Cell Major Histocompatibility Complex Research Articles

Development of a major histocompatibility complex class II conditional knockout mouse to study cell-specific and time-dependent adaptive immune responses in peripheral nerves.

The precise relationship between molecular mimicry and tissue-specific autoimmunity is unknown. Major histocompatibility complex (MHC) class II antigen presenting cell-CD4+ T-cell receptor complex interactions are necessary for adaptive immunity. This study aimed to determine the role of endoneurial endothelial cell MHC class II in autoimmune polyneuropathy. Cryopreserved Guillain-Barré syndrome (GBS) patient sural nerve biopsies and sciatic nerves from the severe murine experimental autoimmune neuritis (sm-EAN) GBS model were studied. Cultured conditional ready MHC Class II antigen A-alpha chain (H2-Aa) embryonic stem cells were used to generate H2-Aaflox/+ C57BL/6 mice. Mice were backcrossed and intercrossed to the SJL background to generate H2-Aaflox/flox SJL mice, bred with hemizygous Tamoxifen-inducible von Willebrand factor Cre recombinase (vWF-iCre/+) SJL mice to generate H2-Aaflox/flox; vWF-iCre/+ mice to study microvascular endothelial cell adaptive immune responses. Sm-EAN was induced in Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+, H2-Aaflox/flox; +/+, H2-Aa+/+; vWF-iCre/+ and untreated H2-Aaflox/flox; vWF-iCre/+ adult female SJL mice. Neurobehavioral, electrophysiological and histopathological assessments were performed at predefined time points. Endoneurial endothelial cell MHC class II expression was observed in normal and inflamed human and mouse peripheral nerves. Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+ mice were resistant to sm-EAN despite extensive MHC class II expression in lymphoid and non-lymphoid tissues. A conditional MHC class II knockout mouse to study cell- and time-dependent adaptive immune responses in vivo was developed. Initial studies show microvascular endothelial cell MHC class II expression is necessary for peripheral nerve specific autoimmunity, as advocated by human in vitro adaptive immunity and ex vivo transplant rejection studies.

Abstract C106: IMPACT restrains immuno-metabolic GCN1 signaling to govern pancreatic cancer metastasis

Abstract Introduction: Metastatic outgrowth and colonization requires that disseminated tumor cells simultaneously compensate for alterations in nutrient availability, counteract oxidative stress, and evade host innate and adaptive immune surveillance. The mechanistic underpinnings of how these vital processes are coordinated is not understood. Experimental procedures: Using intrasplenic injection (liver metastases) and tail vein injection (lung metastases), we employed a gain-of-function cDNA screen in mice to identify regulators of colonization. In doing so, we identified GCN1 signaling as indispensable to the disseminated pancreatic cancer cell. Summary of unpublished data: Specifically, we demonstrate that alterations in nutrient availability encountered in target organs (liver, lung) trigger pancreatic cancer cells to utilize GCN1 stress signaling to upregulate the expression of serine, folate, and methionine pathway biosynthetic enzymes together with amino acid transporters through the integrated stress response effector ATF4. These pathways act in concert to facilitate acquisition of metabolites critical for cellular functions including maintenance of redox homeostasis. Surprisingly, we found that GCN1 also functions in the nucleus, where it interacts with HNRNPK to destabilize the transcripts encoding natural killer (NK) cell activation ligands and key regulators of major histocompatibility complex (MHC) class I molecules. Intriguingly, we identified an endogenous protein rheostat, IMPACT, for GCN1’s dual functions and show that IMPACT expression is lost in human pancreatic metastases through DNA methylation. IMPACT overexpression in pancreatic cancer cell lines inhibited the integrated stress response effector ATF4 to retard nutrient uptake and activated the expression of NK cell ligands and MHC class I molecules to fuel anti-tumor immune responses. Conversely, IMPACT knockout enabled successful acquisition of metabolic intermediaries in response to nutrient alterations and suppressed anti-tumor immune response to accelerate metastatic outgrowth. Accordingly, bioinformatic analyses of human pancreatic cancer showed that while the expression of GCN1 increases in metastatic disease, the expression of IMPACT is lost, correlating with significantly shorter survival of GCN1-high, IMPACT-low expressing tumors. Finally, IMPACT overexpression synergized with immune checkpoint inhibitors in mice to eliminate macrometastases formation. Conclusion: In summary, we have identified IMPACT as an immunometabolic checkpoint that restrains GCN1-mediated metabolic plasticity and GCN1-mediated suppression of innate and adaptive immune surveillance. We propose that drugs that can restore IMPACT expression or IMPACT mimetic agents will have therapeutic value for patients with pancreatic cancer. Citation Format: Surajit Sinha, Abir Panda, Zeribe Nwosu, Rodrigo Neves Das, Xu Ke, Elke van Beek, Alexander J. Rossi, Reed I. Ayabe, James McDonald, Michael M. Wach, Samantha Ruff, Priyanka P. Desai, David Sun, Martha E. Teke, Emily A. Verbus, Areeba Saif, Shreya Gupta, Tahsin Khan, Leila Sarvestani, Carrie E. Ryan, Jacob Lambdin, Kirsten Remmert, Emily Smith, Kenneth Luberice, Stephie Lux, Imani A. Alexander, Tracey Pu, Allen Luna, Sarfraz R. Akmal, Shahyan Rehman, Ashley Rainey, Hanna Hong, Yuri Lin, Samantha Sevilla, Gasmi Billel, Sivasish Sindiri, Todd Prickett, King Chan, Eileen Li, Xiaolin Wu, Nicholas D. Klemen, Giorgio Trinchieri, Costas A. Lyssiotis, Jeremy Davis, Pankaj K. Singh, Steven A. Rosenberg, Michael B. Yaffe, Filippo Giancotti, Ethan M. Shevach, Jonathan M. Hernandez. IMPACT restrains immuno-metabolic GCN1 signaling to govern pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C106.

