bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryptococcus neoformans activates the inflammasome and its capsule plays an important role in this process (P1357) (68.9)
Authors: Saavedra Pedro , Capparelli Manuella , Matos Larissa , Zamboni Dario , Casadevall Arturo , Bocca Anamelia , Magalhaes Kelly
Published: January 2023

Cryptococcus neoformans is an encapsulated fungus widely distributed around the globe and capable of causing pulmonary infection and meningitis in immunocompromised individuals. Its capsule is well known for conferring C. neoformans the ability to evade host immune system, leading to its proliferation. Regarding the importance of initial innate immune response to cryptococcal infections, we decided to investigate if C. neoformans is capable of activating the inflammasome as well as the role of its capsule in this pathway. In order to verify whether C. neoformans can trigger inflammasome activation, peritoneal (PMs), bone marrow-derived dendritic cells (BMDCs) or the human monocyte cell line (THP-1) were pretreated or not with caspase-1 and potassium efflux inhibitors and infected with wild type C. neoformans (B3501) or its acapsular mutant (Cap67). Our results showed that acapsular mutant of C. neoformans, but not the wild type, induced IL-1β secretion in both murine and human infected cells. Besides, IL-1β secretion seemed to be dependent of fungal viability as heat-killed fungi elicited lower levels of this cytokine compared the living microorganism. In addition, acapsular C. neoformans triggered caspase-1-dependent and potassium efflux independent inflammasome activation. Taken together, these data suggest that C. neoformans activates the inflammasome and its capsule plays an important role in this process.
The Involvement of BLK in Development of Splenic B Cells and B Cell Adhesion and Migration (69.11)
Authors: Wu Ying-Yu , Haque Mohammad , Da Huining , Alarcon-Riquelme Marta
Published: January 2023

The BLK gene has been identified as genetically associated with systemic lupus erythematosus (SLE). BLK genetic variants involved in risk for SLE correlate with reduced expression of BLK in human B-cells. Recent studies have found that BLK deficiency dampened MZ B cell development, but enhanced its functional response to BCR stimulation. However, reduction of MZ B cells cannot explain why decreased expression of BLK is associated with SLE and why the TI-humoral response is increased in BLK-KO mice. In light of these recent discoveries, we performed a more comprehensive characterization of splenic B cell subsets and their biological functions. We report that BLK-KO mice have accumulated numbers of T1 B cells and splenic B1 B cells. B1 B cells from BLK-KO mice express higher levels of VLA4. Mature B cells and transitional B cells have normal in vitro adhesion to VCAM1 in the absence of stimulation. Nevertheless, BCR-engaged BLK-KO MZ B cells adhered less to VCAM1, whereas BCR-crosslinked BLK-KO T1 B cells had increased adhesion to VCAM1. Moreover, BLK-KO MZ B cells showed a reduced CXCR5-mediated chemotaxis, while BLK-KO T1 B cells had an increased CXCR5-mediated chemotaxis. Thus, this study demonstrates that BLK may have a novel role in the development of splenic B1 B cells and implies a previously unappreciated role for the involvement of BLK in B-cell adhesion and migration.
Molecular analysis of age-associated B cells (ABCs) (69.7)
Authors: Scholz Jean , Hao Yi , Russell Lisa , Maul Robert , Paley Michael , Wherry E. , Gearhart Patricia , Cancro Michael
Published: January 2023

Age-associated B cells (ABCs) are a phenotypically and functionally distinct mature B cell subset that accumulates with age. Unlike follicular (FO) B cells, ABCs do not rely on B lymphocyte stimulator (BLyS) to survive, and are likely derived from mature B cells that exhaustively expand throughout the animal’s lifespan. Because ABCs respond to innate but not adaptive stimuli, produce cytokines, and potentiate Th17 polarization, they may alter the quality of immune responses in aged animals, thereby contributing to age-associated increases in susceptibility to infection and inflammation. In order to further investigate ABC origins, relationships to other B cell subsets, and roles in immunosenescence, we have undertaken detailed gene expression and BCR repertoire analyses. Microarray results show striking differences in gene expression patterns between ABCs and FO B cells from aged and young mice. Heavy-chain gene analysis indicates that ABCs display a diverse VH repertoire, with a mutation frequency ~6-fold higher than that observed among FO B cells, indicating previous activation and concomitant somatic hypermutation. Despite this, the ratio of CDR to FWR mutations indicates a lack of antigen-mediated selection. Taken together, our results suggest that ABCs are derived from a diverse progenitor pool and reflect prior activation; however, expansion of the ABC subset either does not include selection based on CDR specificity, or involves aberrant selection.
Antigen density determines the pattern of pMHC cross-reactivity of CD4+ T cell responses during viral infection(69.8)
Authors: Vanguri Vijay , Smith Rebecca , Shah Shivanee , Paczosa Michelle , Huseby Eric
Published: January 2023

The activation of the CD4+ T cell immune response to pathogens is determined by the interaction between the T cell receptor and its cognate pathogen-derived peptide presented by MHC-II molecules on antigen-presenting cells (APCs). In vitro, whether a given CD4+ T cell will undergo activation is determined by the potency and density of the activating ligand. We created an in vivo murine model to identify how ligand potency and ligand density impact the overall cross-reactivity of an anti-viral CD4+ T cell response. We observed graded CD4+ T cell responses after immunization by strong, medium, or weak affinity ligands, with phenotypic differences evident within 24 hours and with long-term effects on the repertoire and clonal expansion. In addition, vaccinia viruses that expressed a high density ligand on the surface of APCs generated an expanded profile of CD4+ T cell specificities compared to that generated in response to lower density antigen. This occurred because medium affinity CD4+ T cells were able to be activated and expanded in the presence of high but not low antigen density.
Differential requirement for CCR7 expression in primary and secondary CD8 T cell responses to infection (69.9)
Authors: Sharma Naveen , Lefrançois Leo
Published: January 2023

