bluuebunny/crossref_metadata_2025 huggingface dataset:
Fates of Commensal Specific, Innate-like CD8+ T cells in the Gut
Authors: Farley Taylor K , Krishnamurthy Siddharth , Powrie Fiona , Belkaid Yasmine
Published: August 2023

Barrier tissues such as the skin and gut are host to a variety of microbes collectively termed the microbiota. To maintain host homeostasis, sufficient exposure to the microbiota is necessary, however, diseases such as Inflammatory Bowel Disease (IBD), have been described as occurring amidst breakdown in the normal communication between host and microbe. Fundamentally, IBD is a disease of aberrant immune activation to the microbiota and epithelial barrier dysfunction. At the skin barrier, we have previously described the H2-M3 restricted, CD8+ T cell response in mice after topical exposure to a commensal microbe that aids in barrier maintenance and epithelial repair. However, the residence of these H2-M3 restricted, commensal specific T cells in the gut, and potential contribution to barrier function in this tissue is unknown. We have now identified H2-M3 restricted CD8+ T cells populating the lamina propria and intraepithelial compartments of both the colon and small intestine following novel commensal exposure. Further, during inflammatory conditions these cells drastically expand and produce pro-inflammatory Type 1 cytokines. Following these discoveries, we have developed novel tetramer reagents to track and interrogate the function of H2-M3 restricted T cell responses to Helicobacter hepaticus, a common gut commensal with known roles in perpetuating colitis in immunocompromised animals. Utilizing these tools, we hope to uncover how these non-classically restricted immune responses to the same commensal under homeostatic or inflammatory conditions differ, to identify potential targets to correct aberrant reactivity to the microbiota during colitis.

NIH-Oxford Cambridge Scholars Program
Vaccination prior to SARS-CoV-2 infection reduces autoreactive antibody induction
Authors: Lin Chun-Yang , Castellaw Ashley , Brice David ,   SJTRC Study Team, Schultz-Cherry Stacey , Thomas Paul , Wolf Josh , McGargill Maureen
Published: August 2023

SARS-CoV-2 infection leads to diverse outcomes ranging from asymptomatic infection to death. Several studies reported elevated autoreactive antibodies in SARS-CoV-2-infected individuals compared to healthy controls, suggesting SARS-CoV-2 infection promotes autoantibodies, which could increase the risk of autoimmunity. However, it is not known whether autoreactive antibodies observed after infection were present in individuals prior to infection. Further, the prevalence, duration, and vaccine impact on autoreactive antibodies are unresolved. Since elevated autoreactive antibodies precede the onset of autoimmune disease, it is important to investigate the impact of SARS-CoV-2 infection on self-reactive antibodies. We established a longitudinal study to collect baseline PBMC and plasma samples from participants at enrollment, before SARS-CoV-2 infection or vaccination. Participants were screened weekly by nasal swab PCR to detect asymptomatic infection. Additional samples were collected after confirmed SARS-CoV-2 infection and assessed for autoreactive antibodies predictive of various autoimmune disorders. Preliminary analysis showed that a subset of individuals exhibited an increase in autoreactive antibodies following infection, while other individuals unexpectedly displayed decreased levels. Notably, individuals infected after vaccination displayed fewer changes in autoreactive antibodies compared to infection prior to vaccination. Our data suggest that SARS-CoV-2 infection increases specific autoreactive antibody levels but does not induce a widespread increase in all autoreactive antibodies. Moreover, SARS-CoV-2 vaccination prior to infection reduced autoantibody induction.

Supported by grants from NIH CEIRS HHSN272201400006C and Household Respiratory Virus SARS-CoV-2 Transmission and Immunity Sub-Study (HRTS) 3U01AI144616-02S1
Thymic innate lymphoid cells promote thymocyte differentiation of transplanted hematopoietic progenitors in immunodeficient mice
Authors: Houques Chloe , Machado Alice , Pouzolles Marie , Harly Christelle , Taylor Naomi , Zimmermann Valérie S
Published: August 2023

Patients with combined immunodeficiencies can be cured by intravenous transfer of allogeneic hematopoietic stem and progenitor cells (HSPC). However, complications such as an impaired and delayed T cell differentiation are unfortunately common, notably due to the defective thymic architecture in these patients and the requirement of HSPC homing to the thymus. To circumvent these problems, we have developed an innovative approach based on the direct targeting of HSPC into the thymus by intrathymic (IT) injection. Using ZAP70-deficient mice as a paradigm, we previously showed that IT injection of HSPC can support a long-term thymus-autonomous differentiation. Here, we show that the IT injection of WT HSPC results in a rapid engraftment and differentiation. Donor-derived thymocytes expand >100-fold, accounting for >13% of thymus cellularity by 3 weeks post IT transfer and importantly, engraftment occurs in the absence of cytoreductive conditioning. While mature CD4 thymocyte differentiation occurs in 3 weeks, the differentiation of mature Treg thymocytes requires 4 weeks. The kinetics of thymic Treg development parallels the generation of a thymic medulla, with the appearance of mature medullary thymic epithelial cells at 3–4 weeks post transplantation. Notably, the generation of the thymic medulla is associated with the differentiation of donor-derived RORγT+ innate lymphoid cells (ILC). While ILC are not required for physiological thymopoiesis, we find that they are critical for T cell differentiation in ZAP70-deficient mice presenting with an absence of the thymic medulla. These results may open new therapeutic avenues, promoting the differentiation of ILC via IT-mediated HSPC transfer, enhancing T cell differentiation.