Development of innovative multi-epitope mRNA vaccine against Pseudomonas aeruginosa using in silico approaches.

The rising issue of antibiotic resistance has made treating Pseudomonas aeruginosa infections increasingly challenging. Therefore, vaccines have emerged as a viable alternative to antibiotics for preventing P. aeruginosa infections in susceptible individuals. With its superior accuracy, high efficiency in stimulating cellular and humoral immune responses, and low cost, mRNA vaccine technology is quickly replacing traditional methods. This study aimed to design a novel mRNA vaccine by using in silico approaches against P. aeruginosa. The research team identified five surface and antigenic proteins and selected their appropriate epitopes with immunoinformatic tools. These epitopes were then examined for toxicity, allergenicity and homology. The researchers also checked their presentation and identification by major histocompatibility complex cells and other immune cells through valuable tools like molecular docking. They subsequently modeled a multi-epitope protein and optimized it. The mRNA was analyzed in terms of structure and stability, after which the immune system's response against the new vaccine was simulated. The results indicated that the designed mRNA construct could be an effective and promising vaccine that requires laboratory and clinical trials.

Interleukin-22 suppresses major histocompatibility complex II in mucosal epithelial cells.

Major histocompatibility complex (MHC) II is dynamically expressed on mucosal epithelial cells and is induced in response to inflammation and parasitic infections, upon exposure to microbiota, and is increased in chronic inflammatory diseases. However, the regulation of epithelial cell-specific MHC II during homeostasis is yet to be explored. We discovered a novel role for IL-22 in suppressing epithelial cell MHC II partially via the regulation of endoplasmic reticulum (ER) stress, using animals lacking the interleukin-22-receptor (IL-22RA1), primary human and murine intestinal and respiratory organoids, and murine models of respiratory virus infection or with intestinal epithelial cell defects. IL-22 directly downregulated interferon-γ-induced MHC II on primary epithelial cells by modulating the expression of MHC II antigen A α (H2-Aα) and Class II transactivator (Ciita), a master regulator of MHC II gene expression. IL-22RA1-knockouts have significantly higher MHC II expression on mucosal epithelial cells. Thus, while IL-22-based therapeutics improve pathology in chronic disease, their use may increase susceptibility to viral infections.

