bluuebunny/crossref_metadata_2025 huggingface dataset:
A repertoire independent and cell intrinsic defect in murine GVHD induction by effector memory T cells (169.6)
Authors: Juchem Kathryn , Anderson Britt , Zhang Cuiling , McNiff Jennifer , Demetris Anthony , Caton Andrew , Shlomchik Warren , Shlomchik Mark
Published: January 2023

Graft versus host disease (GVHD) is a major complication of allogeneic stem cell transplantation (alloSCT). In murine models of alloSCT, naïve T cells (TN) cause GVHD, while effector memory T cells (TEM) do not. Why TEM fail to cause GVHD is unknown and whether central memory T cells (TCM) are also unable to cause GVHD is controversial. It has been proposed that a more-restricted T cell receptor repertoire within the memory T cell (TM) pool may limit the ability of TM to recognize alloantigens. To address the role of repertoire, we developed a novel T cell receptor transgenic (Tg) GVHD model in which naive CD4+ TS1 Tg T cells, which recognize an epitope of influenza hemagglutinin (HA), are transferred, along with syngeneic bone marrow, into irradiated transgenic recipients that ubiquitously express HA. We then converted TS1 TN to TM and tested these for GVHD induction. TEM caused minimal, transient GVHD whereas TN and TCM caused potent GVHD. TEM engrafted and accumulated in the spleen to the same extent as TN, but failed to accumulate in the colon. Within the colon, TEM were dividing less actively than TN, and in co-transfer experiments TN out-competed TEM. TEM induced GVHD in recipients that lack expression of PD-L1 and PD-L2, indicating that PD-1, which is unregulated on TS1 cells post transplant, limits the ability of TEM to induce GVHD. Thus, cell intrinsic properties independent of repertoire explain the impairment of TEM while TCM clearly can cause GVHD.
Swine leukocyte antigen diversity in commercial breeding boars: molecular characterization and potential implications on animal health (170.1)
Authors: Ho Chak-Sum , Galina-Pantoja Lucina , Mellencamp Martha , Lee Jun-Heon , Smith Douglas
Published: January 2023

The highly polymorphic swine MHC antigens (SLA) are among the most important determinants of swine immune responses to disease and vaccines, yet their diversity in commercial swine breeding stock has not been studied at the molecular level. Using PCR-based typing assays, diversity of the class I (SLA-1, -2, -3) and class II (DRB1, DQB1, DQA) genes was surveyed in ten pure-line, pedigreed boar populations (n = 413) of a major commercial pig breeder. Of a maximum possible of 623 SLA haplotypes of these loci, 44 class I and 30 class II haplotypes were identified. The three most common class I and class II haplotypes constituted a combined frequency of 33% and 36%, respectively. Half of the studied animals (48% and 53%, respectively) had at least a copy of either of these class I or class II haplotypes. Prevalence of SLA homozygosity varied significantly among boar lines, ranging between 5-70%; overall one in five animals was homozygous for all six SLA loci surveyed. Limited MHC diversity, which has been associated with poor disease outcomes in other species, may result from unintentional selection of the SLA region which links closely to many favorable production traits. Molecular breeding techniques, including the assessment of SLA, may allow breeders to unlink SLA from production traits and reduce its negative pressure. Monitoring SLA diversity in breeding and commercial herds (produced by 2, 3, or 4-way crosses of breeding stock) will ensure robust immune response.
Comparing immune response times and pathogen replication rates across vertebrate host species: a case study in West Nile Virus (170.17)
Authors: Moses Melanie , Banerjee Soumya
Published: January 2023

The vast majority of biological rates and times decrease systematically with increasing animal body size. For example, circulation times, a range of developmental times, and life span are systematically slower in larger animals. We use empirical data and ordinary differential equation (ODE) models of disease progression to show that immune response is independent of host body size, but that viral pathogen replication rates in vivo are systematically slower for larger animals. The models focus on West Nile Virus, but we suggest that this pattern holds more generally. We examine how the sizes and numbers of lymph nodes and the size of the lymphocyte repertoire enable immune responses that are similar across animals with vast differences in body size. We discuss how body size influences pathogenesis, epidemic spread of multi-host pathogens and the metabolic cost of immunity. Comparative studies of immune response and pathogen replication have implications for human health and animal disease.
Innate immune responses differ in baboons, chimpanzees and humans. (170.18)
Authors: Brinkworth Jessica , Pechenkina Kate , Silver Jack , Goyert Sanna
Published: January 2023

Innate immunity is the first host defense against invading pathogens and involves the activation of immune cells via Toll-like receptors (TLRs). As close human relatives sharing genomic identity, chimpanzees (98.6%) and baboons (~92%) are important biomedical models. Despite strong genetic relatedness, all three species very differently manifest infectious diseases characterized by strong innate immune activation (HIV-1, hepatitis). To examine potential differences in innate immune responses to TLR-detected pathogens, fresh whole blood from humans, chimpanzees and baboons was stimulated for 90 minutes with Pam3CSK4, Lipomannan from Mycobacterium smegmatis, LPS from E. coli, and LcrV from Yersinia pestis. Immune activation was assessed by measuring cytokine/chemokine induction using real-time PCR. Major differences in cytokine/chemokine induction patterns were observed between species. Baboons preferentially express the chemokine CXCL2, whereas chimpanzees and humans preferentially express CCL3 in response to all agonists. In contrast, chimpanzees initiate a strong IL-8 (CXCL8) response to all agonists while humans and baboons minimally respond with IL-8. Anti-inflammatory responses also differ between species; baboons more strongly induce IL-10 and humans and chimpanzees preferentially induce IL1RN in response to challenge. These observations suggest inter species differences in cell activation and trafficking that may impact the use of these animals as biomedical models.
Identification of myeloid-derived suppressor cells in dogs (170.2)
Authors: Goulart Michelle , Pluhar Elizabeth , Husbands Brian , Ohlfest John
Published: January 2023