Since naïve and memory T cells localize in both red and white pulp of the spleen, we wished to test the relative contribution of priming in each location. To do so, we employed TCR transgenic CD8 T cells (OT-I) that either lacked CCR7 or constitutively expressed CCR7 (CD2-CCR7). After Listeria infection, CCR7-/- CD8 T cells did not enter the T cell zone (PALS) while CD2-CCR7 OT-I cells were confined nearly exclusively to the PALS. The response of CCR7-/-CD8 T cells was blunted compared to WT and CD2-CCR7 OT-I cells and was characterized by skewing toward KLRG1highCD127low short-lived effector cells (SLEC) and away from KLRG1lowCD127high memory precursor effector cells (MPEC). Transfer studies showed that CCR7-/- OT-I cells competed with endogenous ova-specific CD8 T cells, suggesting that some priming occurred in the red pulp in normal conditions. Interestingly, CCR7-/-, CD2-CCR7 and WT OT-I memory cells responded equally well to challenge infection indicating a low stringency for localization for memory CD8 T cell activation. These results suggested that antigen presentation in the red pulp promoted SLEC generation while white pulp priming promoted MPEC development, perhaps due to differences in the inflammatory milieu. Along with our demonstration that naïve and memory CD8 T cell activation exhibited distinct anatomical requirements, our findings implied that local environmental cues regulated the development of primary and secondary CD8 T cell responses to infection.
Identifying third signal cytokines for development of differently functioning CD8 T cells in Mycobacterium tuberculosis infection (70.4)
Authors: Sun Tao , Einarsdottir Thorbjorg , Flynn JoAnne
Published: January 2023

Mycobacterium tuberculosis (Mtb), causing tuberculosis (TB), is estimated to infect one third of the human population, and kills about 1.8 million people per year. CD8 T cells are the essential components in the adaptive immunity against Mtb. We previously demonstrated that Mtb-specific CD8 T cell populations are divided into two distinct types: either cytotoxic or IFN-γ secreting. However, the third cytokine signals that prime CD8 T cells to evolve into these phenotypes, or to develop capability to have both cytotoxic and IFN-γ producing functions are not known. We found that naïve CD8 T cells adoptively transferred into mice during Mtb infection were primed into more IFN-γ+ IL-2+ TNFα+ and CD107+ IFN-γ+ phenotypes, compared with cells transferred before infection. We also observed dynamic changes in levels of multiple cytokines within tissues over the course of infection. We hypothesize that the relative balances between pro- and anti-inflammatory cytokines contribute to the different phenotypes of CD8 T cells. Here we are using an in vitro OT-I T cell priming system together with lung and lymph node homogenates, as well as individual cytokines identified in those homogenates for effects on CD8 T cell priming and phenotypes. We will also examine the cytokine effects on CD8 T cells in vivo. In summary, our study contributes to a better understanding in how CD8 T cell responses are modulated during Mtb infection, which will help with design of better therapies or vaccines.
STAT4 deficiency impairs CD4 T cell responses, yet enhances pathogen-specific antibody production during Pneumocystis murina lung infection (70.7)
Authors: Myers Riley , Trevor Jennifer , Nelson Michael , Dunaway Chad , Steele Chad
Published: January 2023

Although it is clear that the loss of CD4+ T cells is a predisposition to Pneumocystis pneumonia, the underlying mechanism for host defense is not well understood. Mice deficient in IFN-αR, IL-12p35, or IL-23p19 have delayed clearance of Pneumocystis murina, all of which activate the pro-Th1 transcription factor STAT4. In order to investigate the role of STAT4 in host defense against P. murina, C57BL/6 wild-type (WT) and STAT4-/- mice were infected intratracheally with P. murina cysts and fungal burden was assessed 28 days later. Despite a reduction in the total number of leukocytes in the lung on day 14 after infection in STAT4-/- mice, P. murina clearance at day 28 was not affected by the absence of STAT4. On day 14 after P. murina infection, STAT4-/- mice showed increased production of the Th2-type cytokine IL-5 relative to WT mice. In addition to CD4+ T cells, humoral immune responses are also critical for the clearance of P. murina, and because IL-5 has been shown to be important in antibody (Ab) production, we examined pathogen-specific Ab production in WT and STAT4-/- mice. STAT4-deficient mice had increased production of anti-P. murina IgG1, IgG2b, and IgG2c leading to enhanced opsonic killing by alveolar macrophages ex vivo compared to serum of WT mice. These data suggest that increased anti-P. murina Ab production, which enhances alveolar macrophage-mediated killing, may overcome T cell defects in the absence of STAT4, resulting in unimpaired clearance of P. murina.
Pregnancy establishment depends on a pre-existent population of thymic-derived uterine Foxp3+ cells while pregnancy maintenance relies on de novo converted regulatory T cells (71.1)
Authors: Zenclussen Ana , Teles Ana , Leber Anne , Tadokoro Carlos
Published: January 2023

