bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid and systemic innate immune responses to an adenoviral HIV vaccine (45.27)
Authors: Andersen-Nissen Erica , Zak Daniel E , Duerr Ann C , Peterson Eric R , Robertson Michael N , DeRosa Stephen C , McElrath M. Juliana , (HVTN) and the NIAID-sponsored HIV Vaccine Trials Network
Published: January 2023

Understanding innate immune responses to candidate HIV vaccines will guide vaccine design. We assessed the early innate response in volunteers enrolled in HVTN 071, a trial of intramuscularly delivered Merck adenovirus serotype 5 (MRKAd5) gag/pol/nef- the same vaccine used in the Step trial. Samples were collected pre-vaccination and at 4-6 hours, days 1, 3, and 7 (n=11) post-vaccination or at day 1 post-vaccination (n=24). Cytokine and chemokine levels in the serum were measured by multiplex assay and cell populations were analyzed by multiparameter flow cytometry. Serum levels of IP-10, MCP-1, TNF-α, IL-6, IL-1RA, IFN-γ, and IL-10 were similar at baseline and 4-6 hours post-vaccination, showed up to 10-fold increases over baseline on day 1 post-vaccination, and returned to baseline levels by day 3. Dendritic cell concentrations increased in 4 hours; monocytes increased gradually and peaked at day 3. In contrast, T, B, and NK cell blood concentrations decreased sharply at day 1 post-vaccination, suggesting massive trafficking to the vaccination site. Moreover, PBMC gene expression analysis by microarray consistently revealed strong induction of inflammatory and antiviral genes. Together, these data demonstrate robust early immune activation after vaccination. Future studies of innate immune responses to other candidate HIV vaccines may elucidate why the vaccine used in the Step trial did not show efficacy.
Reduced Th17 cells in a patient with recurrent staphylococcal disease and neutralizing autoantibodies against IL-6 (47.33)
Authors: PUEL Anne , de Beaucoudrey Ludovic , Chrabieh Maya , Picard Capucine , Gendrel Dominique , Casanova Jean-Laurent
Published: January 2023

We investigated a patient presenting with two severe episodes of staphylococcal cellulitis and abscesses. We identified in the patient's serum high titers of autoantibodies against IL-6, able to specifically almost completely neutralized IL-6 ex vivo and in vitro. The existence of a relationship between IL-6 deficiency and susceptibility to S. aureus recently gained credence from the identification of patients with STAT3 deficiency and an autosomal dominant form of the hyper-IgE syndrome, as these patients often present recurrent staphylococcal skin abscesses and show defective responses in particular to IL-6 and IL-10. A defect in the Th17 population differentiation has been reported in STAT3 deficient patients. In order to determine the role of IL-6 in Th17 differentiation in human, we investigated the presence of this population ex vivo and in vitro in our patient. We observed a strong decrease of Th17 positive cells both ex vivo and after differentiation in vitro, strongly suggesting an important role of IL-6 in Th17 development in vivo in human
Clinical-electrophysiologic-pathologic correlations in severe murine experimental autoimmune neuritis (48.14)
Authors: Ubogu Eroboghene E , Yosef Nejla , Xia Robin H
Published: January 2023

Severe murine experimental autoimmune neuritis (sm-EAN) is a reliable animal model of Guillain-Barré Syndrome. There is insufficient data characterizing the inflammatory infiltrates in this model or correlating neuromuscular severity with electrophysiological parameters. Sm-EAN was induced in 8-12 week old female SJL/J mice as previously published. Disease onset occurred at 9-13 days, with maximal severity between 26-32 days post-induction (p.i.). There was residual mild weakness >2 months p.i. Severe multifocal or diffuse demyelination with some mild axonal loss was observed at peak severity, associated with mononuclear cell infiltrates (F4/80+ macrophages, CD3+ and CD4+ T-lymphocytes and CD19+ B-cells). There was increased expression of CCL2-CCR2, CCL5-CCR1, CCR5 and CXCL10-CXCR3 at peak severity. Residual infiltrates and rudimentary 'onion bulb' formation were seen during the recovery phase. Serial electrophysiological studies of the dorsal caudal tail nerve and bilateral sciatic nerves showed statistically significant reductions in mean compound motor action potential (CMAP) amplitudes and conduction velocity and increases in mean CMAP durations compared to controls. Regression analyses demonstrated correlations (r2=0.78-0.99) between neuromuscular severity and the above parameters, with stronger correlations seen with the dorsal caudal tail motor nerve responses.
Targeted depletion of lymphotoxin-alpha-expressing Th1 and Th17 cells inhibits autoimmune disease (48.22)
Authors: Grogan Jane L , Chiang Eugene , Yu Xin , Kolumam Ganesh
Published: January 2023