Supported by grants from ANR (CHIC) and AFM-Telethon
Role of paracrine mTOR signaling in regulating thymus size and function
Authors: Wedemeyer Sarah A , Jones Nicholas , Wimberly Kymberly , Xiao Yangming , Griffith Ann V
Published: August 2023

The thymus is the primary site of T cell generation, where the unique thymic stromal microenvironment governs T cell differentiation. However, the size of the thymus declines precipitously relatively early in life, resulting in declining production of naïve T cells. Homeostatic mechanisms drive a shift toward an oligoclonal T cell memory, leaving the elderly less responsive to vaccines and new infections. Preventing or reversing age-associated thymic atrophy therefore holds potential for extending the health span. Mechanisms governing thymic atrophy have been difficult to identify because the primary targets of atrophy, cortical thymic epithelial cells (cTECs), are rare and difficult to isolate. Characterization of the transcriptional response of thymic stromal cells during age-related atrophy and experimental regeneration suggested that paracrine mTOR signaling in cTECs promoted by ligands expressed in medullary TECs (mTECs) may be required to maintain the cellular architecture of the cortical stroma and preserve thymus size. To test these hypotheses, we generated two new tissue-specific transgenic mouse lines overexpressing mTOR-activating growth factors (IGF1 and FGF21) in most mTECs. Ongoing flow cytometry and confocal microscopy experiments indicate faithful transgene expression in mTECs, and suggest that mTEC-derived growth factors promote temporally distinct patterns of mTORC1 and mTORC2 signaling in cTECs to promote thymus growth and maintenance with age. This work was supported by NIH R21AI154109 and T32 AI138944-05.

Supported by grants from NIH (R21 AI154109, T32 AI138944-05)
NDRG3 is a novel regulator of T and B cell development with phosphorylation-specific activity and phenotypes
Authors: Boone D Tyler , Abdullah Leena , Huang Yina H
Published: August 2023

Common lymphoid progenitors (CLPs) in the bone marrow give rise to both T and B cells, but the mechanisms underpinning downstream cell fate decisions have yet to be fully elucidated. We have identified N-Myc Downstream Regulated Gene 3 (NDRG3) - a highly conserved regulator of Wnt/B-catenin signaling - as a novel regulator of lymphocyte cell fate decisions and a substrate of Akt kinase. To determine the effect of NDRG3 phosphorylation, hematopoietic stem cells (HSCs) overexpressing a phospho-null (S331A) or phospho-mimetic (S331E) mutant of NDRG3 were transferred into lethally irradiated mice followed by analysis of the reconstituted immune populations. S331A-expressing cells developed a 3:1 preference for the T cell fate while those expressing S331E had a 9:1 preference for the B cell fate. To further probe the effects of NDRG3 phosphorylation, we developed a mouse model that utilizes IL-7Ra-driven Cre to express phospho-null NDRG3-S331A from the endogenous locus. NDRG3-S331A expressing thymocytes displayed a faster transition through the preTCR and TCR checkpoints. Given the importance of TCF1 in thymocyte development, NDRG3-S331A mice were crossed to TCF1-GFP and TCF1-RE-GFP reporter mice to examine effects on the Wnt/TCF1 signaling pathway. In the spleen, T cells were increase at the expense of B cells. Within the B cell population, NDRG3-S331A cells exhibited a 4:1 preference for the marginal zone to follicular B cell fate that was associated with increased levels of IRF4 and IRF8 transcription factors, which are necessary for proper B cell development. These data reveal that NDRG3 controls the developmental kinetics and pathways governing T and B cell development and maturation in a phosphorylation-dependent manner.

Supported by NIH grant R01 AI089805 and a training fellowship from the Burroughs Wellcome Fund.
Aire Drives Steroid Hormone Biosynthesis by Medullary Thymic Epithelial Cells
Authors: Donahue Kaitlynn , Taves Matthew , Ashwell Jonathan D
Published: August 2023

Thymocytes progress toward mature T cells through a series of antigen-specific selection steps facilitated by thymic epithelial cells (TECs) in the cortex, the site of positive selection, and medulla, the site of negative selection. TECs produce glucocorticoids (GCs), which enhance positive selection and increase the fitness of the T cell antigen-specific repertoire, but which TEC subpopultion does this, and how it is regulated, is unknown. We generated a reporter mouse that expresses a fusion protein containing Cyp11b1, the enzyme that mediates the last step in de novo GC production, and the fluorescent tag mScarlet knocked into the endogenous Cyp11b1 locus. We found that Cyp11b1 is exclusively expressed in medullary TECs (mTECs), consistent with the notion that GC antagonize negative selection. This is seemingly at odds with the fact that mTECs facilitate negative selection via the transcription factor Aire, which drives promiscuous expression of tissue-restricted antigens (TRAs) vital for establishing central immune tolerance. We found that Cyp11b1 is primarily expressed in Aire+ and post-Aire mTECs and is absent in mTECs of Aire-deficient mice. Transcriptomic analysis showed that most genes of the GC biosynthetic pathway are expressed in mTECs in an Aire-dependent manor. Moreover, we found that their actual steroid product was produced by WT but not Aire-deficient thymi. We propose that in addition to its well-established role of inducing TRAs that promote negative selection, Aire has an obverse function in inducing production of GC that antagonize negative selection, which together expand and enhance the TCR repertoire.
Sequential and interwoven requirements for HEB and Id3 in fetal gamma-delta T cell commitment and functional programming
Authors: Anderson Michele K , Selvaratnam Johanna , Rocha Juliana , Rajan Vinothkumar , Wang Helen , Reddy Emily , Guidos Cynthia , Zuniga-Pflucker Juan Carlos
Published: August 2023