Pregnancy represents a challenge for the maternal immune system; it has to be alert against pathogens while tolerating paternal alloantigens in fetal structures. The maternal immune system is aware of these alloantigens, actively tolerating them to ensure the survival of the fetus. Regulatory T cells (Treg) are reportedly involved in allotolerance towards paternal antigens. In mouse models, the transfer of antigen-specific Treg can protect from immunological rejection of the fetuses. However, Treg generation and indispensability for pregnancy remained unclear. Treg accumulate during the receptive phase of the estrous cycle as documented by in vivo imaging using a 2-photon microscope. Treg depletion prior to and during very early pregnancy (foxp3.DTR mice) leads to impaired implantation, while their depletion at later time points does not interfere with gestation. Helios+ cells leave the thymus and accumulate in blood and uterus-draining lymph nodes during early pregnancy. At later stages, Foxp3+ Treg are generated de novo in the periphery as we demonstrated in a Rag-model of cell transfer. While naturally occurring Treg are needed for pregnancy establishment, their absence at later stages is compensated by the generation of Foxp3+ cells from Foxp3- cells. Our data provides new insights in the generation/ function of Treg during pregnancy, which is essential to understand natural mechanisms of tolerance acquisition.
Molecular characterization of wheat antigens involved in celiac disease (71.13)
Authors: Srinivasan Bharani , Focke-Tejkl Margit , Swoboda Ines , Constantin Claudia , Mittermann Irene , Pahr Sandra , Vogelsang Harald , Huber Wolf-Dietrich , Valenta Rudolf
Published: January 2023

Wheat gliadins trigger a hypersensitive immune response in genetically susceptible individuals causing celiac disease (CD). The small intestinal mucosa of CD patients is characterised by presence of numerous infiltrating CD4+ T cells. These T cells proliferate and secrete IFN-γ when stimulated with gliadin extracts and are implicated in the mucosal damage and production of anti-gliadin antibodies. Peptide sequences in the Pro(P) and Glub(Q) rich regions of gliadins are known to be T cell-stimulatory but due to the heterogeneity of gliadin sequences, several potential stimulatory epitopes and the individual protein antigens are yet to be identified and characterized. Thus our aim was to identify and characterize wheat antigens with ability to initiate and sustain celiac disease. Due to their complex biochemistry the three subtypes of gliadins α/β, γ, and ω-gliadins are difficult to isolate into pure fractions. Hence, we developed a method wherein the alcohol extracted gliadins was sub-fractionated ion-exchange chromatography and each sub-fraction’s reactivity to serum IgA, from clinically well defined CD (active/diet) patients and non-CD patients was analyzed. We identified and generated recombinant gliadins that were CD specific. Epitope mapping studies revealed major immune-reactive regions. Recombinant gamma-gliadins will be useful for characterizing the immune response to wheat antigens and to develop reliable diagnostic and therapeutic strategies.
Increased small intestinal and colonic permeability, and loss of villus tip surface area, correlates with microbial translocation and immune activation in HIV (71.5)
Authors: Stubblefield Park Samantha , Sung Hannah , Funderburg Nicholas , Meddings Jon , Levine Alan
Published: January 2023

As early diagnosis and viremic control of HIV becomes more prevalent, the clinical focus has turned to preventing co-morbidities that include cardiovascular, hepatic, neurological, and metabolic abnormalities and immune exhaustion. Immune activation, as measured by leukocyte activation and soluble inflammatory mediators in circulation, is increased in HIV patients, even those on effective HAART therapy, and is a better predictor of disease progression and of all-cause mortality than viral load. Intestinal permeability, which often leads to microbial products in the bloodstream, may provide a cause for chronic inflammation in HIV patients. Here, we provide corroborating evidence of small intestinal and colonic permeability in a cohort of HIV patients with increased LPS, determined by measuring the excretion of various saccharides. Levels of lactulose and mannitol (small intestinal permeability) and sucralose (colonic permeability) were elevated (p=0.0014 and p<0.001, respectively) in the urine of HIV patients, independent of HAART status. Further analysis of the saccharide excretion profile revealed that HIV patients have a loss of villus tip surface area in the small intestine as compared to controls (p<0.001). These results were correlated to markers of bowel damage and microbial translocation (LPS Binding Protein, EndoCAb, sCD14, I-FABP) and immune activation (CRP, IL-6, IFN-α) and to clinical parameters in hopes of identifying key risk factors for disease progression.
IL-12 induced Fas expression in myeloid-derived cells within tumors delivers a co-stimulatory signal for adoptively transferred CD8+ T cells (74.10)
Authors: Kerkar Sid , Restifo Nicholas
Published: January 2023

Engineering CD8+ T cells to deliver IL-12 to the tumor site can lead to striking improvements in the ability of adoptively transferred T cells to induce the regression of established murine cancers. We have recently shown that IL-12 triggers an acute- inflammatory environment that reverses dysfunctional antigen presentation by myeloid- derived cells within tumors and leads to an increase in the infiltration of adoptively transferred antigen-specific CD8+ T cells. Here, we find that local delivery of IL-12 increased the expression of Fas within tumor-infiltrating macrophages, dendritic cells, and myeloid-derived suppressor cells and that these changes were abrogated in mice deficient in IL-12 receptor signaling. Importantly, up- regulation of Fas in host mice played a critical role in the proliferation and anti-tumor activity of adoptively transferred IL-12 engineered CD8+ T cells. We also observed higher percentages of myeloid-derived cell populations within tumors in Fas deficient mice, indicating the resistance for stromal collapse in the absence of signaling through the death receptor Fas. Taken together, these results describe the requirement for co-stimulatory reverse signaling through Fasl on T cells that successfully infiltrate tumors, a mechanism likely triggered by the induction of Fas expression on myeloid-derived cells by IL-12 and the subsequent collapse of the tumor stroma.
Oncogenic collaboration with IL-6 in the induction of spontaneous germinal centers (74.11)
Authors: Rosean Timothy , Tompkins Van , Waldschmidt Thomas , Janz Siegfried
Published: January 2023