Approximately six million dogs are diagnosed with cancer each year in the United States.Naturally occurring malignances in dogs share many features with human cancers that make dogs an excellent outbred spontaneous tumor model. The accumulation of myeloid derived suppressor cells (MDSCs) in tumor-bearing mice and in humans with cancer is known to be a key mechanism of tumor escape from immune surveillance and antitumor immunity. Due to their remarkable ability to downregulate the immune response against cancer cells in mice and in humans, we hypothesized that MDSCs would also play important role in tumor-induced immune suppression in dogs with cancer. Peripheral blood mononuclear cells of dogs with a variety of cancers and of age-matched healthy control dogs were analyzed by flow cytometry. Immunophenotyping revealed that dogs with cancer exhibited a significantly higher percentage of CD11b+ CD14- MHCII- cells compared with healthy dogs. More importantly, these cells have polymorphonuclear granulocyte morphology and demonstrated an ability to suppress T-cell proliferation in a dose dependent manner. Therefore, according to these results, canine MDSCs do exist and can be a potential target for therapeutic interventions in dogs with cancer. Furthermore, the study of MDSCs in dogs can be used to address underlying questions of resistance and evasion of malignant tumors that comprise an important target for translation cancer immunotherapy.
Investigation of IgH isotypes in ancestral mucosal immune model of class switch (170.3)
Authors: Du Christina , Mashoof Sara , Criscitiello Michael
Published: January 2023

The major barrier breached by most pathogens is the gut mucosal surface. Secretory IgA is the predominate immunoglobulin of mucosal surfaces in mammals. Induction of the mucosal immune response by ingestion of antigen usually elicits some localized response, but a poor systemic response. The importance of gut associated lymphoid tissues (or GALT) has been extensively reported in higher vertebrates, but less is known in lower vertebrates. In mammals IgA is the primary immunoglobulin of mucosal immunity. But no IgA has been identified in cold-blooded animals. In higher vertebrates, antigen must stimulate the lymphoid tissues in the intestines to elicit an IgA response, and cytokines from CD4 positive helper T cells are required for B cell switch. It is not known if this is the case in lower vertebrates, or if T cell help evolved before or after class switch recombination. My study will fill in these gaps in our knowledge by inoculating frogs orally and compare this route to intraperitoneal immunized frogs in our unique larval thymectomy model. Oral immunization is a novel approach to eliciting immune response in Xenopus. I propose that IgX will increase with oral inoculation compared to intraperitoneal injection. This would be the first demonstration of class switch upon oral immunization to a mucosal isotype in the first vertebrates that employs our Ig heavy chain switch mechanism, which would shed light on the most fundamental aspects of our humoral adaptive immune system.
Evolution of the T cell receptor: expression of non-conventional TCRδ chains in Xenopus (170.7)
Authors: Jackson Shawn , Undieh Ada , Steiner Harold , Parra Zuly , Miller Robert , Ohta Yuko , Flajnik Martin
Published: January 2023

γδ T cell receptors (TCR) that have structural differences from conventional γδ T cells in mammals have been observed in non-eutherian vertebrates. Three different types of V regions are present in the frog Xenopus tropicalis genomic TCRδ locus (Eur. J. Immunol. 40: 2319, 2010), most similar to Vδ, Vα, and VHδ (evolutionarily derived from the V regions of Ig H chains). We have begun a survey of the tissue distribution of TCRδ expression in X. laevis tadpoles and adults by 5’ RACE PCR. The deduced amino acid sequences predicted from X. tropicalis genome sequences were aligned with those from X. laevis TCRδ V RACE clones, revealing similar Vδ, Vα, and VHδ families in X. laevis. Analyses of the TCRδ RACE nucleotide sequences have identified the putative D and J gene segments in X. laevis as well. Although we find representatives of all TCRδ V sub-families in all tested adult tissues, the distribution of the data suggests that the expressed repertoire is skewed in some tissues, especially in the skin, where a majority of recovered sequences were of the Vα type. Interestingly, analyses of the Vδ type indicate a lack of N-nucleotide addition in the CDR3 in adults, suggestive of a population of T cells that is generated during larval life that persists through metamorphosis. Continued study of these unique TCR in Xenopus will illuminate apparent plasticity in TCR evolution, and shed light on how γδ TCR recognize antigen in all vertebrates.
Fli-1 transcription factor affects inflammatory cell infiltration in the lupus nephritis development by regulating expression of inflammatory chemokines (44.12)
Authors: Zhang Xian , Williams Sarah , Suzuki Eiji , Karam Eva , Gilkeson Gary
Published: January 2023