Aberrant autoimmune responses are often underpinned by dysregulated and uncontrolled CD4+ T helper (Th) cell subsets Th1 and Th17. We identify surface lymphotoxin-alpha (LTa) as common to Th1 and Th17 cells and employ a novel strategy to target these subsets using a depleting monoclonal antibody directed to surface LTa. A depleting anti-LTa mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of anti-LTa; inhibited disease, resulting in immunoablation of T cells expressing IL-17, IFN-g; and TNF-a, whereas a decoy LTbR fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of FcgR binding and ADCC activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1b and TNF-a within joints. These data indicate that depleting Th1 and Th17 with anti-LTa mAb may be beneficial in the treatment of autoimmune disease.
IMO-3100, an Antagonist of Toll-Like Receptors 7 and 9, Modulates Gene Expression and Regulatory Networks Induced by Ligands (48.25)
Authors: Jiang Weiwen , Bhagat Lakshmi , Yu Dong , Kandimalla Ekambar R , Agrawal Sudhir
Published: January 2023

In autoimmune diseases, immune complexes containing self-nucleic acids have been shown to induce inflammatory responses through TLR7 and 9. IMO-3100 is a novel DNA-based antagonist of TLR7 and 9 that is shown to inhibit TLR7- and 9-mediated immune responses. To elucidate inhibitory effects of IMO-3100 on TLR7 and 9 agonist-mediated gene expression, we treated human PBMCs with a TLR7 or TLR9 agonist with or without IMO-3100, isolated total RNA, carried out gene expression profiling on human inflammatory response and autoimmunity microarray, and analyzed data to identify changes in regulatory networks. IMO-3100 alone showed insignificant up-regulation of a small number of genes. TLR9 agonist significantly up-regulated a number of genes, including IP-10, MyD88, MCP-1, IL-6, IRF7, IL-17A, NF-κB1, and RIPK2, which regulate immune responses, NF-κB signaling, inflammation, and chemotaxis networks. TLR7 agonist showed a different gene expression profile compared with TLR9 agonist. The genes that were significantly up-regulated by TLR7 agonist include IP-10, IRF7, IFN-α1, IFN-α4, IFN-γ, IL-15Ra, IL-17A, IL-6, and TIRAP, which regulate host defense response, chemotaxis, and cytokine and chemokine signaling pathways. Co-incubation of IMO-3100 with either TLR agonist resulted in inhibition of genes that were up-regulated with either TLR agonist alone. These results indicate that TLR7 and 9 agonists induce different gene expression profiles and IMO-3100 is a potent inhibitor of these responses. IMO-3100 is a lead candidate identified for development for potential applications in autoimmune diseases.
TLR2 dependent activation of IL-6/IL-17 axis in the development of Collagen-Induced Arthritis (48.28)
Authors: Ortmann Rob , Sisk Bryan , Krogstad Aric , Kannan Krishna
Published: January 2023

Toll-like receptors (TLRs) are pattern-recognition receptors for foreign microbial products which initiate host defense responses. Several lines of evidence suggest that abnormal TLR signaling may lead to excess production of pro-inflammatory cytokines and development of Th-17 cells, a pathogenic T helper cell subset implicated in Collagen-Induced Arthritis (CIA). In this study, we examined the TLR2 dependent cytokine milieu that promotes Th-17 polarization and the development of disease. Using C57BL/6 (B6), DBA/1, and B6 TLR2-KO mice, we examined the role of TLR2 signaling in the development of pro-inflammatory responses during various phases of arthritis development. We observed that TLR2-KO mice were resistant to disease whereas both B6 and DBA/1 mice developed CIA 4 to 5 weeks after immunization. Ex vivo culture of spleen cells and draining lymph node cells demonstrated significantly elevated levels of IL-6, TNF-α, IFN-γ and IL-17 in response to type II collagen in DBA/1 and B6 mice as compared to TLR2 deficient animals. Importantly, IL-6 levels were elevated both before and after arthritis developed. Also, progressively higher levels of IL-17 and Th-17 cells were found as disease developed, and this increase was not seen in TLR2 deficient animals. Together, our results demonstrate a role for TLR2 mediated control of the IL-6/Th-17 axis in the development of collagen-induced arthritis.
Inhibition of TNFR1 signaling by P60 PLAD protein ameliorates skin lesions in lupus MRL/lpr mice (50.24)
Authors: Deng Guo-Min , Liu lena , Tsokos George C
Published: January 2023