Donor plasmacytoid dendritic cells limit graft-versus-host disease through vasoactive intestinal polypeptide expression

AbstractVasoactive intestinal polypeptide (VIP), an anti-inflammatory neuropeptide with pleiotropic cardiovascular effects, induces differentiation of hematopoietic stem cells into regulatory dendritic cells that limit graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We have previously shown that donor plasmacytoid dendritic cells (pDCs) in bone marrow (BM) donor grafts limit the pathogenesis of GVHD. In this current study we show that murine and human pDCs express VIP, and that VIP-expressing pDCs limit T-cell activation and expansion using both in vivo and in vitro model systems. Using T cells or pDCs from transgenic luciferase+ donors in murine bone marrow transplantation (BMT), we show similar homing patterns of donor pDCs and T cells to the major sites for alloactivation of donor T cells: spleen and gut. Cotransplanting VIP-knockout (KO) pDCs with hematopoietic stem cells and T cells in major histocompatibility complex mismatched allogeneic BMT led to lower survival, higher GVHD scores, and more colon crypt cell apoptosis than transplanting wild-type pDCs. BMT recipients of VIP-KO pDCs had more T helper 1 polarized T cells, and higher plasma levels of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α than recipients of wild-type pDCs. T cells from VIP-KO pDC recipients had increasing levels of bhlhe40 transcripts during the first 2 weeks posttransplant, and higher levels of CyclophilinA/Ppia transcripts at day 15 compared with T cells from recipients of wild-type pDCs. Collectively, these data indicate paracrine VIP synthesis by donor pDCs limits pathogenic T-cell inflammation, supporting a novel mechanism by which donor immune cells regulate T-cell activation and GVHD in allogeneic BMT.

Major histocompatibility complex class I molecule expression by pancreatic cancer cells is regulated by activation and inhibition of the epidermal growth factor receptor.

Pancreatic cancer is one of the deadliest neoplasms, with a dismal 5-year survival rate of only 10%. The ability of pancreatic cancer cells to evade the immune system hinders an anti-tumor response and contributes to the poor survival rate. Downregulation of major histocompatibility complex (MHC) class I cell-surface expression can aid in immune evasion by preventing endogenous tumor antigens from being presented to cytotoxic T cells. Earlier studies suggested that epidermal growth factor receptor (EGFR) signaling can decrease MHC class I expression on certain cancer cell types. However, even though erlotinib (a tyrosine kinase inhibitor that targets EGFR) is an approved drug for advanced pancreatic cancer treatment, the impact of EGFR inhibition or stimulation on pancreatic cancer cell MHC class I surface expression has not previously been analyzed. In this current study, we discovered that EGFR affects MHC class I mRNA and protein expression by human pancreatic cancer cell lines. We demonstrated that cell-surface MHC class I expression is downregulated upon EGFR activation, and the MHC class I level at the surface is elevated following EGFR inhibition. Furthermore, we found that EGFR associates with MHC class I molecules. By defining a role in pancreatic cancer cells for activated EGFR in reducing MHC class I expression and by revealing that EGFR inhibitors can boost MHC class I expression, our work supports further investigation of combined usage of EGFR inhibitors with immunotherapies against pancreatic cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Predicting TCR-Epitope Binding Specificity Using Deep Metric Learning and Multimodal Learning.

Understanding the recognition of specific epitopes by cytotoxic T cells is a central problem in immunology. Although predicting binding between peptides and the class I Major Histocompatibility Complex (MHC) has had success, predicting interactions between T cell receptors (TCRs) and MHC class I-peptide complexes (pMHC) remains elusive. This paper utilizes a convolutional neural network model employing deep metric learning and multimodal learning to perform two critical tasks in TCR-epitope binding prediction: identifying the TCRs that bind a given epitope from a TCR repertoire, and identifying the binding epitope of a given TCR from a list of candidate epitopes. Our model can perform both tasks simultaneously and reveals that inconsistent preprocessing of TCR sequences can confound binding prediction. Applying a neural network interpretation method identifies key amino acid sequence patterns and positions within the TCR, important for binding specificity. Contrary to common assumption, known crystal structures of TCR-pMHC complexes show that the predicted salient amino acid positions are not necessarily the closest to the epitopes, implying that physical proximity may not be a good proxy for importance in determining TCR-epitope specificity. Our work thus provides an insight into the learned predictive features of TCR-epitope binding specificity and advances the associated classification tasks.

