bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple and fast method for the isolation of untouched mouse panDCs from spleen (100.43)
Authors: Tabatabaei-Zavareh Nooshin , Luong Wendy , Thomas Terry , Fairhurst Maureen
Published: January 2023

Dendritic cells (DCs) control immune responses through their robust antigen presenting activity. Steady-state mouse spleen contains three major DC subsets with distinct functional and phenotypic properties including CD8+ and CD8− conventional DCs (cDCs), and plasmocytoid DCs (pDCs). These subsets are present at a very low frequency (< 4%). Typically, elaborate purification protocols such as FACS-based cell sorting or expansion in culture are needed to obtain enough DCs for subsequent studies. Here, we describe a negative selection method to isolate all DC subsets (panDC) from mouse spleen. This method uses immunomagnetic, column-free cell separation technology (EasySepTM). Briefly, non-DCs are labeled for depletion with biotinylated antibodies and cross-linked to magnetic particles using bispecific antibody complexes. The unwanted cells are then removed using an EasySepTM magnet. The selection steps can be fully automated using RoboSepTM . The panDCs are assessed by flow cytometry and defined as Lin−CD11c+ (cDCs) or Lin−CD11cloPDCA-1+ (pDCs). PanDC purities of 80 ± 7% (n=8) are achieved. The rare pDCs are enriched 36-fold with purity of 11.4 ± 1.4% as compared to 0.3 ± 0.1% in the start spleen. Both CD8+ and CD8− cDC subsets are represented in the cDC fraction. The freshly isolated DCs are not activated but upregulate maturation markers upon stimulation with LPS. This method enables fast, easy isolation of panDCs required for immune regulation studies.
Characterization of HLA-DM susceptibility of MHC II-peptide complex in antigen presentation and epitope selection (100.54)
Authors: Yin Liusong , Calvo-Calle Mauricio , Stern Lawrence
Published: January 2023

HLA-DM (DM) is a non-classic major histocompatibility complex II (MHC II), which mediates the exchange of peptides loading to MHC II during antigen presentation. However, the role of DM-mediated peptide exchange in epitope selection is still unclear. In this study, we addressed this question systematically using overlapping peptides from vaccinia virus protein A10L. We measured the binding affinity, intrinsic half life, DM-mediated half life and immunogenecity of 126 A10L peptides bound to HLA-DR1. Correlation coefficient analysis shows that immunogenecity of peptides is very weakly correlated with either binding affinity or intrinsic half life, while strongly correlating with half life in the presence of DM. Moreover, receiver operating characteristics analysis (ROC) indicates that DM-mediated half life is a very strong predictor for epitopes, while binding affinity and intrinsic half life can only mildly predicts epitopes. Taken together, our present quantitative and qualitive analysises demonstrate that DM-susceptibility is a strong and independent factor governing peptide’s immunogenicity.
TIM-3- and TIM-4-mediated phagocytosis of apoptotic cells induces cytokine production in peritoneal macrophages (100.55)
Authors: Bu Xia , DeKruyff Rosemarie , Umetsu Dale , Freeman Gordon
Published: January 2023

TIM-3 and TIM-4 mediate phagocytosis of apoptotic cells by recognizing phosphatidylserine on the surface of the apoptotic cell but are expressed by different cell populations, suggesting each has distinct functions in regulating immune responses. TIM-4 is exclusively expressed on APCs while TIM-3 is expressed on some T cell and APC subsets. We found that TIM-4 was constitutively expressed on mouse resident peritoneal macrophages (PMϕ) and played a predominant role in phagocytosis of apoptotic cells. Following phagocytosis of apoptotic cells, PMϕ secreted CCL2 (MCP-1), CCL3 (MIP-1 α), CCL4 (MIP-1β), CCL7 (MCP-3), CCL9 (MIP-1γ), CXCL-2 (MIP-2), CCL19 (MIP-3), CCL22 (MDC), IL-10 and TGF-β. mRNA levels of these cytokines increased very early (peaking at 0.5-4 hours), but the peak of protein level occurred 48-72 hours after phagocytosis. When TIM-4 on PMϕ was blocked by TIM-4 antibody, phagocytic activity was dramatically decreased and cytokine expression reduced. Following in vivo thioglycollate treatment, expression of TIM-4 by PMϕ was gradually lost, whereas expression of TIM-3 increased. TIM-3-mediated phagocytosis by thioglycollate-induced PMϕ resulted in a generally similar cytokine production pattern compared with TIM-4 mediated phagocytosis by PMϕ, but also resulted in the production of IL-1β and TNF-α. In summary, both TIM-4 and TIM-3 mediated phagocytosis of apoptotic cells and promoted macrophage production of cytokines that further regulates the immune response.
Infection with murine gamma herpesvirus 68 modifies experimental autoimmune encephalomyelitis (101.1)
Authors: Casiraghi Costanza , Horwitz Marc
Published: January 2023