Multiple myeloma (MM) is the second most common hematological malignancy in the United States, and represents one percent of all cancers. Despite ongoing research, MM remains incurable, with an average life expectancy of five to eight years after diagnosis. In order to better understand MM development, our lab has created a novel mouse model of plasmacytoma (PCT) which mimics key features of MM. This mouse model contains two transgenes. The first recapitulates a T(12:15) Myc translocation, while the second is a copy of human IL-6. The resultant mouse, designated iMyc/IL-6, develops PCT around three months of age with full tumor penetrance. Interestingly, both IL-6 and iMyc/IL-6 mice develop robust spontaneous germinal centers (GC) by two months of age. Notably, the addition of the iMyc oncogene in iMyc/IL-6 mice results in an increased frequency of GC B cells. Correlating with the development of spontaneous GCs, both IL-6 and iMyc/IL-6 mice develop a robust follicular T helper (Tfh) cell response which becomes more pronounced as mice develop tumors. These results may provide important clues into the role of the T cell response in the development of MM. Our current research is focused on elucidating the role of the Tfh response in the development of PCT.
TSLP driven inflammation fosters breast tumor development (74.2)
Authors: Marches Florentina , Wu Te-Chia , Xu Kangling , Pedroza Alexandro , Zurawski Sandy , Banchereau Jacques , Zurawski Gerard , Liu Yong-Jun , Palucka Karolina
Published: January 2023

Solid tumors are often associated with aseptic inflammation. There are two types of inflammation that have opposing effects on tumors, chronic inflammation that promotes cancer survival and metastasis, and acute inflammation which triggers cancer destruction. Chronic inflammation is often linked with the presence of type 2-polarized macrophages induced by Th2 cytokines, IL-4 and IL-13. Our studies have demonstrated the presence in breast cancer tumors of inflammatory Th2 cells, which produce IL-13, IL-4, and TNF. These CD4+ T cells appear to play a key role in the disease as they accelerate breast tumor development in a xenograft model through the production of IL-13. Breast tumors appear to play a critical role in conditioning the infiltrating myeloid DCs (mDCs) to induce such inflammatory Th2 cells. Our recent results suggest that thymic stromal lymphopoetin (TSLP) plays a role in mDCs conditioning. Breast cancer cell lines and primary tumors from patients show TSLP protein expression. TSLP-neutralizing antibodies block the upregulation of OX40L by mDCs exposed to tumor supernatant and consequently block mDCs capacity to generate inflammatory Th2 cells in vitro. Targeting pattern recognition receptors on mDCs renders them resistant to TSLP signal. These mDCs don't phosphorylate STAT6, don't express OX40L and turn on the secretion of IL12p70. The final outcome is the block of inflammatory Th2 and tumor rejection. Thus, TSLP could serve as therapeutic target in breast cancer.
Involvement of Toll-like Receptor 2 (TLR2) in self-renewal of mouse mammary cancer initiating cells (CIC) (74.8)
Authors: Cavallo Federica , Conti Laura , Lanzardo Stefania , Arigoni Maddalena , Forni Guido , Calogero Raffaele
Published: January 2023

CIC are a subpopulation of cancer cells with stem cell properties that may be responsible for tumor progression, metastasis, resistance to therapy, and tumor recurrence. It is thus conceivable that the identification of the molecular pathway and antigens directly involved in the maintenance of the stem-cell like features of CIC could open a new and promising therapeutic opportunity for anti-cancer interventions. We enriched CIC from a mammary carcinoma cell line derived from a tumor of ErbB2 transgenic mice (TuBo cells) generating subsequent passages of mammospheres. We compared their transcriptional profiling with that of the bulk population of TUBO cells and focused our attention on a cluster of 183 up-regulated transcripts. Among them there were growth factors and immune system-related genes, including toll-like receptor 2 (TLR2). The inhibition of TLR2 signaling strongly impaired in vitro mammospheres generation and in vivo tumorigenicity, demonstrating the involvement of TLR2 in CIC self-renewal. In conclusion, this study provides a genomic characterization of mammary CIC and shows that TLR2 might have a role in initiation and progression of breast cancers.
Dysregulation in p38 signalling contributes to altered phenotype and function in Nrf2 deficient dendritic cells (P5025)
Authors: Aw Yeang Han Xian , Al-Huseini Laith , Sethu Swaminathan , Hamdam Junnat , Goldring Christopher , Park Kevin , Sathish Jean
Published: January 2023