CRIPSR/Cas9 Technology: Hypoimmunogenic Pluripotent Stem Cells Evade Immune Rejection in Fully Immunocompetent Allogeneic Recipients

Purpose Induced pluripotent stem cells (iPSCs) are promising candidates for regenerative medicine. To overcome the allogeneic immune barrier, we created hypo-immune iPSCs using CRISPR/Cas9 editing strategies and demonstrate long-term survival of allografts without the need of immunosuppression. Methods Hypoimmunogenic mouse and human iPSCs were generated via CRISPR/Cas9 technology by knocking out major histocompatibility complex (MHC) class I and class II and overexpression of the “don't eat me” signal CD47. Unmodified and hypoimmunogenic mouse and human iPSCs were transplanted into allogeneic BALB/c mice or humanized mice, respectively. Cellular and antibody responses as well as innate immune responses were analyzed and in vivo bioluminescence imaging was performed to assess cell survival. Results After allogeneic transplantation, hypoimmunogenic mouse and human cells showed a significantly decreased immune response in ELISPOT assays, comparable to the level of syngeneic transplants. No donor-specific antibodies were detected in mice and humanized mice receiving hypoimmunogenic cells demonstrating a lack of immune recognition. According to the ‘missing-self’ hypothesis, lack of target cell MHC class I expression facilitates NK cell-mediated cytotoxicity. Our data show the existence and functionality of the immune checkpoint signal regulatory protein-α (SIRPα) in innate immune cells which is modulated by CD47 overexpression. The lack of immune activation after transplantation of hypoimmunogenic iPSCs resulted in graft survival of transplanted mouse and human iPSCs in all allogeneic recipients with an observation period of 50 days. Conclusion Hypoimmunogenic iPSCs might serve as an unlimited cell source for the generation of universally-compatible “off-the-shelf” cell grafts or tissues in future clinical applications without the need of immunosuppression.

Experimental Platform Using the Amphibian Xenopus laevis for Research in Fundamental and Medical Immunology.

The amphibian Xenopus constitutes a powerful, versatile, and cost-effective nonmammalian model with which to investigate important contemporary issues of immunity relevant to human health such as ontogeny of immunity, self-tolerance, wound healing, autoimmunity, cancer immunity, immunotoxicology, and adaptation of host immune defenses to emerging pathogens. This model system presents several attractive features: an external developmental environment free of maternal influence that allows for easy experimental access from early life stages; an immune system that is remarkably similar to that of mammals; the availability of large-scale genetic and genomic resources; invaluable major histocompatibility complex (MHC)-defined inbred strains of frogs; and useful tools such as lymphoid tumor cell lines, monoclonal antibodies, and MHC tetramers. Modern reverse genetic loss-of-function and genome-editing technologies applied to immune function further empower this model. Finally, the evolutionary distance between Xenopus and mammals permits distinguishing species-specific adaptation from more conserved features of the immune system. In this introduction, the advantages and features of Xenopus for immunological research are outlined, as are existing tools, resources, and methods for using this model system.

Two to Tango: Co-evolution of Hominid Natural Killer Cell Receptors and MHC.

Natural killer (NK) cells have diverse roles in hominid immunity and reproduction. Modulating these functions are the interactions between major histocompatibility complex (MHC) class I molecules that are ligands for two NK cell surface receptor types. Diverse killer cell immunoglobulin-like receptors (KIR) bind specific motifs encoded within the polymorphic MHC class I cell surface glycoproteins, while, in more conserved interactions, CD94:NKG2A receptors recognize MHC-E with bound peptides derived from MHC class I leader sequences. The hominid lineage presents a choreographed co-evolution of KIR with their MHC class I ligands. MHC-A, -B, and -C are present in all great apes with species-specific haplotypic variation in gene content. The Bw4 epitope recognized by lineage II KIR is restricted to MHC-B but also present on some gorilla and human MHC-A. Common to great apes, but rare in humans, are MHC-B possessing a C1 epitope recognized by lineage III KIR. MHC-C arose from duplication of MHC-B and is fixed in all great apes except orangutan, where it exists on approximately 50% of haplotypes and all allotypes are C1-bearing. Recent study showed that gorillas possess yet another intermediate MHC organization compared to humans. Like orangutans, but unlike the Pan-Homo species, duplication of MHC-B occurred. However, MHC-C is fixed, and the MHC-C C2 epitope (absent in orangutans) emerges. The evolution of MHC-C drove expansion of its cognate lineage III KIR. Recently, position −21 of the MHC-B leader sequence has been shown to be critical in determining NK cell educational outcome. In humans, methionine (−21M) results in CD94:NKG2A-focused education whereas threonine (−21T) produces KIR-focused education. This is another dynamic position among hominids. Orangutans have exclusively −21M, consistent with their intermediate stage in lineage III KIR-focused evolution. Gorillas have both −21M and −21T, like humans, but they are unequally encoded by their duplicated B genes. Chimpanzees have near-fixed −21T, indicative of KIR-focused NK education. Harmonious with this observation, chimpanzee KIR exhibit strong binding and, compared to humans, smaller differences between binding levels of activating and inhibitory KIR. Consistent between these MHC-NK cell receptor systems over the course of hominid evolution is the evolution of polymorphism favoring the more novel and dynamic KIR system.

