bluuebunny/crossref_metadata_2025 huggingface dataset:
The frequency of circulating HSV-2 antigen-specific T cells and antibodies is stable and remains unchanged by HSV-2 reactivation in infected individuals (105.54)
Authors: Skoberne Mojca , Larson Shane , Zielinski Veronica , Long Deborah , Warren Terri , Lundberg Amy , Hetherington Seth , Flechtner Jessica
Published: January 2023

HSV-2 infection is characterized by chronic intermittent shedding of virus and episodic clinical outbreaks. T cells and antibodies play a critical role in controlling the infection. While patients possess T cell reactivity to multiple HSV-2 antigens, reactivity to specific antigens may be associated with control of clinical disease. Recurrence of clinical disease is episodic, but little is known about the temporal variation in peripheral T cell responses to HSV-2. T cell and antibody responses to two antigens previously associated with asymptomatic infection were studied among 27 HSV-2 seropositive individuals with a history of HSV outbreaks by IFN-γ ELISPOT and endpoint titer antibody ELISA, respectively. Peripheral blood was collected 4 times, 3 weeks apart and within 48 hours of any genital lesion outbreak. Patients were categorized as high or low responders. None of high and 9 of 23 low T cell responders experienced clinical outbreaks. Immune responses did not vary significantly during the study period or during clinical outbreaks. The correlation of immune response and subclinical disease is currently under evaluation. These results indicate that in chronic HSV-2 infection, levels of peripheral T cells and antibodies remain consistent over time and are not boosted during viral outbreaks. This supports the hypothesis that a vaccination strategy to increase T cell responses to select HSV-2 antigens may provide a therapeutic benefit to the patients.
Immunization with MOG fused to a novel single chain fragment variable (scFv) for murine DEC-205 suppresses allergic experimental encephalomyelitis (EAE) in mice. (106.12)
Authors: Ring Sabine , Enk Alexander , Mahnke Karsten
Published: January 2023

The antigenreceptor DEC-205 is expressed by Dendritic cells (DC) and greatly increases antigen presentation. We have shown that in vivo loading of DC with antigen via anti-DEC-205:antigen conjugates induces immunosuppressive regulatory T-cells (Treg). Therefore we set out to create single chain fragment variables (scFv) specific for DEC-205 fused with the EAE antigen MOG, to target non-activated DC for induction of tolerance to EAE in vivo. A non-binding anti-b-gal scFv:MOG was created as control. ScFv:MOG fusions proteins were expressed and purified, and immunohistochemical staining of cytospins from isolated CD11c+ cells displayed a positive staining for DEC-scFv, colocalizing with MHC class II. For functional testing DEC-scFv:MOG was injected into mice. Lymph node DC were isolated thereafter and cocultivated with MOG-specific (2D2) T-cells. Strong T-cell proliferation was induced by DC from mice injected with DEC-scFv:MOG in contrast to controls. When analyzing the T-cell populations in DEC-scFv:MOG injected mice, we found enhanced numbers of CD4+CD25+FoxP3+ Treg (16% of CD4) as compared to control animals (10% of CD4). Most importantly, when EAE was induced in either DEC-scFv-MOG-injected or in control mice, none of the DEC-scFv:MOG injected mice developed EAE symptoms. In contrast, all animals in the control group developed a severe EAE. Thus, these data indicate that targeting of MOG to steady state DC in vivo prevents EAE by a Treg-driven mechanism.
Internalization of Poly(dG)-antigens by dendritic cells (106.14)
Authors: Narciso Jo Erika , Toussaint Hélène , Erdos Geza , Donahue Cara , Falo Louis
Published: January 2023

Effective vaccine design depends in part on the ability to target specific antigens (Ag) to Ag presenting cells (APCs), including dendritic cells (DCs). Further, to generate effective antitumor and antiviral immunity, vaccine platforms should enable efficient MHC class I and class II restricted antigen processing leading to synergistic CD4 and CD8 T-cell responses. To facilitate targeted vaccine delivery, we have developed Poly(dG)-protein Ags. These Ag conjugates have the potential to greatly increase vaccine efficiency by inducing spontaneous aggregation of Ag into particulate complexes with intrinsic adjuvant function. The resulting particulate structure includes exposed phosphate groups with affinity for scavenger receptors expressed by APCs. We have successfully conjugated Poly(dG) residues to the model Ag ovalbumin (OVA) and found that Poly(dG)-OVA can be rapidly endocytosed, and efficiently presented to class II-restricted T cells, and cross-presented to class I-restricted T cells in vitro and in vivo. We compared DCs internalization of Poly(dG) conjugated and unconjugated Ag using confocal microscopy and found that Poly(dG)-OVA bound to the cell surface of DCs with higher affinity and was internalized several times more efficiently than Ag alone. These results suggest that coupling Poly(dG) to Ags enables efficient APCs targeting. Understanding the mechanisms of internalization, processing, and presentation of Poly(dG)-Ags will contribute to future vaccine design.
Biodegradable polymer, PLGA, is an efficient vehicle for class II MHC-restricted antigen presentation and the induction of DC maturation (106.15)
Authors: Lee Chong-Kil , Park Dong-Eun , Kim Uji , Park Chan-Su , Kim Ki-Hyang , Im Sun-A
Published: January 2023

