tfh-cells was partially decreased by in vitro addition of belatacept or tacrolimus (by ~60%). Baseline expressions and proportions of activated CD86+ B-cells, plasmablasts, and transitional B-cells after donor antigen stimulation did not differ between belatacept- and tacrolimus-treated patients. Donor antigen-driven CD86 upregulation on memory B-cells was not fully prevented by adding belatacept in vitro (~35%), even in supratherapeutic doses. In contrast to tacrolimus, belatacept failed to inhibit donor antigen-driven plasmablast formation (~50% inhibition vs. no inhibition, respectively, p < 0.0001). In summary, donor antigen-driven Tfh-B-cell crosstalk is similar in cells obtained from belatacept- and tacrolimus-treated patients. Belatacept is, however, less potent in vitro than tacrolimus in inhibiting Tfh-cell-dependent plasmablast formation.

Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets.

Recurrent Staphylococcus aureus infections are common, despite robust immune responses. S. aureus infection elicited protective antibody and T cell responses in mice that expressed the Major Histocompatibility Complex (MHC) of the H-2d haplotype, but not H-2b, demonstrating that host genetics drives individual variability. Vaccination with a-toxin or leukotoxin E (LukE) elicited similar antibody and T cell responses in mice expressing H-2d or H-2b, but vaccine-elicited responses were inhibited by concomitant infection in H-2d-expressing mice. These findings suggested that competitive binding of microbial peptides to host MHC proteins determines the specificity of the immunodominant response, which was confirmed using LukE-derived peptide-MHC tetramers. A vaccine that elicited T cell and antibody responses protected mice that expressed H-2d or H-2b, demonstrating that vaccination can overcome MHC-restricted immunodominance. Together, these results define how host genetics determine whether immunity elicted by S. aureus is protective and provide a mechanistic roadmap for future vaccine design.

Intrarenal B cells in human renal allografts indicate transplant recipients with a poor prognosis, but how these cells contribute to rejection is unclear. Here we show using single-cell RNA sequencing that intrarenal class-switched B cells have an innate cell transcriptional state resembling mouse peritoneal B1 or B-innate (Bin) cells. Antibodies generated by Bin cells do not bind donor-specific antigens nor are they enriched for reactivity to ubiquitously expressed self-antigens. Rather, Bin cells frequently express antibodies reactive with either renal-specific or inflammation-associated antigens. Furthermore, local antigens can drive Bin cell proliferation and differentiation into plasma cells expressing self-reactive antibodies. These data show a mechanism of human inflammation in which a breach in organ-restricted tolerance by infiltrating innate-like B cells drives local tissue destruction.

Despite numerous advances in the definition of a role for regulatory T cells (Tregs) in facilitating experimental transplantation tolerance, and ongoing clinical trials for Treg-based therapies, critical issues related to the optimum dosage, antigen-specificity, and Treg-friendly adjunct immunosuppressants remain incompletely resolved. In this study, we used a tractable approach of MHC tetramers and flow cytometry to define the fate of conventional (Tconvs) and Tregs CD4+ T cells that recognize donor 2W antigens presented by I-Ab on donor and recipient antigen-presenting cells (APCs) in a mouse cardiac allograft transplant model. Our study shows that these endogenous, donor-reactive Tregs comparably accumulate in the spleens of recipients undergoing acute rejection or exhibiting costimulation blockade-induced tolerance. Importantly, this expansion was not detected when analyzing bulk splenic Tregs. Systemically, the distinguishing feature between tolerance and rejection was the inhibition of donor-reactive conventional T cell (Tconv) expansion in tolerance, translating into increased percentages of splenic FoxP3+ Tregs within the 2W:I-Ab CD4+ T cell subset compared to rejection (~35 vs. <5% in tolerance vs. rejection). We further observed that continuous administration of rapamycin, cyclosporine A, or CTLA4-Ig did not facilitate donor-specific Treg expansion, while all three drugs inhibited Tconv expansion. Finally, donor-specific Tregs accumulated comparably in rejecting tolerant allografts, whereas tolerant grafts harbored <10% of the donor-specific Tconv numbers observed in rejecting allografts. Thus, ~80% of 2W:I-Ab CD4+ T cells in tolerant allografts expressed FoxP3+ compared to ≤10% in rejecting allografts. A similar, albeit lesser, enrichment was observed with bulk graft-infiltrating CD4+ cells, where ~30% were FoxP3+ in tolerant allografts, compared to ≤10% in rejecting allografts. Finally, we assessed that the phenotype of 2W:I-Ab Tregs and observed that the percentages of cells expressing neuropilin-1 and CD73 were significantly higher in tolerance compared to rejection, suggesting that these Tregs may be functionally distinct. Collectively, the analysis of donor-reactive, but not of bulk, Tconvs and Tregs reveal a systemic signature of tolerance that is stable and congruent with the signature within tolerant allografts. Our data also underscore the importance of limiting Tconv expansion for high donor-specific Tregs:Tconv ratios to be successfully attained in transplantation tolerance.