Expression of Fli-1, a member of the Ets family of transcription factors, is implicated in the development of nephritis in murine models of systemic lupus erythematosus (SLE). Expression of monocyte chemotactic protein-1 (MCP-1) and Chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES) plays an important role in the inflammatory cell infiltration. In this study, we examined the role of Fli-1 on chemokine production and inflammatory cell infiltration in conjunction with nephritis development in NZM2410 mice, an animal model of SLE. We generated Fli-1 heterozygous knockout NZM2410 mice (Fli1+/-; Fli-1 homozygous knockout is embryonic lethal) and wild-type (WT) littermate (Fli1+/+) mice as controls. Expression of MCP-1 and CCL5 in the kidneys from 18-week old Fli-1+/- NZM2410 mice was significantly decreased compared to that from WT littermates. The number of macrophages, neutrophil granulocytes, T cells and B cells in the kidneys from 34-week Fli-1+/- NZM2410 mice decreased by 44 - 75% compared to WT littermate controls. The productions of MCP-1 in endothelial cells transfected with specific Fli-1 siRNA were significantly reduced compared to cells transfected with negative control siRNA. Our data indicate that Fli-1 plays an important role in production of inflammatory chemokine MCP-1 and inflammatory cell infiltration in the lupus nephritis development.
Role of interferon-producing killer dendritic cells in the development of autoimmune disease (44.14)
Authors: Voynova Elisaveta , Bolland Silvia
Published: January 2023

Interferon-producing killer dendritic cells (IKDCs) share functional characteristics with NK cells and DCs. We investigated their role in the autoimmune disease developed by transgenic mice that overexpress TLR7 (TLR7tg). We determined that a cell population resembling IKDCs expands in TLR7tg mice through their intrinsic sensitivity to TLR7 stimulation. We confirmed that they are bona fide NK cells by their ability to kill cells lacking MHC I molecules in vitro. These cells were also able to present antigen to naïve T cells in in vitro assays. Adoptive transfer of IKDCs isolated from TLR7tg mice into wild type mice induced priming and activation of naïve T cells. In vitro stimulation with TLR7 agonists of IKDCs derived from TLR7tg resulted in the production of TNFa, MCP-1, IFNa and high levels of IL-10. We also tested the requirement of cytokines that are essential for NK cell development. We found that the expansion of IKDCs in TLR7tg mice was IL15 dependent but IL18 independent. We observed that lack of IL15 reduced many pro-inflammatory subsets (Ly6C+monocytes, granulocytes, DCs and memory T cells) and delayed the development of glomerulonephritis in TLR7tg mice. These results reveal expansion of IKDCs in autoimmune inflammatory conditions and suggest a dual role for these cells in both the priming of autoreactive T cells and the release of inflammatory cytokines.
The inflammasome and caspase-1 involvement in systemic sclerosis fibroblasts mediates fibrosis and myofibroblast differentiation (44.33)
Authors: Artlett Carol , Sassi-Gaha Sihem , Rieger Judy , Boesteanu Alina , Feghali-Bostwick Carol , Katsikis Peter
Published: January 2023

Systemic sclerosis (SSc) is a chronic idiopathic disease of unknown etiology that is characterized by fibrosis of the skin and visceral organs mediated by activated myofibroblasts. The recently identified inflammasomes are cytosolic receptors that tightly regulate the activity of caspase-1 and downstream signaling molecules such as IL-1β and IL-18. The NLRP3 inflammasome has been implicated in the development of idiopathic pulmonary fibrosis; however the role of the inflammasome has not been studied in SSc dermal or lung fibrosis. We identified increased expression of 40 genes associated with the inflammasome or downstream signaling molecules in SSc fibroblasts. Inhibition of caspase-1 in SSc dermal and lung fibroblasts abrogated secretion of collagens, and IL-1β and IL-18. In fibrotic SSc lesions, persistently activated fibroblasts called myofibroblasts mediate the excessive secretion of collagen and other extracellular matrix proteins in both the dermis and visceral organs. We observed decreased expression of the myofibroblast protein α-smooth muscle actin in SSc dermal fibroblasts treated with the caspase-1 inhibitor. NLRP3-/- and ASC-/- mice were resistant to bleomycin-induced skin fibrosis suggesting a key role for the inflammasome in in vivo dermal fibrosis. We report that innate immune signaling contributes to SSc fibrosis via the activation of the inflammasome and caspase-1 and suggest that inflammasome activation may play an important role in the pathogenesis of SSc.
Regulated release of nitric oxide by fibroblastic reticular cells controls the frequency and pathogenicity of autoreactive T cells in a model of intestinal tolerance (44.37)
Authors: Lukacs-Kornek Veronika , Malhotra Deepali , Fletcher Anne , Pinner Sophie , Tayalia Prakriti , Turley Shannon
Published: January 2023

Fibroblastic reticular cells (FRCs) are non-hematopoietic stromal cells of lymphoid organs wielding significant immunological impact. They exert important effects on the migration and homeostasis of naïve T cells however, their influence on activated T cells is undescribed. Here we report that nitric oxide (NO) release by FRCs profoundly inhibits T cell proliferation without altering early activation signals. Mechanistically, the expression of nitric oxide synthase 2 and production of NO paralleled the activation of T cells and required a tripartite synergism between IFNgamma, TNFalpha, and direct contact with activated CD8 T cells. We have also assesed the role of NOS2 in the iFABP-tOVA transgenic mouse model in which a truncated, cytosolic form of ovalbumin (tOVA136-336) is expressed as a self-antigen by mature intestinal epithelial cells under the control of the intestinal fatty acid binding protein (iFABP) promoter. In this model, where FRCs play a major role in the induction of CD8 T cell tolerance, FRC-derived NO controlled the magnitude of the response at the level of T cell proliferation. The percentage and absolute number of self-reactive CD8 T cells was increased in NOS2-/- iFABP mice, resulting in increased weight loss and more severe ileocolitis. Our study illuminates a hitherto unrecognized role of FRCs in regulating T cell responses that may have important implications for inflammatory bowel diseases.
Characterization of B cell repertoires in Sjögren’s syndrome by microengraving (44.48)
Authors: Nguyen Cuong , Ogunniyi Adebola , Love Christopher
Published: January 2023