Nrf2 is a redox sensitive transcription factor vital for the maintenance of cellular homeostasis. Dendritic cells (DCs) are potent APCs responsible for the initiation of an immune response. Our previous study highlights the role of Nrf2 in DC function (increased costimulatory receptors expression and T cell stimulatory capacity) and intracellular signaling (such as ROS, NFκB and MAPKs). The p38 MAPK pathway plays a pivotal role in the up regulation of costimulatory receptors (CD86 & MHC II). However, it is unclear how Nrf2 affects the integrity of this pathway. In the current study using bone marrow-derived immature DCs from Nrf2 deficient (KO) and wild type mice, we investigate the effect of Nrf2 on various signaling events in the p38 pathway and associated functional responses. Our results indicate that the loss of Nrf2 in DCs resulted in elevated basal p38 phosphorylation. Furthermore, an increase in basal phosphorylation of CREB and ATF-1 - downstream targets of p38, was also observed. This was coupled with increased IL-10 cytokine production at basal conditions and also upon exposure to LPS. Increased costimulatory receptor expression, antigen specific T cell activation propensity and LPS stimulated IL-10 production observed in the KO DCs can be reversed by pharmacological inhibition of p38. These results suggest that deficiency of Nrf2 in DCs leads to dysregulation of the p38 signaling pathway contributing to the altered phenotype and function of these DCs.
Monocytes express the serine protease granzyme B - potential involvement in antigen processing and presentation. (P5033)
Authors: Schuldhaus Christopher , Beyer Thamara , Kaltenmeier Christof , Lindner Stefanie , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: January 2023

In recent years, dendritic cells derived from monocytes (mo-DC) have been extensively studied as novel cell-therapeutic candidates for the treatment of various tumors. In order to elicit tumor-specific CTL responses, DC need to be capable of cross-presenting antigens. Recently, the serine protease granzyme B (GrB) was found to be crucially involved in cleavage and cross-presentation of antigens. We therefore hypothesized that monocytes may be able to express GrB under certain circumstances, comparable to other DC precursors including mDC and pDC. Using various methods including FACS analysis, ELISpot and Western immunoblot we found that monocytes can express GrB depending on various external stimuli. Importantly, we found that the TLR4 ligand LPS appears to play a central role in the induction of GrB in monocytes, and that the presence of antigen-stimulated B cells synergistically enhances GrB expression in monocytes. Moreover, when maturing monocytes into mo-DC, the IL-2 family cytokine IL-7 further supported the expression of GrB in the emerging mo-DC population. This is the first report describing the expression of GrB protein in monocytes and mo-DC in response to various cytokine and TLR stimuli. Our results may open novel approaches to the improvement of mo-DC-related cellular vaccines based on the expression of certain proteases such as GrB.
Understanding MHCII-restricted T cell receptor cross-reactivity to foreign peptides (P5011)
Authors: Nelson Ryan , Beisang Dan , Taylor Justin , Jenkins Marc
Published: January 2023

CD4+ T cells bearing unique T cell receptors (TCRs) recognize antigens in the form of peptides bound to major histocompatibility complex class II proteins (p:MHCII). In mice, structural studies of peptides bound to the MHCII molecule I-Ab have revealed that p:I-Ab complexes consist of a core peptide of at least 9 amino acids. Five of the positions within the nonamer core interact with the peptide binding groove of I-Ab and orient the 4 TCR contact amino acids so that they are accessible for TCR recognition. However, the rules that govern this TCR recognition and cross-reactivity for p:MHCII complexes are largely unknown. Using a p:MHCII-tetramer based approach, we tested the limits of MHCII-restricted TCR cross-reactivity by studying two nonamer peptides with 4 identical TCR contact amino acids but different I-Ab anchor residues. To our surprise, we found that 80% of T cell clones could not discriminate between the two peptides. When we expressed one of the peptides as a self peptide in a transgenic mouse model, we found that the T cell response to immunization with the other peptide was decreased by over 50%. These results could explain how TCR cross-reactivity can account for repertoire selection as well as autoimmunity induced by molecular mimicry.
HLA-F and MHC-I open conformers cooperate in an MHC-I antigen cross presentation pathway. (P5015)
Authors: Geraghty Daniel , Goodridge Jodie , Lee Ni , Burian Aura , Pyo Chul-woo , Tykodi Scott , Warren Edus , Yee Cassian , Riddell Stanley
Published: January 2023

Peptides that are presented by MHC class I (MHC-I) are processed from two potential sources - newly synthesized endogenous proteins for direct presentation on the surface of most nucleated cells and exogenous proteins for cross-presentation typically by professional antigen presenting cells. Here we present data that implicates the non-classical HLA-F and open conformers of MHC-I expressed on activated cells in a pathway for the presentation of exogenous proteins by MHC-I. This pathway is distinguished from the conventional endogenous pathway by its independence from TAP and Tapasin and its sensitivity to inhibitors of lysosomal enzymes, and further distinguished by its dependence on MHC-I allotype-specific epitope recognition for antigen uptake. Thus, our data from in vitro experiments collectively support a previously unrecognized model of antigen cross-presentation mediated by HLA-F and MHC-I open conformers on activated lymphocytes and monocytes, which may significantly contribute to the regulation of immune system functions and the immune defense.
HIV-1 envelope glycoprotein variable loop 4 and 5 are indispensable for envelope structural integrity (P5017)
Authors: Tingting Yuan , Zhang Mei-Yun , Li JingJing
Published: January 2023