Recent findings have identified Epstein-Barr virus (EBV) as a putative environmental trigger of multiple sclerosis (MS) but the mechanisms by which this virus causes MS remains elusive. We used murine gamma herpesvirus 68 (MγHV), the murine homolog to EBV, to examine how MγHV infection could aid in the enhancement of an autoimmune reaction against the CNS. C57Bl/6 mice were infected with MγHV and five weeks later, when the virus is cleared and has established latency, EAE was induced. Mice previously infected with MγHV developed a more severe EAE course showing both signs of paralysis and other neurological symptoms such as ataxia. MγHV EAE mice had higher levels of IFN-γ and TNF-α in the serum when compared to EAE mice and showed pronounced CD4 and CD8 T cell infiltrations both in the spinal cord and the brain parenchyma. On the other hand, CD4 T cells were less prominent in the brain parenchyma of uninfected EAE mice and CD8 T cells were absent. CD4 and CD8 T cells isolated from the CNS of MγHV EAE mice produced higher levels of IFN-γ accompanied by IL-17 suppression, whereas CD4 T cells isolated from the CNS of EAE mice produced high levels of IL-17 and lower IFN-γ levels. The absence of IL-17 production in infected mice suggests that the disease might be triggered by different T cell subsets than typical EAE. In conclusion, the ability of MγHV to elicit a more pathogenic T cell response could represent a new mechanism through which EBV triggers autoimmunity.
The role of Epstein-Barr Virus Latent Membrane Protein 2A in Experimental Autoimmune Encephalomyelitis (101.12)
Authors: Chang Rhoda , Miller Stephen , Longnecker Richard
Published: January 2023

Multiple Sclerosis (MS) is an inflammatory, autoimmune disease of the central nervous system. The cause of MS is still unknown but epidemiological and immunological studies have implicated Epstein-Barr Virus (EBV), which infects B cells, as a possible etiological agent. Of particular interest is EBV Latent Membrane Protein 2A (LMP2A). Previous work in our lab has demonstrated that LMP2A enhances the expansion and differentiation of B cells upon antigen stimulation, revealing a potential contribution of LMP2A in autoimmunity. As B cells may be a key player in MS, we wanted to examine the role of LMP2A in the animal model Experimental Autoimmune Encephalomyelitis (EAE). Recombinant MOG protein was immunized into transgenic mice in which B cells express LMP2A. LMP2A mice show increased severity and incidence of disease, and faster onset of disease. The explanation of this difference is not due to lymphocyte recruitment into the CNS or differences in serum antibody levels. Further in vitro analysis will address the role of LMP2A in lymphocyte activation and antigen presentation.
Establishment of a sustained delayed type hypersensitivity model with mono-arthritic manifestations in C57Bl/6J mice (101.17)
Authors: Nansen Anneline , Atkinson Sara , Usher Pernille , Markholst Helle , Haase Claus
Published: January 2023

RA is a progressive, inflammatory and destructive autoimmune disease, characterized by chronic synovial joint inflammation. Various RA models of both mono-and polyarthritis have been developed in different inbred strains of mice. However, establishment of sustained arthritis models in the C57Bl/6J mice has been difficult. As most genetically modified mice are bred on the C57Bl/6J background, there is a need to explore new approaches of obtaining robust models in this mouse strain. We have established a sustained DTH model with monoarthritic manifestations in C57Bl/6J mice. The model builds on a classical transient DTH model, where a DTH reaction is elicited by antigen injection into the right hind footpad at 7 day post Antigen sensitisation. Interestingly, we find that administration of a single low dose of a monoclonal antibody cocktail of anti-collagen type II antibodies before elicitation of the DTH reaction results in a sustained footpad swelling that is maintained for at least two weeks. Histopathological examination revealed inflammation and bone destruction with characteristics of arthritis with only the Antigen challenged footpad being affected (monoarthritis). This was not observed in mice receiving an isotype antibody cocktail. Moreover, no mice receiving anti-CII antibodies alone developed polyarthritis. Similar results were obtained using Balb/C mice thus the presence of anti-CII antibodies appears crucial for development of sustained joint tissue inflammation.
PPARγ is required for CD4+ T cell proliferation, survival, and disease-mediation in lymphopenia-associated autoimmunity. (101.21)
Authors: Housley William , Adams Catherine , Clark Robert
Published: January 2023

Ligands for the nuclear hormone receptor PPARγ ameliorate many autoimmune conditions in both mice and humans. Recent reports have also suggested that PPARγ is important in regulatory T cell function and in preventing the development of pathogenic Th17 cells. In contrast to this role of PPARγ in suppressing autoimmunity, we now find that CD4+ T cell PPARγ expression is required for development of autoimmunity under lymphopenic conditions. PPARγ-deficient (PPARγ-/-) CD4+ T cells are unable to mediate disease in both murine graft-versus-host disease and an adoptive transfer model of colitis. In the colitis model, PPARγ is required for the accumulation and cytokine production of CD4+ T cells in the spleen, mesenteric lymph nodes, and colonic lamina propria. This defect is specific for CD4+ T cells as PPARγ-/- CD8+ T cells accumulate equally to wild type CD8+ T cells. Mechanistically, PPARγ-/- CD4+ T cells show diminished in vivo proliferation as assessed via Ki67 staining, but no difference in activation marker expression demonstrating that PPARγ is required for proliferation and cytokine production but not for activation. Endogenous PPARγ-/- CD4+ T cells recover normally after mice are sub-lethally irradiated demonstrating that loss of proliferative potential requires antigen-induced activation as well as lymphopenia. Our results identify a novel function for PPARγ in the proliferation and survival of CD4+ T cells in lymphopenia-associated autoimmunity.
MHC Dextramers enhance the detection sensitivity of antigen-specific autoreactive CD4 T cells (101.22)
Authors: Upadhyaya Bijaya , Massilamany Chandirasegaran , Gangaplara Arunakumar , Reddy Jay
Published: January 2023

Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 cells, detection of autoreactive cells by major histocompatibility complex (MHC) class II tetramers is challenging because of their low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers designated ‘MHC Dextramers’ that contain a large number of peptide-MHC complexes per dextramer molecule. We prepared MHC Dextramers for proteolipid protein (PLP) 139-151 using soluble IAs molecules by coupling them to an activated dextran backbone (kindly provided by Immudex, Denmark). We used PLP-reactive T cells generated from immunized SJL mice to validate the reagents by flow cytometry. Using Theiler’s murine encephalomyelitis virus 70-86 Dextramers as controls, we showed that the MHC Dextramers detect PLP-reactive T cells with specificity, and the detection sensitivity was enhanced by 6-fold when compared with uncoupled tetramers. The MHC Dextramers also allowed us to enumerate the frequencies of antigen-specific cells from immunized mice ex vivo . Similar results were obtained when the antigen-sensitized cells were enumerated in A/J mice immunized with cardiac myosin heavy chain (Myhc)-α 334-352 using IAk MHC Dextramers. Taken together, the data suggest that MHC Dextramers can be efficiently used to track the generation of self-reactive CD4 cells in various experimental systems.
Memory cells specific for myelin oligodendrocyte glycoprotein govern the transfer of experimental autoimmune encephalomyelitis (101.26)
Authors: Williams Jessica , Kithcart Aaron , Smith Kristen , Shawler Todd , Cox Gina , Whitacre Caroline
Published: January 2023

Multiple sclerosis (MS) is a chronic, inflammatory disease of the central nervous system thought to be initiated by CD4+ T cells specific for myelin antigens. Experimental autoimmune encephalomyelitis (EAE), a model of MS, is induced in susceptible mouse strains by immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). MOG35-55-specific T cell receptor transgenic (2D2) mice are susceptible to EAE following immunization with MOG peptide. To better understand the role of T cells in EAE initiation, we explored several approaches for effecting adoptive transfer of EAE using 2D2 T cells. Surprisingly, despite robust proliferation and production of IFN-γ and IL-17 following a variety of culture conditions, EAE was not transferred. Only when 2D2 whole splenocyte preparations contained CD44+ T cells and were Th1-differentiated using MOG peptide and IL-12 did they reliably transfer EAE. Further, we found that CD44hiCD62Llo effector/memory CD4+ T cells are likely responsible for disease transfer due to up-regulation of the adhesion molecule CD44 following differentiation. We also observed a sex dimorphism in 2D2 mice with males transferring more severe EAE, associated with a larger population of effector/memory CD4+ T cells in males compared to females. Given the importance of MOG in MS pathogenesis, mechanistic insights into adoptively transferred EAE by MOG-specific Th1 cells could prove valuable in MS research.
Tissue-expressed coinhibitory B7 molecules can control both adaptive and innate immunity (101.28)
Authors: Zang Xingxing
Published: January 2023

Some new members of the B7/CD28 families were discovered over the last several years and their functions in vivo need to be elucidated. We show that B7x (B7-H4 or B7S1) was expressed in pancreatic b cells but not in immune cells. Using a CD8 T cell-mediated diabetes model, we demonstrate that mice lacking B7x developed more severe diabetes than the control, conversely mice overexpressing B7x in b cells were diabetes free. Pathogenically effector CD8 T cells were capable of migrating to the pancreas but failed to kill target cells and destroy tissue when encountering local B7x. In response to the local B7x these CD8 T cells achieved an overlap yet distinct gene-expression profile from anergic or exhausted CD8 T cells. Our findings reveal that B7x in the periphery abrogates CD8 T cell-mediated tissue destruction. B7-H1 (PD-L1) is widely expressed in immune cells and is important in the coinhibition of T cell function via binding its receptor PD-1. We show that both human and mice intestinal epithelium expressed B7-H1 and mice deficient for B7-H1 were highly susceptible to gut injury and associated mortality and morbidity. Defective B7-H1 led to loss of epithelial integrity and systemic dispersion of commensal bacteria during intestinal inflammation. B7-H1 expressed on parenchymal cells but not on hematopoietic cells controlled intestinal inflammation, which was not dependent on PD-1, B7-1(CD80), and the adaptive immunity.
The role of Th40 cells in EAE mice. (101.29)
Authors: Carter Jessica , Vaitaitis Gisela , Wagner David
Published: January 2023

Auto-aggressive T cells are responsible for the onset and perpetuation of autoimmune diseases, including Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). A subset of CD4 T cells, CD4+CD40+ (Th40) cells, have an expansive role in the Non-Obese Diabetic (NOD) mouse model of T1D. Not only do Th40 cells increase in number with the onset of disease, but they are necessary and sufficient in transferring T1D from NOD to NOD.scid mice. Because Th40 cells have an imperative role in the development of disease in T1D, we considered that Th40 cells might also be involved in Experimental Autoimmune Encephalomyelitis (EAE), a neurological disease which mimics human MS. When given myelin oligodendrocyte glycoprotein (MOG) and pertussis toxin (PT), C57Bl/6 mice develop neurological symptoms and impaired motor functions similar to those in human MS patients. Data shows an increase, not only in CD4 T cells, but also in Th40 cells in mice receiving induction of EAE. This does not occur in their control counterparts. Further, purified splenic Th40 cells from asymptomatic disease induced C57Bl/6 mice rapidly transfer EAE to C57Bl/6.scid recipients with only 1.2x106 cells, demonstrating that Th40 cells alone can induce EAE. Also, we show cerebral infiltrates of immune cells in localized regions of the brain of the Th40 recipient mice, suggesting that these lesions are similar to the lesions found in human MS patients.
Loss of T cell CD98hc specifically ablates T cell clonal expansion and protects from autoimmunity. (102.16)
Authors: Cantor Joseph , Slepak Marina , Ege Nil , Chang John , Ginsberg Mark
Published: January 2023

