bluuebunny/crossref_metadata_2025 huggingface dataset:
Tertiary lymphoid tissue with germinal centers and T follicular helper cells in immunologically privileged retinas of mice with chronic autoimmune uveitis (P4172)
Authors: Kielczewski Jennifer , Horai Reiko , Caspi Rachel
Published: January 2023

Mice transgenic for a retina-specific T cell receptor develop spontaneous autoimmune uveitis. In contrast to induced models of uveitis the disease is prolonged and the mice display distinct retinal lesions that resemble tertiary lymphoid tissue. Here we investigate these lesions for presence of germinal center (GC) and T follicular helper cell (TfH) markers. Retinas were immunohistochemically stained for CD4+ T cells, CD8+ T cells, and B cells, as well as the GC markers PNA and GL-7. The retinal lesions stained positively for CD4+ T cells and B cells and also for PNA and GL-7. Laser capture microdissection was then used to isolate the retinal lesions and microarray analysis was performed to assess expression of GC- and TfH-associated genes. The microdissected lesions consistently showed upregulation of germinal center markers and T follicular helper cell markers with a 4 fold increase in Btla, Fas, and CXCL13, a 3.5 fold increase in CXCR5, and a doubling in CXCR4 compared to control retina. Heat map analysis revealed a similar gene expression profile of the retinal lesions to that of germinal centers present in 5 day-immunized spleen and lymph nodes. We conclude that despite the immunologically privileged status enjoyed by the eye, chronic autoimmune uveitis precipitates development of tertiary lymphoid tissue in the retina, expressing genes and immunological markers characteristic of GC and TfH.
IL-23 controls autoimmunity by maintaining the integrity of marginal zone barrier that clears apoptotic bodies (P4178)
Authors: Li Hao , Hsu Hui-Chen , Wu Qi , Yang PingAr , Cua Daniel , Mountz John
Published: January 2023

Th17 cells, which are maintained by IL-23, are important for the spontaneous germinal center (GC) response in autoimmune BXD2 mice. Here, we unexpectedly found that BXD2-Il23-/- mice exhibit accelerated development of spontaneous GCs and production of pathogenic autoantibodies compared with wild-type (WT) BXD2 mice. Administration of adenovirus expressing IL-23 to BXD2-Il23-/- mice at early age delayed the disease onset. Despite a decline of Th17 cells in the spleen of BXD2-Il23-/- mice, there was a severe impairment in clearance of apoptotic bodies and an accelerated age-related loss of marginal zone macrophages (MZMs). In the spleens of both B6 and BXD2 mice, IL-23R was expressed primarily by MZMs which is the major cell that clears apoptotic debris in the spleens. Absence of IL-23 and loss of MZMs in BXD2-Il23-/- mice are associated with elevated production of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Selective depletion of MZMs by repeated injection of clondronate liposome to BXD2-WT, but not BXD2-Ifnar-/- mice, mimicked the phenotypes of BXD2-Il23-/-. Our results suggest a novel mechanism that IL-23 can act as a double edge sword to control autoimmunity in that over-expression of IL-23 can provoke autoimmunity through the maintenance of Th17 yet a complete deficiency of IL-23 induced autoimmunity through the gradual loss of marginal zone barrier, defective clearance of apopototic debris and elevated induction of type I IFNs.
Ceramide analogue displays stimulatory activity on dendritic cells to promote T cell responses upon virus infection (P6115)
Authors: Pritzl Curtis , Seo Young-Jin , Stokes Zach , Hahm Bumsuk
Published: January 2023

The ceramide family of lipids has been shown to play various important roles in both cell structure and signaling in a diverse array of cell types, including immune cells. However, very little is known regarding how ceramide affects the activation of dendritic cells (DCs) in response to viral infection. In this study, we demonstrate that a synthetic ceramide analogue (C8) stimulates DCs to increase the expansion of virus-specific CD8 T cells upon virus infection. Exogenously supplied C8 ceramide elevated the expression of DC maturation markers such as MHC class I and the co-stimulatory molecule B7-2 following infection with the immune suppressive Clone 13 strain of lymphocytic choriomeningitis virus (LCMV) in vitro. In addition, the ceramide analogue enhanced the production of IL-12 from DCs infected by the virus. Importantly, ceramide-conditioned, LCMV-infected DCs displayed a heightened activity to promote proliferation of virus-specific, CD8 T cells when compared to virus-infected DCs. Furthermore, locally instilled, but not systemically administered, ceramide analogue via intranasal inoculation significantly increased virus-reactive CD8 T cell responses in vivo. Collectively, these findings provide new insights into ceramide-mediated regulation of DC responses against virus infection and help us establish a foundation for novel immune-stimulatory therapeutics.
2D affinity measurements of primary and secondary polyclonal CD4+ effector T cells in lymphocytic choriomenengitis infection (P6121)
Authors: Andargachew Rakieb , Evavold Brian
Published: January 2023