Microengraving is a novel technology that uses an array of microfabricated wells to isolate and characterize large numbers of single cells. Using printing technique, secreted antibodies can be captured and characterized on glass slides. Using microengraving, we profiled the antigenic repertoires of B cells reacting against salivary gland (SG) tissues in Sjögren’s syndrome (SjS), an autoimmune disease targeting the exocrine glands. Cells from spleens and cervical lymph nodes (CLN) of normal C57BL/6 (B6) and SjS-susceptible (SjSs) C57BL/6.NOD-AecAec2 mice were isolated and loaded onto microwells. Capture slides preincubated with mouse Igs were used for printing. Detection antibodies included IgG1, IgG3, and SG lysates of B6 and SjSs mice that were labeled with different fluorescent dyes. Data indicated an increase in the frequency of IgG1 antibodies against SG lysates of B6 and SjSs mice when printed using CLN cells of SjSs mice but not B6 mice. In contrast, an increase in the frequency of IgG3 antibodies against SG tissues of B6 and SjSs mice was only observed when printed with SjSs mice splenocytes. These data demonstrate the diversity in the isotypic repertoire of antibodies during autoimmune process, and further revealed a dichotomy in peripheral tolerance. The result supports the generation of self-reactive B cell repertories during the autoimmune process. Furthermore, the use of micrograving of single cells might allow for identification of novel biomarkers.
Role of PD-L1/PD-L2 expression by human brain endothelial cells in regulating T cell responses in the context of Multiple Sclerosis. (44.6)
Authors: Pittet Camille , Ifergan Igal , Prat Alexandre , Arbour Nathalie
Published: January 2023

T lymphocytes have been implicated in the CNS injury observed in Multiple Sclerosis (MS), an inflammatory disease of the central nervous system (CNS). Engagement of Programmed Death-1 (PD-1), expressed on activated T cells, by its ligands (PD-L1 or PD-L2) suppresses T cell responses. We have previously shown that PD-L1 is upregulated on human CNS glial cells upon inflammation both in vitro and in MS lesions. We observed that several infiltrating CD8 T cells are devoid of PD-1 in MS lesions. Whether human brain endothelial cells (HBECs) which sit at the CNS entrance also express PD-L1 or PD-L2 and thereby block PD-1+ cells migration into CNS has not been established. We assess the expression of PD-L1 and PD-L2 by HBECs and establish its impact on immune responses. Under basal condition, PD-L2 is already highly expressed by HBECs, while PD-L1 is not detected. After stimulation with pro-inflammatory cytokines mimicking MS CNS environment, both proteins are significantly upregulated to similar levels. Blocking PD-L1/2 on HBECs leads to enhanced alloreactive CD4 and CD8 T cell responses in coculture assays and increased migration of CD4 and CD8 T cells using an in vitro model of the blood brain barrier. Our results show that PD-L1 and PD-L2 are both expressed by HBECs upon pro-inflammatory stimuli, and that HBECs-provided PD-L1/2 molecules have a role in dampening T cell responses, as well as in blocking T cell migration into the CNS.
Leptin serum levels in different subtypes of Multiple sclerosis: does leptin play a role in progress of Multiple sclerosis? (44.9)
Authors: Zarkesh-Esfahani Sayyed Hamid , Bahrami Ehsan , Maghzi Amir Hadi , Etemadifar Masoud , Kardi Mohammad
Published: January 2023

Background: Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS resulting in accumulating neurological disability. Recent evidences have strongly indicated that leptin has influence on the immune system and maybe involved in the pathogenesis of autoimmune diseases such as MS. Objectives: To compare serum leptin levels among healthy volunteers and patients with different subtypes of MS and to investigate its association with disease progression. Material and Methods: Leptin concentrations in sera of 114 healthy volunteers and 136 patients with different types of MS, including 93 relapsing remitting (RR), 18 primary progressive (PP), 13 secondary progressive (SP), and 12 progressive relapsing (PR) after matching for sex, age and BMI were measured using ELISA technique. Results: Statistically significant difference was observed between healthy volunteers and MS patients (18.55±23.55 vs 38.86±39.59 ng/ml, p<0.001), SP and PP (25.80±22.05 vs 70.64±53.01, p<0.01), and also between RR and PP (42.10±42.35 vs 70.64±53.01, p<0.05). There was a tight correlation between Progression Index and leptin serum concentrations (F=21.7, P<0.001). Conclusion: Leptin serum level is significantly higher in the sera of MS patients compared to healthy sex, age and BMI matched group. Patients with progressive forms have elevated leptin levels that seem to be associated with their disease progression. Our data suggests a pro-inflammatory role for leptin in MS.
CD4 T cells must be present during B cell development for B cells to respond to a T-dependent foreign antigen (45.19)
Authors: Eisenberg Robert , Choudhury Arpita
Published: January 2023

