Regulation of the BCR signalosome by the class II peptide editor, H2-M, affects the development and repertoire of innate-like B cells.

The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.

Generating Discretionary Income in an Academic Department of Pathology.

The 2021 Association of Pathology Chairs Annual Meeting included a chairs' session and a premeeting discussion-group webinar sponsored by the Senior Fellows Group (former chairs of academic departments of pathology who have remained active in the Association of Pathology Chairs) focused on generating discretionary income for departments. Discretionary income was defined as revenue that can be used by the department with few, if any, restrictions. Such income is particularly desirable given limitations on departmental budgets. Four discussion-group panelists presented the funds-flow model in their respective institutions and how they derived and used discretionary income. Discretionary income was obtained from both external sources (eg, philanthropy, indirect cost recovery, partnerships with outside entities, medical education courses, research laboratory agreements, clinical trials) and internal sources (eg, core facilities, institutional programmatic support, institutional incentive programs). Significant departmental variations were associated with differences in institutional financial structure and policies, revenue-generating capabilities of the department and individual faculty, practice plan policies, donor intentions, and geographic market forces. Most finances were dependent upon a robust funds-flow model. Uses of discretionary funds included salary support, recruitment expenses (including start-up packages), research equipment, space renovation, social events, support of academic programs, and travel. Panelists also discussed particular challenges of discretionary-fund generation and use during the coronavirus disease 2019 pandemic. Notably, each institution had its own unique methodology for generating discretionary income, and no obvious standard approach was identified. The 2 moderators emphasized the importance of identifying and understanding opportunities, issues, and institutional culture surrounding generation and use of discretionary funds.

Targeting transcriptional coregulator OCA-B/Pou2af1 blocks activated autoreactive T cells in the pancreas and type 1 diabetes.

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.

HLA-DM catalytically enhances peptide dissociation by sensing peptide-MHC class II interactions throughout the peptide-binding cleft.

Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.

Elimination of Osteosarcoma by Necroptosis with Graphene Oxide-Associated Anti-HER2 Antibodies.

The prognosis for non-resectable or recurrent osteosarcoma (OS) remains poor. The finding that the majority of OS overexpress the protooncogene HER2 raises the possibility of using HER2 as a therapeutic target. However, clinical trials on the anti-HER2 antibody trastuzumab (TRA) in treating OS find no therapeutic benefit. HER2 overexpression in OS is not generally associated with gene amplification, with low-level expression regarded as HER2 "negative", as per criteria used to classify breast cancer HER2 status. Nevertheless, active HER2-targeting approaches, such as virus-based HER2 vaccines or CAR-T cells have generated promising results. More recently, it has been found that the noncovalent association of TRA with nanomaterial graphene oxide (GO) generates stable TRA/GO complexes capable of rapidly killing OS cells. TRA/GO induces oxidative stress and strong HER2 signaling to elicit immediate degradation of both cIAP (cellular inhibitor of apoptosis protein) and caspase 8, leading to activation of necroptosis. This is an attractive mechanism of cancer cell death as chemo/apoptosis-resistant tumors may remain susceptible to necroptosis. In addition, necroptosis is potentially immunogenic to promote tumor immunity, as opposed to apoptosis that tends to silence tumor immunity. Currently, no established anticancer therapeutics are known to eliminate cancers by necroptosis. The aim of this article is to review the rationale and mechanisms of TRA/GO-mediated cytotoxicity.

Peptidomic analysis of type 1 diabetes associated HLA-DQ molecules and the impact of HLA-DM on peptide repertoire editing.

HLA-DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII-bound peptides in human embryonic kidney 293T cells expressing HLA-DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM-resistant, DM-dependent, or DM-sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ-peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T-cell epitopes to DM editing.

Association of rituximab with graphene oxide confers direct cytotoxicity for CD20-positive lymphoma cells.