T cells have been shown to undergo affinity/avidity maturation through selective expansion of higher affinity/avidity (tetramer positive) cells, resulting in the narrowing of the antigen specific repertoire over the course of primary and secondary immune responses. Using a highly sensitive two dimensional micropipette adhesion frequency assay (2D) to measure polyclonal CD4+ T cell affinities, we determined if affinity maturation occurred between primary and secondary peak effectors in lymphocytic choriomenengitis virus (LCMV) infection, and if measured affinities correlated with functional avidity. The 2D assay detected 4-5 fold more antigen specific high and low affinity cells than tetramer in both primary and secondary infection, but both 2D and tetramer indicated a 2-3 fold decrease in the frequency of LCMV specific CD4+ cells in the homologous viral rechallenge response. The range of affinities of the antigen specific repertoire was more restricted during secondary infection contracting to a narrower sixteen fold from the wider hundred fold range of the primary response. By functional avidity, secondary effectors were more sensitive to antigen despite a 3 fold decrease in their measured average affinity. Therefore, in this homologous viral rechallenge model, affinity maturation did not occur between primary and secondary effectors, and affinity did not correlate with functional avidity.
Cell intrinsic gp130 signaling is vital for virus specific CD4 T cell survival and interleukin 21 production during late stages of chronic viral infection (P6131)
Authors: Harker James , Dolgoter Aleksandr , Zuniga Elina
Published: January 2023

Infection with chronic viral infections such as human immunodeficiency virus-1 and hepatitis C virus significantly increases the risk of developing cancer. CD4 T cells number and function are critical in determining the outcome of chronic viral infections in both mice and humans, but little is known about the signals that promote their survival or helper functions in this highly immunosuppressive environment. Here we showed that exclusive ablation of the common interleukin-6 family co-receptor, gp130 in T cells profoundly compromised the numbers of virus-specific CD4 T cells, their differentiation into T follicular helper cells and IL-21 production at late (but not early) stages of a chronic murine viral infection. This was accompanied by a marked reduction in both virus-specific CD8 T cells and antibodies and a severe failure to control the virus. Notably, cell-intrinsic signaling by the IL-6 family cytokine IL-27 was necessary for CD4 T cell survival while IL-6 was required for optimal T follicular helper cell differentiation. Contrastingly IL-6 and IL-27 were both capable of inducing IL-21 production by virus specific CD4 T cells, but neither was essential in vivo. Our data situates direct gp130 signaling in CD4 T cells at the center of antibody and CD8 T cell mediated immune responses during an established chronic viral infection, highlighting its potential as a therapeutic target against such infections.
Competition for antigen selects T cell responses that undergo memory inflation and maintains clonal dominance during MCMV infection (P6091)
Authors: Snyder Christopher , Turula Holly , Farrington LIla , Grey Finn , Hill Ann
Published: January 2023

Cytomegalovirus (CMV) establishes a life-long, persistent/latent infection. Continuous immune surveillance by CMV-specific CD8+ T cells results in their accumulation over time, a process called “memory inflation”. These “inflationary” T cells migrate systemically and comprise the largest T cell populations in humans, making CMV-based vaccines attractive. Using recombinant murine (M)CMV expressing the SIINFEKL peptide, we found that SIINFEKL-specific T cells inflated at the expense of T cells specific for other MCMV-derived peptides. However, co-infecting mice with viruses expressing and lacking SIINFEKL enabled T cells with multiple specificities to inflate, implying T cell competition for antigen at the level of the infected target. Such competition was also observed among SIINFEKL-specific T cells. Adoptively transferred OT-I T cells, which have a high affinity for the SIINFEKL peptide, were preferentially enriched shortly after infection and inhibited the inflation of host-derived T cells specific for SIINFEKL. Importantly, we observed sporadic, late rejection of OT-Is as a result of minor histocompatibility differences, and only such rejection enabled host-derived T cells to inflate robustly. These data imply that relative clonal abundance is actively maintained by competition. Together, these data show that T cell competition for antigen during MCMV infection dictates which T cell responses inflate and even which clones dominate within a response.
T cell responses against structural proteins develop rapidly during acute simian varicella virus infection and are maintained during viral latency (P6133)
Authors: Haberthur Kristen , Kraft Aubrey , Meyer Christine , Asquith Mark , Dewane Jesse , Messaoudi Ilhem
Published: January 2023

Varicella zoster virus (VZV) is the etiological agent of varicella and herpes zoster. Clinical observations suggest that successful control of VZV during both primary infection and reactivation is mediated by cellular rather than humoral immunity. Numerous studies have characterized T cell responses directed against select VZV open reading frames (ORF), but a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. We identified several SVV ORFs targeted by T cell responses in majority of animals examined during both acute and latent infection. We show that structural proteins are targeted by both CD4 and CD8 T cells, and immediate early/early viral proteins are primarily targeted by CD8 T cells. Also, while both CD4 and CD8 T cell responses are detected during acute SVV infection, latent infection elicits primarily CD8 T cell responses. These results suggest that CD4 T cell immunity may wane faster than CD8 T cell immunity. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs to be further investigated as potential components of second-generation VZV vaccines designed to boost T cell immunity.
Pro-apoptotic Bim regulates NK cell contraction and the generation of the NK memory pool following viral infection (P6106)
Authors: Min-Oo Gundula , Bezman Natalie , Madera Sharline , Sun Joseph , Lanier Lewis
Published: January 2023