We have previously shown that normal B cells from C57BL/6 (B6) mice must develop in the presence of syngeneic CD4 T cells if they are to be capable of producing antinuclear autoantibodies in response to allohelp from bm12 CD4 T cells (Clin Immunol 136, 105, 2010). We have now determined that B cells require a similar ‘nurturing’ from CD4 T cells during ontogeny, if they are to become capable of responding to a T-dependent foreign antigen. B6.CD4-/- mice given B6 CD4 T cells do not respond to OVA immunization. In addition, B cells isolated from B6.CD4-/- mice and transferred to B6.rag1-/- mice along with B6 CD4 T cells completely failed to respond to immunization with OVA in alum, whereas B cells isolated from CD4-sufficient mice responded strongly. Autoreconstituting B cells in B6.CD4-/- mice irradiated to 500 rads on day 0 require the presence of CD4 T cells administered on day 1 in order to be able to respond to OVA + B6 T cells on day 21. B cells from B6.CD4-/- mice are also deficient in stimulating the antigen-specific proliferation of OVA-specific OTII T cells in vitro. This deficiency could not be accounted for by phenotypic differences. Overall, our data reveal a role for CD4 T cells in the development of B cells before they encounter antigen. This ‘nurturing’ role allows the B cells to stimulate antigen specific T cells to proliferate to antigen and then respond themselves by the production of specific antibody.
Induction of antigen-specific B cell tolerance through the development of a versatile platform to target siglecs (45.4)
Authors: Macauley Matthew , Pfrengle Fabian , Paulson James
Published: January 2023

CD22 and SiglecG are proposed to be negative regulators of B cell receptor signaling through their ability to recognize sialic acid-containing glycans. Recent studies have shown that multivalent presentation of high affinity CD22 and SiglecG ligands in cis with a T-independent antigen can tolerize B cells (Duong et al., J. Exp. Med., 2010). Here we describe studies aimed at showing the generality of this phenomenon. To realize this aim, a novel liposomal nanoparticle platform was developed for presentation of both high affinity Siglec ligands and antigen. Chemical conjugation of the Siglec ligand and antigen to this platform is facile and, importantly, this platform is highly compatible with in vivo studies. Using this new platform, robust B cell tolerance is achieved toward both T-independent and T-dependent antigens in mice. Mechanistic studies provide clear evidence that: i) CD22 and SiglecG are the targets; ii) apoptosis of B cells is the mode of tolerance; iii) tolerization is highly selective to B cells containing a BCR that is specific for the targeted antigen. These studies reinforce the hypothesis that CD22 and SiglecG are critical components of a B cell that allows it to distinguish self from non-self. This method has clear potential for treatment of autoimmune diseases and allergies by antigen specific tolerization of B cells.
Cannabinoid receptor 2 plays a critical role in marginal zone B cell retention and T-independent humoral responses (45.6)
Authors: Basu Sreemanti , Ray Avijit , Dittel Bonnie
Published: January 2023

Marginal zone (MZ) B cells play a very important role in the early defense against blood borne pathogens by producing natural IgM and inducing T-independent (TI) humoral responses. Lack of MZ B cells has been associated with higher susceptibility to bacterial infections. However, the development/maintenance of these cells is not fully understood. Mice deficient in cannabinoid receptor (CB) 2, a Gi protein-coupled receptor highly expressed on B cells, showed a significant reduction in MZ B cells. We found that CB2 deficiency correlated with significantly decreased natural and TI IgM levels (post-immunization) in CB2-/- mice. We investigated the mechanism by which CB2 regulates MZ B cell numbers. Analysis of mixed BM chimeras showed a B cell intrinsic CB2 requirement, although additional data suggested no involvement of CB2 in MZ B cell development or survival. We next studied whether CB2 regulates MZ B cell adhesion/retention. Interestingly, WT B cells showed significantly higher adhesion to ICAM-1+VCAM-1, critical for MZ B cell retention in the splenic MZ, compared to CB2-/-. 2-AG, an endogenous CB2 ligand further enhanced WT B cell adhesion and blocking of CB2 signaling in WT mice with a CB2-antagonist dislodged MZ B cells from the spleen, increasing their numbers in the blood. Unmanipulated CB2-/- mice also exhibited increased MZ B cells in the blood compared to WT. Collectively, our study identifies a crtical role of CB2 in MZ B cell retention and TI humoral responses.
The interplay between TGFβ and cγc cytokine signaling in CD8+ T cell biology (46.19)
Authors: Sanjabi Shomyseh , Pham Joey , Mosaheb Munir , Ishigame Harumichi , Flavell Richard
Published: January 2023

Role of TGFβ signaling in peripheral tolerance is well appreciated. However, little is known about the role of TGFβ signaling in cytotoxic T lymphocytes during an inflammatory immune response. As naive CD8+ T cells become activated, they undergo clonal expansion followed by contraction where 90-95% of these cells die through apoptosis and the remaining 5-10% form long-lasting memory T cells. The effector cells that die during this process are called short-lived effector cells (SLECs) and the ones that survive to become memory cells are called memory precursor effector cells (MPECs). The apoptotic process during contraction is crucial as it resets T cell homeostasis, promotes protective immunity by allowing the formation of long-lived memory cells, and aids to limit autoimmunity. The survival of the effector T cells is directly proportional to the level of Bcl-2 they have. In effector CD8+ T cells, Bcl-2 can be induced in response to both IL-7 and IL-15, with IL-7 being in general a stronger inducer of Bcl-2 than IL-15. We have shown that while IL-15 promotes the survival of SLECs during expansion and contraction of effector CD8+ T cells, TGFβ promotes their apoptosis. The opposing effect of these two cytokines converges at the Bcl-2 promoter, where IL-15 induces Bcl-2 expression, while TGFβ reduces the amount of this anti-apoptotic molecule. The molecular mechanism by which these two cytokines control the expression of Bcl-2 is currently being investigated.
CXCR3 expression affects the balance of effector and memory CD8 T cell generation (46.6)
Authors: Hu Joyce , Kagari Takashi , Clingan Jonathan , Matloubian Mehrdad
Published: January 2023