Solid organ transplantation is the preferred treatment for end-stage organ failure. Although transplant recipients take life-long immunosuppressive drugs, a substantial percentage of them still reject their allografts. Strikingly, barrier organs colonized with microbiota have significantly shorter half-lives than non-barrier transplanted organs, even in immunosuppressed hosts. We previously demonstrated that skin allografts monocolonized with the common human commensal Staphylococcus epidermidis (S.epi) are rejected faster than germ-free (GF) allografts in mice because the presence of S.epi augments the effector alloimmune response locally in the graft. Here, we tested whether host immune responses against graft-resident commensal microbes, including S.epi, can damage colonized grafts independently from the alloresponse. Naive hosts mounted an anticommensal T cell response to colonized, but not GF, syngeneic skin grafts. Whereas naive antigraft commensal T cells modestly damaged colonized syngeneic skin grafts, hosts with prior anticommensal T cell memory mounted a post-transplant immune response against graft-resident commensals that significantly damaged colonized, syngeneic skin grafts. Importantly, allograft recipients harboring this host-versus-commensal immune response resisted immunosuppression. The dual effects of host-versus-commensal and host-versus-allograft responses may partially explain why colonized organs have poorer outcomes than sterile organs in the clinic.

Monoclonal antibodies (abs) targeting the programmed cell death 1 (PD-1) immune checkpoint pathway have revolutionized tumor therapy. Because T-cell-directed PD-1 blockade boosts tumor immunity, anti-PD-1 abs have been developed for examining T-cell-PD-1 functions. More recently, PD-1 expression has also been reported directly on cancer cells of various etiology, including in melanoma. Nevertheless, there is a paucity of studies validating anti-PD-1 ab clone utility in specific assay types for characterizing tumor cell-intrinsic PD-1. Here, we demonstrate reactivity of several anti-murine PD-1 ab clones and recombinant PD-L1 with live B16-F10 melanoma cells and YUMM lines using multiple independent methodologies, positive and negative PD-1-specific controls, including PD-1-overexpressing and PD-1 knockout cells. Flow cytometric analyses with two separate anti-PD-1 ab clones, 29F.1A12 and RMP1-30, revealed PD-1 surface protein expression on live murine melanoma cells, which was corroborated by marked enrichment in PD-1 gene (Pdcd1) expression. Immunoblotting, immunoprecipitation, and mass spectrometric sequencing confirmed PD-1 protein expression by B16-F10 cells. Recombinant PD-L1 also recognized melanoma cell-expressed PD-1, the blockade of which by 29F.1A12 fully abrogated PD-1:PD-L1 binding. Together, our data provides multiple lines of evidence establishing PD-1 expression by live murine melanoma cells and validates ab clones and assay systems for tumor cell-directed PD-1 pathway investigations.

The current paradigm indicates that naive T cells are primed in secondary lymphoid organs. Here, we present evidence that intranasal administration of peptide antigens appended to nanofibers primes naive CD8+ T cells in the lung independently and prior to priming in the draining mediastinal lymph node (MLN). Notably, comparable accumulation and transcriptomic responses of CD8+ T cells in lung and MLN are observed in both Batf3KO and wild-type (WT) mice, indicating that, while cDC1 dendritic cells (DCs) are the major subset for cross-presentation, cDC2 DCs alone are capable of cross-priming CD8+ T cells both in the lung and draining MLN. Transcription analyses reveal distinct transcriptional responses in lung cDC1 and cDC2 to intranasal nanofiber immunization. However, both DC subsets acquire shared transcriptional responses upon migration into the lymph node, thus uncovering a stepwise activation process of cDC1 and cDC2 toward their ability to cross-prime effector and functional memory CD8+ T cell responses.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.