Apoptosis is critical in lymphocyte development and homeostasis; moreover, this pathway eliminates activated lymphocytes and prevents immune pathology during viral infection. Pro-apoptotic factor Bim controls LCMV-specific T cell contraction and the formation of memory T cells. Natural killer (NK) cells also undergo antigen-driven expansion to become long-lived memory cells after mouse cytomegalovirus (MCMV) infection. Bim expression is decreased in memory NK cells; therefore, we examined the role of Bim in regulating the MCMV-driven memory NK pool. Ly49H+ Bim-/- NK cells respond similarly to wild-type cells early after MCMV. However, following the m157-driven expansion phase, Bim-/- Ly49H+ NK cells show impaired contraction and significantly outnumber wild-type cells by day 14. Importantly, the inability to reduce the Ly49H+ effector pool leads to a larger Bim-/- NK memory subset; these Bim-/- memory cells display a less mature phenotype (CD11blo,CD27hi) and lower levels of NK memory markers, KLRG1 & Ly6c. Furthermore, compared to wild-type memory NK cells, Bim-/- cells are functionally impaired in response to receptor-mediated stimulation, despite equal responses in naïve cells. These results suggest that apoptosis of antigen-specific effector NK cells is critical in generating a potent memory pool. Further studies are required to elucidate the mechanisms that drive apoptosis in most activated NK cells, but spare a small subset to become long-lived memory cells.
The inhibitory receptor NKG2A on NK cells controls CD8+ T cell responses to hepatic viral infection (P6109)
Authors: Krueger Peter , Hahn Young
Published: January 2023

NKG2A is an inhibitory receptor expressed on a variety of liver lymphocytes, including NK and activated CD8+ T cells. NKG2A recruits the phosphatase SHP1 and inhibits signaling cascades necessary for immune cell activation when bound to its ligand, the nonclassical MHC-I Qa1b/Qdm complex. Elevated NKG2A expression on circulating NK cells is associated with chronic hepatitis C virus infections in humans; however, the impact of NKG2A+ NK cells in host immunity and chronic infections has not been elucidated. Using NKG2A-/- mice infected with adenovirus-ova, we have observed that loss of NKG2A signaling on NK cells leads to an increase of antigen-specific CD8+ T cell numbers with enhanced IFNγ production in the liver, but not the spleen, from days 6 through 40 post infection. Importantly, intrinsic NKG2A signaling on CD8+ T cells does not appear to control their proliferation or IFNγ expression. Furthermore, blockade of NKG2A-Qa-1b interaction on NK cells by the administration of Qa1b neutralization antibody or NK cell depletion prior to infection leads to increased hepatic CD8+ T cell numbers, suggesting NK cells are actively dampening CD8+ T cell cellularity and functions. Taken together, these results indicate that NK cell-mediated NKG2A signaling plays a pivotal role in controlling CD8+ T cell responses to hepatic viral infection.
IL-15 mediated NK cell responses during rhinovirus infection requires type I interferon signalling (P6111)
Authors: Jayaraman Annabelle , Johnston Sebastian , Bartlett Nathan
Published: January 2023

Rhinoviruses (RV) are a global burden on respiratory health, particularly for those with chronic respiratory diseases such as asthma. The lack of treatments for RV-induced diseases inspires further studies into immune mechanisms of disease. IL-15 and NK cells are critical for defence against many viruses, however their role during RV infections has not been defined. Hypothesis IL-15, induced by type I interferons (IFNs), is critical for NK cell responses in the lungs during RV infection. Aims Use mouse models of RV infection with modulation of IL-15 or type I IFN signalling and assess airway inflammation and viral load. Results RV-1B infection induced upregulation of IL-15 gene and protein expression at day 1 in lung tissue (2.5-fold P<0.001) and was proceeded by an activated (increased CD69, GranzymeB and IFN-γ expression) NK cell response in the lungs at days 2-4. Neutralisation of airway IL-15 with a monoclonal antibody at the time of infection inhibited NK and CD8+ T cell responses. In type I IFN receptor knockout mice, deficient RV-induced IL-15 expression (P<0.01) was associated with deficient NK and CD8+ T cell responses and increased viral load. This study demonstrates the necessity of type I IFN signalling for increased IL-15 expression during RV infection, which is important for NK and CD8+ T cell antiviral responses. These data identify type I IFNs and IL-15 as potential therapeutics to prevent/treat RV-induced diseases such as asthma exacerbations.
Optimal germinal center B cell activation and T-dependent antibody production is dependent on Complement Receptor 1 (P1461)
Authors: Donius Luke , Orlando Christopher , Handy Jennifer , Weis Janis , Weis John
Published: January 2023

Rapid identification of antigens during a primary immune response requires timely detection, opsonization, and delivery to immune follicles for B cell activation. Evidence demonstrates complement sequestration of antigen is important for efficient detection and antibody production by B cells. Follicular dendritic cells (FDC), which promote B cell activation and maturation by organizing germinal centers and retaining antigen, as well as B cells, are exclusive expressers of the Complement Receptor 2 (Cr2) gene products Cr1 and Cr2. We show here that Cr1 is the nearly exclusive product of the Cr2 gene on FDC and, in contrast, Cr2 is the predominant product on B cells. Consistent with FDC restricted expression of Cr1, immunization of Cr1 deficient (Cr1KO) mice demonstrated deficiencies in antibody and B cell responses to T-dependent but not T-independent (TI) antigens. Cr1KO mice exhibit a reduction in antigen specific IgM and IgG3 during primary immunization with TNP-KLH, and do not generate activated germinal center B cells as readily as wildtype mice in response to sheep red blood cell. Cr1KO mice generate effective immunity to Streptococcus pneumoniae, which typically is controlled by TI induced antibody against the bacterial polysaccharide capsule. In total the results of this study define a specific role for Cr1 in B cell germinal center maturation, and defines the Cr1KO mouse as a new tool for studying activation of B cells during primary immune responses.
The role of NCOR co-repressors in lymphocyte development and function (P1471)
Authors: Manlove Luke , Farrar Michael
Published: January 2023