Non-Hodgkin lymphoma (NHL) is one of the most common hematologic malignancies among adults for which the chimeric monoclonal anti-CD20 antibody (Ab) rituximab (RTX) is used as first-line therapy. As RTX itself is not directly cytotoxic but relies on host immune effector mechanisms or chemotherapeutic agents to attack target cells, its therapeutic capacity may become limited when host effector mechanisms are compromised. Currently, refractory disease and relapse with NHL are still common, highlighting the need for novel anti-CD20 antibody strategies with superior therapeutic efficacy over current protocols. We hypothesized that making RTX directly cytotoxic might improve the therapeutic efficacy. Graphene oxide (GO) has recently emerged as a highly attractive nanomaterial for biomedical applications; and several studies have reported cytotoxic effect of GO on benign and malignant cells in vitro. Herein, we report that RTX can be stably associated with GO, and that GO-associated RTX (RTX/GO) demonstrates remarkably high avidity for CD20. Binding of GO-associated RTX to CD20-positive lymphoma cells induces CD20 capping and target cell death through an actin dependent mechanism. In vivo, GO-associated RTX, but not free RTX, quickly eliminates high-grade lymphomas in the absence of host effector mechanisms in a xenograft lymphoma mouse model. Our findings represent the first demonstration of using GO-associated antibody as effective cytotoxic therapy for human B cell malignancies in the absence of chemotherapy, and these findings could have important clinical implications.

Type 1 diabetes associated HLA-DQ2 and DQ8 molecules are relatively resistant to HLA-DM mediated release of invariant chain-derived CLIP peptides.

HLA-DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4(+) T cells. Individuals expressing HLA-DQ2 or DQ8, and DQ2/8 trans-dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM-mediated editing of CLIP was further confirmed by HPLC-MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D-associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D-susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2-CLIP complexes are highly resistant to DM editing, whereas DQ8-CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM-binding region. Our findings show that T1D-susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D-susceptible DQ molecules to DM editing and preferential presentation of T1D-associated autoantigenic peptides may contribute to the pathogenesis of T1D.

MHC variation sculpts individualized microbial communities that control susceptibility to enteric infection.

The presentation of protein antigens on the cell surface by major histocompatibility complex (MHC) molecules coordinates vertebrate adaptive immune responses, thereby mediating susceptibility to a variety of autoimmune and infectious diseases. The composition of symbiotic microbial communities (the microbiota) is influenced by host immunity and can have a profound impact on host physiology. Here we use an MHC congenic mouse model to test the hypothesis that genetic variation at MHC genes among individuals mediates susceptibility to disease by controlling microbiota composition. We find that MHC genotype significantly influences antibody responses against commensals in the gut, and that these responses are correlated with the establishment of unique microbial communities. Transplantation experiments in germfree mice indicate that MHC-mediated differences in microbiota composition are sufficient to explain susceptibility to enteric infection. Our findings indicate that MHC polymorphisms contribute to defining an individual's unique microbial fingerprint that influences health.

Expression of the mouse MHC class Ib H2-T11 gene product, a paralog of H2-T23 (Qa-1) with shared peptide-binding specificity.

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP(+) and TAP(-) cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1-restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function.

Biological function of HLA-DO (H2-O).

HLA-DO (H2-O) is a highly conserved nonpolymorphic major histocompatibility complex class II (MHCII) like molecule expressed in B lymphocytes, dendritic cells, and thymic epithelial cells. The biological function of DO has been elusive. Recent studies using site-directed mutagenesis, crystallography, and enzyme kinetics demonstrate that DO functions strictly as an inhibitor rather than modifier of DM function. DO stably binds to DM at the catalytic site to block DM interaction with MHCII. While the new data establish the molecular mechanism of DO function, the reason that professional antigen-presenting cells (APCs) express DO to generate DO-DM complexes that are functionally inactive remains unclear. Despite the finding that DO inhibits DM, antigen presentation by H2-O-/- APCs is inefficient compared to wild-type (WT) APCs, and H2-O-/- mice are partially immunodeficient and spontaneously develop auto-antibodies to nuclear antigens. The results of functional studies raise the question of how an inhibitor of DM enhances antigen presentation and promotes immunity. In this review, we analyze the related findings from previous and recent studies. The integration of the all of the data allows us to propose a model explaining how DO enhances antigen presentation by inhibiting DM function.

Structural Characteristics of HLA-DQ that May Impact DM Editing and Susceptibility to Type-1 Diabetes.