The antigen presentation and maturation activities of antigen-encapsulated biodegradable nanoparticles were compared with that of antigen-antibody immune complexes. Dendritic cells (DCs) were allowed to uptake ovalbumin (OVA) via pinocytosis, phagocytosis or endocytosis, and then the levels of MHC-restricted OVA peptide presentation were measured using OVA specific CD4 and CD8 T cells. Poly(D,L-lactic acid-co-glycolic acid, PLGA)-encapsulated OVA (pOVA) and OVA-IgG complexes (OVA-IC) were equally efficient in inducing MHC class I and II-restricted OVA peptide presentation. Comparison of the amount of OVA peptide presentation showed that class II-restricted presentation pathways were robust process compared to class I-restricted presentation pathways. Maturation inducing activities of pOVA and OVA-IC were also examined. pOVA was as efficient as or better compared to OVA-IC in inducing the expression of cell surface molecules such as class II, CD80, CD86, and the secretion of cytokines IL-6, IL-1β and TNF-α. These results show that the biodegradable polymer, PLGA, is an efficient vehicle not only for class II-restricted antigen presentation but also for induction of DC maturation. The present study also shows that in exogenous antigen presentation by DCs, cross presentation is a much weaker phenomenon compared to MHC class II-restricted presentation.
Intestinal dendritic cells survey circulatory antigens prior to induction of regulatory CD8α/β T cells (106.2)
Authors: Song Joo Hye , Change Sun Young , Guleng Bayasi , Cotoner Carmen , Arihiro Seiji , Karp Christopher , Kweon Mi-Na , Sharpe Arlene , Bhan Atul , Terhors Cox , Reinecker Hans-Christian
Published: January 2023

Background: Antigens in the intestine transition from fenestrated capillaries through the lamina propria into the draining lymphatics, but whether or how this process controls mucosal immune responses is unclear. CX3CR1+ DCs are uniquely positioned for the recognition of environmental and circulatory antigens in the lamina propria as they interact with both the epithelium and with the capillary vessel system in the lamina propria. Results: We demonstrate that small intestinal CX3CR1+ dendritic cells (DCs) acquire and process both circulatory and luminal antigens in the lamina propria. Through cross-presentation of antigen, CX3CR1+ DCs induce differentiation of IL-10 secreting CD8α/β T regulatory cells, that is dependent upon Programmed Death ligand 1/2 and its counter-structure PD1 of CD8 T cells. Differentiated CD8+ T regulatory cells represent a subset of intraepithelial lymphocytes (IELs) which express the specific adhesion molecule CD103. The CD8+ Tregs inhibit antigen specific CD4+ T cell activation through IL-10 secretion and prevent CD45RBhigh CD4 T cell-mediated small intestinal inflammation. Conclusion: Our finding shows that the lamina propria CX3CR1+ DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of self- and intestinally- absorbed antigens leading to the induction of CD8+T regulatory cells, which govern tolerance in the mucosal immune system.
Central and overlapping role of cathepsin B and Inflammasome Adaptor ASC In antigen presenting function of human dendritic cells (106.22)
Authors: Dhodapkar Kavita , Guo Xiuyang
Published: January 2023

Inflammasomes are molecular platforms that trigger processing of proinflammatory cytokines. Inflammasomes have also been implicated in regulating adaptive immunity. How inflammasome activation relates to the maturation and antigen presentation in human dendritic cells (DCs) is unknown. Here we show that DC maturation stimuli lead to rapid but transient activation of caspase-1 in human monocyte-derived DCs. RNAi mediated inhibition of the inflammasome component ASC leads to marked inhibition of the capacity of lipopolysachharide (LPS)-matured DCs to stimulate antigen-specific T cells. RNAi mediated inhibition of Cathepsin B (CatB) also similarly inhibits the capacity of human DCs to stimulate immunity. The defective ability of ASC or CatB deficient DCs to stimulate T cells is independent of inflammasome-mediated processing of inflammatory cytokines such as IL1 and includes DCs loaded with preprocessed peptide. Gene expression profiles of ASC or CatB deficient human DCs show marked overlap with down-regulation of several genes implicated in DC function, but differ from those recently shown to be altered in ASC deficient murine DCs. These data demonstrate an important role for ASC and CatB in regulating function of human DCs. In addition to their well-studied roles in the cytosol (such as inflammasome and lysosome function), surprisingly overlapping effects of these proteins on gene expression may also contribute to their roles in the immune system.
The MHC class I antigen processing components TAP and ERAAP sculpt the MHC class II-restricted self peptidome and modulate the CD4+ T cell receptor repertoire impacting T helper responses to microbial pathogens. (106.24)
Authors: Spencer Charles , Dragovic Srdjan , Niu Xinnan , Sette Alessandro , Link Andrew , Joyce Sebastian
Published: January 2023

Humans deficient in TAP or ERAAP function present with recurrent infections despite elevated CD4+ T cell numbers. As CD4+ T cells regualte multiple immune response, their actions are critical to anti-microbial responses. Recently, studies have identified a role for TAP and ERAAP in generating influenza, Listeria and minor histocompatibility antigen peptides recognized by CD4+ T cells. Though the MHC class I-restricted self peptidome is known to be regulated by TAP and ERAAP, the affect on MHC class II-restricted self peptidome is unknown. Here we show that the MHC class II-restricted self peptidome is dramatically altered by the activities of TAP and ERAAP. The CD4+ T cell repertoire correspondingly had profound alterations in the diversity of the TCR CDR3 region that defines antigen specificity. This altered CD4+ T cell repertoire led to altered class II-restricted peptide recognition pattern in TAP-deficient mice. Though some peptides were recognized at an enhanced magnitude, the recognition of others was either lost or newly acquired. Taken together these data suggest that TAP and ERAAP sculpt class II-restricted peptidomes and thereby affect the CD4+ T cell repertoire. The recognition of a new, yet overlapping, immunome by TAP deficient CD4+ T cells demands further investigation to understand the extent of interaction between the MHC class I and class II antigen processing pathways and their effects on anti-microbial responses particularly in TAP deficient individuals.
HLA-DO promotes bacterial superantigen binding to MHC class II molecules (106.27)
Authors: Pezeshki Abdul Mohammad , Azar Georges , Denzin Lisa , Mourad Walid , Thibodeau Jacques
Published: January 2023