Biomaterials exhibiting precise ratios of different bioactive protein components are critical for applications ranging from vaccines to regenerative medicine, but their design is often hindered by limited choices and cross-reactivity of protein conjugation chemistries. Here, we describe a strategy for inducing multiple different expressed proteins of choice to assemble into nanofibres and gels with exceptional compositional control. The strategy employs 'βTail' tags, which allow for good protein expression in bacteriological cultures, yet can be induced to co-assemble into nanomaterials when mixed with additional β-sheet fibrillizing peptides. Multiple different βTail fusion proteins could be inserted into peptide nanofibres alone or in combination at predictable, smoothly gradated concentrations, providing a simple yet versatile route to install precise combinations of proteins into nanomaterials. The technology is illustrated by achieving precisely targeted hues using mixtures of fluorescent proteins, by creating nanofibres bearing enzymatic activity, and by adjusting antigenic dominance in vaccines.

In the past decade, appreciation of the important effects of commensal microbes on immunity has grown exponentially. The effect of the microbiota on transplantation has only recently begun to be explored; however, our understanding of the mechanistic details of host-microbe interactions is still lacking.

Pancreatic beta-cells proliferate following administration of the beta-cell toxin streptozotocin. Defining the conditions that promote beta-cell proliferation could benefit patients with diabetes. We have investigated the effect of insulin treatment on pancreatic beta-cell regeneration in streptozotocin-induced diabetic mice, and, in addition, report on a new approach to quantify beta-cell regeneration in vivo.

Recurrent Staphylococcus aureus infections are common, suggesting that immunity elicited by these infections is not protective. We previously reported that S. aureus skin infection (SSTI) elicited antibody-mediated immunity against secondary SSTI in BALB/c mice. In this study, we investigated the role of humoral immunity and the IgG-binding proteins Sbi and SpA in S. aureus SSTI. We found that B lymphocyte-deficient μMT mice were highly susceptible to infection, compared with congenic BALB/c mice. Importantly, transfer of immune serum protected μMT mice, demonstrating an appropriate response to protective antibody. We found that deletion of sbi, but not spa, impaired virulence, as assessed by skin lesion severity, and that Sbi-mediated virulence required B lymphocytes/antibody. Furthermore, neither Sbi nor SpA impaired the elicited antibody response or protection against secondary SSTI. Taken together, these findings highlight a B lymphocyte/antibody-dependent role of Sbi in the pathogenesis of S. aureus SSTI, and demonstrate that neither Sbi nor SpA interfered with elicited antibody-mediated immunity.

Limiting CD4+ T cell responses is important to prevent solid organ transplant rejection. In a mouse model of costimulation blockade-dependent cardiac allograft tolerance, we previously reported that alloreactive CD4+ conventional T cells (Tconvs) develop dysfunction, losing proliferative capacity. In parallel, induction of transplantation tolerance is dependent on the presence of regulatory T cells (Tregs). Whether susceptibility of CD4+ Tconvs to Treg suppression is modulated during tolerance induction is unknown. We found that alloreactive Tconvs from transplant tolerant mice had augmented sensitivity to Treg suppression when compared with memory T cells from rejector mice and expressed a transcriptional profile distinct from these memory T cells, including down-regulated expression of the transcription factor Special AT-rich sequence-binding protein 1 (Satb1). Mechanistically, Satb1 deficiency in CD4+ T cells limited their expression of CD25 and IL-2, and addition of Tregs, which express higher levels of CD25 than Satb1-deficient Tconvs and successfully competed for IL-2, resulted in greater suppression of Satb1-deficient than wild-type Tconvs in vitro. In vivo, Satb1-deficient Tconvs were more susceptible to Treg suppression, resulting in significantly prolonged skin allograft survival. Overall, our study reveals that transplantation tolerance is associated with Tconvs' susceptibility to Treg suppression, via modulated expression of Tconv-intrinsic Satb1. Targeting Satb1 in the context of Treg-sparing immunosuppressive therapies might be exploited to improve transplant outcomes.

The current paradigm that subunit vaccines require adjuvants to optimally activate innate immunity implies that increased vaccine reactogenicity will invariably be linked to improved immunogenicity. Countering this paradigm, nanoparticulate vaccines have been reported to act as delivery systems for vaccine antigens and induce immunity without the need for exogenous adjuvants or local inflammation; however, the mechanisms underlying the immunogenicity of nanoparticle vaccines are incompletely identified. Here, we show that antigens displayed on self-assembling nanofiber scaffolds and delivered intranasally are presented by CD103+ and CD11b+ lung dendritic cells that up-regulate CD80 and migrate into the draining lymph node (LN). This was accompanied by a nearly exclusive priming and accumulation of antigen-specific TH17 cells occurring independently in both LN and lung. Thus, self-assembling peptide nanofiber vaccines may represent a novel, needle- and adjuvant-free means of eliciting protective immunity against fungal and bacterial infections at skin and mucosal barrier surfaces.