CD98hc (CD98 heavy chain, 4F2 antigen, Slc3a2) is a lymphocyte activation antigen with unclear function in T cells. Deletion of CD98hc in B cells leads to complete failure of B cell proliferation, plasma cell formation, and antibody secretion. Here we examined the role of T cell CD98 in protective and auto- immune responses by specifically deleting it in mouse T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with reduced T cell clonal expansion. However, initial T cell homing to pancreatic islets was unimpaired. In contrast to B cells, CD98-null T cells showed only moderately impaired antigen-driven proliferation in vitro and nearly normal homeostatic proliferation. Furthermore, these cells were activated by antigen and produced cytokines (CD4) and efficiently killed targets (CD8). The integrin binding domain of CD98hc was necessary and sufficient for full clonal expansion, highlighting the role of adhesive signaling in T cell proliferation and autoimmune disease. When CD98-null T cells were pre-expanded in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. The integrin binding domain of CD98hc is thus required for antigen-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.
L. amazonensis infection differentially affects migration of phagocyte populations from inflammatory site to the draining lymph node (102.27)
Authors: Hermida Micely , Malta Rafaela , Doria Priscila , Taguchi Angela , Utsch Lara , Maciel Maira , Mengel José , dos Santos Washington
Published: January 2023

Leishmania infection interferes with integrin function. In this work we use a model of chronic peritonitis to study the effect of L. amazonensis infection upon the ability of phagocytes populations to migrate from an inflammatory site to the draining lymph node (dLN). First we showed that macrophage recruitment into the peritoneum and migration to the dLN were both maximal 4 days after thioglycollate injection into the peritoneum. At this time point, cell tracking assays using cell transfer between MHC-mismatched mice showed that 1% of the leukocytes injected into the peritoneum migrated to the dLN after 24h. In the mice injected with cells cultivated with medium alone adoptive cells represented 14% of the peritoneal cells. Among these leukocytes 57% were macrophages, 2% were neutrophils and 35% were myeloid dendritic cells. In the LN, 22% of the migrating cells were macrophages, 4% were neutrophils and 25% were myeloid dendritic cells. In the mice injected with leukocytes co-cultivated with Leishmania adoptive cells represented 12% of the peritoneal cells. Among these cells 44% were macrophages, 1% were neutrophils and 46% were dendritic cells. In the LN 19% of the migrating cells were macrophages, 2% were neutrophils and 12% were myeloid dendritic cells. Our data show that: (1) A variety of phagocytes are able to migrate from the inflammatory site to the dLN. (2) Populations of Leishmania-infected dendritic cells appear to be retained in the peritoneum.
Latent CMV and EBV infections and the mobilization of viral-specific and senescent T-cells with exercise (102.6)
Authors: Simpson Richard , Spielmann Guillaume , Bigley Austin , Rector Jerrald , Morrison Mark , LaVoy Emily , Lowder Thomas
Published: January 2023

Exercise mobilizes senescent T-cells into the blood but the impact of latent CMV and EBV infections on this response is unknown. This study aimed to determine if: (1) CMV or EBV specific T-cells are mobilised in response to exercise; and (2) the impact of CMV and EBV on the exercise-responsiveness of senescent T-cells. 24 healthy males (age 28.5 ± 5) completed a 30-min cycling protocol at 85% of maximum power. PBMCs isolated before, immediately after and 1hour after exercise were labelled with MHC class I Pentamers specific to CMV (pp65) or EBV (LMP-2) and assayed for Naïve (KLRG1-/CD28+/CD57-), effector-memory (KLRG1+/CD28+/CD57-) and senescent (KLRG1+/CD28-/CD57+) surface markers. Exercise increased CMV and EBV-specific T-cell numbers by 158% and 200% respectively, and altered the proportions of senescent (+44%) and naïve (-20%) CD8+ T-cells. CMV+ had a greater (+136%) proportion of senescent and a lower (-33%) proportion of naïve CD8+ T-cells compared to CMV-, and demonstrated a greater egress of senescent T-cells in response to exercise. Senescent markers on both CMV and EBV-specific T-cells increased with exercise (+53% and +76% respectively). To conclude, CMV and EBV specific CD8+ T-cells are mobilized into blood with acute exercise and those infected with CMV demonstrate a greater relative egress of senescent T-cells during exercise recovery. Future work will determine the potential impact of exercise in altering senescent T-cell frequency in those infected with CMV
The XS106 cell line: profile of TLR ligand responses reveals a Langerhans cell-like phenotype (102.7)
Authors: Dearman Rebecca , Eaton Laura , Kimber Ian
Published: January 2023

The XS106 dendritic cell (DC) line, isolated from the epidermis of newborn A/J mice, has been characterized with respect to membrane marker expression and responsiveness to Toll like receptor (TLR) ligand (lipopolysaccharide [LPS], peptidoglycan [PGN], CpG, flagellin, R848) activation. Resting XS106 cells were positive for MHC class II, CD80, CD86, CD40, CD11c, CD11b and CD103 but did not express detectable Langerin or EpCAM. Cells expressed constitutively high levels of type 2-associated chemokines CCL17 and CCL22 but little of the type 1-associated chemokines CXCL9 and CXCL10. Stimulation (24h) with LPS, flagellin and PGN markedly up-regulated membrane marker and CCL17 and CCL22 expression, whereas both CpG and R-848 were without significant effect. Enhanced type 1 chemokine expression was inducible by culture with recombinant interferon-γ. Using a functional assay, cells migrated towards CXCL12, but not to CCL19 (ligands promoting migration to the dermis and lymph nodes, respectively). Migration was abrogated if cells were activated with LPS or PGN, but not CpG. Thus, XS106 cells have a relatively immature DC membrane marker phenotype which is not identical to that reported for either of the main skin DC populations (Langerhans cells (LC) or dermal (d)DC). The TLR ligand response pattern shows that XS106 are similar to LC and dDC with regard to their preferential type 2 chemokine profile and their pattern of migration is LC-like.
Dendritic cells elicit allergic immune responses in a CCR7-dependent fashion (103.10)
Authors: Saban Daniel , Schlereth Simona , Lee Hyunsoo
Published: January 2023