LAG-3 inhibits the activation of CD4+ T cells that recognize stable pMHCII through its conformation-dependent recognition of pMHCII.

The success of tumor immunotherapy targeting the inhibitory co-receptors PD-1 and CTLA-4 has indicated that many other co-receptors might be potential druggable targets, despite limited information about their functional differences. Here we identified a unique target selectivity for the inhibitory co-receptor LAG-3 that was intrinsic to its immunoregulatory roles. Although LAG-3 has been reported to recognize major histocompatibility complex (MHC) class II, it did not recognize MHC class II universally; instead, we found that it selectively recognized stable complexes of peptide and MHC class II (pMHCII). LAG-3 did not directly interfere with interactions between the co-receptor CD4 and MHC class II or between the T cell antigen receptor and MHC class II. Instead, LAG-3 preferentially suppressed T cells responsive to stable pMHCII by transducing inhibitory signals via its intracellular region. Thus, LAG-3 might function more selectively than previously thought and thereby maintain tolerance to dominant autoantigens.

Autophagy Proteins in Phagocyte Endocytosis and Exocytosis.

Autophagy was initially described as a catabolic pathway that recycles nutrients of cytoplasmic constituents after lysosomal degradation during starvation. Since the immune system monitors products of lysosomal degradation via major histocompatibility complex (MHC) class II restricted antigen presentation, autophagy was found to process intracellular antigens for display on MHC class II molecules. In recent years, however, it has become apparent that the molecular machinery of autophagy serves phagocytes in many more membrane trafficking pathways, thereby regulating immunity to infectious disease agents. In this minireview, we will summarize the recent evidence that autophagy proteins regulate phagocyte endocytosis and exocytosis for myeloid cell activation, pathogen replication, and MHC class I and II restricted antigen presentation. Selective stimulation and inhibition of the respective functional modules of the autophagy machinery might constitute valid therapeutic options in the discussed disease settings.

αβ T cell receptor germline CDR regions moderate contact with MHC ligands and regulate peptide cross-reactivity.

αβ T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface.

Cardiomyocytes Derived from MHC-Homozygous Induced Pluripotent Stem Cells Exhibit Reduced Allogeneic Immunogenicity in MHC-Matched Non-human Primates.

SummaryInduced pluripotent stem cells (iPSCs) can serve as a source of cardiomyocytes (CMs) to treat end-stage heart failure; however, transplantation of genetically dissimilar iPSCs even within species (allogeneic) can induce immune rejection. We hypothesized that this might be limited by matching the major histocompatibility complex (MHC) antigens between the donor and the recipient. We therefore transplanted fluorescence-labeled (GFP) iPSC-CMs donated from a macaque with homozygous MHC haplotypes into the subcutaneous tissue and hearts of macaques having heterozygous MHC haplotypes (MHC-matched; group I) or without identical MHC alleles (group II) in conjunction with immune suppression. Group I displayed a higher GFP intensity and less immune-cell infiltration in the graft than group II. However, MHC-matched transplantation with single or no immune-suppressive drugs still induced a substantial host immune response to the graft. Thus, the immunogenicity of allogeneic iPSC-CMs was reduced by MHC-matched transplantation although a requirement for appropriate immune suppression was retained for successful engraftment.

B cell modulation in transplantation.