Autoreactive CD4+ T cells initiate the chronic autoimmune disease Type-1 diabetes (T1D), in which multiple environmental and genetic factors are involved. The association of HLA, especially the DR-DQ loci, with risk for T1D is well documented. However, the molecular mechanisms are poorly understood. In this review, we explore the structural characteristics of HLA-DQ and the role of HLA-DM function as they may contribute to an understanding of autoreactive T cell development in T1D.

Immunodeficiency and autoimmunity in H2-O-deficient mice.

HLA-DO/H2-O is a highly conserved, nonpolymorphic MHC class II-like molecule expressed in association with H2-M in thymic epithelial cells, B lymphocytes, and primary dendritic cells. The physiological function of DO remains unknown. The finding of cell maturation-dependent DO expression in B lymphocytes and dendritic cells suggests the possibility that H2-O functions to promote the presentation of exogenous Ag by attenuating presentation of endogenous self-peptides. In the current study, we report that H2-O(-/-) mice spontaneously develop high titers of IgG2a/c antinuclear Abs (ANAs) with specificity for dsDNA, ssDNA, and histones. Reconstitution of RAG1(-)(/)(-) mice with T and B cells from H2-O(-)(/)(-) or wild-type mice demonstrated that production of ANAs requires participation of CD4(+) T cells from H2-O(-)(/)(-) mice. Bone marrow chimeras demonstrated that loss of H2-O expression in thymic epithelial cells did not induce ANAs, and that lack of H2-O expression in bone marrow-derived cells was sufficient to induce the autoimmune phenotype. Despite production of high titers of autoantibodies, H2-O(-/-) mice exhibit a delayed generation of humoral immunity to model Ags (OVA and keyhole limpet hemocyanin), affecting all major T-dependent Ig classes, including IgG2a/c. Ag presentation experiments demonstrated that presentation of exogenous Ag by H2-O(-/-) APC was inefficient as compared with wild-type APC. Thus, H2-O promotes immunity toward exogenous Ags while inhibiting autoimmunity. We suggest that H2-O, through spatially or temporally inhibiting H2-M, may enhance presentation of exogenous Ag by limiting newly generated MHC class II molecules from forming stable complexes with endogenous self-peptides.

Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity.

Foxp3 plays an essential role in conferring suppressive functionality to CD4(+)/Foxp3(+) regulatory T cells (Tregs). Although studies showed that Foxp3 has to form cooperative complexes with NFAT to bind to target genes, it remains unclear whether NFAT is available in the nucleus of primary Tregs for Foxp3 access. It is generally believed that NFAT in resting cells resides in the cytoplasm, and its nuclear translocation depends on calcineurin (CN) activation. We report that a fraction of NFAT protein constitutively localizes in the nucleus of primary Tregs, where it selectively binds to Foxp3 target genes. Treating Tregs with CN inhibitor does not induce export of NFAT from the nucleus, indicating that its nuclear translocation is independent of CN activity. Consistently, Tregs are resistant to CN inhibitors in the presence of IL-2 and continue to proliferate in response to anti-CD3 stimulation, whereas proliferation of non-Tregs is abrogated by CN inhibitors. In addition, PMA, which activates other transcription factors required for T cell activation but not NFAT, selectively induces Treg proliferation in the absence of ionomycin. TCR interaction with self-MHC class II is not required for PMA-induced Treg proliferation. Tregs expanded by PMA or in the presence of CN inhibitors maintain Treg phenotype and functionality. These findings shed light on Treg biology, paving the way for strategies to selectively activate Tregs.

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus.

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)ß(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of ß(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.

Utility of characteristic QTOF MS/MS fragmentation for MHC class I peptides.

Systematic investigation of cellular process by mass spectrometric detection of peptides obtained from proteins digestion or directly from immuno-purification can be a powerful tool when used appropriately. The true sequence of these peptides is defined by the interpretation of spectral data using a variety of available algorithms. However peptide match algorithm scoring is typically based on some, but not all, of the mechanisms of peptide fragmentation. Although algorithm rules for soft ionization techniques generally fit very well to tryptic peptides, manual validation of spectra is often required for endogenous peptides such as MHC class I molecules where traditional trypsin digest techniques are not used. This study summarizes data mining and manual validation of hundreds of peptide sequences from MHC class I molecules in publically available data files. We herein describe several important features to improve and quantify manual validation for these endogenous peptides--post automated algorithm searching. Important fragmentation patterns are discussed for the studied MHC Class I peptides. These findings lead to practical rules that are helpful when performing manual validation. Furthermore, these observations may be useful to improve current peptide search algorithms or development of novel software tools.