The related co-repressors NCOR1 and NCOR2 interact with transcription factors required for lymphocyte development and function. To examine the role of NCOR2 in lymphocytes, we generated CD4-Cre x Ncor2FL/FL and Mb1-Cre x Ncor2FL/FL mice. Deletion of Ncor2 in T cells had a modest effect on T cell development. Specifically, in mixed bone marrow chimeras comparing Ncor2-deficient versus wild type T cells, we observed that loss of Ncor2 impairs the Double Positive to Single Positive transition, suggesting an effect on positive or negative selection. We are currently generating CD4-Cre x Ncor1FL/FL x Ncor2FL/FL mice to determine whether NCOR1 and NCOR2 play partially redundant roles in thymocyte development. In addition, we are examining the impact that NCOR2 deficiency has on T cell function. Unlike T cell development, deletion of Ncor2 alone in B cells led to a dramatic reduction in mature B cells at the T2 to mature B cell transition. We are currently characterizing NCOR2 at the molecular level to determine how NCOR2 modulates transcription and B cell development. Finally, we are generating Mb1-Cre x Ncor1FL/FL and Mb1-Cre x Ncor1FL/FL x Ncor2FL/FL mice to assess redundancy between the NCOR1 and NCOR2 proteins in B cell development. We suspect that NCOR1 and NCOR2 may have redundant roles in modulating transcription in lymphocytes, and that ablation of both co-repressors will lead to pronounced developmental blocks in both B and T lymphocytes.
Human CARD11 deficiency causes profound combined immunodeficiency (P3325)
Authors: Warnatz Klaus , Baerbel Keller , Buchta Mary , Kienzler Anne-Kathrin , Elpeleg Orly , Somech Raz , Cohen Sivan , Shachar Idit , Miosge Lisa , Schlesier Michael , Fuchs Ilka , Enders Anselm , Eibel Hermann , Grimbacher Bodo , Stapansky Polina
Published: January 2023

Patients with primary immunodeficiency present an excellent opportunity to understand the role of specific molecules in the human immune system. Recently we had the chance to investigate a young girl with a complete CARD11 deficiency due to a homozygous deletion of exon 21. Clinically the child presented with increased susceptibility to respiratory tract infections at 6 months, before she was diagnosed at 13 months with Pneumocystis jirovecii revealing her combined immunodeficiency. The immunological characterization showed severe hypogammaglobulinemia, but normal T-, B- and NK-cell numbers. While there were only subtle changes in the T cell compartment, B-cell differentiation was blocked at the transitional stage. In the absence of CARD11 protein expression activation of the canonical NF-κB pathway after antigen receptor or PMA stimulation was abrogated, while CD40 signaling in B cells was preserved. CARD11 deficient T cells were severely impaired in the up-regulation of ICOS, OX40, cytokine production and proliferation after TCR stimulation. On B cells BAFF receptor expression was reduced and after activation ICAM1 and CD25 were not induced while CD86 induction was comparable to control cells. Immunoglobulin production was intact after anti-IL-21/CD40L stimulation, demonstrating that plasma cell differentiation was not dependent on intact CARD11. Thus CARD11 plays a crucial, non-redundant role in the antigen specific immune response in humans.
Autoimmune susceptibility gene critically influences CD39 T cell expression and function in modulating human inflammation (P3313)
Authors: Moncrieffe Halima , Ursu Simona , Pesenacker Anne , Gordon-Smith Sophie , Zheng Dongling , Wedderburn Lucy
Published: January 2023

Background: Purinergic signaling is an important mechanism of immunomodulation. The ectoenzyme CD39 is a key regulator of extracellular ATP levels and catalyses hydrolysis of ATP to AMP. The A allele of the CD39 SNP rs10748643 is associated with both susceptibility to the autoimmune condition Crohn’s disease and low CD39 RNA transcript expression levels in HapMap transformed immune cell lines. Aim: To investigate the association between CD39 genetic polymorphism, protein expression and function in human healthy and autoimmune arthritis inflamed tissues. Results: We demonstrate increased CD39 protein on T cells in the human inflamed arthritis joint compared to peripheral blood. SNP rs10748643 AA individuals had a greatly reduced CD39 T cell expression and ATPase capacity compared to GG individuals (p<0.001). An electrophoretic mobility shift assay revealed a decrease in nuclear protein-DNA complex with rs10748643 AA probe compared to the GG probe. Bioinformatic analysis revealed AA individuals had ablation of an Sp1 transcription factor binding site, previously shown to be important in CD39 upregulation. Discussion: This is the first study to show how a specific SNP genotype, previously found in increased frequencies in patients with Crohn’s autoimmune disease and associated with reduced CD39 mRNA expression, is associated with functional modulation of the immune system.
In vivo trafficking, persistence and efficacy of Lewis-Y chimeric antigen receptor T cells in patients with Lewis-Y positive acute myeloid leukaemia (P4354)
Authors: Neeson Paul , Ritchie David , Khot Amit , Peinert Stefan , Tai Tsin , Honemann Dirk , Gambell Peter , Westerman David , Westwood Jennifer , Scott Andrew , Kravets Lucy , Dickinson Michael , Trapani Joseph , Smyth Mark , Darcy Phillip , Kershaw Michael , Prince H.
Published: January 2023