In response to infection, activated CD8 T cells differentiate in vivo into a heterogeneous population of cells consisting of short-lived, terminally differentiated effector cells and memory precursor cells that give rise to long-lived memory cells. The strength and duration of antigenic stimulation and inflammatory signals influence CD8 T cell differentiation and the balance between these two fates. However, cell extrinsic cues, such as chemokines, that affect the localization and hence the exposure of CD8 T cells to antigenic and inflammatory signals are not completely understood. Here, we find that the inflammatory chemokine receptor CXCR3 is upregulated on activated CD8 T cells and influences their differentiation. CXCR3 guides activated CD8 T cells towards marginal zone areas where they are exposed to antigenic and potentially inflammatory stimuli. In the absence of CXCR3 expression, activated CD8 T cells are localized to the T zones of the splenic white pulp where they are less likely to receive antigenic stimulation. The increased exposure to antigen is associated with an increase in the generation of short-lived effector cells while the decreased exposure to antigen is associated with a quantitative and qualitative enhancement of memory cells. Our findings demonstrate that chemokine receptors and T cell positioning in secondary lymphoid organs can influence the balance of effector and memory T cell generation.
Comparison of antibody gene mutation patterns in first attack optic neuritis and transverse myelitis leading to Multiple Sclerosis (47.18)
Authors: Ligocki Ann , Rounds William , Cowell Lindsay , Chaudhary Parul , Xiang Diane , Guidry Paula , Scheuermann Richard , Racke Michael , Courtney Ardith , Frohman Elliot , Greenberg Benjamin , Monson Nancy
Published: January 2023

Multiple Sclerosis (MS) is the leading disease of the central nervous system and a significant, costly cause of disability in young adults. Patients at risk for MS often present with a single demyelinating event, typically called a “clinically isolated syndrome” (CIS). A CIS can occur in the brain, spinal cord (Acute Partial Transverse Myelitis, APTM), or optic nerve (Optic Neuritis, ON) at similar rates and present a challenging diagnostic and therapeutic dilemma. Stratifying CIS patients most likely to develop MS is a complicated process, but desirable since treatment with the appropriate immunomodulatory agents early in the disease course can delay long-term disability. Thus, a primary focus of our laboratory has been to develop a completely novel type of biomarker that can identify CIS patients that will develop MS. This unique biomarker is based on a pattern of antibody gene mutations (i.e. antibody gene signature or “AGS”) that is exclusive to B cells from the cerebrospinal fluid and brain lesions of MS patients that initially presented with either TM or ON. In fact, the AGS is consistently elevated in patients with one attack of ON that go on to develop definite MS, and can predict conversion to MS with 91% accuracy. More recently, we are testing the hypothesis that the AGS serves as a predictor for conversion to MS in CIS patients that present with TM. Preliminary data demonstrate that the AGS is also prevalent in CIS patients presenting with TM.
Overexpression of short CYLD leads to autoimmunity and production of autoantibodies (47.20)
Authors: Reissig Sonja , Hövelmeyer Nadine , Weih Debra , Weih Falk , Nikolaev Alexei , Waisman Ari
Published: January 2023

CYLD is a tumour suppressor gene which plays an important role in NF-kB signaling. To analyze the function of CYLD in vivo we used the CYLDex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a naturally occurring short splice variant of the cyld gene (sCYLD). This overexpression resulted in splenomegaly and lymphadenopathy. Further, the B cell numbers in spleen and lymph nodes were increased at the expense of T cells. CYLDex7/8 mice showed a strong reduction of CD4 single positive (SP) and CD8 SP T cells in the thymus and periphery. In in vitro and in vivo studies we could show that CD4+ T cells display a hyperactive phenotype accompanied with an increased production of inflammatory cytokines. We could also demonstrate a defect in negative selection of CYLDex7/8 thymocytes by using the HY-TCR transgenic system. mTECs in the thymus, which are important for the negative selection of autoreactive T cells, were drastically reduced in number. Since abnormal T cell responses are often associated with chronic inflammations, we examined if CYLDex7/8 mice develop immunological abnormalities. Importantly, we could observe that CYLDex7/8 mice crossed onto TCR transgenic background developed severe colonic inflammation accompanied with high production of nuclear autoantibodies.
Impact of TLR9 on hyper-responsive ITAM signaling in a mouse model of SLE (47.24)
Authors: Mills Robyn , Lam Viola , Tobler Diana , Oksenberg Nir , Cresalia Nicole , Anderson Mark , Mao Jian-Hua , Weiss Arthur , Hermiston Michelle
Published: January 2023

Anti-nuclear autoantibody (ANA) production by autoreactive B cells is a hallmark symptom of the autoimmune disease Systemic Lupus Erythematosus (SLE), which has a complex genetic etiology with environmental influences. In the CD45E613R knock-in mouse model of SLE, mice homozygous for an activating point mutation in CD45 develop high titer anti-dsDNA autoantibodies on the BALB/c (BA) and not the C57BL6/J (B6) or 129/Sv genetic backgrounds despite hyper-responsive ITAM signaling in several immune lineages in each genetic background. A screen for genetic modifiers of autoantibody production identified the candidate gene Tlr9. Sequence analysis of B6 and BA alleles of Tlr9 indicate several point mutations, resulting in five amino acid substitutions in TLR9. Splenic CD11b+ myeloid cells from BA express lower levels of TLR9 and BA-derived BMDCs are less responsive to synthetic TLR9 ligand CpG DNA. BA B cells are also less responsive to TLR9-mediated signaling than B6 B cells despite similar levels of TLR9 protein, indicating the BA TLR9 allele is hypo-responsive. Genetic ablation of TLR9 in CD45E613R mice on the non-autoreactive B6 genetic background augments disease, with mice developing splenomegaly and increased frequencies of memory T cells by 3 months of age. Some aged animals also developed ANAs, supporting the hypothesis that reduced TLR9 responsiveness permits increased autoreactivity in the context of hyper-responsive ITAM signaling.
Regulation of the NKG2D ligand Mult1 by shedding (48.22)
Authors: Deng Weiwen , Nice Timothy , Raulet David
Published: January 2023