HLA-DO (H2-O in mice) is an intracellular non-classical MHC class II molecule. It forms a stable complex with HLA-DM (H2-M in mice) and by regulating its activity shapes the peptide repertoire. Using a stable cell line overexpressing HLA-DO (HeLa-CIITA-DO), we show here that HLA-DO improved binding of SEA and TSST-1 superantigens (SAgs) to MHC class II molecules. Binding of SEB was not affected in these conditions. Exogenous pulsing of Class II-associated invariant chain peptide (CLIP) elucidate that CLIP is a major player in this process. Accordingly, HLA-DO knock-down using specific siRNA, decreased SEA and TSST-1 binding in HeLa-CIITA-DO cells. Furthermore, silencing of DM increased SEA and TSST-1 binding in HeLa-CIITA and 293-CIITA cells. Shutting down of Ii reversely decreased binding of SEA and TSST-1 in 293-CIITA cells. However, in HeLa-CIITA, Ii depletion just decreased SEA binding but does not affect TSST-1 binding. H2-O did not show the same boosting effect on SAgs binding in splenocytes. In conclusion, our results show that HLA-DO can improve SEA and TSST-1 binding in line with a role for CLIP in SAgs binding.
HLA-DO and its effect on peptide binding to MHC Class II (106.29)
Authors: Poluektov Yuri , Kim AeRyon , Sadegh-Nasseri Scheherazade
Published: January 2023

True function of HLA-DO (DO), an MHC Class II accessory molecule remains a mystery. The leading model on it function proposes that DO inhibits the effects of DM. Most of those studies were have used in vivo mouse models, looking at the endpoint of peptide presentation, not taking into account the individual steps in the process. To directly study DO function, we designed a recombinant soluble DO that included leucine zipper for better dimerization of the DOα- and β- chains and expressed it in insect cells. Kinetic experiments involving DR1 and the peptide with or without DM and DO revealed unexpected results. We found that; a) DO diminishes the binding of two peptides that are DM-sensitive, b)DO has positive effects on binding of peptides that form DM-resistant complexes, c) DO only has an effect on peptide association and virtually no effect on peptide dissociation, and d) DO functionsdirectly on DR molecules.We propose that DO binds to the peptide-receptive MHC II molecules that DM generates either by dissociating the bound peptides, or opening of the empty groove. The role for DO then is to enforce another layer of control on peptide selection. In support of this hypothesis, using BIACore Surface Plasmon Resonance, we have measured direct binding of DO to an open conformation of HLA-DR1 in real time, while no binding to a closed DR1 conformation wasdetected.
miRNAs regulate epidermal Langerhans cell cross-presentation (106.3)
Authors: Mi Qing-Sheng , Xu Ying-Ping , Qi Rui-Qun , Zhou Li
Published: January 2023

Langerhans cells (LCs) are skin-residential dendritic cells (DCs) with a life cycle distinct from other types of DCs and place very important roles in skin immunity and tolerance. MicroRNAs (miRNAs), evolutionarily conserved small non-coding RNAs that repress target genes, regulate immune cell development and function. Recent studies from our laboratory and others indicated that miRNA deletion in DC-lineage interrupts the homeostasis and function of epidermal LCs with normal conventional DCs, suggesting that miRNAs are required for LC development and function. However, the roles of individual miRNAs in regulating LC development and function are still completely unknown. miRNAs miR-150 and miR-223 are highly expressed in mature lymphocytes and myeloid cells, respectively. We have found that miR-150 and miR-223 are also expressed in LCs and the expression of miR-150 is significantly down-regulated during in vitro LC maturation. Using miR-150 and miR-223 knockout mouse models, we have found that lack of miR-150 or lack of miR-223 does not disturb the development, maturation, migration and phagocytic capacity of epidermal LCs. However, lack of miR-150 significantly reduces the capacity of LCs to cross-present a soluble antigen to antigen-specific CD8+ T cells, while lack of miR-233 significantly increases the capacity of LC cross-presentation. Thus, our data indicate that miR-150 and miR-223 are not required for LC development, but differentially regulate LC cross-presentation.
Tapasin-assisted peptide loading and the nature of the HLA-A2(T134K) assembly defect (106.36)
Authors: Rizvi Syed , Raghavan Malini
Published: January 2023

MHC class I molecules are assembled in endoplasmic reticulum (ER) with the help of a peptide loading complex (PLC). The PLC includes specific assembly factors, the transporters associated with antigen processing (TAP) and tapasin, and generic ER chaperones, calreticulin and ERp57. Previous studies have shown that the T134K mutation of HLA-A2 (A2) interferes with cell-surface expression. It has been suggested that reduced cell-surface expression of A2(T134K) is due to impaired interactions of A2(T134K) with tapasin. We expressed the A2(T134K) in a tapasin-deficient cell line (M553), and compared assembly and surface expression of the mutant relative to that of wild-type A2. In M553 cells, the A2(T134K) was profoundly impaired in tapasin-induced cell surface expression. Surprisingly, the A2(T134K) was able to bind to tapasin and TAP. However, compared with A2, assembly of A2(T134K) with β2m was reduced, and ER retention of the mutant was correspondingly enhanced, consistent with impairment in intracellular peptide loading. Similar association kinetics were observed for the interactions of purified empty soluble versions of A2 or A2(T134K) with peptides. On the other hand, both peptide-deficient and peptide-loaded forms of the A2(T134K) were found to be more thermostable compared to wild-type A2. These findings indicate that stabilizing conformational changes induced by the A2(T134K) mutation interfere with tapasin-assisted optimization of the A2(T134K) peptide repertoire.
Murine MHC class Ib gene T11: a functional paralog of T23/Qa-1? (106.42)
Authors: Chen Lili , He Xiao , Kumanovics Attila , Fairbanks Jared , Jensen Peter
Published: January 2023