Second generation chimeric antigen receptor (CAR) T cells were used to treat patients with acute myeloid leukemia (AML) in a phase I clinical study. Autologous T cells were genetically modified to express a CAR which re-directed T cell effector function to the LeY tumor associated carbohydrate antigen on AML cells. CAR-T cell therapy safety, AML disease response, and CAR-T cell trafficking and persistence post-infusion were investigated. Five patients received GMP grade CAR-T cells (LeY-T). Post infusion, no patients experienced grade 3 or 4 toxicities. Patient AML responses to LeY-T cell infusion included a transient cytogenetic response and a reduction in peripheral blood leukemic blast count. In all patients, LeY-T cells trafficked thru peripheral blood, and persisted in the bone marrow. In one patient, leukemia cutis was associated with trafficking of the LeY-T cells to the skin at sites of AML blast infiltration. Despite LeY-T cells being present at the disease site, relapse with LeY-expressing AML blasts occurred in all patients (range 29 days to 23 months) post-infusion. Further studies indicated LeY-T cell CAR expression was downregulated post-infusion in vivo, this was also observed post-LeY antigen exposure and long term culture in vitro. This study provides important safety and feasibility data to support the application of CAR-T cell therapy to treat AML. Furthermore, we provide a potential mechanism for tumor escape from LeY-T cell surveillance in vivo.
Differential signaling responses may promote proliferation and survival of chimeric antigen receptors linked to 4-1BB:CD3ζ. (P4356)
Authors: Posey Avery , Kawalekar Omkar , June Carl
Published: January 2023

Chimeric antigen receptors have been explored clinically with more disappointments than successes. T lymphocytes modified with CARs bearing the CD137 (4-1BB) costimulation domain have been successful in patients with acute and chronic leukemia. We hypothesized that the initial signaling events after CAR-target ligation might identify mechanisms responsible for prolonged persistence in vivo. Human T cells were electroporated with mRNA to express anti-mesothelin chimeric antigen receptors containing either the CD28:CD3ζ domains, 4-1BB:CD3ζ domains or CD3ζ domain alone. CAR T cells expressing the 28ζ domains showed a rapid calcium flux after stimulation with surrogate antigen (mesothelin-Fc fusion protein) that was similar to TCR stimulation via endogenous CD3ζ, but no detectable calcium flux was observed for BBζ or ζ alone CARs. BBζ and ζ alone CARs showed a similar calcium response to 28ζ CAR T cells when stimulated with OKT3. 28ζ CAR T cells also show an intense increase in both proximal and distal phosphoproteins, compared to a moderate increase from BBζ and ζ alone CARs. Here we report that CARs containing 28ζ demonstrate accelerated signaling compared to BBζ and ζ only CARs. These results may have implications for sustainability and effectiveness of CAR-T cells in future clinical studies.
T-bet dictates CD8+ T cell tolerance versus immunity following antigen recognition (P4368)
Authors: Jackson Stephanie , Yuan Jinyun , Berrien-Elliott Melissa , Meyer Jennifer , Teague Ryan
Published: January 2023

Efforts to overcome tolerance to improve adoptive immunotherapy for cancer have been hindered because the T cell intrinsic pathways that regulate whether tumor/self-reactive T cells are directed toward tolerance versus immunity remain largely undefined. Our lab studies a murine model of tolerance in which adoptively transferred tumor/self-reactive CD8+ T cells undergo multiple rounds of division following engagement of tumor/self-antigen, but fail to produce effector molecules and are progressively deleted without providing anti-tumor immunity. Here, we demonstrate that T cells engaging antigen within this tolerizing environment fail to express the T-box transcription factor, T-bet, which is induced under immunizing conditions. We further establish that restoring T-bet expression in these T cells within the tolerizing environment by vaccination with Listeria monocytogenes corresponds with their acquisition of effector function and the prolonged survival of leukemia-bearing hosts. Utilizing T-bet deficient T cells, we confirmed that T-bet is required for this rescue of effector function and for therapeutic anti-tumor immunity within a tolerizing environment. We are now utilizing T cells with constitutive T-bet expression to determine if T-bet alone is sufficient to overcome tolerance. Our results identify T-bet as a CD8+ T cell-intrinsic regulator of effector responses within a tolerizing environment and have important implications for immunotherapy in patients with cancer.
Redirection of TH17 cells with a chimeric antigen receptor containing the ICOS costimulatory domain enhances function, antitumor activity and persistence of TH17 cells (P4350)
Authors: Guedan Sonia , Carpenito Carmine , Chen Xi , Madar Aviv , McGettigan Shannon , Frigault Matthew , Scholler John , Scholler Nathalie , Bonneau Richard , June Carl
Published: January 2023

Adoptive transfer of TH17 cells polarized and expanded in vitro is an attractive therapy for the treatment of cancer. CD278, the inducible costimulator (ICOS) has been shown to be critical for the sustained expansion of human TH17 cells after their primary activation. We hypothesized that the incorporation of the ICOS intracellular domain in a CAR can promote the Th17 phenotype after antigen priming and enhance the antitumor activity of engineered T cell therapies. Th17 polarized cells were engineered to express a CAR comprised of a single-chain variable fragment that binds mesothelin fused to the T cell receptor- ζ signal transduction domain in tandem with the CD28, CD137 (4-1BB) or CD278 (ICOS) intracellular domains. Upon antigen recognition, Th17 cells redirected with the CAR:ICOS maintained a core molecular signature of Th17 cells, secreted higher amounts of IL17A, IL17F and CCL20 and expressed higher levels of CD161, NKG2D, IL1R-1 when compared with CD28 and 4-1BB CARs. When transferred into NSG mice with large tumors, Th17/Tc17 cells redirected with the CAR: ICOS mediated enhanced anti tumor responses and showed increased persistence. Our studies indicate that redirection of Th17 cells with a CAR encoding the ICOS intracellular domain is critical for obtaining potent Th17 cells with enhanced function and persistence. The design of novel ICOS-based CARs has the potential to augment antitumor effects in clinical trials.
Effect of combined treatment of selective serotonin reuptake inhibitors plus immunomodulator on circulating cytokines levels in patients with major depressive disorder along 52 weeks of follow up (P4521)
Authors: Pavon Lenin , Hernandez Maria Eugenia , Perez-Tapia Sonia Mayra , Mendieta Danelia , Bojalil Rafael , Estrada-Garcia Iris , Estrada-Parra Sergio
Published: January 2023