Skin disease is the second most common manifestation in patients with systemic lupus erythematosus (SLE). The etiology of skin injury remains unclear. There is urgent need to understand better the pathogenesis of skin lesions and to develop effective, non-toxic therapy for cutaneous lupus erythematosus. MRL/lpr mice develop pathology similar to human SLE including skin lesions. To understand whether tumor necrosis factor (TNF) contributes to the pathogenesis of cutaneous lupus erythematosus, we treated lupus MRL/lpr mice with TNF receptor (R) defined PLAD protein. P60 PLAD inhibits the function of TNFR1, whereas P80 PLAD inhibits the function of TNFR2. We found that P60 PLAD protein significantly inhibited skin injury but P80 PLAD failed to do so in MRL/lpr mice. Both PLAD proteins promoted the development of proteinuria. Immunohistochemistry studies of skin lesions demonstrated that TNFR1 but not TNFR2 in expressed in the skin lesion of MRL/lpr mice. P60 PLAD significantly inhibited NF-kB, MCP-1 and iNOS expression by skin keratinocytes. Our results indicate that TNFR1 contributes to the development of skin lesions in lupus-prone mice suggest that it can be targeted successfully to treat cutaneous SLE.
Beneficial Therapeutic Effect of Cellcept® in Systemic Lupus Erythematosis (SLE); Suggested Mechanism of Action (50.26)
Authors: nuhaily Samer , Yei Soonpin , Zarieh Ata , Meza Miguel , Zhang Hongying (Hannah) , Incardona Francesca , Esber Henry J , Damaj Bassam B
Published: January 2023

Cellcept® inhibits proliferative responses of T- and B-lymphocytes to both mitogenic and allospecific stimulation and suppresses antibody formation in B-lymphocytes. MPA prevents the glycosylation of lymphocyte and monocyte glycoproteins that are involved in adhesion to endothelial cells. Although Cellcept® is prescribed "off label" to treat SLE and shows great benefits, the exact mechanism of action is not fully understood. MRL/lpr developing glomerular disease mice that exhibit similar pathology to human SLE were used to study the therapeutic effects of Cellcept®. Although Cellcept® at 100mg/kg doses significantly decreased the proteinurea levels in the treated mice, it had marginal effects on the ds-DNA antibody levels. In addition, Cellcept® reduced the levels of B cells in the spleens of treated mice consistent with its immunosuppressant benefit. Histopathological evaluation demonstrated that Cellcept® reduced glomerulonephritis but not vasculitis in kidneys of treated mice. Furthermore, it was shown that Cellcept® lowered the number of monocytes in kidneys; this is the most likely mechanism of action responsible for the less kidney damage and lower proteinurea levels. The reduction in the number of monocytes in kidneys is the first major indication that the beneficial effect of Cellcept® to the kidney function in patients with SLE might be due to the inhibition of mocnocyte infiltration responsible for the destruction of nephritic tissues and not through the action on B cells by lowering the ds-DNA antibody levels.
APLP2 Diverts MHC-Peptide Complexes to Clathrin-Mediated Endocytosis and Lysosomal Degradation (78.14)
Authors: Tuli Amit , Sharma Mahak , Capek Haley , Naslavsky Naava , Caplan Steve , Solheim Joyce
Published: January 2023

The amyloid precursor-like protein 2 (APLP2) is a secreted protein that is ubiquitously expressed. We have shown previously that APLP2 associates with peptide-bound forms of H-2Kd, and influences the endocytosis, surface expression, stability, and turnover of Kd. Our new findings indicate that APLP2 is internalized in a clathrin-dependent manner, as a dominant negative dynamin II mutant and the C-terminal tail of AP180 (both inhibitors of clathrin-dependent endocytosis) block its internalization. However, endocytosis of Kd is not blocked by these inhibitors, consistent with findings by others that MHC class I molecules are internalized mainly through a clathrin-independent pathway. Furthermore, the APLP2 cytoplasmic tail contains overlapping tyrosine-based motifs that can potentially bind to adaptor protein AP-2. Mutation of the tyrosine in this sequence severely impaired endocytosis of APLP2. This APLP2 mutant, unlike wild-type APLP2, failed to enhance the endocytosis of Kd molecules. Also, we found that APLP2 and Kd bind at the plasma membrane (PM) and are internalized together. Upon increased APLP2 expression, Kd molecules were predominantly directed to the lysosomes rather than recycled to the PM. These findings suggest a model in which APLP2 binds Kd at the PM, facilitates uptake of Kd in a clathrin-dependent manner, and routes the endocytosed Kd to the lysosomal degradation pathway. Thus, APLP2 has a multi-step trafficking function which influences the expression of MHC-peptide complexes at the PM. [NIH GM57428 & GM74876]
Dendritic cell antigen presentation drives simultaneous cytokine production by effector and regulatory T cells in inflamed skin (78.24)
Authors: McLachlan James B , Catron Drew M , Moon James J , Jenkins Marc K
Published: January 2023

Effector (Teff) and regulatory (Treg) T cells produce cytokines that balance immunity and immunopathology at sites of infection. It is not known how this balance is achieved. Here, we show that Treg and Teff cells specific for the same foreign peptide:major histocompatibility complex II (pMHCII) ligand accumulated preferentially in a subcutaneous site injected with the relevant antigen plus an adjuvant. Some of the Treg cells in this site were producing IL-10 twelve days after injection while a similar fraction of the Teff cells were producing IFN-γ. Acute ablation of Treg cells increased the fraction of IFN-γ-producing Teff cells, indicating that Teff function was limited by the Treg cells. Production of cytokines by both populations was driven by pMHCII presentation by local CD11bhigh dermal dendritic cells. Therefore, balanced production of microbicidal and suppressive cytokines in inflamed skin is achieved by simultaneous dendritic cell antigen presentation to Teff and Treg cells.
Regulation of Th1 and Th2 phenotypes by γ-secretase inhibitor in an animal model for asthma (79.10)
Authors: Kang Jin Hyun , Kim Do Jin , Park Choon-Sik , Chung Il Yup
Published: January 2023