Two major developmental steps are necessary for the generation of innate IL-17 producing γδ T cells. First, T cell precursors in the fetal thymus commit to either the αβ or the γδ T cell lineage. This is followed by a second stage of differentiation into subsets that can produce either IL-17 (γδT17) or IFNγ (γδT1) cells. Both developmental events are dependent on TCR signal strength, but how this is interpreted at the molecular level has been unclear. Id3, which antagonizes the activity of HEB transcription factors, is upregulated in proportion to TCR signal strength, providing an important link between TCR signaling and gene expression. We previously found that γδ T cells in mice lacking HEB were impaired in IL-17 production. To better understand the molecular basis of this defect, we conducted single cell RNA-sequencing on fetal thymic γδ T cells. In WT cells, the ratio of HEB to Id3 was highest in populations undergoing γδ T cell commitment, whereas later populations were dominated by Id3. Comparing scRNA-seq datasets from WT and HEB-deficient γδ T cells revealed a profound shift in TCRγ and TCRδ chain expression. This was accompanied by a decrease in key regulators of early γδ T cell development. Id3 expression was also severely decreased. Accordingly, Id3-deficient mice were impaired in their ability to generate γδT17 cells. However, γδ T cells from Id3-deficient mice showed a decrease in second stage γδT17 regulators, rather than the first stage regulators defective in HEB-deficient mice. Therefore, distinct ratios of HEB and Id3 are required during the γδ T cell commitment, and HEB is instrumental in inducing Id3 to enable functional programming of γδT17 cells in the fetal thymus.

Supported by grants from CIHR (201610PJT), NSERC (RGPIN-2020-05596) and NIH (1P01AI102853-06)
The ER aminopeptidases, ERAP1 and ERAP2, Synergize to Self-Modulate their Respective Activities
Authors: Martin-Esteban Adrian , Rodriguez Jesus Contreras , Peske David J , de Castro Jose Antonio Lopez , Shastri Nilabh , Sadegh-Nasseri Scheherazade
Published: August 2023

Critical steps in Major Histocompatibility Complex Class I (MHC-I) antigen presentation occur in the endoplasmic reticulum (ER). In general, peptides that enter the ER are longer than the optimal length for MHC-I binding. The final trimming of MHC-I epitopes is performed by two related aminopeptidases, ERAP1 and ERAP2 in humans that possess unique and complementary substrate trimming specificities. While ERAP1 efficiently trims peptides longer than 9 residues, ERAP2 preferentially trims peptides shorter than 9 residues. Using a combination of biochemical and proteomic studies followed by biological verification, we demonstrate that the optimal ligands for either enzyme act as inhibitors of the other enzyme. Specifically, the presence of octamers reduced the trimming of long peptides by ERAP1, while peptides longer than nonomers inhibit ERAP2 activity. We propose a mechanism for how ERAP1 and ERAP2 synergize to modulate their respective activities and shape the MHC-I peptidome by generating optimal peptides for presentation.

This work was supported by grants ROI AI121174, R-37AI-060040, ROI AI-130210 from the National Institutes of Health (NIH) and SAF2017-86578-R from the Ministry of Science, Innovation, and Universities of the Government of Spain and by an institutional grant of the Fundación Ramón Areces to the CBMSO.
Recombinant Surface Immunogenic Protein from S. agalactiae and hemocyanin from Fisurella latimarginata promote cross-presentation dependence on the TLR-4 signaling
Authors: Diaz-Dinamarca Diego A , Salazar Michelle , Manubens Augusto , Salazar Fabián , Troncoso Mayarling F , Lavandero Sergio , Diaz Janepsy , Vasquez Abel E , Becker María Inés
Published: August 2023

Improved vaccine adjuvants are needed to address infectious diseases, cancer, and future pandemics. Toll-like receptors (TLRs) have been researched as targets of vaccine adjuvants because they modulate the innate immune system and activate adaptive immunity. TLR-based adjuvants induce cross-presentation of exogenous antigens to CD8 T lymphocytes. TLR4 signaling recruits the adapters myeloid differentiation marker 88 (MyD88), which mediates the recycling of endoplasmic reticulum fusion to the phagosome as well as the TIR-domain-containing adapter-inducing interferon-β (TRIF), which is essential for translocation from the endosome to the cytosol.

Given the role of TLR4 agonists, most studies have used ligands based on lipopolysaccharides, but these have side effects (especially fever). Here we propose using TLR4 protein-based agonists (TLR-4-PBA), which have multiple advantages as adjuvants: They are naturally biocompatible and biodegradable substances that induce minimal reactogenicity and toxicity. We studied mollusk hemocyanin from Fissurella latimarginata (FLH) and the recombinant surface immunogenic protein from Streptococcus agalactiae (rSIP). These two proteins differ significantly in their structure, and we show Th1 immunomodulatory properties.

Using mouse bone marrow-derived dendritic cells pulsed with ovoalbumin (OVA), we then showed that rSIP and FLH promote cross-presentation in OT-I CD8+ T lymphocytes. This effect depends on TLR4 and the recruitment of MyD88 and TRIF. Studies are in progress to evaluate IRF3 and NF-κ B activation. Our results illustrate how these TLR-4-PBAs could act as a clinically useful vaccine adjuvant for innate immunity and Th1 adaptive immune response.