DCs are key in mounting allergic immunity by mobilizing from allergen exposed sites to lymphoid tissues for T cell activation. However, whether the CCR7-CCL19/21 axis promotes DC mobilization and Th2 responses in allergy is incompletely understood. We thus sought to determine if CCR7 expression by DCs promotes allergy. Allergic conjunctivitis (AC) was evaluated in wild-type (WT) mice immunized via adoptive transfer with OVA/Alum-primed (WT) T cells. Mice were injected subconjunctivally (SCJ) with exogenous WT or CCR7 KO DCs (or PBS), and immediately instilled with OVA-loaded eye drops to induce AC. Clinical signs were scored biomicroscopically, and sera were sampled for OVA-specific IgE analysis. T cells were isolated for cytokine analyses to in vitro recall stimulation. Lastly, leukocytosis of collagenase-digested conjunctival cells was FACS analyzed. Upregulated CCR7 expression was confirmed of SCJ injected WT DCs found in lymph nodes of immunized mice following instillation of PE-OVA eye drops. Mice with WT DCs showed significant augmentation of clinical signs relative to sham controls. This was corroborated by a 2-fold increase in mast cell and eosinophil infiltration, 4-fold increase in IL-13 and IL-4, and an 8-fold increase in IgE (p<0.05). Strikingly, this augmentation was completely reversed in hosts with CCR7 KO DCs, and this abrogation was consistently seen in all parameters. Thus, our study suggests that DCs promote allergic immunity in a CCR7-dependent fashion.
Galectin-3 modulates T helper responses by regulating dendritic cell cytokine expression (103.11)
Authors: Fermin Agnes , Chen Huan-Yuan , Hsu Daniel , Wang Li Chieh , Wan Lei , Liu Fu-Tong
Published: January 2023

Dendritic cells play a critical role in the initiation and maintenance of inflammatory responses. Galectin-3, a protein found in DCs, is a carbohydrate-binding protein implicated in several cellular processes. In mouse models of asthma and atopic dermatitis, galectin-3-deficient (gal3-/-) mice had significantly fewer infiltrating eosinophils and displayed lower Th2 but higher Th1 responses compared to wild-type mice, suggesting that galectin-3 plays a key role in allergic inflammation. Given the ability of DCs to direct the T lymphocyte response, we hypothesized that galectin-3 may affect immune responses by altering DC functions and tested the hypothesis by comparing wild-type and gal3-/- DCs. OT-II CD4+ cells cultured with OVA-pulsed gal3-/- DCs generated higher Th1 responses relative to gal3+/+ DCs, and the differences were diminished following IL-12 neutralization. Moreover, gal3-/- DCs expressed more IL-12p35 mRNA than gal3+/+ DCs, indicating that gal3 may modulate IL-12 at the transcriptional level or through upstream signaling pathways. T cells co-cultured with gal3-/- DCs also secreted more IL-17 than cells cultured with gal3+/+ DCs, suggesting that gal3 may also negatively regulate Th17 responses. Furthermore, we observed higher IL-6 secretion and IL-23p19 expression in gal3-/- DCs, which may contribute to the enhanced Th17 polarization induced by these cells. We conclude that gal3 may regulate DC cytokine expression, thereby modulating T helper responses.
G0S2, an early response gene, regulates quiescence in naïve T cells (104.10)
Authors: Yamada Takeshi , Lee Ping-Hsien , Burns Audrea , Lacorazza Daniel
Published: January 2023

The G0/G1 switch gene 2 (G0S2) was identified in a screen using mitogen-activated human blood mononuclear cells (DNA and Cell Biology 9, 579, 1990). G0S2 is a small basic protein expressed in hematopoietic cells that is upregulated in autoimmune and inflammatory processes. However, the ability of G0S2 to control T cell proliferation has yet to be determined. We found that G0S2 transcription was suppressed following TCR activation in naïve T cells, suggesting its role in T cell quiescence. Also, inhibition of MAPK, PI3K, mTOR and Ca2+/calcineurin pathways abrogated G0S2 gene suppression, which suggests that the aforementioned pathways are involved in the suppression of G0S2. Moreover, gain-of function experiments, using retroviral gene transfer and BM transplantation, showed that ectopic G0S2 expression inhibited proliferation of T cells during homeostasis and upon TCR activation. Conversely, silencing G0S2 expression enhanced T cell proliferation, indicating that G0S2 maintains quiescence of naïve T cells. We further identified nucleolin as a protein partner of G0S2 by Co-IP and mass-spectrometry. This data suggests that G0S2 interferes with nucleolin-mediated stabilization of mRNAs of genes required for proliferation control. Collectively, our study uncovered an important role of G0S2 in the mechanism of T cell quiescence.
CD5 through CK2 activation regulates B-1a B-cell survival, proliferation, and T-independent type 2 antibody response. (104.15)
Authors: Cashman Kevin , Sestero Christine , De Sarno Patrizia , Raman Chander
Published: January 2023