MHC class Ia molecules, H2-K/D/L in mouse, select CD8 T cells during development and present peptides to appropriate CD8 T cells in immune responses. Mouse MHC class Ib H2-Q, T, M regions contain more than 20 protein-encoding genes, but only few have been studied. Qa-1, encoded by H2-T23 gene, is one of the best-studied MHC class Ib. Qa-1 presents peptide generated from MHC Ia to NKG2A/CD94 receptors and provides inhibitory signal to NK cells. Here, we report the study of another MHC class Ib gene, H2-T11. T11 is 95% identical in DNA level and 87% identical in protein level to T23. T11 mRNA was detected in spleen, thymus and uterus in B6 mice, and its expression pattern is more limited than that of T23. Recombinant T11 was refolded with β2m, with or without the exogenous Qdm peptides. The refolded T11 had a typical secondary structure of MHC class I proteins, shown by circular dichroism analysis. The refolded T11 bound Qdm in vitro and could be recognized by a Qa-1 specific mAb. T11 was ectopically expressed on the surface of several cell lines, including a Tap deficient one. The T11 expressing cells could not present insulin to Qa-1 restricted, insulin-specific T cell hybridoma, in contrast to T23 expressing cells. Our results indicated that despite a high similarity to T23, T11 might have different function from T23/Qa-1. We are in the process of eluting naturally bound peptides from T11 expressing cells, and using T11/Qdm tetramers to look for its potential ligand(s).
Characterization of a human CD1d-knockin mouse (106.44)
Authors: Yuan Weiming , Wen Xiangshu , Rao Ping , Kim Seil , Cresswell Peter
Published: January 2023

CD1d-restricted natural killer T (NKT) cells regulate the immune system in response to a broad range of diseases. The CD1d/NKT antigen presentation pathway is largely conserved between human and mouse, however, there is distinct difference between the two species. To better study human CD1d antigen presentation in an in vivo setting, we have generated a human CD1d knock-in (KI) mouse. We have expressed human CD1d (hCD1d) in place of mouse CD1d (mCD1d). The expression of hCD1d was verified on CD4+CD8+DP thymocytes and thymic dendritic cells, which are involved in NKT cell positive and negative selections, respectively, in thymus. CD1d-α-GalCer tetramer+ invariant NKT (iNKT) cells have been shown in thymus, spleen and liver. However, reduced numbers of iNKT cells were observed in these organs compared to that in wild-type mice. Among these iNKT cells, Vβ8 has been over-represented comparing with wild-type mouse, suggesting hCD1d preferentially selects for mouse Vβ8 chain, which is highly homologous to human Vβ11 chain. In vitro presentation of various glycolipids by BMDCs from hCD1d KI mice showed that hCD1d is functional in presenting different groups of lipids. Furthermore, lipid administration in the KI mice showed that in vivo hCD1d-restricted NKT cells are functional. In summary, our hCD1d KI mouse can be a novel model for in vivo studying hCD1d-specific antigen presentation in antitumor and antimicrobial immunity as well as autoimmune diseases.
Similar repertoires of T follicular helper and conventional CD4 T cells are generated in response to protein immunization. (P772) (106.47)
Authors: Leddon Scott , Sant Andrea
Published: January 2023

T follicular helper (Tfh) cells potentiate high-affinity, class-switched B cell responses, the predominant correlate of protection for vaccines. However, little is known about the epitope specificities of Tfh cells generated during polyclonal responses. To date, studies of epitope-specific Tfh cells have relied on either the transfer of TcR transgenic cells or assays involving reagents to stain TcR (tetramer or antibody). In order to comprehensively evaluate polyclonal responses generated from the natural endogenous TcR repertoire, we developed a sorting strategy to separate Tfh cells from CD44highnon-Tfh cells and measured epitope-specific responses with cytokine-specific ELISPOT assays. The immunodominance hierarchies of both Tfh and non-Tfh cells generated in response to immunization with several model antigens (MalE, HEL, and OVA) were remarkably similar. Strikingly, the frequency of cytokine producing Tfh cells at the peak of expansion, independent of the epitope measured, was approximately 6 to 12-fold higher then non-Tfh cells. In agreement with previous studies of unfractionated CD4 cells from our group, increasing the kinetic stability of peptide-MHC class II complexes enhances the priming of Tfh cells. By understanding the parameters that control epitope specific priming, vaccines may be tailored to enhance or focus Tfh responses to facilitate optimal B cell responses.
A novel form of T cell antagonism by trapping of immunological synapse (106.49)
Authors: Carreño Leandro , Riquelme Erick , González Pablo , Espagnolle Nicolas , Riedel Claudia , Valitutti Salvatore , Kalergis Alexis
Published: January 2023