FONDECYT 1201600 (MIB); FONDEF ID21I10370 (AEV), Instituto de Salud Pública de Chile Funding (AEV, DD-D); ANID/PhD Nacional program 21210946 (MLS) and 21200880 (DD-D); FONDAP 15130011 (MT, SL).
The antigen presentation of MCMV specific Qa-1-restricted CD8+ T cells
Authors: Reilly Shanelle , Demers Delia , Fugere Celine , Brossay Laurent
Published: August 2023

Murine cytomegalovirus (MCMV) is a well-established model system for human CMV (HCMV), a ubiquitous herpesvirus that affects over 70% of people worldwide. Although the classical CD8+ T cell response to MCMV has been well characterized, little is known about the non-classical CD8+ T cell response. Our laboratory recently demonstrated that Qa-1-restricted CD8+ T cells are sufficient to protect mice from MCMV induced lethality. Our recent data shows that this protective T cell population has limited αβ TCR diversity. To identify the MCMV peptide(s) presented by Qa-1, we utilized bioinformatic tools that predict MHC binding, including NetMHC 4.0 and T Cell Epitope Prediction tools available on the Immune Epitope Database and Analysis Resource. Using these tools, we queried the entire MCMV coding sequence to identify potential nonamers predicted to bind to Qa-1. Furthermore, these candidates were also queried to identify protease cleavage sites to ensure that they are predicted to exist in vivo. Approximately 40 potential peptides were selected and tested in an in vitro screen using Qa-1 expressing cell lines. Our results suggest that several candidate peptides bind to Qa-1 as strongly as well-defined canonical binding partners. Taken together, these data suggest that an MCMV-responsive T cell population with limited αβ TCR diversity recognizes MCMV-derived peptides presented by Qa-1. This population is likely to be of importance for viral protection when classical CD8+ T cell immunity is compromised.

This work is supported by the NIH Research Grant AI046709. Shanelle Reilly is supported by research supplement R01 3AI046709-18S1 to promote diversity.
Mass spectrometric profiling of HLA-B44 peptidomes provides evidence for Tapasin-mediated Tryptophan editing
Authors: Kaur Amanpreet , Surnilla Avrokin , Zaitouna Anita J , Basrur Venkatesha , Mumphrey Michael B , Cieslik Marcin , Carrington Mary N , Nesvizhskii Alexey I , Raghavan Malini
Published: August 2023

Activation of CD8+ T cells against pathogens and cancers involves the recognition of antigenic peptides presented in complex with human leukocyte antigen (HLA) class-I proteins. Peptide binding to HLA-I proteins is coordinated by a multi-protein complex called the peptide loading complex (PLC). Tapasin, a key PLC component, facilitates the binding and optimization of HLA-I peptides. However, different HLA-I allotypes have variable requirements for tapasin for their assembly and surface expression. HLA-B*44:02 and HLA-B*44:05, which differ only at position 116 of their heavy chain sequences, fall at the opposite ends of the tapasin-dependency spectrum. HLA-B*44:02 (D116) is highly tapasin-dependent, whereas HLA-B*44:05 (Y116) is highly tapasin-independent. To better understand the influences of tapasin upon HLA-B44 peptidome compositions, we undertook mass spectrometric comparisons of HLA-B*44:02/05 peptidomes under tapasin-sufficient and/or tapasin-deficient conditions. Analyses of the HLA-B*44:05 peptidomes in the presence vs. absence of tapasin reveal that tapasin increases the frequency of C-terminal tryptophan residues. In the presence of tapasin, C-terminal tryptophans are also more highly represented among peptides unique to B*44:02 and those shared between B*44:02 and B*44:05, compared with peptides unique to B*44:05. Overall, our findings demonstrate that tapasin influences the C-terminal composition of HLA-I-bound peptides. For the HLA-B44 family, the presence of tapasin or high tapasin-dependence of an allotype results in better binding of peptides with C-terminal tryptophans, consistent with the role for tapasin in stabilizing an open conformation to accommodate bulky C-terminal residues.

Supported by grants from NIH to Malini Raghavan (R21 AI164025, R21 AI126054 and R01AI044115)
Expanded presentation of Lyme autoantigens and identification of a novel immunogenic CD4+ T cell epitope from Borrelia burgdorferi MCP4 in murine Lyme arthritis
Authors: Danner Rebecca , Pereckas Michael , Rouse Joseph , Wahhab Amanda , Lochhead Robert B
Published: August 2023

Lyme arthritis (LA), caused by Borrelia burgdorferi (Bb), is often accompanied by autoimmune T and B cell responses, but the mechanisms of infection-induced autoimmunity are unclear. We used an immunopeptidomics approach to identify potential Lyme autoantigens and immunogenic Bb antigens, which were validated by histology and testing of T cell reactivity. C57BL/6 (B6) mice, which develop mild inflammatory LA, and B6 Il10−/− (IL10 KO) mice, which develop severe, persistent LA, were infected with Bb for 4 or 16 weeks, and MHCII-bound peptides from inguinal and popliteal lymph nodes were identified by LC-MS/MS. Joint inflammation, fibrosis, and vascular remodeling were analyzed by H&E, Masson’s trichrome, and anti-CD31 immunohistochemistry, respectively. Six Bb peptides were identified by LC-MS/MS, of which one epitope from methyl-accepting chemotaxis protein 4 (MCP4) was an immunogenic CD4+ T cell antigen. Over 10,000 self peptides, particularly from proteins involved in cholesterol metabolism, tissue damage, and vascular inflammation were identified in infected mice. Presentation of peptides from previously identified human Lyme autoantigens apolipoprotein B-100, fibronectin, and type V collagen were expanded in infected mice, suggestive of epitope spreading. Consistent with immunopeptidomics data, joints showed increased inflammatory infiltrate, fibrosis and neovascularization in infected mice, compared with joints from uninfected mice. In conclusion, this immunopeptidomics approach revealed key insights into potential mechanisms of infection-induced autoimmunity in LA and indentified a novel immunogenic CD4+ T cell antigen from Bb MCP4.