CD5 expression is the characteristic feature of B-1a B-cells and B10 regulatory B-cells; however, the physiological role of this receptor on the development, persistence, and/or function of these B-cell populations remains undefined. To begin addressing this question we generated a CD5 mutant knock-in mouse (CD5ΔCK2BD) in which the serine threonine kinase, CK2, binding domain in the CD5-cytoplasmic tail was deleted. CD5 plays a critical role in regulating T-cell survival following antigen receptor engagement and this activity is dependent on CK2 activation. In the CD5ΔCK2BD mouse, the ability to activate CK2 is abrogated. B-1a B-cells in the CD5ΔCK2BD mouse turnover at a rate two-fold greater than that in WT mice. This increased turnover rate compensated for the decreased B-cell survival to maintain a near normal B-1a B-cell number in the peritoneal cavity. However, total serum natural IgM and IgA was significantly lower than that in WT mice. CD5ΔCK2BD B-1a B-cells proliferated poorly in response to anti-IgM stimulation but not LPS stimulation. The CD5ΔCK2BD mouse also exhibited an impaired anti-phosphatidylcholine (PC) response following immunization with heat killed S. pneumoniae, a prototypic B-1a B-cell response. Immune responses to T-independent type 1 antigen were not altered in the CD5ΔCK2BD mouse. These data provide the first evidence that CD5 has a direct role in regulating B-1a B-cell physiology.
Mechanisms of defective T cell-dependent B cell responses with age (104.17)
Authors: Yu Mingcan , Li Guangjin , Lee Won-Woo , Yuan Ming , Weyand Cornelia , Goronzy Jörg
Published: January 2023

The ability to generate immune responses after vaccinations declines with age. Efforts to improve vaccine responses have focused on identifying immunogenic vaccines and novel adjuvants. We hypothesized that signaling pathways in T cells can be directly targeted to optimize responses in the elderly. Gene expression profiling of CD4 memory T cell from 65 to 85 year-old compared to young adults stimulated with myeloid dendritic cells plus superantigen showed an overexpression of the dual-specific phosphatase 4 (DUSP4) on days 2 and 3 in the elderly which was confirmed by qPCR (p<0.001), Western blotting (p=0.03) and DUSP4 promoter reporter gene assays (p<0.001). Overexpression of DUSP4 in young T cells shortened CD40L (p<0.001) and ICOS (p<0.001) expression and decreased IL-4 (p<0.001), IL-17A (p<0.001) and IL-21 (p<0.001) production. Conversely, DUSP4 silencing increased expression. Improvement was more pronounced in elderly T cells for CD40L (p<0.001), IL-4 (p=0.007) and IL-21 (p=0.04). CD4 memory T cells from 65 to 85 year-old individuals failed to support differentiation of young adult B cells into CD38+IgD- B cells in vitro (p=0.004). DUSP4 silencing in CD4 T cells restored this ability (p=0.02). The role of DUSP4 expression in CD4 T cells in regulating T cell-dependent B cell responses was confirmed in in vivo studies using CD4 knockout mice reconstituted with DUSP4 overexpressing or control OT-II CD4 T cells and in healthy volunteers undergoing influenza vaccination.
Intestinal inflammation in Gimap5 deficient mice: a possible link between loss of FoxO expression and impaired Treg function (104.18)
Authors: Aksoylar Halil , Lampe Kristin , Hoebe Kasper
Published: January 2023

We previously identified an N-ethyl-N-nitrosourea (ENU) induced mutant, sphinx, which lacks CD8+ T cells and natural killer cells in the peripheral lymphoid organs due to a recessive mutation in Gimap5 gene. Gimap5sph/sph mice developed spontaneous intestinal inflammation that is dependent on CD4+ T cells and commensal microflora. Gimap5sph/sph CD4+ T cells displayed a progressive lymphopenia, adopted a CD44hi CD62Llow activated phenotype and produced increased levels of IL-17. Lymphopenia of CD4+ T cells was accompanied with the progressive loss of FoxO1 and FoxO3 proteins. FoxO transcription factors have recently been implicated in regulatory T cell (Treg) development and function. Here we show that Gimap5sph/sph CD4+ T cells had a selective decline of the FoxP3+ Treg subset with time. Moreover, immune suppressive capacity of Gimap5sph/sph Treg cells was impaired in correlation with the loss of FoxO proteins. Further, adoptive transfer of wild type Treg cells into Gimap5sph/sph mice prevented the development of intestinal inflammation. Together, these results show a possible link between impaired homeostasis of T cells, particularly the Treg subset and loss of FoxO proteins which leads to intestinal inflammation in the absence of Gimap5 function.
Role of MS4a4B in regulation of T cell apoptosis (104.20)
Authors: Xu Hui , Yan Yaping , Zhang Guangxian , Rostami Abdolmohamad
Published: January 2023