Major Depressive Disorder (MDD) is a psychiatric illness that commonly displays a deficit of serotonergic neurotransmission in the central nervous system. MDD patients also show alterations in alterations in cortisol and cytokines levels. Those patients are treated with selective serotonin reuptake inhibitors (SSRIs). The aim of this study was to assess the efficacy of the combined treatment with SSRIs plus an immunomodulator -dialyzable leukocyte extract (DLE) - in restoring the balance of both the serotoninergic system and the endocrinoimmune parameters. A total of 34 patients diagnosed with MDD received anti-depressant treatment with SSRIs plus DLE. As control 31 patients received only SSRIs. Proinflammatory cytokines (Th1) IL-1β, IL-2, IFN-γ, anti-inflammatory cytokines (Th2) IL-13, IL-10, and 24-h urine cortisol were determined at weeks (W) 0, 5, 20, 36 and 52 of treatment. The maximum reduction in cortisol levels in SSRIs group was only 30% (W52). By contrast, the treatment with SSRIs plus DLE decreased 53% the cortisol levels from W20. Reduced cortisol in patients treated with SSRI plus DLE correlated with reductions in the concentration of Th2-cytokine and increases in Th1-cytokines at the end of the study. These results suggest that the immune-stimulating action of DLE, in association with the effects of SSRIs, allowed the immune system to balance the ratio of Th1/Th2 cytokines associated and decreased levels of cortisol.
Development of pattern recognition receptor ligands as malaria vaccine adjuvants (P4532)
Authors: Chang Sandra , Chang Martin , Posner Jourdan
Published: January 2023

A critical obstacle to malaria vaccine development is the lack of appropriate adjuvants to elicit potent and protective immunity. Recent insights into the role of pattern recognition receptors (PRRs) in dendritic cell (DC) activation and identification of follicular helper T (Tfh) cells, as specialized providers of B cell help, enable a novel approach to adjuvant selection. We hypothesize that specific combinations of PRR agonists will be effective adjuvants by promoting the differentiation of Tfh cells. We evaluated PRR ligands that activate through distinct intracellular signaling pathways: a Toll-like receptor (TLR) agonist signaling through the MyD88-dependent pathway (R848), TLR agonists signaling through the TRIF-dependent pathway (p(I:C) and GLA); an agonist of Nod1 (C12-iE-DAP) that signals through RIP2; and an agonist for Mincle (synthetic trehalose-dibehenate) that signals through FcRγ Src-family kinase. R848 was a strong inducer of DC activation in vitro, particularly when combined with p(I:C), inducing significantly higher cytokine RNA (IL-12, TNFα) and protein (IL-12, TNFα, IL-6, IFN-γ), producing higher DC expression of CD86, and activating both pDCs and cDCs. IL-6, a potent Tfh stimulator, was maximal in DC cultures stimulated with R848 combinations. Ongoing studies will evaluate the ability of specific PRR combinations to polarize naïve CD4+ T cells to differentiate into Tfh cells in vitro and in vivo and provide help for P. falciparum blood-stage vaccines.
Flow cytometric analysis of bone marrow and spleen cells in adjuvant-treated animals (P4502)
Authors: Yang Ya-Wun , Chang Chih-Feng
Published: January 2023

Despite the poorly understood mechanisms, vaccine adjuvants have been extensively used to enhance the immunogenicity of specific antigens in vaccination. To examine the effect of adjuvants on the differentiation of the hematopoietic system, C57BL/6J mice were immunized with ovalbumin (OVA) with alum, followed by flow cytometric analysis of cells in the bone marrow and spleens after vaccination. Our data showed that treatment of animals with alum upregulated the expression of AA4.1 in the Dump(CD3/CD8/Gr-1/F4/80)(-)CD24(+) population, and a concomitant increase of both B220(-)CD11b(+) and CD24(+)CD43(+) cells in the spleen, indicating the differentiation effect on the hematopoietic cell development. Our results also showed that Dump(-)B220(-)CD24(+) population in the bone marrow were significantly elevated, suggesting the potential induction of myeloid differentiation from proB populations in the adjuvant-treated animals. Similar results were obtained in the complete-Freund's adjuvant (CFA)-treated mice. Treatment of animals with CFA was found to elevate the lineage2 (CD3/CD19/NK1.1/B220/Ter119)(-)CD11b(+) population at the expense of lineage2(+) cells, suggesting the potential differentiation of hematopoietic stem cells towards the myeloid lineage from the lymphoid progenitors. These results raised the potential effect of vaccine adjuvants on modulation of hematopoietic hierarchy, explaining their actions of mechanism on stimulation of the specific immunological responses.
BPV VLPs presenting HIV-1 epitopes from membrane-proximal external region of gp41 induced HIV-specific mucosal and systemic cross-clade neutralizing antibodies (P4507)
Authors: Zhai Yougang , Zhong Zhenyu , Zariffard Mohammadreza , Spear Gregory , Qiao Liang
Published: January 2023