Supported by grants from NIAID (R21AI148982)
QuickSwitch™: an MHC tetramer platform for making fast and reliable custom tetramers for T cell staining
Authors: Poluektov Yuri O , Delcommenne Marc C
Published: August 2023

The use of MHC tetramers to identify antigen specific T cells has become widespread among immunologists since the late 90s. Tetramers have become so ubiquitous that many companies and research facilities can ship and deliver some of the most commonly used tetramers within a matter of days. But when it comes to the uncommon tetramers or tetramers with antigenic peptides that have only recently been identified, it may be quite a challenge to find the needed tetramer. In this case, most researchers are left with trying to produce their own tetramer or having another facility do it for them, which can become a lengthy process. In an effort to help those researchers, we have developed the QuickSwitch™ MHC tetramer. This MHC tetramer comes loaded with a place-holder peptide (or exiting peptide) that can be exchanged for most well-binding peptides of a given MHC haplotype. This way, researchers are able to make a tetramer with any peptide they have available in a matter of hours. More importantly, our MHC tetramer platform comes with a means to monitor how much of the available peptide has exchanged with the exiting peptide and quantify the MHC occupancy. This allows the user to not only determine if the peptide in question can make a viable MHC tetramer, but also identify the strong and weak binding peptides for that MHC haplotype. This piece of information can be invaluable in future studies, in particular for vaccine development.

MBL International
Structures of antibody/MHC-I complexes reveal deficiencies in structure prediction programs
Authors: Boyd Lisa , Jiang Jiansheng , Ahmad Javeed , Natarajan Kannan , Margulies David H
Published: August 2023

The remarkable success of structural prediction programs such as AlphaFold2 and RosettaFold have revolutionized the contemporary approach to protein structure determination. However, intrinsically disordered proteins (IDPs) and protein/protein complexes provide a greater challenge to such algorithms. In particular, there has been limited success in the structural prediction of antibody/protein antigen complexes and TCR/MHC complexes, in part because the database of such structures is limited. Here, we present several high-resolution X-ray structures of anti-MHC/MHC-I complexes consistent with previous exon-shuffling and epitope mapping data that contradict computational predictions. Specifically, two mAb previously mapped to the alpha3 domain of H2-D(d) or H2-L(d)/D(b) respectively are shown to bind in distinct orientations, either of which may sterically block CD8 binding. In addition, a peptide dependent, but peptide non-specific anti-H2-D(d) mAb binds to loops of the alpha2 domain. These results substantiate the need to determine experimental structures of antibody/protein complexes as we proceed to develop avenues for the de novo design of specific antibodies. Supported by the Intramural Research Program of the NIAID, NIH

Supported by the Intramural Research Program of the NIAID, NIH
Endo-lysosomal assembly variations among Human Leukocyte Antigen class I (HLA-I) allotypes
Authors: Raghavan Malini , Olson Eli , Ceccarelli Theodora
Published: August 2023

The extreme polymorphisms of HLA-I proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes (CTL). The canonical endoplasmic reticulum (ER) HLA-I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA-I molecules. We hypothesized variable influences of HLA-I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA-I are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B* 08:01 or HLA-B*35:01 being among the most resistant or sensitive respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in redistribution of HLA-B*35:01, but not HLA-B* 08:01, to LAMP1+ compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA-I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA-I expression and increase the efficiency of cross-presentation.

Supported by grants NIH R01 AI044115 T32GM008353 and T32AI007413
Inhibited MHC Class I and MHC Class II antigen processing and presentation upon Sars-CoV-2 infection
Authors: Nanaware Padma Pandurang , Cruz John , Khaja Muneeruddin , Shaffer Scott A , Stern Lawrence
Published: August 2023

Major histocompatibility (MHC) bound viral peptides are the immune signatures to initiate the selective and cross-reactive T cell responses. Identifying the antigenic targets of adaptive immunity to SARS-CoV-2 is a key towards understanding the pathogenesis of Sars-CoV-2 and the rapidly evolving mutants. We isolated the immunopeptidomes to identify naturally processed and presented MHC-II (HLA-DR, HLA-DP) and MHC-I (HLA-ABC) bound canonical and out-of-frame viral peptides. We identified 5 HLA-DR canonical epitopes (13 peptides) from spike protein, 2 HLA-DP canonical epitopes (4 peptides) from spike and membrane proteins, 1 canonical MHC-Class I peptides and 5 out-of-frame MHC-I peptides from different ORF proteins. The viral peptides were presented in lower abundances as compared to host peptides obscuring the identification of the MHC bound viral peptides. Some of these peptides were shown to recall T cell responses in COVID-19 donors in other reported studies. We observed the downregulation of surface and total HLA-ABC, HLA-DR and HLA-DP expression upon Sars-CoV-2 infection possibly restricting the presentation of viral peptides and therefore delaying the immune response. The mechanisms by which SARS-CoV-2 evades adaptive immune responses mediated by MHC-I are studied in detail by different groups. Hence, we further investigating the modulators of the MHC-II pathway using proteomics-based approach. We observed significant downregulation of CD74 protein, CIITA protein and cathepsins in infected cells affecting the processing and presentation of viral peptides. These results may help in understanding significantly altered antigen presentation in Sars-CoV-2 infected cells.
Structural classification of peptide/HLA complexes enables accurate high-throughput modeling
Authors: Gupta Sagar , Nerli Santrupti , Kandy Sreeja Kutti , Mersky Glenn L , Sgourakis Nikolaos G
Published: August 2023