T cell activation results from productive T cell receptor (TCR) engagement by a cognate peptide-MHC (pMHC) complex on the antigen presenting cell (APC) surface, a process leading to the polarization of the T cell secretory machinery towards the interface of APC. We have previously shown that the half-life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters determining T cell activation. Here, by using APLs conferring different half-lives to the TCR/pMHC interaction we have tested how this parameter can control T cell polarization. We observed that only TCR/pMHC interactions with intermediate half-lives can promote the assembly of synapses that lead to T cell activation. Strikingly, intermediate half-life interactions can be competed out by short half-life interactions, which can efficiently promote T cell polarization and antagonize T cell activation that was induced by activating intermediate half-life interactions. However, short TCR/pMHC interactions fail at promoting phosphorylation of signaling molecules at the T cell-APC contact interface, which are needed for T cell activation. Our data suggest that while intermediate half-life pMHC ligands promote assembly of activating synapses, this process can be inhibited by short half-life antagonistic pMHC ligands, which promote the assembly of non activating synapses.
Particles such as an asian sand dust activate autophagic and apoptotic key molecules in CD11b positive cells. (106.6)
Authors: Yoshida Yasuhiro , Song Yuan , Ichinose Takamichi
Published: January 2023

Asian sand dust (ASD) is derived from air pollutants in East China and has a long history of appearing in Japan. Recently, it is knowing that ASD contains microbial materials, sulfate (SO(4)(2-)) and nitrate (NO(3)(-)), therefore there is increasing concern for its possible adverse health effects. Although it has been reported that ASD induces inflammatory responses in the lung tissue and causes respiratory diseases such as eosinophilia in mice, the effect of ASD on the peripheral lymphoid organ are not fully understood. In this study, we examine the effect of ASD on immune system. ICR mice were administered intratracheal ASD (0, 0.2, 0.02 mg/mice), and were sacrificed at 4 weeks (1 time per week and total 4 times) after administration. Spleen cells were prepared and cell lysates of splenocytes were resolved in western blotting. Immuno-blotting demonstrated that ASD induced autophagic-maker LC3 and p62 expression and also induced PARP cleavage suggesting caspase-dependent apoptosis in splenocytes. In addition, ASD could increase the expression of TNF-α mRNA analyzed by Real-time PCR. As same as in vivo, we observed ASD enhanced TNF-α production induced by ConA in splenocytes. Taken together, ASD induced systemic inflammation resulting in autophagic and apoptotic phenomena in splenocytes, suggesting that ASD administration not only triggered pulmonary inflammation but also affected peripheral lymphoid tissue.
The human invariant chain p35 favors formation of nonameric complexes with MHC class II molecules (107.1)
Authors: Cloutier Maryse , Fortin Jean-Simon , Thibodeau Dr Jacques
Published: January 2023

The invariant chain (Ii) associates with MHCII alpha beta (αβ) chains after biosynthesis. Although previous studies described (αβIi)3 nonameric complexes, a recent report suggested that Ii trimers could only bind a single MHCII heterodimer at once. (Roche et al, 1991, Koch et al, 2011) We have investigated this discrepancy in the context of two human Ii isoforms, p33 and p35. The latter includes an ER retention motif (RXR) that is masked by MHCII β cytoplasmic tail upon its association with Ii. Using transfected HEK293T cells and flow cytometry, we monitored ER egress of various combinations of MHCII and Ii variants. First, we showed that MHCII only masked Ii RXR motif if associated directly (in cis) to the p35 subunit. Second, our results indicated that while p33 homotrimers may exit the ER as pentamer with one MHCII, p35-p33 mixed-trimers require cis masking of every Ii RXR motif before egress, thereby favoring heptamers and nonamers formation. Indeed, sucrose gradient of cell extracts confirmed larger complexes with p35 compared to p33. Altogether, our data argue for a kinetic model in which p33 homotrimers are free to exit the ER at any time, independently of the number of associated MHCII. In contrast, ER retention of Ii trimer containing p35 allows for binding of multiple MHCII until every RXR motifs are inactivated.
HCV and HIV stimulate production of the inflammatory cytokine IL-18 through activation of the Nalp3 inflammasome in monocytes (108.12)
Authors: Chattergoon Michael , Levine Jordana , Cox Andrea
Published: January 2023

Activation of the inflammasome-complex leads to production of the pro-inflammatory, inflammasome-associated cytokines IL-18 and IL-1β. These cytokines amplify the innate antiviral immune response and skew the developing adaptive immune response. We previously reported that IL-18 is the earliest host response to hepatitis C virus (HCV) infection and remains elevated at low levels in chronic infection. Our group has also reported an association between IL-18 SNPs and likelihood of HCV clearance. Despite these observations, the role of IL-18 in control of viruses capable of chronic infection is unknown. We now show that IL-18 is also elevated in persons with uncontrolled HIV and to a lesser extent in those with natural immunologic control of the virus. To understand inflammasome activity during chronic viral infection, we studied the interaction of HCV or HIV infected plasma and PBMC subsets from HIV and HCV uninfected subjects. We determined that (1) monocytes cultured with plasma from HIV or HCV viremic individuals produce IL-18 and other pro-inflammatory cytokines; (2) the virion is the inflammasome stimulating component within plasma, (3) siRNA knockdown of Nalp3 in monocytes modulates HCV and HIV induction of IL-18. Our data elucidate mechanisms by which HCV and HIV activate the inflammasome complex and demonstrate the production of inflammatory cytokines through the interaction of HIV and HCV and monocytes.
NEMO recruitment of polyubiquitinated TBK1 to MAVS builds signalosome to regulate innate anti-viral immunity (108.3)
Authors: Wang Lingyan , Li Shitao , Berman Michael , Kong Young-Yun , Dorf Martin
Published: January 2023