HIV-1 envelope glycoprotein (Env) is a trimer of non-covalently linked heterodimer of gp120 and gp41 subunits. Gp120 is composed of five conserved regions (C1-5) that are interspersed with 5 variable regions (V1-5). Despite less conserved nature of the sequences, variable loops may be functionally important for virus entry and constitute neutralization determinants. To investigate the importance of loops for Env biology, we generated a series of loop deletion mutants and investigated the effect of each loop deletion on Env structural integrity, and on Env expression, cell surface display, virus packaging and entry into permissive cells. We found that loop V4 and V5 were important for envelope structural integrity. Replacement of V4 or V5 with a flexible linker led to loss of the binding of CD4 binding site antibodies and CD4-induced antibodies to the Env, but enhanced the exposure of the membrane-proximal external region. Although replacement of V4 or V5 with the flexible linker did not affect Env expression in cells, it significantly affected Env display on cell surface, leading to the failure in virus packaging and subsequent entry into permissive cells. HIV-1 Env variable loop V4 and V5 are indispensable for Env structural integrity. Deletion of V4 and V5 significantly affected Env cell surface display, virus packaging and subsequent virus entry into the cells.
Low GILT expression is associated with poor survival in diffuse large B cell lymphoma (P5057)
Authors: Phipps-Yonas Hannah , Cui Haiyan , Haddock Ellen , Deymier Martin , Lybarger Lonnie , Roe Denise , Hastings Karen
Published: January 2023

Diffuse large B-cell lymphoma (DLBCL) is the most common form of aggressive non-Hodgkin lymphoma. Since low MHC class II expression is associated with poor survival in DLBCL, we examined whether expression of other components in the MHC class II antigen processing pathway may be important in DLBCL biology. Gamma-interferon-inducible lysosomal thiol reductase (GILT) reduces protein disulfide bonds in the endocytic compartment exposing additional epitopes for class II binding and facilitating antigen presentation. In four independent gene expression profiling cohorts with a total of 585 DLBCL patients, low GILT expression was significantly associated with poor overall survival. In contrast, low expression of MARCH1, an E3 ligase that regulates class II trafficking, and HLA-DRA, a classical MHC class II gene, were associated with poor survival in one of four cohorts. The association of low GILT expression with poor survival is independent of established clinical and molecular prognostic factors, the International Prognostic Index and cell of origin classification, respectively. GILT protein expression was detected in both tumor cells and tumor-infiltrating antigen presenting cells. These studies demonstrate that GILT expression levels correlate with clinical outcome and underscore that antigen processing is critical to patient survival in DLBCL.
Identification and functional characterization of the antigen presenting cells recognized by tumor-associated regulatory T cells in genetically engineered mouse models of prostate cancer (P5065)
Authors: Shen Lynn , Nishi Saki , Savage Peter
Published: January 2023

The enrichment of Foxp3+ regulatory T cells (Tregs) constitutes a central mechanism of immune regulation, but has also been associated with poor patient prognosis in a variety of human cancers. Previous studies have suggested that Treg development, specificity, and mechanism of action are dependent upon antigen recognition; however, little is known about the identity of the antigen presenting cells (APCs) that are directly recognized by Tregs in vivo. Previously, we have identified an endogenously occurring monospecific Treg population, termed “MJ23 Tregs,” that is highly enriched in the prostate tumors of TRAMP mice. Using MJ23 T cells as a probe, we have determined that despite the presence of multiple MHC class II-expressing cell types within tumor lesions, the antigen recognized by MJ23 Tregs is exclusively presented by a distinct subset of dendritic cells (DCs). In order to determine the functional significance of this interaction, ongoing work aims to determine the impact of DC ablation on Treg localization and function, and on prostate cancer development. In all, our work will determine the extent to which the activity of Tregs within tumor lesions is coordinated in trans by the APC.
Transcriptional and functional conservation between human Langerhans cells and mouse thymic DCs (P5067)
Authors: Klechevsky Eynav , Artyomov Maxim , Munk Adiel , Cella Marina , Tung Thomas , Lam Samuel , Schreiber Robert , Klechevsky Eynav
Published: January 2023

The characterization of human DC subsets is essential for the design of new vaccines and for better translation of mouse immunological data to the clinic. Previously, we showed that human epidermal Langerhans cells (LCs) were highly efficient at priming effector CD8+ T cell responses. However, the ability of mouse LCs to cross-present antigens and drive potent CTL responses is controversial. We used expression profiling to find the human LC equivalent in the mouse. Our studies suggest that the mouse equivalent to the human LC are the two mouse thymic DC subsets. Our studies also reveal a distinct genetic signature of over 25 genes, that could identify and predict which DC subsets can cross-present. The cross-presentation signature was present in the human blood CD141+ DCs and correctly identified the ability of other distinct human and mouse DC subsets to cross present. We confirmed these predictions with the functional ability of DCs to efficiently induce antigen-specific effector CD8+ T cells. Our studies may help bridge differences between mouse and human DCs and facilitate the application of these data to the clinic.
Intracellular detection of antibody-bound viruses and bacteria by TRIM21 (P4159)
Authors: McEwan William , Tam Jerry , Watkinson Ruth , Bidgood Susanna , Hauler Felix , Mallery Donna , James Leo
Published: January 2023