The class I major histocompatibility complex (MHC-I) proteins display 8 to 15 residue peptides derived from endogenous proteins on the cell surface for immune surveillance. Accurate structural modeling of peptides bound to HLA (the human MHC) proteins has been mired by the inability to capture the central residues of the peptide. Here, an analysis of high-resolution X-ray crystal structures revealed that diverse peptide conformations arise as a result of local changes in the backbone configuration. As a result, we established an internal-coordinate based system to assess structural similarity between peptide backbones and found that peptide/HLA complexes, encompassing a wide range of allotypes and peptide sequences, present a discrete set of peptide backbone conformations. Leveraging these representative backbones and a regression model trained on a physically realistic scoring function, we developed RepPred, a structural modeling approach which predicts near native nonamer/HLA-A*02:01 complexes with high accuracy and throughput. Our method significantly outperformed five state-of-the-art peptide/HLA modeling approaches on the basis of structural accuracy. Thus, the structural information obtained from RepPred can be used to address long-standing immunology challenges such as predicting peptide immunogenicity.

Supported by grants from NIH (R01AI143997, R35GM125034, U01DK112217).
Nanoparticle geometry, immune memory, and antigen presentation determine the cross-reactive antibody response against sarbecoviruses
Authors: Wang Eric , Cohen Alexander A , Caldera Guzman Luis F , Bjorkman Pamela J , Chakraborty Arup K
Published: August 2023

In response to the threat of emerging SARS-CoV-2 variants and SARS-like betacoronaviruses (sarbecoviruses), mosaic nanoparticles presenting receptor-binding domains (RBDs) of multiple sarbecoviruses have been developed to elicit cross-reactive antibodies that target conserved regions of the RBD. To better understand vaccination with these antigens, we developed a computational model of the humoral response to nanoparticle and spike antigens using a combination of molecular dynamics, Markov processes, and population dynamics simulations. Avidity is currently only accounted for by introducing a second arm on rate, but additional effects of avidity will be considered in the future. Our model agrees with titers measured from nanoparticle and spike vaccinations in animals as well as clinical data from mRNA vaccines. We then used our model to predict the effect of previous vaccinations with wildtype spike on nanoparticle vaccinations. Compared to naïve individuals, previously vaccinated individuals produce a higher cross-reactive titer on the first nanoparticle vaccination and a lower cross-reactive titer on the second. This difference is due to expansion of cross-reactive B cells and competition with strain-specific B cells from previous vaccinations. Furthermore, we find that a cocktail of homotypic nanoparticles produces the same cross-reactive titers as the mosaic nanoparticle, which is because cross-reactive B cells can bind a greater fraction of presented antigen and can survive successive rounds of selection when the nanoparticle density is low.

Supported by grants from the National Science Foundation (1745302), NIH (1-R61- AI161805-01), and the Ragon Institute of MGH, MIT, and Harvard.
Universal Candida cell-surface-specific monoclonal antibodies protect mice against multidrug-resistant Candida auris bloodstream infections
Authors: Rosario-Colon Jonothan A , Eberle Karen , Xin Hong
Published: August 2023

Candida species are the leading cause of life-threatening fungal infections in humans, especially among immunocompromised and hospitalized individuals. Of utmost concern is the emerging “superbug” Candida auris which has the potential to disseminate into bloodstream infections with up to 60% mortality in susceptible populations. Importantly, C. auris is the first inherently multidrug-resistant fungal pathogen, which contributes to a delay in effective treatment and increased mortality rates. Consequently, C. auris is the first fungal pathogen to cause a serious global public health threat.

Our lab has identified and isolated a panel of universal monoclonal antibodies (mAbs) specific to Candida cell wall epitopes that share high homology among various human pathogenic Candida species. Using an established murine model of disseminated infection, we showed that passive transfer of mAb C3.1 (anti-β-1,2-mannotriose) significantly extended survival and reduced fungal burdens and inflammation during early infection. We also showed that mAbs C3.1 and 9F2 (anti-phosphoglycerate kinase 1) significantly enhanced phagocytosis of C. auris yeast by macrophages and inhibited the proliferation of multiple C. auris clinical isolates in vitro.

Since there are currently no approved antifungal vaccines or antibodies and current treatments are inadequate, critically needed antifungal antibodies can provide long-awaited effective therapies against this deadly pathogen. Our future directions include developing humanized mAbs to evaluate their protective efficacy in genetic mouse models of human immunodeficiency. We will then utilize Fc engineering to derive human IgG1 variants for enhanced complement-based effector efficacy.