RIG-I-like receptors (RLRs) are intracellular sensors utilized by nearly all cell types for recognition of viral RNA, initiation of antiviral defense, and induction of type I interferons (IFN). TBK1 is a critical kinase implicated in RLR-dependent IFN transcription. The E3 ligases Mind bomb 1 (MIB1) and Mind bomb 2 (MIB2) conjugate TBK1 with K63-linked polyubiquitin on residues K69 and K154. TBK1 polyubiquitination is critical for recruitment of the downstream adaptor, NEMO. After viral infection NEMO and IRF3 are independently recruited to MAVS. NEMO bridges TBK1 to MAVS through ubiquitin binding domains. The assembly of the NEMO/TBK1 complex on MAVS leads to activation of TBK1 kinase activity and phosphorylation of the transcription factor, IRF3. In contrast, activation of NF-kB is not impaired by MIB deficiency. The combined results refine current views of RLR signaling, define the role of TBK1 polyubiquitination and detail the assembly of the MAVS signalosome.
Bacterial DNA-induced anti-apoptotic c-IAP-2 protects human monocytic cells against HIV-Vpr-mediated mitochondrial membrane depolarization and apoptosis through TRAF 1/2 via sequential inhibition of pro-apoptotic Caspase 8, Bid and Bax (108.7)
Authors: Saxena Mansi , Busca Aurelia , Kumar Ashok
Published: January 2023

Monocytic cells are important HIV reservoirs and exhibit marked resistance to apoptosis upon infection, thereby hindering clearing of pathogens. However, the mechanism underlying the development of resistance to apoptosis in these cells is poorly understood. Using Vpr-52-96 amino acids peptide (Vpr), a HIV accessory protein, as a model for apoptosis, we have previously shown that prior treatment with CpG or E.coli DNA prevented Vpr-mediated apoptosis by induction of anti apoptotic c-IAP-2 gene via calmodulin-dependent kinase II (CAMK-II) activation in human monocytes and promonocytic THP-1 cells. Herein, we show that c-IAP-2 induced by bacterial DNA in a CaMK-II dependant manner, protects against Vpr-mediated mitochondrial depolarization. Vpr-induced mitochondrial depolarization and apoptosis is mediated by TRAF1/2 downregulation and subsequent activation of proapoptotic caspase-8, bid and bax. Treatment of cells with bacterial DNA, through c-IAP-2 induction, prevented these effects of Vpr, thereby protecting against Vpr-mediated mitochondrial depolarization and apoptosis. Overall, these results suggest a key role played by bacterial DNA-induced c-IAP-2 in preventing Vpr-mediated mitochondrial depolarization and apoptosis through prevention of TRAF 1/2 downregulation and sequential inhibition of caspase 8, bid and bax. Furthermore, strategies based on suppressing c-IAP-2 by targeting CaMK-II, TRAF1/2 or Caspase-8 may be helpful in controlling reservoir formation.
Altered class switch recombination junctions in patients with deficiency in Mlh1 and Brca1 (109.6)
Authors: Bjorkman Andrea , Du Likun , Lindblom Annika , Pan-Hammarstrom Qiang
Published: January 2023

Class switch recombination (CSR) is a B-cell specific process that results in the change of immunoglobulin isotype from IgM to IgA, IgG or IgE. It involves the creation of DNA double strand breaks (DSBs) in switch regions, which are sensed and repaired by DNA damage response proteins and the non-homologous end-joining (NHEJ) pathway. Here we have studied CSR in patients with heterozygous mutations in both the mismatch repair (MMR) gene Mlh1 and the breast cancer susceptibility gene Brca1 and in patients with mutations in either Mlh1 or Brca1 alone. Brca1 is involved in several processes ranging from DNA DSB repair, particularly through homologous recombination, and cell cycle checkpoint control to ubiquitination and chromatin remodeling. Its function in NHEJ as well as in CSR is unknown. The MMR pathway normally repairs mismatches arising in the DNA, while during CSR it seems to be involved in the creation of DSBs by processing of mismatches in the switch regions. To study CSR in the patients we have analyzed the in vivo generated recombination switch junctions, formed after the repair of two switch regions during CSR. We have found that the switch recombination junctions are altered in all patients, suggesting the importance of these proteins during CSR in human B cells.
Antigenic ligand density modulates execution of naïve CD8+ T cell response programme (110.1)
Authors: Gund Rupali , Ebenezer Chitra , Thyagarajan Krishnamurthy , Chaudhry Ashutosh , George Anna , Bal Vineeta , Rath Satyajit
Published: January 2023

Upon brief exposure to antigenic stimulation, naïve CD8+ T lymphocytes execute an autonomous programme of several rounds of proliferation and maturation into effector cytotoxic cells. We have studied the role of ligand density as a modifier of this program using T cell receptor (TCR)-transgenic CD8+ T cell activation by short-duration stimulation in vitro. Over the entire ligand density range studied, we observe a graded increase in the frequency of responder naïve T cells that upregulate the expression of CD69, an early activation marker. However, the levels of induced CD69 are similar at all ligand densities. When CD69+ T cells are purified at early stages and cultured, most undergo successive proliferation cycles, showing that the execution of the autonomous programme during CD8+ T cell response is independent of ligand density. However, at very low ligand densities, the efficiency with which CD69+ T cells enter proliferation is poor and slow, leading to lower cell yields. At these ligand densities, post-activation TCR internalization is also inefficient. At any given ligand density, if activated CD69+ T cells that have internalised TCR are separated from those that have not internalised TCR, TCR-high cells show a lag in proliferation regardless of the ligand density. Together, these data indicate that ligand density quantitatively modulates the lag phase of the autonomous proliferation-differentiation programme of CD8+ T cells via TCR internalization-dependent mechanism.
Differential localization of T-bet and Eomes within distinct human CD8 T-cell memory populations (110.10)
Authors: McLane Laura , Cosma Gabriela , Paley Michael , Wherry E. , Makedonas George , Betts Michael
Published: January 2023