γ-Secretase inhibitor (GSI) has been used to effectively block Notch signaling, which is implicated in the differentiation and functional regulation of T helper (Th) effector cells. We investigated a therapeutic potential of GSI against development of Th2-mediated inflammatory airway disease and the possible mechanism. Intranasal administration of GSI led to inhibition of asthma phenotypes including airway inflammation, goblet cell metaplasia, and airway hyperresponsiveness. When BAL cells from inflamed airways of mice were stimulated via TCR- or non TCR-pathways in the presence of GSI, Th2 cytokine production was decreased while Th1 cytokine production was increased. Furthermore, GSI treatment of CD4+ T cells that were purified from BAL cells resulted in markedly elevated IFN-g production and declined IL-5 production. However, this analogy was not exactly applied to splenic CD4+ T cells isolated from both naïve and asthma-induced mice: both IL-5 and IFN-g productions were inhibited by GSI, indicating that CD4+ T cells differentially responded to GSI depending on places where they reside. These data suggest that GSI is of therapeutic benefit against asthma by modulating Th effector responses.
Alternatively activated macrophages participate in the recruitment of eosinophils to the lung in a murine model of allergic lung inflammation (79.2)
Authors: Ford Andrew Q. , Smith Elizabeth , Noben-Trauth Nancy , Keegan Achsah D.
Published: January 2023

The IL-4 receptor α (IL-4Rα) chain has a broad expression pattern and is involved in IL-4 and IL-13 signaling, allowing it to influence various pathological components of allergic lung inflammation. We've previously reported that IL-4Rα expression on both bone marrow derived and non-bone marrow derived cells contributes to the severity of allergic lung inflammation. We found a correlation between the expansion of cells expressing the IL-4Rα, CD11b, and IAd and the degree of eosinophilia in ovalbumin challenged mice, suggesting a possible role for monocytes in the development of disease. To determine the role of CD11b+ cells in allergic lung inflammation, CD11b cells were developed in vitro from IL-4Rα positive and negative mice and transferred to IL-4RαxRAG2 knockout mice; TH2 cells were provided exogenously. Mice receiving IL-4Rα+, CD11b+ cells showed a marked increase in the recruitment of eosinophils to the lung after challenge with ovalbumin as compared to mice receiving IL-4Rα-, CD11b+ cells. Furthermore, an increase in cells expressing the macrophage marker F4/80 and the alternatively activated macrophage marker Ym-1 were recruited to the lungs of mice receiving IL-4Rα+, CD11b+ cells. These results suggest that stimulation of macrophages through IL-4Rα leads to their alternative activation and participation in the recruitment of eosinophils to the lung. (PHS AI38985)
Epicutaneous antigen challenge of orally sensitized mice elicits allergic dermatitis by redirecting α4β7 gut homing T cells to the skin (79.7)
Authors: Oyoshi Michiko K , ElKhal Abdallah , Scott Jordan E , He Rui , Wurbel Marc , Campbell James , Geha Raif S
Published: January 2023

Patients with atopic dermatitis (AD) can develop skin flares after ingestion of food allergens or cutaneous contact with these antigens. We examined the mechanism by which cutaneous exposure to allergens in orally sensitized individuals may elicit allergic skin inflammation. Wild-type (WT) or CC chemokine receptor (CCR)4-/- mice were fed intragastrically once a week for eight weeks 5 mg ovalbumin (OVA) and 10 μg cholera toxin (CT) in 200 μL saline or with CT alone in saline. Mice were then epicutaneously (EC) challenged with 100 μg OVA or saline. Mice were sacrificed a week later and analyzed for allergic skin inflammation. For adoptive transfer, CD4+α4β7+ T cells were purified from mesenteric lymph node (MLN) of orally sensitized WT or CCR4-/- mice and administered to WT recipients followed by EC challenge with OVA. Mice orally immunized with OVA then EC challenged with OVA developed allergic skin inflammation characterized by dermal infiltration with eosinophils and CD4+ T cells and upregulation of Th2 cytokines mRNA expression. In contrast to WT mice, orally immunized CCR4-/- mice failed to develop allergic skin inflammation in response to EC challenge. Purified CD4+α4β7+ T cells from MLN of orally immunized WT mice, but not CCR4-/- mice, transferred allergic skin inflammation to WT recipients. These results suggest that CD4+α4β7+ T cells educated in the gut can be reprogrammed in vivo to home to the skin in a CCR4 dependent manner and cause allergic skin inflammation in response to cutaneous antigen exposure.