Supported by grants from NIH /NIAID (R21AI168929) and the Department of Defense (PR171482)
Development of a next generation pertussis vaccine: An unbiased biochemical approach to identify novel CD4+ T cell recognized antigens from Bordetella pertussis
Authors: Shamseldin Mohamed Medhat , Hernandez Gabrielle , Verzani Eva , Hall Jesse M , Caution Kyle J , Carr Steven A , Deora Rajendar , Abbelin Jenn , Dubey Purnima
Published: August 2023

Waning acellular pertussis vaccine (aPV) efficacy has brought about the reemergence of pertussis despite global vaccine coverage. Decreasing vaccine efficacy is correlated with the emergence of vaccine escape mutants lacking bacterial antigens expressed in current laboratory strains. Recent work shows that systemic and mucosal CD4+ T cells are necessary for improved vaccine efficacy and increased duration of immunity. However, the antigens recognized by these T cells are undefined. We hypothesize that the efficacy and duration of aPV-induced immunity will be improved by the addition of new bacterial antigens that induce a strong CD4+ T cell response. Here we used the natural antigen processing machinery of mouse bone marrow derived dendritic cells to identify peptides derived from the laboratory Bp strain Bp536 and a clinical isolate, H762, that are presented on MHC Class II. Bioinformatics analysis selected the highest affinity peptides for both mice and human MHC Class II. The immunogenicity of each peptide was tested ex vivo using ELISA and an Activation Induced Marker (AIM) assay. We stimulated splenic T cells from convalescent or whole cell vaccine (wPV) immunized mice and human PBMCs. This unbiased biochemical approach generated a list of Bp-specific MHC Class II epitopes that are recognized by CD4+ T cells. These data suggest that Bp expresses multiple novel CD4+ T cell recognized antigens that may improve vaccine efficacy and the duration of protection when incorporated in a next generation aPV.
Potent suppression of viral rebound after analytical treatment interruption in ART-suppressed SHIV-infected animals by a CD4 binding site bNAb
Authors: Fox Madison L , Shrader Hannah , Hait Sabrina Helmold , Talana Chloe Adrienna , Lovelace Sarah , Choe Misook , Del Prete Gregory , McCarthy Elizabeth , Paul-Todd John , Woodward Ruth , Koup Richard , Pegu Amarendra
Published: August 2023

Broadly neutralizing antibodies (bNAbs) are promising immunotherapeutic biological agents against HIV-1 that can be used to complement small-molecule combination antiretroviral therapy (cART) drugs for the treatment of HIV-1 infection. There have been several analytical treatment interruption (ATI) studies utilizing bNAbs in ART-suppressed HIV-1-infected patients in which bNAbs are infused during an ART cessation period. To model these clinical studies, bNAbs can be administered to ART-suppressed SHIV-infected rhesus macaques (RM). However, delivery of human bNAbs to RMs induces formation of anti-drug antibodies (ADA) which shorten the half-life of the bNAb and limit the time during which effectiveness can be assessed. In this study, we infused six ART-suppressed SHIV-infected RMs with four 6–10 mg/kg doses of VRC01v23, a next generation CD4-binding site bNAb, every 2–4 weeks for a total duration of 10 weeks. Prior to bNAb treatment, three 50 mg/kg doses of an anti-CD20 antibody were given two weeks apart in order to deplete peripheral B cells and eliminate ADA. Five of six animals had persistent B cell depletion and showed no evidence of ADA after infusions of VRC01v23 and cessation of ART. No viral rebound was observed in these five animals. The sixth animal developed ADA against VRC01v23 resulting in rapid clearance of VRC01v23 from circulation and viral rebound. These data demonstrate that B cell depletion can limit the development of ADA against human bNAbs allowing for the assessment of long-term therapeutic potential of human bNAbs in RMs.

Supported by intramural NIAID funding
Combination adjuvants and antigens provide heterosubtypic protection against lethal influenza infection
Authors: Brown Deborah M , Briere Jenna , Cheang Hoi Lam , Custer Dylan , Reiley William C , Laferriere Ethan
Published: August 2023

Influenza A virus (IAV) is a negative strand RNA virus that undergoes antigenic drift and shift, leading to serologically distinct strains that can cause tens of thousands of deaths annually in the US. The wide variety of hemagglutinin (HA) and neuraminidase (NA) proteins necessitates yearly reformulation of the vaccine to protect against novel seasonal and pandemic strains. Current IAV vaccines do not contain adjuvants, however, immune modulators such as pattern recognition receptor (PRR) agonists may enhance antigen presentation to T cells and provide broader protection against IAV strains that differ in HA and NA outer coat proteins. We set out to investigate whether TLR4 (lipopolysaccharide) and TLR3 (poly IC) agonists in combination with HA (H3N2) and nucleoprotein (NP) or NP and matrix (M1) proteins could provide protection against lethal challenge with mismatched IAV strain H1N1. Mice given H3 and NP with LPS + pIC in a prime/boost strategy demonstrated 80% survival after heterosubtypic H1N1 challenge while H3 + NP in PBS showed 20% survival. Similar results were seen using LPS + pIC in combination with NP and M1 protein with 90% survival. High endpoint titers of anti-NP antibodies (that also bound H1N1 virus) correlated with heterosubtypic protection suggesting a role for non-neutralizing antibodies in promoting survival. In addition, LPS and pIC enhanced dendritic cell (DC) function in vitro and increased migratory monocyte DC in the lymph node in vivo, coupled with early upregulation of T cell marker CD69. This strategy may provide increased efficacy over current IAV vaccines by inducing non-neutralizing antibodies and enhanced T cell responses to internal proteins.