MS4a4B is a membrane protein that highly expressed in T cells. We have demonstrated previously that MS4a4B has an inhibitory role in T cell proliferation. Interestingly, a decreased level of MS4a4B is associated with apoptotic T cells. In this study, we further analyzed the role of MS4a4B in T cell proliferation and apoptosis. We co-cultured MS4a4B+ EL4 cells (GFP+) with MS4a4B- EL4 cells (GFP-) and compared cell proliferation by flow cytometry. We found that MS4a4B inhibited EL4 cell proliferation during the early stage of co-culture. However, the percentage of MS4a4B+ EL4 cells was enhanced, suggesting that MS4a4B prolonged EL4 cell survival. To determine if MS4a4B expression reduced apoptosis in EL4 cells, we analyzed apoptotic cells during MS4a4B+/MS4a4B- cell co-culture. The results showed that the apoptosis percentage in MS4a4B+ EL4 cells was markedly lower than in MS4a4B- EL4 cells. To determine whether MS4a4B protected T cells from activation-induced cell death (AICD), we knocked down MS4a4B expression in mouse T cell line (T32 cells) and primary T cells by MS4a4B shRNA vector and MS4a4B siRNA. Knockdown of MS4a4B promoted anti-CD3/anti-CD28-induced apoptosis in T32 cells and primary T cells. Machinery analysis showed that over-expression of MS4a4B decreased the activity of caspase 3/7, caspase 8 and caspase 9. Our data suggest that MS4a4B not only has an inhibitory role in T cell proliferation but also plays a role in regulation of T cell apoptosis.
Circulating antigen presenting cells in chronically infected HBV patients do not constitutively cross-present HBV antigens (105.22)
Authors: Gehring Adam , Haniffa Muzlifah , Konduru Sastry , Ginhoux Florent , Bertoletti Antonio
Published: January 2023

Persistent exposure to antigen is thought to play a primary role in dysfunction of virus-specific T cells in chronic infection. Our study sought to determine whether antigen presenting cells (APCs) in the blood of chronic HBV patients constitutively cross-present circulating HBV antigen to virus-specific CD8 T cells. We isolated dendritic cells, CD14 or CD16 monocytes and B cells from HLA-A2+ healthy donors and chronic HBV patients with 10^8 IU HBV DNA/ml serum. APCs were co-incubated with TCR-redirected T cells specific for HLA-A2 restricted HBc18-27 and HBs183-91 epitopes and activation was monitored by staining for IFN-γ. APCs were also stained with TCR-like antibodies specific for the same peptide/HLA-A2 complexes. We did not observe any HBc18-27- or HBs183-91-specific T cell activation following incubation with sorted healthy donor APCs. Nor did we see T cell activation after co-culture with APCs isolated from chronic HBV patient PBMCs. Staining with TCR-like antibodies confirmed that there were no detectable HBc18-27- or HBs183-91-HLA-A2 complexes constitutively present on the cell surface. IFN-γ production and TCR-like antibody staining was observed with peptide loaded APCs. Our data suggests that APCs in the peripheral blood of chronically infected patients do not constitutively cross-present antigen to virus-specific CD8 T cells and therefore may not contribute to T cell dysfunction.
Fitness costs and diversity of CTL response determine the rate of CTL escape during the acute and chronic phases of HIV infection (105.23)
Authors: Ganusov Vitaly , Goonetilleke Nilu , Liu Michael , Ferrari Guido , Shaw George , McMichael Andrew , Borrow Persephone , Korber Bette , Perelson Alan
Published: January 2023

Human immunodeficiency virus type 1 (HIV-1, or HIV) often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. We used single genome amplification and sequencing of complete HIV genomes to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set-point at 1 year after the infection. We found that the rate of viral escape from CTL responses in a given patient decreases dramatically from the acute infection to the viral set point. Using a novel mathematical model that tracks the dynamics of viral escape at multiple epitopes we show that a number of factors could potentially contribute to a slower escape in the chronic phase of infection, such as a decreased magnitude of epitope-specific CTL responses, an increased fitness cost of escape mutations, or an increased diversity of the CTL response. In the model, increasing the diversity of the CTL response over time can reduce the rate of viral escape from a given CTL response, particularly if CD8+ T cells compete for killing of infected cells or control virus replication non-lytically. Our mathematical framework of viral escape from multiple CTL responses can be used to predict the breadth and magnitude of HIV-specific CTL response that needs to be induced by vaccination to prevent (or reduce) viral escape following HIV infection.
Memory T cell stability in the wake of heterologous viral infections (105.35)
Authors: Che Jenny , Welsh Raymond
Published: January 2023

CD8 T cell memory is generally stable but can be reduced by heterologous infections. CD44hi CD8 and CD4 T cells undergo attrition early during infection with type I IFN-inducing pathogens or after exposure to toll receptor agonists. This attrition coincides with the early reduction of bona fide virus-specific CD8 and CD4 memory T cells and can explain the long-term loss in memory T cells that occurs after heterologous infection. An increase in the percentage of apoptotic cells was detected by TUNEL assay in both CD44hi CD8 and CD44hi CD4 T cells at day 3 post-infection with lymphocytic choriomeningitis virus (LCMV), a strong type I IFN inducer. Infection with LCMV significantly reduced vaccinia (VV)-specific CD8 memory T cells, which were further reduced by heterologous infection with murine cytomegalovirus (MCMV). MCMV also reduced LCMV-specific CD8 and CD4 memory T cells, but LCMV memory was not further eroded by persistent MCMV infection over a 48-week period, even though MCMV-specific memory cells increased in number due to memory inflation. This argued that the early IFN-dependent attrition had a greater effect on memory loss than did competition later on by inflating memory cells. A caveat, however, is that MCMV memory inflation was somewhat impaired by a prior LCMV infection, possibly due to reduced latent MCMV load caused by heterologous immunity. These results illustrate the dynamic nature of T cell memory in the wake of acute and persistent viral infections.
Maintenance of serological memory in virus-infected T cell-deficient mice (105.36)
Authors: Raval Forum , Lamothe Ben , Szomolanyi-Tsuda Eva
Published: January 2023