Two conserved epitopes, located in the membrane-proximal external region (MPER) of gp41 of the human immunodeficiency virus type 1 (HIV-1) envelope spike, are promising targets for vaccine design in efforts to elicit 2F5 and 4E10-like anti-HIV-1 broadly neutralizing antibodies. Since most HIV-1 infections initiate at mucosal surfaces, the induction of mucosal neutralizing antibodies is necessary and of utmost importance to counteract HIV-1 infection. Here, we utilized a mucosal vaccine vector, bovine papillomavirus (BPV) virus-like particles (VLPs), as a platform to present HIV-1 neutralizing epitopes by inserting the extended 2F5 (ELLELDKWASLWN) or 4E10 epitope (NWFDITNWLWYIK) or the MPER domain into surface loops of BPV L1 respectively. In order to enhance the presentation of the neutralizing epitopes, the modified 4E10 and 2F5 epitope were presented by inserting two alanines (AA) in N-terminal, C-terminal or both terminals of the epitopes. The chimeric VLPs presenting MPER domain resembled the HIV-1 natural epitopes better than the chimeric VLPs presenting unmodified single ones. Moreover, N-terminal modified 2F5 epitope further enhanced the epitope presentation when inserted in D-E loop. Oral immunization of mice with the chimeric VLPs displaying 2F5 or MPER elicited epitope-specific serum IgGs and mucosal secretory IgAs. The antibodies induced by chimeric VLPs presenting MPER domain are capable of neutralizing clade B and clade C HIV-1 viruses, although modest.
Associating long-lived liver CD49b+CD8b+ T cells with long-lasting immunity against malaria (P4481)
Authors: Jang Ihn , Chen Lin , Peng Xiaohong , Choi Kimberly , Gibson Claire , Wang Ruobing
Published: January 2023

The sterile and long-lasting immunity against malaria can be induced by vaccination with genetically attenuated parasites (GAP). We have demonstrated that GAP-specific CD8+ T cells are key in protective immunity against pre-erythrocytic stages in mice. Here, we further characterized the CD8+ T cell phenotype and function associated with the longevity of the sterile protection induced by PyFabb/f- GAP. We demonstrated that vaccination of mice with Fabb/f- GAP resulted in a significant increase of effector/memory CD8+ T cells characterized as CD8bhi or CD8blo T cells expressing integrin CD49b primarily detected in the liver, a primary infection site. More importantly, CD49b+CD8blo T cell subset persisted for a long period and strongly associated with GAP-induced prolonged protection in Balb/c mice. In contrast, Fabb/f- GAP induced a short-lived CD49b+CD8bhi T cells but failed to induce a sustainable CD49b+CD8blo T cells leading to a short-term protection in C57Bl/6 mice. In understanding the biological effects of these CD8 T cell subsets, ex vivo functions and transcriptional analysis are underway. The predictive contributions of short-lived and long-lived CD8 T cells induced by GAP vaccination in protective efficacy against malaria would be highlighted by our findings.
Activated mesenchymal stem cells amplify antibiotic activity against chronic Staphylococcus aureus infection (P5056)
Authors: Johnson Valerie , Webb Tracy , Dow Steven
Published: January 2023

Background/Rationale: Antimicrobial resistance is one of the greatest challenges facing the medical community today and new interventions are needed. Recent studies have shown that mesenchymal stem cells (MSC) exhibit antimicrobial activity when activated by Toll-like receptor (TLR) ligands in vitro. We hypothesized that activated MSC could enhance the activity of conventional antibiotics in common wound infections. Methods: MSC derived from adipose tissue of mice were expanded in vitro and activated with TLR ligands to assess the effects on antimicrobial activity and production of antimicrobial peptides (AMP). The effects of MSC treatment in vivo were assessed using a mouse model of chronic S. aureus biofilm infection. Mice were treated with a series of 4 MSC i.v. injections, with or without antibiotic treatment, and bacterial load assessed over 14 days via IVIS imaging and bacterial culture of the wound site. Results: Activation of MSC with TLR ligands significantly increased production of the AMP CXCL10 and inhibited bacterial growth in vitro. The most potent TLR agonist in vitro was the TLR3 agonist polyI:C. In the chronic S. aureus infection model, activation of MSC with polyI:C resulted in decreased bacterial counts. Conclusions: Treatment with activated MSC induced AMP production and enhanced antibiotic therapy in vivo. These results suggest that stem cell therapy using activated MSC may be an effective means to enhance antibiotic therapy for chronic infections.
Co-transplantation of mesenchymal stem cell and fibroblast reduces inflammation and corrects defective dermal remodeling (P5062)
Authors: Yates Cecelia , Rodrigues Melanie , Wells Alan
Published: January 2023