Funded by The Atopic Dermatitis and Vaccinia Network NIH/NIAID contract N01 AI40030
Flow cytometric cellular analyses on the developmental, activation, and proliferation states of B cells in rainbow trout (81.3)
Authors: Barr Maggie , Mott Katrina , Rosato Pamela , Zwollo Patty
Published: January 2023

Teleosts, including rainbow trout, provide an important animal model for the immunologist as they lack bone marrow and lymph nodes and utilize the kidney, spleen, and blood as immune organs. The developmental, activation, and proliferation stages of trout B cells and the location of these processes within the immune tissues are not well defined. Expression patterns of B cell transcription factors can be used as markers to investigate the B cell stages. In this study, the transcription factors Pax5 and XbpI, as well as RAG1, total IgM, secreted IgM, and BrdU incorporation were used as markers in trout B cells. Pax5 is expressed in early B cell development through the plasmablast stage, but is absent from plasma cells. In contrast, XbpI is induced in activated B cells and is expressed throughout plasmablast and plasma cell stages. Trout immune cells were LPS-activated in culture, fixed and permeabilized, and stained cells were analyzed by flow cytometry. Preliminary data suggests that each of the immune organs contains a plasmablast (Pax5+/XbpI+/total IgM+/ BrdU+) population either prior to, or upon LPS activation in culture. In the spleen, most of such cells are present at day 4, while blood has most plasmablasts on day 2. Both anterior and posterior kidney contain relatively high frequencies of plasmablasts immediately after isolation, but frequencies decrease upon culturing. This study supports the concept that flow cytometric analysis can be used to determine the location and differentiation state of B cells within the immune organs of the rainbow trout. This research was funded by a grant from the NIH.
Alternative splicing of Pax5 in immune tissues of rainbow trout (Oncorhynchus mykiss) (81.4)
Authors: Talauliker Raaj M , Harman Alice , Zwollo Patty
Published: January 2023

Pax5 is a master regulator of B cell development and regulates gene expression of a host of genes involved in cell maturation and activation. Previous studies on alternative splicing of Pax5 have reported on different isoforms that differ in DNA binding (encoded by exons 2 and 3) and trans-activation (TA) potential (encoded by exons 7-10). Our lab explores alternative splicing of Pax5 as a viable way of regulating downstream gene targets and hence, progression of the B cell program in rainbow trout. cDNAs from four trout immune tissues (anterior and posterior kidney, spleen and blood) were screened by nested PCR to search for alternatively spliced Pax5 transcripts. Several splice forms, lacking exons 2, 8 or 9, were uncovered. Using quantitative real-time PCR, the change in expression of each isoform is currently being analyzed in immune tissues stimulated in culture with LPS. This study will set the framework to better understand how alternative splicing can potentially regulate expression of target genes during B cell differentiation. This research is supported by a grant from the NIH and a graduate research grant from the A&S GSA.
Characterization of an IgM linear epitope involved in interaction with the catfish soluble FcRI receptor (81.6)
Authors: Nayak Deepak K , Wilson Melanie , Miller Norman , Bengtén Eva
Published: January 2023

The IgM binding properties of a recently identified soluble Fc receptor (IpFcRI) from channel catfish, Ictalurus punctatus, were examined. Recombinant (r) IpFcRI protein was used to assess binding to native as well as reduced IgM in latex bead immunoassays and Western blot analyses. rIpFcRI was found to recognize catfish native IgM, as well as reduced IgM heavy (μ) chain. To map the IpFcRI docking site, IgM constant (Cμ) domains Cμ1, Cμ2, Cμ3, and Cμ4 were expressed in E. coli and only the rCμ3 and rCμ4 were found to retain IpFcRI binding properties. Subsequently, rCμ3 and rCμ4 were split into three sub-regions in order to locate the binding site residues. By sequence analyses an octapeptide (FxCxVxHE) present in both Cμ3 and Cμ4 was identified as the putative IpFcRI binding site. Using overlapping oligopeptides, IpFcRI was shown to bind this octapeptide motif in dot blot analyses. In addition, since this motif is also present in other vertebrate immunoglobulins (Ig), including rainbow trout, chicken and mouse, Igs from various species were examined, and IpFcRI binding was shown only in Igs that contained this specific motif. Supported by NIH grant R01 AI19530 and UMMC IRSP 59908.
Rag deletion in peripheral T cells prevents TCR revision (82.10)
Authors: Hale Scott , Fink Pamela J
Published: January 2023

In Vβ5 TCR transgenic (Tg) mice, CD4+Vβ5+ T cells are reactive to a peripherally expressed endogenous superantigen. Most self-reactive CD4+ T cells are deleted in the periphery, but through the process of TCR revision, some lose Vβ5 surface expression, re-express Rag genes, undergo rearrangement of endogenous TCRβ, and express a newly formed TCR. As Vβ5 Tg mice age, TCR revision results in the accumulation of post-revision CD4+TCRβ+Vβ5- cells.