B cells are cells of the lymphoid lineage characterized by the surface expression of antibody/immunoglobulin (Ig) as the B cell receptor (BCR). B cells are the precursors of antibody-forming plasma cells. It has recently been appreciated that subpopulations of B cells may also act as immunoregulatory cells. These ‘regulatory’ B cells were first described in the mouse and subsequently in humans 1,2, and mediate immunoregulation via the production of interleukin (IL)-10 and IL-35 3. B cells bind antigen via their BCR, and the outcome of this interaction depends upon the affinity of the BCR for the antigen and the environmental context. For example, B cell activation can be facilitated by the presence of complement components (C3b and C3d binding to CD21/CR2). B cells may also receive proactivation and survival signals via cytokines, including BAFF (B cell-activating factor belonging to the tumour necrosis factor family, also known as BLyS). Over-expression of BAFF is sufficient to drive abnormal B cell survival, hypergammaglobulinaemia and a lupus-like autoimmune disease in mice independent of T cell help 4. If the BCR aggregation is sufficient to induce signalling, the B cell internalizes bound antigen, processes it and presents in the context of major histocompatibility complex (MHC) class II. At the border between the B cell follicle and T cell zone of secondary lymphoid organs, the B cell MHC–peptide complex may be recognized by a cognate T cell. Following this T–B interaction, B cells return to the follicle to form the germinal centre, where they undergo somatic hypermutation and class-switch recombination. Here, B cells with a higher affinity for antigen are positively selected and differentiate into either memory B cells or plasma cells. A subset of CD4 T cells located within B cell follicles and characterized by expression of the transcription repressor Bcl-6 [known as T follicular helper (Tfh) cells] are essential for the development of germinal centre B cells 5. A small proportion of plasma cells arising from the germinal centre become established as long-lived plasma cells in the bone marrow. Given the potent and widespread effects of B cell activation, it is critical that there are also stringent control measures to prevent inappropriate B cell responses. To this end, the B cell expresses a number of inhibitory receptors, for example FcγRIIB, CD22, CD72 and PIR-B 6. In recent years, there has been increasing interest in how B cells, plasma cells and their associated antibody respond to allografts 7. Sensitized patients with preformed human leucocyte antigen (HLA) antibodies have an increased risk of acute and chronic antibody-mediated rejection (AMR), which significantly impacts allograft longevity 8. Furthermore, it is now well established that the appearance of de-novo donor-specific antibodies (DSAs) is associated with AMR and chronic allograft attrition 9. Outside their remit of antibody production, there is an appreciation that B cells may play a role in acute cellular rejection, also known as T cell-mediated rejection (TCMR). In contrast to these negative effects of B cells and antibody on the allograft, there is a growing body of evidence that B cells may be beneficial for long-term graft survival; a number of studies have shown a B cell transcriptomic signature in tolerant transplant recipients 10,11 and an up-regulation of B cell biomarkers in rejection-free transplant recipients 12 that may be due to the effects of ‘regulatory’ B cells. Possible strategies to target B cells in transplantation include: (i) B cell depletion; (ii) modulation of B cell activation; (iii) increase B cell inhibition; and (iv) enhancing the generation of regulatory B cells.

Fish oil disrupts B cell MHC class II lateral organization in the murine immunological synapse

Fish oil enriched long chain n‐3 polyunsaturated fatty acids disrupts the lateral organization of proteins on the T cell side of the immunological synapse. However, the impact of fish oil on the antigen presenting cell side of the immunological synapse is unclear. We previously demonstrated that B cells isolated from mice fed fish oil suppressed B cell microdomain clustering as well as the ability to activate naïve CD4+ T cells through antigen presentation. In this study, using a combination of quantitative imaging methods, we tested the effect of fish oil on the lateral organization of B cell major histocompatibility complex (MHC) class II molecules involved in forming the immunological synapse. Fish oil had no impact on pre‐synaptic B cell MHC II clustering. However, fish oil did suppress the recruitment of MHC class II to the synapse. As a consequence, T cell PKCθ recruitment to the synapse was also diminished. The effects were independent of changes in B‐T cell adhesion. Finally, we compared the impact from fish oil to cholesterol depletion using methyl‐β‐cyclodextrin treatment and found similar results. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B cells compared to T cells. Furthermore, the data establish MHC class II lateral organization on the B cell side of the immunological synapse as a novel molecular target of fish oil.NIH R15AT006122

T Cell Receptor Specificity, Cross-Reactivity, and MHC Restriction are Inextricably Linked and Result from Cooperative Engagement of the Composite Peptide/MHC Surface