Many viruses can induce strong acute T cell-independent (TI) B cell responses due to their complex arrays of repetitive antigenic determinants on virions, coupled with the induction of innate cytokines, but T cell help is thought necessary for the development of long term humoral immunity in the form of long-lived plasma cells and memory B cells. However, we have found that T cell deficient (TCRbxd KO) mice persistently infected with polyomavirus (PyV) develop long-lasting high serum antiviral IgG responses, and we questioned whether these mice could generate TI B cell memory. TCR bxd KO PyV-infected mice had very low numbers of antibody secreting cells within the bone marrow compared to wild type (WT) mice. Moreover, by using a functional adoptive transfer assay, memory B cell responses were not detected in T cell deficient mice. In SCID mice reconstituted with TCRbxd KO mouse splenocytes, antiviral serum IgG gradually decreased, presumably due to a lack of supply of naïve B cells from bone marrow. In contrast, CD4 T cell deficient mice lack T cell help but have only short-lived antibody responses, perhaps due to control of persistent antigen by CD8 T cells. Therefore, we hypothesized that a high level of the persistent PyV antigen may subvert immunological tolerance and activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses in T cell deficient mice. Here, we provide evidence for that hypothesis.
Type I Interferon produced by infected cells but not plasmacytoid dendritic cells is required for effective CD8 T cell responses and clearance of a viral infection (105.37)
Authors: Clingan Jonathan , Ostrow Kristin , Hosiawa-Meagher Karoline , Chen Zhijian , Locksley Richard , Matloubian Mehrdad
Published: January 2023

Following viral infection, multiple innate immune pathways can be engaged leading to the production of type I interferons (IFN) and other inflammatory mediators. Two major pathways involved in the sensing of virus include the Toll-like receptor (TLR) pathway, particularly important in plasmacytoid dendritic cells (pDC), and the mitochondrial antiviral signaling protein (MAVS) pathway, activated in infected cells. Although both of these pathways can be activated during a viral infection, the relative contribution of these pathways to the quality of adaptive immune responses and virus control in vivo is not well understood. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we show that while both the TLR and MAVS pathways become activated in pDC and infected cells, respectively, the MAVS pathway contributes more greatly to the type I IFN response, and is required for the production of a high quality CD8 T cell response. There were fewer effector CD8 T cells from MAVS-deficient mice, and these cells had altered expression of activation markers and exhibited a more exhausted phenotype. MAVS-deficient mice also exhibited delayed clearance of virus compared to wild-type mice, or mice deficient in signaling through nucleic acid sensing TLRs. These results demonstrate that although multiple pathways can be activated in response to LCMV, type I IFN derived from infected cells is crucial for an optimal CD8 T cell response and rapid control of infection.
Visualize immune responses during murine cytomegalovirus infection (105.38)
Authors: Qiu Zhijuan , Khanna Kamal
Published: January 2023

Cytomegalovirus (CMV) belongs to herpesviridae family, and can cause severe disease in immunocompromised individual. In the infected host CMV establishes a life-long latent infection even in the presence of concomitant anti-viral immunity. In humans and in mice, CMV infection leads to chronic clonal expansion of antigen specific CD8 and CD4 T cells, a phenomenon known as T cell inflation. The mechanisms that contribute to this phenomenon remain poorly defined. Furthermore, the source of antigen presentation to T cells during latency is not known. Here we used a combination of in-situ MHC class I tetramer staining and adoptive transfer studies to visualize the anatomy of the virus specific CD8 T cells in lymphoid and non-lymphoid organs of mice during primary and latent murine CMV (MCMV)-OVA infection. Our imaging studies revealed a complex spatial and temporal map of antigen presentation in the context of antigen specific CD8 T cell localization during acute and latent CMV infection. These data suggest that viral reactivation likely occurs intermittently and regionally within individual organs. Our studies provide important clues towards the mechanisms responsible for T cell inflation following CMV infection.
Suppression and enhancement of HIV replication in macrophages by CD8+ lymphocytes (105.43)
Authors: Kavanagh Daniel , Rodriguez-Garcia Marta , Levi Karen , Kaufmann Daniel
Published: January 2023

CD8+ cytolytic T cells can suppress HIV replication by a variety of lytic and nonlytic mechanisms, and the relative clinical importance of particular CTL functions is not fully understood. Therefore studies have utilized various in vitro viral suppression assays as an indication of the antiviral efficacy. We measured antiviral activities of CD8+ lymphocytes from elite controllers who spontaneously suppress HIV without medication, from chronically infected HIV+ donors, and from HIVneg donors. Bulk CD8+ lymphocytes containing a CD56+ fraction suppressed HIV replication in autologous macrophages, even for HIVneg donors. CD56-depleted CD8 T cells from HIV+ donors also suppressed HIV replication in macrophage targets, with elite controller T cells exerting significantly greater suppression than progressor T cells. CD56-depleted CD8 T cells from HIVneg donors did not suppress but rather enhanced HIV-replication specifically in macrophages. Finally, when CD8 T cells and HIV-infected macrophages from elite controllers were separated by a transwell membrane, CD8 T cells did not suppress but rather enhanced HIV replication, indicating that HIV suppression was cell-contact dependent while soluble factors may be responsible for enhanced HIV replication. In summary CD8+CD56+ cells have innate antiviral activity in both elite controllers and uninfected donors, while CD8+ T cells have specific antiviral activity in HIV+ donors. In some cases inflammatory CTL may enhance viral replication.