Natural Killer group 2D (NKG2D) is an activating receptor expressed by NK cells and some T cells that recognizes a diverse family of ligands, including the Rae-1 family, H60, and Mult1 in the mouse, and the MICA and ULBP families in humans. The binding of NKG2D to its ligands plays an important role in immunosurveillance of tumors and infectious pathogens, but disregulation of this system has been tied to autoimmunity and chronic inflammatory disease. Intriguingly, some of human NKG2D ligands such as MICA and MICB can be shed from tumor cells and have been detected in sera from patients with various types of cancer. Here we report that the mouse NKG2D ligand Mult1 protein is shed from the surface of cells and that the resulting soluble Mult1 can still bind to NKG2D. While cell-bound Mult1 causes downregulation of surface NKG2D, soluble Mult1 did not alter NKG2D expression on NK cells in vitro. Furthermore, shedding of Mult1 could be blocked by the general matrix metalloproteinase (MMP) inhibitor BB94. Finally, soluble Mult1 is elevated in the sera from Eu-Myc transgenic mice, a strain that develops B lymphomas. This study represents the first instance of NKG2D ligand shedding in the mouse, which provides an important animal model to probe the mechanisms underlying shedding and its impact on immune recognition.
Effect of complement C5a in tumor progression and the local tumor microenvironment (48.27)
Authors: Gunn Lacey , Ding Chuanlin , Hansen Richard , Hu Xiaoling , Cai Yihua , Aggarwal Deep , Qi Chunjian , Ma Yungfeng , Yan Jun
Published: January 2023

Promoting complement activation may enhance immunological mechanisms of anti-tumor antibodies for tumor destruction. However, complement activation components, such as C5a, trigger inflammation which can promote tumor growth. It has been demonstrated, in one tumor model, that C5a in the tumor can inhibit anti-tumor CD8 T cell responses through effects on myeloid-derived suppressor cells. We address the role of C5a on tumor growth by transfecting tumor cells with mouse C5a: SKOV-3 human ovarian carcinoma and RMA murine lymphoma. The two models allow us to study C5a in immunocompromised and immunocompetent hosts. In vitro growth kinetics of C5a, control vector (CV), or wild-type cells revealed no significant differences. In both models, tumor-bearing mice with C5a-transfected tumor cells have significantly less tumor burden as compared to CV tumors. In the immunocompromised model, DX5 CD11b NK cells and F4/80 macrophages infiltrated C5a expressing tumors with significantly greater frequency while VEGF and arginase production were significantly less. The syngeneic model revealed C5a leads to a greater tumor influx of CD11c and CD4 T cells. A greater percentage of CD4 T cells infiltrating RMA C5a tumors demonstrated an activated status, CD44hi CD62L low. Thus our studies demonstrate tumor C5a chemoattracts innate NK cells and macrophages while downregulating negative mediators, and promotes infiltration of important antigen-presenting cells and activated CD4 T effector cells.
Tumors adapt rapidly to thwart vaccine-induced T cell attack but this adaptive response can be overcome by manipulation of costimulation and immune checkpoints (48.30)
Authors: McGray Aj , Bernard Dannie , Kelly Ryan , Jha Mayank , Gregory Caitlin , Bassett Jennifer , Wan Yonghong , Bramson Jonathan
Published: January 2023

We have developed a prototypic recombinant adenovirus vaccine encoding dopachrome tautomerase (rHuAd5-hDCT) that leads to moderate suppression of B16F10 melanoma growth through a CD8+ T cell- and IFN-γ-dependent mechanism. The immunogenicity of rHuAd5-hDCT was improved by concomitant 4-1BB stimulation leading to enhanced control of tumor growth; however, most tumors ultimately relapsed. Gene expression analysis revealed a rapid adaptation of the tumor environment within 5 days of vaccination. Intratumoral IFN-γ and TNF-α expression increased rapidly following vaccination but, paradoxically, expression was almost completely attenuated at the time of highest infiltration by vaccine-induced CD8+ T cells. We also noted robust, yet transient, induction of PD-L1 and PD-L2 in the tumor following vaccination, which correlated with the attenuation of IFN-γ and TNF-α expression. Blockade of PD-1 signaling in combination with rHuAd5-hDCT enhanced effector cytokine expression in the tumor and improved growth control, but the tumors ultimately relapsed. Strikingly, the combination of PD-1 blockade and 4-1BB stimulation with rHuAd5-hDCT produced a synergistic enhancement in intratumoral T cell activity, resulting in complete regression despite no increase in the functionality or number of tumor infiltrating CD8+ T cells. These results punctuate the need to understand the dynamic changes within the tumor to optimize cancer immunotherapy.
NKT cell mediated responses to mantle cell lymphoma (48.35)
Authors: Sun Wenji , Kimball Amy , Hester Danubia , Webb Tonya
Published: January 2023