T cell receptors (TCRs) recognize peptides bound and presented by major histocompatibility complex (MHC) proteins using multiple complementarity determining region (CDR) loops. While numerous analyses have illuminated structural and biophysical aspects of TCR recognition, how the distribution of binding free energy within TCR-pMHC interfaces promotes unique TCR recognition features, including MHC restriction and the apparent dichotomy of specificity and cross-reactivity, remains unclear. Utilizing double mutant cycles, here we performed a comprehensive structural and thermodynamic deconstruction of the interaction between the A6 TCR and the Tax peptide presented by the class I MHC HLA-A2. In contrast with general expectations, we observed that the central regions of the peptide and its interactions with the hypervariable CDR3 loops contribute little to specificity, instead promoting by dynamic effects the cross-reactivity that is a hallmark of TCR recognition. We also observed that TCR restriction towards HLA-A2 results from not from conserved interactions with the germline loops, but instead from strong interactions with the hypervariable CDR3 loop of the α chain. Formation of these latter interactions, however, is dependent upon the unique structural properties of the peptide, highlighting that TCR specificity towards peptide and MHC can emerge from the need to engage a unique, composite peptide/MHC interface with tightly coupled structural properties.

Effects of Repeated Lipopolysaccharide Stimulation on the Development of Antigen-presenting Cells and T Cells Pool in Hen Vagina

The aim of this study was to determine how the mucosal immunity mediated by T cells is developed in the vagina. Antigen-presenting cells and T cells play important roles in the early process of host defense. Mature T cells and immature dendritic cells expressing CC chemokine receptor (CCR) 6. In contrast, immature T cells and mature dendritic cells express CCR7. It was examined whether repeated antigen stimulation is effective for development of the pool of antigen-presenting cells and T cells in the vagina. White Leghorn hens were intravaginally injected 1 (single injection group) or 5 times (repeated injection group) in 10 days with saline (control) or lipopolysaccharide (LPS). The vagina was collected 1 day after the final injection. Frozen sections of them were immunostained for CD4+, CD8+, TCRγδ+ T cells and major histocompatibility complex (MHC) class II+ cells. Expressions of CCR6, CCR7 and MHC class II were analyzed by quantitative RT-PCR. The frequency of CD4+, CD8+, TCRγδ+ T cells were significantly increased in LPS-repeated injection group, however that of MHC class II+ cells was not changed significantly. The expression of CCR6 was significantly upregulated in LPS-repeated injection group, however, the expression of CCR7 and MHC class II was not affected. The densities of the each T cell subset and MHC class II+ cells, as well as expressions of chemokine receptor, MHC class II were not affected by single LPS injection or saline injection. These results suggest that the repeated antigen stimulation may not affect antigen-presenting ability by antigen-presenting cells, whereas it may lead influx of T cell subsets in the vagina. These accumulated T cells pool may contribute to form prequiescence of host immunity in the vaginal mucosa.

Correlation between lipid deposition, immune-inflammatory cell content and MHC class II expression in diffuse intimal thickening of the human aorta

Inflammatory reactions driven by an accumulation in the intima of immune-inflammatory cells and focal lipid depositions are the hallmarks of atherogenesis. It is commonly accepted that immune-inflammatory cell accumulation and lipid deposition are associated with the very earlier stage of atherosclerosis but no study has yet focused on the determination of quantitative values of this association. The present study examined correlations between lipid deposition, immune-inflammatory cell content and major histocompatibility complex (MHC) class II molecule HLA-DR expression in diffuse intimal thickening (DIT), which is thought to represent the earliest macroscopic manifestation of atherosclerosis. In parallel consecutive tissue sections of DIT, lipids were examined by chromatographic analysis (including triglycerides, cholesteryl esters, free cholesterol and phospholipids), histochemically, using Oil Red O staining, and by electron microscopy. Immune-inflammatory cells and HLA-DR expression were examined immunohistochemically in consecutive sections of the same tissue specimens. The study revealed that lipids exhibited a non-uniform distribution throughout the intima. In the juxtaluminal sublayer, lipids were localized both intracellularly and extracellularly, whereas in the juxtamedial musculoelastic sublayer, lipids were present predominantly along elastic fibers. Lipid deposits were found to positively correlate with HLA-DR expression (r=0.79; p<0.001). The study also identified a positive correlation between lipid deposition and immune-inflammatory cell content but the correlation values varied between different sublayers of the tunica intima. The correlation between lipid deposition and immune-inflammatory cell content in the juxtaluminal sublayer of the intima was notably stronger (r=0.69; p<0.001) than in the juxtamedial musculoelastic layer (r=0.28; p<0.001). The findings of the present study support a view that lipid accumulation in the intima plays a role in the initiation of inflammatory reaction and that at the pre-lesional stage in the development of atherosclerosis, lipid-associated immune cell activation might occur primarily in the juxtaluminal portion of the intima.