Supported by Trudeau Institute and Clarkson University.
Evolution of the Structure and Organization of the Mammalian Immunoglobulin Loci
Authors: Sampson Jordan M , Miller Robert D
Published: August 2023

There are three extant lineages of mammals: placentals (e.g. humans and mice), marsupials (e.g. kangaroos and opossums), and monotremes (e.g. platypus). Of the three lineages, the marsupials are unusually and uniformly limited in their immunoglobulin heavy chain (IgH) isotype content. All marsupials studied have only one IgM, IgG, IgE, and IgA and lack IgD. Comparatively, there is a greater complexity of IgH isotypes and subclasses in both placentals and monotremes. Marsupials also have a greater complexity in their light (L) chain V genes than IgH chains. Transposable elements (TEs), like retroelements, have been hypothesized to contribute to gene copy variation and genome evolution. Recently, these TEs have been found in the IgH loci. Here we investigate the impact these TEs have on the evolution of marsupial IgH and IgL loci. We have found that some of the TEs in the opossum IgH locus are short and non-functional, potentially contributing to the restriction seen in the locus. Conversely, the TEs in the L chain loci are longer and may explain the differences in complexity in both the V and C regions between the H and L chain loci.

Supported by National Science Foundation Award IOS-2103367.
Does the upper respiratory tract microbiome change in dairy calves fed milk replacer containing probiotics?
Authors: Chitko-McKown Carol G , McDaneld Tara G , Kritchevsky Janice E , Bryan Keith A , Eicher Susan
Published: August 2023

Our overall hypothesis is that feeding probiotics to dairy calves will alter overall immunity and may additionally change the microbiome in the respiratory tract as well as the gut. A group of 20 dairy calves were split into two treatment groups: Control (N=10, milk replacer), and Probiotic (N=10, milk replacer + 0.5 g/day Bovamine Dairy). On day 0 birth weight was obtained and the calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group. Blood was collected along with nasal and tonsil swabs on days 0, 7, 14, 21, 28, 42, and 48. Calves were monitored daily for fecal, nasal, and ocular discharge scores. Our preliminary data showed no significant differences (P<0.5) between the Control and Probiotic groups in immune cell populations in peripheral blood. DNA was extracted from nasal and tonsil swabs to evaluate the bacterial populations in the upper respiratory microbiome. Hypervariable regions 1 through 3 along the 16S ribosomal RNA gene were amplified by PCR and sequenced by Illumina MiSeq to determine the bacterial taxa present. Evaluation of these samples revealed a greater percentage of operational taxonomy units (OTU) were classified at the genus level in the tonsil compared to the nasal samples. The microbiome of the tonsils was more stable over time points compared to the nasal samples. Evaluation of each timepoint for nasal and tonsil samples identified bacterial taxa that changed in relative abundance with the addition of the probiotic compared to the control diet. Addition of probiotic changed the relative abundance of OTU (P<0.01) in the nasal and tonsil sampling sites and at multiple timepoints. Analyses are ongoing.

Project supported by fund from the USDA Agricultural Research Service.
LAIR-1 expression on SIV-specific CD8 T cells is associated with T cell impairment and disease progression
Authors: Govindaraj Sakthivel , Sharma Asish Arunkumar , Ali Syed , Babu Hemalatha , Busman-Sahay Katheleen , Bosinger Steven , Estes Jacob , Sekaly Rafick-Pierre , Villenger Francois , Amara Rama Rao , Velu Vijayakumar
Published: August 2023

Functional impairment of CD8 T cells is characteristic of chronic HIV infection. HIV-specific CD8 T cells undergo exhaustion, lose effector function and fail to control the virus. Leukocyte-associated Ig-like receptor-1 (LAIR-1), a transmembrane glycoprotein, expressed on human blood mononuclear leukocytes and act as an inhibitory receptor of cytotoxic cells. However, the role of LAIR-1 expression on virus-specific CD8 T cells during SIV/HIV infection remains unknown.

We examined a total of 14 macaques infected with the Simian Immunodeficiency Virus (SIVmac251) and studied the dynamics of LAIR-1+ SIV-specific CD8 T cells for their phenotype and function using flow cytometry. We performed immunohistochemistry to stain for LAIR-1 in lymph nodes and used RNA seq to study the transcriptomic profile of LAIR-1+ CD8 T cells.

Here we report that LAIR-1 is significantly up regulated on SIV-specific CD8+T cells during chronic SIV infection. and the expression of LAIR-1 on total and SIV-specific effector memory CD8 T cells correlated directly with the SIV viral RNA levels. In addition, the level of LAIR-1 in the lymph node was higher compared to naïve animals. Transcriptionally, the LAIR-1+ CD8 T cells exhibited an heightened type I IFN-signaling coupled with reduced TCR signaling, cell cycling and cell metabolism pathways. Indeed, In vitro blockade of LAIR-1 using LAIR-1-Fc/anti-LAIR-1 antibody resulted in enhanced proliferation of SIV-specific CD8 T cells with cytotoxic function.

These data indicate that the immunoregulatory LAIR-1/Collagen pathway is operative during SIV infection and serve as a foundation in vivo trials of LAIR-1 blockade to potentially enhance/restore antiviral SIV-specific CD8 T cells for the HIV cure strategy.

This work was supported in part by NIH grants CFAR ERASE AIDS-R03, R01AI148377 (to VV), U19 AI109633 (to RRA, macaque core to VV),