Cutting edge: HLA-DM functions through a mechanism that does not require specific conserved hydrogen bonds in class II MHC-peptide complexes.

HLA-DM catalyzes peptide dissociation and exchange in class II MHC molecules through a mechanism that has been proposed to involve the disruption of specific components of the conserved hydrogen bond network in MHC-peptide complexes. HLA-DR1 molecules with alanine substitutions at each of the six conserved H- bonding positions were expressed in cells, and susceptibility to DM catalytic activity was evaluated by measuring the release of CLIP. The mutants alphaN62A, alphaN69A, alphaR76A, and betaH81A DR1 were fully susceptible to DM-mediated CLIP release, and betaN82A resulted in spontaneous release of CLIP. Using recombinant soluble DR1 molecules, the amino acid betaN82 was observed to contribute disproportionately in stabilizing peptide complexes. Remarkably, the catalytic potency of DM with each beta-chain mutant was equal to or greater than that observed with wild-type DR1. Our results support the conclusion that no individual component of the conserved hydrogen bond network plays an essential role in the DM catalytic mechanism.

Direct expansion of human allospecific FoxP3+CD4+ regulatory T cells with allogeneic B cells for therapeutic application.

Compelling evidence from animal studies has demonstrated that allospecific FoxP3(+)CD4(+) regulatory T (Treg) cells expanded ex vivo can be used as effective therapeutic tools in the treatment of allograft rejection and graft-vs-host disease. Despite the promising results from animal studies, there remain major barriers to developing Treg cell-based immunotherapy in humans. Currently, no effective approach has been established for selective expansion of human allospecific Treg cells ex vivo. Additionally, the very low frequency of Treg cells present in human peripheral blood could pose a formidable challenge to obtaining a sufficient number of Treg cells from a single donor for ex vivo expansion for therapeutic utilization. Extending our recent finding that mouse B cells preferentially induce expansion of alloreactive Treg cells, we report herein that human Treg cells can be expanded ex vivo with allogeneic B cells. The expanded Treg cells express very high levels of FoxP3, maintain anergic phenotype, and are potent suppressors capable of inhibiting the alloproliferation of third-party responder T cells at very low Treg-to-T effector cell ratio in an alloantigen-specific manner. The alloantigen specificity demonstrated by B cell-expanded Treg cells is not determined by the HLA haplotypes of the Treg cells, but it is induced and determined by the haplotype of the B cells used to expand them. Our findings represent a significant advance in the development of Treg cell-based immunotherapy in humans and raise the possibility of using third-party Treg cells for therapeutic applications.

Identification of naturally processed ligands in the C57BL/6 mouse using large-scale mass spectrometric peptide sequencing and bioinformatics prediction.

Most major histocompatibility complex (MHC) class I-peptide-binding motifs are currently defined on the basis of quantitative in vitro MHC-peptide-binding assays. This information is used to develop bioinformatics-based tools to predict the binding of peptides to MHC class I molecules. To date few studies have analyzed the performance of these bioinformatics tools to predict the binding of peptides determined by sequencing of naturally processed peptides eluted directly from MHC class I molecules. In this study, we performed large-scale sequencing of endogenous peptides eluted from H2K(b) and H2D(b) molecules expressed in spleens of C57BL/6 mice. Using sequence data from 281 peptides, we identified novel preferred anchor residues located in H2K(b) and H2D(b)-associated peptides that refine our knowledge of these H2 class I peptide-binding motifs. The analysis comparing the performance of three bioinformatics methods to predict the binding of these peptides, including artificial neural network, stabilized matrix method, and average relative binding, revealed that 61% to 94% of peptides eluted from H2K(b) and H2D(b) molecules were correctly classified as binders by the three algorithms. These results suggest that bioinformatics tools are reliable and efficient methods for binding prediction of naturally processed MHC class I ligands.