In mice, two T-box transcription factors, T-bet and Eomes, drive the differentiation of CD8 T-cell lineages; however, little is known regarding their role in human CD8 T-cell differentiation. Here, we characterized T-bet and Eomes expression and localization within human CD8 memory T-cell populations. We find T-bet and Eomes are broadly expressed in human memory CD8 T cells, with increasing levels of T-bet and Eomes strongly correlating with differentiation from central memory to effector memory and effector subpopulations. Interestingly, T-bet levels directly correlate to subcellular localization, with a higher propensity for nuclear expression of T-bet within T-bethi cells and predominately cytoplasmic expression in T-betlo cells. In contrast, Eomes appears to be localized solely to the nucleus. In murine CD8 T cells, T-bet can be nuclear, cytoplasmic, or both; however, Eomes is detectable in both compartments suggesting differential regulation compared to humans. Additionally, in humans, Eomes tightly correlated with nuclear T-bet suggesting T-bet localization might impact Eomes expression. Together, our data provide a novel mechanism for CD8 T-cell differentiation in humans and mice based on subcellular localization of T-bet, and reveal the likely presence of previously unknown regulation mechanisms for these critical lineage defining transcription factors.
Defining the early effector CD8 T cell population: plastic cells capable of populating the short-lived and memory pools. (110.11)
Authors: Plumlee Courtney , Obar Joshua , Lefrançois Leo
Published: January 2023

Naïve antigen specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations at the peak of the response, which include memory precursor effector cells (MPEC) and short-lived effector cells (SLEC). Shortly after infection, a third population called early effector cells (EEC) predominate in the antigen specific response and give rise to both the MPEC and SLEC populations. To understand if EEC populations were pre-committed to either an MPEC or SLEC fate, we purified EEC from mice infected with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV), in which the ratio of EEC/SLEC/MPEC at the peak of the response is known to be different. Transfers of sorted EEC into uninfected hosts revealed that EEC were pre-programmed to differentiate based on early signals received from the different infection settings. Additionally, gene expression analysis revealed that a subset of genes was differentially regulated between EEC derived from VSV or LM infections. Surprisingly, when these same EEC were transferred early into mismatched infected hosts, the transferred EEC were diverted from their original commitment programming. These results delineate a model of differentiation where EEC are programmed to form MPEC or SLEC, but where additional inflammatory signals can alter this program. Understanding the molecular regulation of the development of an EEC to a memory cell will facilitate rational vaccine design.
The CD8+ T cell response to infection is dominated by the progeny of few naïve T cells (110.12)
Authors: Gerlach Carmen , Rohr Jan , Perié Leila , van Heijst Jeroen , van Rooij Nienke , Velds Arno , Urbanus Jos , Hauptmann Michael , Jacobs Heinz , de Boer Rob , Schumacher Ton
Published: January 2023

Following infection, antigen-specific CD8+ T cell numbers increase dramatically. Analysis of antigen-specific T cell responses at the bulk cell level has provided insight into the average number of cell divisions that T cells undergo upon activation. However, it is unclear whether all naïve T cells of a given affinity for antigen produce equal numbers of progeny, or whether the total effector T cell pool is predominantly created by the output of few. To quantify how many daughter cells individual naïve T cells produce, we have generated naïve OT-I T cells harboring unique genetic tags (barcodes) and have measured the number of progeny per labeled cell by second-generation sequencing of barcode sequences after infection. Our data reveal that upon infection, individual OT-I T cells that are recruited into the response produce highly variable numbers of daughter cells. These data indicate that the bulk of pathogen protection provided by an antigen-specific T cell response is due to the progeny of only a handful of cells.
Dissociating markers of senescence and protective ability in memory T cells (110.16)
Authors: Prlic Martin , Sacks Jilian , Bevan Michael
Published: January 2023

A set of surface markers is commonly used to predict the potential of CD8 effector T cells to differentiate into memory cells. Similarly, these surface markers together with TNF family member CD27 are frequently used to predict a memory T cell’s ability to mount a recall response. Expression of these markers changes every time a memory cell is stimulated and repeated stimulation can lead to T cell senescence and loss of memory T cell responsiveness. This is a concern for prime-boost vaccine strategies which repeatedly stimulate T cells to increase memory T cell frequency. The molecular cues that cause senescence are still unknown, but cell division history is likely to play a major role. We sought to dissect the roles of inflammation and cell division history in developing T cell senescence and their impact on the expression pattern of commonly used markers of senescence. We developed a system that allows priming of CD8 T cells with minimal inflammation and without acquisition of maximal effector function, such as granzyme expression, but a cell division history similar to priming with systemic inflammation. Memory cells derived from minimal effector T cells are fully functional upon rechallenge, have full access to non-lymphoid tissue and appear to be less senescent by phenotype upon rechallenge. However, these currently used biomarkers to measure senescence do not predict proliferative potential or protective ability, but merely reflect initial priming conditions.
B7-H1 limits the entry of effector CD8 T cells to the memory pool by up-regulating Bim (110.2)
Authors: Gibbons Rachel , Liu Xin , Dong Haidong
Published: January 2023