Mesenchymal stem cell have been reported to enhance wound healing following transplantation however the mechanism for this response is unknown. The mechanism responsible for enhance wound healing includes the possible restorative paracrine effects of MSC synthesized soluble factors on dermal cell type. This study examines the influence MSC have on matrix remodeling during repair an in vivo system. We recently developed a chronic wound scar model based on the absence of signaling through the CXCR3 system; these mice develop sterile chronic inflammation and hypertrophic scars contributed in part by the defective fibroblast. Using these fibroblasts encapsulated with MSC, we sought to determine whether the fibroblast could overcome endogenous deficiencies and generate a physiological dermal tissue. Xenotransplantation of labeled CXCR3-/- mouse fibroblast and hMSC into wounds of both a CXCR3-/- scarring model revealed altered healing patterns. Results showed an enhanced healing response, correction of matrix regeneration, and reduced inflammation during the first 30 days. Flow cytometric analysis of the wound bed at days 1,3,5 revealed a reduction in CD4+ and CD8+ T-cells. Changes in matrix and soluble signals were also examined histologically. These data suggest there is a connection between the initial inflammatory response and matrix changes that regulates scarring. The current study provides insight into cellular therapies as viable method for antifibrotic treatment.
Resolvin E1 modulates Mac-1 signaling and promotes neutrophil apoptosis and the resolution of acute inflammation (P5064)
Authors: Filep Janos , Gjorstrup Per , El Kebir Driss
Published: January 2023

Resolvin E1 (RvE1), derived from the omega-3 polyunsaturated fatty acid eicosapentaenoic acid inhibits Mac-1 (CD11b/CD18)-mediated neutrophil trafficking. Mac-1also generates ligand-specific outside-in signals to modulate neutrophil apoptosis, a critical control point in the resolution of inflammation. We studied the impact of RvE1 on Mac-1signaling and neutrophil apoptosis. Culture of human neutrophils with RvE1 (0.1-10 nM) mitigated ERK and Akt-mediated survival signals from the Mac-1 ligand myeloperoxidase, leading to Mcl-1 degradation and mitochondrial dysfunction. RvE1 facilitated phagocytosis of C3b-opsonized E. coli and triggered phagocytosis-induced apoptosis. RvE1 through the leukotriene B4 receptor BLT1 evoked ROS generation by NADPH oxidase, leading to activation of caspase-8 and 3. In mice, treatment with RvE1 at the peak of inflammation enhanced the resolution of E. coli pneumonia, E. coli peritonitis-associated and carrageenan plus myeloperoxidase-evoked neutrophil-mediated lung injury through facilitating neutrophil apoptosis and removal of apoptotic neutrophils by macrophages. These actions of RvE1 were prevented by the pan-caspase inhibitor zVAD-fmk. Our results identify a novel resolution circuit, in which RvE1 through BLT1 shifts the balance between Mac-1-generated competing pro- and anti-apoptotic signals toward apoptotic pathways, and consequently enhances resolution of acute lung inflammation.
Serum amyloid A induces M2 macrophage-specific genes expression and promotes efferocytosis of apoptotic neutrophils (P5072)
Authors: Sun Lei , Zhou Huibin , Zhu Ziyan , Yan Qian , Ye Richard
Published: January 2023

Macrophages play an important role in the quality, duration and magnitude of most inflammatory reactions in a polarized manner. However, the determinants that modulate their plasticity remain unclear. Serum amyloid A is an acute-phase protein which induces both anti-inflammatory and pro-inflammatory cytokines during inflammation. In this study, we explored the potential involvement of SAA in modulating macrophage polarization. SAA not only induced the expression of anti-inflammatory M2-specific genes such as IL-10, Arginase-1, Ym-1, Fizz-1, Mrc-1, IL-1Rn, CCL17, but also promoted efferocytosis of apoptotic neutrophils, a process that is critical for the resolution of inflammation. Through experiments with formyl peptide receptor 2 antogonist WRW4 and Fpr2 deficient macrophages, we showed that the induction of M2-specific genes and efferocytosis by SAA were mediated specifically by Fpr2. Additionally, SAA stimulation resulted in the increase expression and activation of the transcription factor interferon regulatory factor 4. Selective inhibitors of ERK activity abrogated SAA induction upon M2 macrophages and efferocytosis by decreasing the expression and activation of IRF4. Knocking down the expression of IRF4 abrogated the effect of SAA on M2 macrophage. Our results demonstrate that SAA can induce M2 macrophage-specific genes and promote efferocytosis via Fpr2-ERK-IRF4 signaling, and suggests an alternative mechanism by which SAA is involved in anti-inflammatory responses.
Transcription factor Fli-1 is involved in regulation of inflammatory mediator production and ECM remodeling in primary dermal fibroblasts (P5088)
Authors: Sztokman Nicole , Zhang Xian
Published: January 2023

Numerous inflammatory and fibrotic disorders arise from process malfunctions in fibroblast regulation of extracellular matrix remodeling and cytokine expression in which the Friend leukemia integration factor 1, an E26 transformation-specific family transcription factor, participates. We have previously shown that Fli-1 regulates inflammatory monocyte chemoattractant protein 1 expression in endothelial cells and Fli-1 is highly expressed in fibroblasts. In this report, we investigated the involvement of Fli-1 in extracellular matrix remodeling and regulation of inflammatory mediators in dermal fibroblasts. We isolated primary dermal fibroblasts from wild type and Fli-1 heterozygous C57/BL6 mice. Expression of Fli-1 in fibroblasts from Fli-1 heterozygous mice was reduced by half compared to expression in wild-type mice. We measured the concentration of MCP-1 in supernatants from dermal fibroblasts after simulation with lipopolysaccharide and found MCP-1 concentrations in Fli-1 heterozygous mice to be significantly increased compared with wild type controls. The concentrations of other inflammatory mediators including matrix metalloproteinase I in the supernatants are currently being studied. Our results suggest that Fli-1 is involved in regulating the expression of inflammatory mediators in dermal fibroblasts.