Antibodies are a key component of the adaptive immune response but their activity has previously thought to be limited to the extracellular environment. We have demonstrated that pathogens traffic surface-bound antibodies to the intracellular compartment where they are recognised and neutralized by the high affinity cytosolic Fc receptor TRIM21. In this study we demonstrate that following detection of pathogen-antibody complexes, TRIM21 is able to synthesise K63-linked ubiquitin chains resulting in signalling via NF-κB, AP-1 and IRF3/5/7 pathways. Detection by TRIM21 is sufficient to induce the production of cytokines including CXCL10, IL-6 and IFN-β. Furthermore a wide range of intracellular pathogens, including Salmonella, non-enveloped DNA viruses and RNA viruses can be detected by TRIM21. TRIM21 therefore provides context-dependent detection of intracellular antibody, mediating potent neutralization and signalling functions.
Abnormal neutrophils present in peripheral blood of Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature (CANDLE) syndrome patients. (P4173)
Authors: Biancotto Angelique , Liu Yin , Pelletier Martin , Goldbach-Mansky Raphaela , McCoy J. Philip
Published: January 2023

CANDLE syndrome (CS) is an autoinflammatory disease caused by autosomal recessive mutations in PSMB8. CANDLE involves dysregulation of the IFN signaling pathway and skin infiltrates of immature neutrophils and other cells. We investigated circulating neutrophils from patients with CS for changes in activation or homing markers and quantified the circulating low density granulocytes.. Compared with healthy donors (HD), or matched, paired unaffected parents (PP) carrying one copy of the mutated gene CS patients had increased proportions of immature CD10- neutrophils (7% in CS vs 2.6% in PP and vs 2.4% in HD). In addition, in CS, the reduced proportion of CD10+ mature neutrophils presented an activated phenotype based on expression of CD64 (8.6% vs 4.5% in PP vs 0.9% in HD). We illustrated changes in the maturation activation pattern of neutrophil by calculating the CD10+/CD10- ratio, and showed reduced ratio in CS patients, ratio=13.3 vs 40.1 in PP vs 44.4 in HD. We observed a reduction of CD62L and CD11b/CD18 expression on mature, and increased expression on the immature population. We observed higher circulating LDGs in PBMC fraction of CS (2%) compared to PP (1.1%) or HD (0.7%). The abnormal phenotype of circulating neutrophils, could explain the migration of immature neutrophils to inflammatory skin tissues; however, the overlap of this population with the increased circulating LDG population and their capacity to generate functional responses, remains still unclear.
Evidence for renewal and reconstitution of splenic marginal zone macrophages in young and aged mice (P4181)
Authors: Witte Pamela , Bahamon Erika , Birjandi Shirin
Published: January 2023

Previously, we showed that the spleens of old mice have reduced frequency of MZM and less ability to clear blood-borne particles. One reason for the demise of MZM with age may be lack of renewal, but little is known about turnover of MZM. Here, turnover of MZM was assessed after giving BrdU (injection or in drinking water). Double staining for MARCO and BrdU in spleens from young mice showed that MZM were turning over, either proliferating in situ or derived from dividing precursors. Next, flow cytometry was used to compare the frequencies of BrdU+ MZM per total MZM, from young and old mice. The frequency of BrdU+ MZM was less in aged mice, suggesting that MZM renew more slowly in old than in young. We are interested in the possibility that MZM can be re-established in the aged spleen by cell transfer. Since CCL21 is required to establish MZM, we first compared young and old splenic tissue for expression and staining patterns of CCL21. The distribution was reduced in the PALS and variable in other areas of old spleens; but, total expression was similar between young and old mice, suggesting that sufficient CCL21 may be available in old spleens. We then asked if young bone marrow cells, transferred to intact old mice, could reconstitute MZM in the spleens of old mice. BrdU-labeled young bone marrow cells were used for transfer. The results showed evidence that a low frequency of MZM, derived from young marrow, could establish in intact old mice.
Selenoprotein K interacts with ASAP2 in macrophages to promote phagocytosis (P4151)
Authors: Norton Robert , Huang Zhi , Fay Jeffrey , Hoffmann Fukun , Hoffmann Peter
Published: January 2023

Selenium is a mineral micronutrient that is essential in a variety of biological systems, including immune responses. Selenium is used for biosynthesis of the amino acid selenocysteine, which is used by selenoproteins to carry out biological functions. In humans, 25 selenoproteins have been identified and Selenoprotein K (SelK) was recently identified as an endoplasmic reticulum transmembrane protein important for calcium flux during the activation of immune cells. ASAP2 is an ARF-GAP that localizes to the phagocytic cup when a macrophage engulfs a pathogen. Implementation of the SH3 hunter software predicted that the SH3-binding domain of SelK associates with SH3 domain containing proteins. Furthermore, in an unbiased screen for SelK binding partners we found that ASAP2 immunoprecipitates with SelK. When we tested mutants of SelK with alterations in the SH3 binding domain, we discovered that this interaction was abolished. This indicates that SelK has a functional SH3 binding domain that interacts directly with ASAP2. It has also been established by our lab that SelK KO macrophages exhibit notably impaired phagocytosis of opsonized beads. Studies are underway to determine if the lack of SelK prevents ASAP2 from localizing to the phagocytic cup.
Nectin and nectin-like molecules: immune regulator, adhesion molecule and virus receptors (P1001)
Authors: Gao George
Published: January 2023