Studies from cancer and chronic infections show that B7-H1 (PD-L1) engagement with its receptor PD-1 enhances apoptosis of effector T cells. It is not clear how B7-H1 regulates T cell apoptosis and the subsequent impact of B7-H1 on T cell memory generation. In immunized B7-H1 deficient mice we observed increased expansion of effector CD8 T cells and delayed T cell contraction followed by protective CD8 T cell memory capable of completely rejecting tumor metastases in lung tissue. Intracellular staining revealed that antigen-primed CD8 T cells in B7-H1 deficient mice express lower levels of the pro-apoptotic molecule Bim. Injection of B7-H1 or PD-1 blocking antibody into immunized mice reduced Bim levels in effector CD8 T cells. In vitro studies show that engagement of activated CD8 T cells by plate-bound B7-H1 fusion protein led to increased cell death and higher levels of Bim expression. Our results suggest that B7-H1 may negatively regulate CD8 T cell memory by enhancing the depletion of effector CD8 T cells through up-regulation of Bim. Our findings would provide a new strategy to target B7-H1 signaling in effector CD8 T cells to achieve protective immunity in the treatment of cancer and infectious diseases.
Differential roles of CD95L and TRAIL in activation-induced cell death of cytotoxic T lymphocytes (110.22)
Authors: Zhang Jimin , Devadas Satish , Shi Yufang
Published: January 2023

Cytotoxic T lymphocytes can be differentiated into type 1 (Tc1) and type 2 (Tc2) subsets as their T helper counterparts. Interestingly, our investigation in activation-induced cell death mechanisms reveals that CD95L mediates cell death in Tc1 lymphocytes and that Tc2 lymphocytes require TRAIL. Cell death in both subsets is also dependent on caspases. Inhibiting CD95L binding to its receptors in Tc1 lymphocytes from wild type mice displays altered Tc1 lymphocyte death, while Inhibiting TRAIL binding to its receptors in Tc2 lymphocytes shows altered Tc2 lymphocyte death. Activation-induced cell death in both Tc1 and Tc2 lymphocyte subsets can be blocked by pan caspase inhibitors. Moreover, Tc2 lymphocytes differentiated from TRAIL knockout mice show alleviated cell death. Rescued Tc2 lymphocytes secrete significantly higher amounts of IL-4, IL-5 and IL-10 while Tc1 lymphocytes produce higher IFN-γ and TNF-α. Our results indicate that CD95L and TRAIL play differential seminal roles in activation-induced cell death of these two cytotoxic T lymphocyte subsets, and that their different sensitivity to activation-induced cell death can alter CD8+ phenotype. With our study we conclude that Tc1 lymphocyte activation-induced cell death is mediated by CD95L while Tc2 lymphocytes undergo TRAIL-dependent activation-induced cell death.
Regulatory T cells are critical for effector-to-memory transition of CD8 T cells (110.24)
Authors: Kalia Vandana , Khan Arif , Penny Laura , Sarkar Surojit
Published: January 2023

Memory differentiation involves a coordinate downregulation of effector functions, and progressive acquisition of memory properties. It is believed that abrogation of antigenic stimulation of T cells following pathogen clearance serves as a passive means for effector CD8 T cells to shut-off their effector functions, and transition into memory. Based on the well-established suppressive functions of Tregs on CD4 T cells, we hypothesized that Tregs aid memory CD8 T cell differentiation by actively suppressing the effector program in memory-fated cells. To test this hypothesis, we evaluated effector-memory transition of Ag-specific cells in the presence or absence of Tregs. Tregs were transiently deleted after Ag clearance using the FOXP3-DTR transgenic mice, following which key memory properties of recall proliferation upon secondary challenge, polyfunctionality and homeostatic proliferation were assessed. Consistent with our hypothesis, these studies revealed that effector program failed to downregulate in the absence of Tregs, and acquisition of all three functional memory properties was significantly hampered in the absence of Tregs. Removal of Tregs even after acquisition of optimal memory properties also led to a decline in memory function. Collectively, these studies present a novel mechanism by which Tregs serve to generate as well as maintain a functional memory CD8 T cell compartment by actively suppressing the effector program following antigen clearance.
Retinoic acid modulation of CD8 effector and memory differentiation. (110.4)
Authors: Allie S. , Zhang Weijun , Tsai Ching-Yi , Lee Yu-Chi , Noelle Randolph , Usherwood Edward
Published: January 2023

A plethora of work implicates important effects of the Vitamin A derivative, retinoic acid (RA), in myeloid differentiation, while fewer studies explore the role of RA on lymphoid cells. Most work has looked at the role of RA on CD4 T cells, focusing on regulatory CD4 T cell differentiation and maintenance of this phenotype along with some Th1 functions. There is little information about the role of RA in CD8 T cell differentiation, and even less on cell-intrinsic effects in the CD8 T cell. In this study we explored the role of RA on effector and memory differentiation in a direct and cell intrinsic manner in the context of vaccinia virus infection. We used dominant negative RA alpha-receptor (RARα DN) expressing mice and bone marrow chimeric mice containing both WT and RARα DN compartments. We observed the loss of the short-lived effector cell phenotype and an enhanced memory precursor phenotype at the effector and contraction phase of the CD8 response in the absence of RA signaling. Further, the lack of RA increased the proportion of central memory CD8s. Vitamin A is used in conjunction with various vaccines (e.g. Measles vaccine), these results will elucidate the effect of RA in shaping the effector and memory response helping to tailor the supplement to better enhance the outcome of vaccination.