Mantle cell lymphoma (MCL) is an aggressive subtype of Non-Hodgkin’s lymphoma that is characterized by the abnormal accumulation of CD20+CD5+ B cells in the lymph nodes, spleen, gastrointestinal tract, bone marrow and blood. Although treatment with combination chemotherapy can be effective, most patients relapse, and the outcome for MCL remains poor. Natural killer T (NKT) cells are important in regulating immune responses to tumors. The observation that lymphoma patients have a reduction in both NKT cell number and function suggests that the anti-tumor effects mediated by these cells may be compromised. In these studies, we have investigated NKT cell activation in response to MCL. We found that CD1d cell surface expression was highly variable in a panel of well-characterized MCL cell lines. Importantly, cell surface expression of CD1d did not directly correlate with the ability of the MCL lines to activate primary NKT cells. Circulating NKT cells in the peripheral blood of MCL patients were not stimulated by autologous B cells, even in the presence of the potent antigen, α-GalCer. However, when NKT cells from healthy donors were co-cultured with purified B cells from MCL patients, the NKT cells specifically recognized the malignant B cells and produced IFN-γ, but not inflammatory or regulatory cytokines. These data suggest that B cell lymphomas may condition autologous NKT cells not to recognize them in order to evade NKT cell mediated anti-tumor responses.
BALB/c mouse model of RSV clinical isolate A2001/2-20 pathogenesis (49.13)
Authors: Stokes Kate , Chi Michael , Sakamoto Kaori , Newcomb Dawn , Huckabee Matthew , Lee Sujin , Pretto Carla , Williams John , Peebles R. , Moore Martin
Published: January 2023

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains (A2, Long, and Line 19) differentially induce airway mucus production in mice. We tested whether RSV strains differ in pathogenesis by screening low passage RSV clinical isolates for mucogenicity and virulence in BALB/c mice. RSV clinical isolates induced variable disease severity. Infection of BALB/c mice with clinical isolate RSV A2001/2-20 (2-20) resulted in greater disease severity and higher lung IL-13 levels than infection with RSV laboratory strains and other clinical isolates. Like the line 19 strain, 2-20 induced airway mucin expression in BALB/c mice and resulted in higher viral antigen levels in the bronchiolar epithelium than A2. 2-20 caused epithelial desquamation, airway hyperresponsiveness, and increased breathing effort in BALB/c mice. We hypothesize that the fusion (F) protein of 2-20 contributes to this phenotype. We generated a chimeric RSV by replacing the F gene of A2 with the F gene of 2-20. A2-2-20F grew to similar levels as 2-20 in mouse and human cells in vitro. In BALB/c mice, the A2-2-20F exhibited viral loads similar to 2-20 over a time course and showed comparable mucin levels to 2-20. We established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease. Identification of virulence factors of RSV 2-20 may contribute to our understanding of RSV disease mechanisms.
P-selectin regulation of T cell immunity during influenza virus infection (49.14)
Authors: Tinoco Roberto , Deiro Mia , Lin Melissa , Bradley Linda
Published: January 2023

Influenza viruses represent a major global health concern as they have the potential to generate worldwide pandemics characterized by increased morbidity and mortality. In response to infection of lung epithelial cells, an immune response is generated that will clear the virus and prevent re-infection with the same virus. T cells are critical for eliminating virus-infected cells and their localization to sites of viral replication is crucial to resolving infection. However, the underlying mechanisms regulating effector and memory T cell responses after influenza virus infection remain unclear. Using a mouse model, we demonstrated that the adhesion molecule P-selectin, is induced on lung endothelium after influenza virus infection. More importantly, virus-specific T cells acquire the ability to bind P-selectin through expression and glycosylation of its ligand PSGL1, which coincided with effector function. P-selectin binding antigen-specific CD4+ T cells produced increased levels of IFN-γ. Similarly, P-selectin binding antigen-specific CD8+ T cells also had increased IFN-γ levels in the lung when compared to non-binding CD8+ T cells. Our findings highlight an important role for the adhesion molecule P-selectin in regulating T cell responses by inducing a differential effector program, which may be important for T cell memory and immunity to influenza virus.
The C3a anaphylatoxin receptor is critical in the pathophysiology of RSV infection (49.2)
Authors: Bera Monali , Lu Bao , Martin Thomas , Cui Shun , Gerard Craig , Gerard Norma
Published: January 2023

Respiratory syncytial virus (RSV) is linked to severe lung disease and may lead to wheezing in infants. RSV infection in mice demonstrates infiltration of inflammatory cells, mucus cell metaplasia, and airway hyperresonsiveness (AHR) to methacholine. Previous studies demonstrated activation of complement by RSV. Other studies showed a requirement for C3 or C3a in AHR associated with antigen sensitization as well as inhalation of particulate matter. We hypothesized, therefore, that C3a is a critical component for the AHR associated with RSV infection. We found that mice lacking the C3aR (C3aR1-/-) do not develop AHR secondary to acute RSV infection in contrast to wild type mice. Further, viral clearance was accelerated in C3aR1-/-mice. The AHR phenotype was restored to wild type when C3aR1-/- mice were transplanted with wild type bone marrow cells,supporting a role for a hematopoetic cell type. T cells were the dominant cell type increased in the lungs of both wild type and C3aR1-/-mice following RSV infection, however, the increases in lung IL-23 and IL-17 were significantly lower in C3aR1-/-mice compared with wild type animals. Additionally the increased levels of the transcription factor RORγT, responsible for regulating IL17 synthesis, seen in wild type mice were greatly reduced in C3aR1-/- mice. Our findings identify a key role for activation of complement anaphylatoxin C3aR in the pathophysiology of RSV disease, and may help identify a therapeutic target in humans.