We now show that post-revision CD4+ T cells undergo extensive homeostatic proliferation in lymphopenic hosts. MHC drives maximal proliferation. Additionally, post-revision CD4+ T cells in Vβ5 Tg mice generate an antigen-specific IFNγ response to Listeria infection.

To further investigate the importance of TCR revision, we devised a strategy for removing the Rag2 gene in peripheral T cells. Mice bearing a floxed Rag2 gene and a Cre transgene driven by the distal lck promoter were crossed to ROSA26 YFP reporter mice, in which YFP reports Cre activity. Adoptive transfer of sorted Vβ5+YFP+ T cells from control Rag+/+ mice results in the generation of post-revision T cells, while Vβ5+YFP+ T cells from Ragfl/fl mice remain Vβ5+.

Our results illustrate that TCR revision contributes to the ongoing immunocompetence of Vβ5 Tg mice, and that Rag expression in peripheral T cells is necessary for the generation of Vβ5-CD4+ T cells in Vβ5 Tg mice.

This work was funded by NIH RO1 Grant #AG13078.
The effects of an aged microenvironment on mature B cell homing and distribution of CXCL13 (82.13)
Authors: Minges Wols Heather A. , Ippolito Jill A. , Birjandi Shirin , Ziegler Nicole , Witte Pamela L.
Published: January 2023

The splenic architecture in many aged mice appears disrupted, with some similarities to the disorganization observed in mice lacking the chemokine, CXCL13. Particularly, small follicles and germinal centers are reported and the marginal zone (MZ) appears disrupted. Changes in the proportion of B cell subsets in bone marrow (BM) and secondary lymphoid tissue suggest that migration patterns may be altered. We tested the ability of young splenic B cells to migrate in an aged microenvironment. Of note, donor B cells significantly increased homing to BM in an old host. Nonetheless, similar numbers of donor follicular B cells were found in the spleens and localized to the follicles of aged vs. young recipients. However, fewer donor MZ B cells migrated into aged spleens. Unexpectedly, CXCL13 protein levels, assessed by ELISA, were greater in spleens from aged mice, but differences were apparent in the histological distribution of CXCL13 protein between young and old spleens. In old spleens, CXCL13 was unevenly distributed, concentrated in patches throughout the follicles, and reduced or absent at the follicular edge. The data suggest that CXCL13 expression may be dysregulated in aged mice. While the consequences of changes in CXCL13 distribution are under investigation, it appears not to directly affect follicular B cell migration and may have more impact on MZ B cells. NIH R01AG013874 and CCC Faculty Development Grant.
Foxo1 links trafficking and survival of naïve T cells through regulation of L-selectin, Ccr7 and IL-7Rα expression (82.17)
Authors: Kerdiles Yann M , Beisner Daniel R , Tinoco Roberto , Dejean Anne S , Castrillon Diego H , DePinho Ronald A , Hedrick Stephen M
Published: January 2023

Foxo transcription factors play an evolutionary conserved role in cellular and organismal adaptation to nutrients and growth factor availability. Here we show that, in accord with its preferential expression in lymphocytes, the transcription factor Foxo1 plays a non-redundant role in peripheral naïve T cell homeostasis. Our experiments demonstrate that in naïve T cells Foxo1 controls the expression of the homing receptors L-Selectin and Ccr7 as well as the transcription factor Klf2, and its deletion is sufficient to alter lymphocyte trafficking in vivo. In addition, Foxo1 deficiency is associated with a severe defect in IL-7Ra expression, correlated with the ability of Foxo1 to directly bind an evolutionarily conserved region upstream of the Il7ra transcriptional start site. Finally, in agreement with the regulatory mechanisms controlling Foxo activity, we show that growth factor withdrawal induces a specific Foxo1-dependent increase in L-selectin, Klf2 and IL7ra expression. Overall, these data support a model where the regulation of Foxo1 activity depending on growth factor availability regulates optimal homing and detection of survival signals.
Examining the Role of AP-1 in T cell Development Through Overexpression of BATF Transcription Factors (82.7)
Authors: Jordan Kimberly L , Betz Briana C , Logan Michael R , Taparowsky Elizabeth J
Published: January 2023

The classical AP-1 dimer of Jun and Fos binds to DNA in a sequence specific manner to activate target gene transcription. The BATF family of basic leucine zipper (bZIP) transcription factors form heterodimers preferentially with Jun proteins. These BATF:Jun dimers bind to the same DNA target sequence as the Jun:Fos heterodimer, but do not activate gene transcription. BATF1 and BATF3, two members of the BATF family, are expressed in lymphoid cells. Our lab has generated transgenic mice overexpressing BATF1 or BATF3 in T cells using the human CD2 promoter. The BATF1 and BATF3 transgenic mice demonstrate an increase in CD4+ T cells and a marked decrease in NKT cell numbers. Additionally, both BATF1 and BATF3 transgenic mice demonstrate a marked increase in circulating immunoglobulin levels. Interestingly, only BATF1 transgenic mice demonstrate decreased numbers of NK cells and increased IL-17 production. Our most recent findings reveal a high frequency (>95%) of lymphoid tumors in BATF1, BATF3, and BATF1/3 transgenic mice. These results suggest that the phenotypes that occur as a result of chronic inhibition of AP-1 activity in the T cell lineage predisposes BATF mice to develop a lymphoproliferative disorder resembling T cell lymphoma.
In vitro derivation of marginal zone B cells from CD23- precursors (82.8)
Authors: Roundy Kirstin M , Weis John H
Published: January 2023