Nectin and nectin-like proteins are cell adhesion molecules that mediate the formation of cell adherens junctions by forming homo- or hetero-dimers. Some members of this protein family can also be used by immune receptors to mediate immune recognition. For instance, nectin-like 2 (Necl-2) is used as a ligand for the immune system by interaction with the immune receptor CRTAM (class-I MHC restricted T-cell associated molecule), which is mainly expressed on the surface of cytotoxic lymphocyte cells. However, the Necl-2/CRTAM binding mode and its relationship to cell adhesion are not known. Here, we report a Necl-2/CRTAM complex structure, demonstrating that Necl-2 binding to CRTAM competes with the dimerization of CRTAM and possibly Necl-2. Necl-2 occupies the CRTAM homodimer interface, making homodimerization impossible. Mutational and functional analyses identified key amino acids (double "lock-and-key") responsible for the binding. Our work illustrates how the cell adhesion molecule Necl-2 competitively binds the immune receptor CRTAM. Otherwise, the nectin proteins can also be employed as the virus receptors, such as herpes simplex virus and measles virus.
Regulation of TNFR:TNFRL interactions mediates CD8+ T cell clonal competiton (P1013)
Authors: Burchill Matthew , Tamburini Beth , Marrack Pippa , Kedl Ross
Published: January 2023

The ability to respond to self and foreign antigens requires a network of signals between antigen presenting cells (APCs) and responding T lymphocytes. In our studies, we demonstrate that access to TNF-ligands confer a competitive advantage to CD8+ T cells in response to foreign antigen. Previous work has demonstrated that immunization of mice deficient in CD27, the receptor for CD70, results in a severely restricted antigen-specific CD8 T cell repertoire. We show that adoptively transferred CD8+ T cells are able to compete against endogenous antigen-specific CD8+ T cells more effectively than CD27-deficient CD8+ T cells. To dissect the mechanism by which CD27 expression confers a competitive advantage to CD8+ T cells, we were able to demonstrate that upon engagement of CD27 by CD70, CD27 is lost from the surface of CD8+ T cells and captured by CD70 expressing dendritic cells, effectively blocking access to CD70. These data demonstrate a novel mechanism by which CD8+ T cells of the same specificity can compete against one another by regulating access to TNF-ligands on APCs. Furthermore, multiphoton microscopy revealed CD27 expression is critical to maintain stable T cell:APC interactions. Collectively, our data demonstrate that T cells compete against each other for access to the TNF-ligands, an interaction which affects the duration and potency of T cell:DC interactions and thus influences the repertoire of responding CD8+ T cells to self or foreign antigens.
GITR potentiates anti-viral T cell immunity by limiting regulatory T cells (P1015)
Authors: Clouthier Derek , Wortzman Michael , Watts Tania
Published: January 2023

The immune system must be tightly balanced to control infection while avoiding immune pathology. We investigated the role of the glucocorticoid-induced tumour necrosis factor receptor (GITR) during infection of mice with persistent lymphocytic choriomeningitis virus (LCMV) clone 13 infection. Infection of wild-type (WT) and GITR-/- mice with LCMV revealed that GITR plays a critical role in T cell responses and viral control. At 8 days post-infection (dpi), GITR-/- mice showed 3-fold fewer LCMV-specific CD8+ T cells, and these cells expressed significantly higher levels of inhibitory receptors Tim-3 and PD-1. These defects became more striking at 45 dpi, when there was also a substantial deficit in multifunctional (IFNγ+CD107a+TNFα+) CD8+ T cells in GITR-/- relative to WT mice. GITR-/- mice had 35-fold higher viral load in the kidney 8 dpi and 10-fold higher at day 45. Depletion of CD4+ T cells ameliorated the immune defects observed in GITR-/- mice, suggesting that a CD4+ population largely contributed to the GITR-/- phenotype. Strikingly, there were 4- to 5-fold more CD4+Foxp3+ regulatory T cells (Tregs) in the spleen and lymph nodes of GITR-/- mice 8 dpi, and only slightly more at 45 dpi. The dramatic increase in Treg numbers in GITR-/- mice 8 dpi is accompanied by a more suppressive phenotype. These studies demonstrate that GITR plays an indirect, yet critical role in CD8+ T cell responses and viral control by limiting the accumulation and function of Foxp3+ Tregs.
Irradiation at a young age induces immune-modulation through NF-κB pathway on spleen cells in old age mice (P3044)
Authors: Yoshida Yasuhiro , Song Yuan , Okazaki Ryuji
Published: January 2023

It is reported that irradiation induces shortened lifespan in mice with dose-dependent. We also previously reported that young-age irradiated mice induces shorten lifespan compared to non-irradiated mice. In addition, the number of lymphocytes in the S- and G2/M- phases was significantly increased in the irradiated mice. However, the detail mechanism is still not fully understood. In the present study, we demonstrated that irradiation at young age induces immune-modulation of splenocytes in old age mice. The p53+/- mice were exposed to 3 Gy at 8 weeks of whole-body irradiation and were sacrificed at about 54 weeks of age. The expression of CDK2 was increased in the splenocytes of the mice irradiated at 8 weeks. Electrophoretic mobility shift assay demonstrated increased activated-NF-κB in the splenocytes of the mice irradiated at 8 weeks but not at 17 and 30 weeks mice. Cell viability assay shows constant activation of splenocytes in the mice irradiated at 8 weeks both female and male mice compared to non-irradiated mice. In contrast to this, splenocytes of the mice irradiated at 17 and 30 weeks were similar activities to non-irradiated mice. Finally, IL-6 production from splenocytes was measured by ELISA and irradiated mice have high production of IL-6. Taken together, these results suggest that young age irradiation induced immune-modulated response in the second lymphoid organ, which may relate to be activation of NF-κB.