B cell development has been portrayed as proceeding in a linear fashion; specifically, newly formed B cells migrate from the bone marrow to the spleen where they develop into transitional B cells (T1 and T2), which give rise to marginal zone (MZ) and follicular mature (FM) B cells. Previously, we hypothesized that MZ B cells can develop from a T1 B cell without passing through a T2 developmental stage. We have developed an in vitro culture system that allows immature splenic and bone marrow cells, when cultured in the presence of B cell activating factor (BAFF) and Notch ligand, delta-like-1 (DL1), to develop into mature FM and MZ B cells. Cells cultured with only Notch or BAFF signaling did not assume a MZ phenotype. To further investigate the ability of immature B cells to develop into MZ B cells, we separated immature cells based on CD23 expression and cultured them in the presence of Notch and BAFF. CD23- populations isolated from total splenocytes or B220+ cells yielded the most MZ-like B cells in culture. When CD23+ cells were cultured, most cells retained expression of CD23 and failed to develop into MZ B cells. However, a small percentage of CD23+ cells decreased CD23 expression, yielding a population of cells with a MZ phenotype. These data suggest that the majority of MZ-like B cells in culture were derived from CD23- cells and support the hypothesis that many, if not all, MZ cells in vivo are derived from a CD23- T1-like intermediate.
The length of the secondary stimulus controls the magnitude of recall responses by CD4+ T cells (83.10)
Authors: Ravkov Eugene V , Williams Matthew A
Published: January 2023

The parameters controlling the generation of robust CD4+ T cell recall responses remain poorly defined. In this study we compared recall responses by CD4+ and CD8+ memory T cells following rechallenge. Homologous rechallenge of mice immune to either lymphocytic choriomeningitis virus (LCMV) or Listeria monocytogenes resulted in robust CD8+ T cell recall responses but poor boosting of CD4+ T cell recall responses in the same host. In contrast, heterologous rechallenge with a pathogen sharing only a CD4+ T cell epitope resulted in robust boosting of CD4+ T cell recall responses. Robust CD4+ and CD8+ T cell recall responses were simultaneously induced following rechallenge with peptide-pulsed dendritic cells, ruling out the possibility that the disparity in CD4+ and CD8+ T cell recall responses could be attributed to competition for growth factors or APCs. Instead, CD4+ T cell recall responses were dependant on the duration of the secondary challenge. Increasing the rechallenge dose resulted in more potent boosting of CD4+ T cell recall responses, and artificially limiting the duration of infection following heterologous rechallenge adversely impacted the magnitude of CD4+ T cell, but not CD8+ T cell, recall responses. These findings suggest that rapid pathogen clearance by secondary CTL following homologous rechallenge prevents optimal boosting of CD4+ T cell responses.
Clonotypic density and cross-reactivity of the CD8+ memory T cell repertoire in adults. (83.12)
Authors: Petrova Galina , Gorski Jack
Published: January 2023

Adult memory T cell repertoires are populated by clonotypes that correspond to an individual's history of antigen exposures. The population density of clonotypes in the memory repertoire is important in light of the greatly reduced production of naïve T cells by the involuted thymus and the continuing need to respond to novel pathogens. We measure clonotypic density by comparing the recall response to the influenza-derived, HLA-A2 restricted, M158-66 peptide, with those to peptides substituted at positions 63 and 65 (TCR contact residues). The PBMC were from individuals who show a complex repertoire in recall response to the M158-66 peptide. All samples were obtained under an IRB approved protocol. Peptide-specific responses were identified by CDR3 spectratype analysis, and the clonotypes identified by sequencing the CDR3. As the substitutions became more pronounced, the M158-66 repertoire was lost and new responding clonotypes were revealed. However, the number of new clonotypes identified was lower than observed for M158-66. Structurally similar substitutions revealed a limited cross-reactivity in M158-66 repertoire. These data indicate that the adult repertoire is populated by T cells that can recognize structurally related epitopes. However, these T cells are sparsely distributed for the peptide:MHC structures generated by our substitutions.
Caveolin-1 expression in CD8 T cells positively regulates CD8 T cell function and burst size (83.15)
Authors: Tomassian Tamar  Kathy , Liu Scot Daren , Miceli M. Carrie
Published: January 2023

Caveolin-1 (Cav-1) is a molecular scaffold involved in lipid raft organization as well as protein and membrane trafficking, cytoskeletal reorganization, and regulation of signal transduction in several cell types. Despite initial reports suggesting that cav-1 is not present in lymphocytes, we have identified cav-1 expression in murine T cells by immunoblotting lysates from purified primary T cell populations. We also show that cav-1 positively regulates TCR/CD28 induced CD8 T cell activation. CD8 T cells from cav-1 knockout (KO) mice hypoproliferate and undergo 2 to 3-fold fewer cell divisions in response to 48 hrs of TCR/CD28 engagement relative to wildtype (WT) CD8 cells. Indeed, cav-1 KO CD8 T cells had a lower percentage of live cells and more cells undergoing apoptosis after 48 and 72 hrs of stimulation. To determine if cav-1 regulates CD8 T cell responsiveness in vivo, WT and cav-1 KO mice were challenged with LCMV. We found that cav-1 KO mice have both a lower percentage and absolute number of virus specific CD8 cells, providing an in vivo correlate to our findings in vitro. Further, to determine if cav-1 function is T cell intrinsic, we adoptively transferred cav-1 KO OT-1 cells into WT hosts and challenged with LM-OVA. Our findings show that cav-1 KO donor cells expanded less than WT donor cells, suggesting that cav-1 expressed by CD8 cells regulates burst size. Currently we are working to assess cav-1 subcellular localization during T cell synaptogenesis and elucidate the molecular basis of its activity. These findings identify cav-1 as a novel positive regulator of CD8 T cell activation and cell survival.

T. Tomassian is predoctoral trainee recipient of the Microbial Pathogenesis Training Grant 2-T32-AI-07323.
Expression of the B cell-specific Src family kinase Blk in γδ T cells (85.13)
Authors: Laird Renee M , Hayes Sandra M
Published: January 2023

There are two T lineages, αβ and γδ, which are defined by the antigen-binding heterodimers contained within their antigen receptors. While it is still not known why two T lineages are conserved in all jawed vertebrates, years of study demonstrate that αβ and γδ T cells recognize different antigens and have unique roles in immunity. We have previously shown that αβ - and γδTCRs differ not only in the subunit composition of their signaling modules but also in their ability to transduce signals. We hypothesize that this difference in signal transduction is due to differences in the signaling pathways coupled to the αβ - and γδTCRs. To test this, we used global gene expression profiling to identify signaling molecules that are differentially expressed in αβ and γδ T cells. Unexpectedly, we found that Blk, a Src family kinase expressed in B cells, is expressed in γδ T cells but not in αβ T cells. We developed an intracellular flow cytometric assay to examine the pattern of Blk expression during T cell development. Using this assay, we detected Blk in CD25+ lineage-negative thymocytes, which contain precursors with the developmental potential to become either αβ or γδ T cells. Blk was also found to be expressed in γδ thymocytes but not in CD4+CD8+ thymocytes, suggesting that Blk expression is retained in γδ lineage cells but turned off in αβ lineage cells. We are currently investigating whether Blk plays a role in the development γδ T cells.
Age-related thymic involution is associated with loss of medullary epithelial subsets and regulated by Foxn1 (86.1)
Authors: Su Dong-Ming , Sun Liguang , Guo Jianfei , Cheng Lili , Tvinnerim Amy
Published: January 2023

Age-related thymic involution results from deterioration of the interaction between hematopoietic progenitors and non-hematopoietic thymic epithelial cells (TECs). The involution may be primarily triggered by deterioration of TECs. Therefore we focus on the role of TEC-autonomous gene Foxn1, which is largely undefined in postnatal thymic function and aging. We generated a conditional Foxn1 knockout mouse (fx). Through a tamoxifen-inducible ubiquitous Cre (uCreERT), a postnatal deletion of Foxn1 caused dramatically acute thymic involution within 5 days. The involution was associated with more impairment in medullary TECs (mTECs) than in cortical TECs, especially MHC-IIhiUEA-1hi and K5+ mTECs. The phenotypes were very similar to the naturally aged murine thymus, in which the MHC-IIhiUEA-1hi mTEC subset had a decrease correlated with age, and coincided with reported results showing that the K5+ mTECs were reduced in the naturally aged thymus (Gui. J. Int. Immuno. 07). A more physiologically relevant progressive loss of Foxn1 via a low level of spontaneous activation of the uCreERT transgene in CreERT-fx/fx and CreERT-fx/+ mice caused the similar effects, including acceleration of thymic involution, reduced K5+ mTECs and MHC-IIhiUEA-1hi mature mTECs with age, beginning at 3 months of age, which is much earlier than that in fx/fx and wild type mice. Combining with previous report that naturally aged mice have gradually reduced Foxn1 mRNA expression, we believe that Foxn1 is involved in age-related thymic involution through impairment of specific epithelial cell subpopulations. It causes deterioration of the postnatal thymic microenvironment, thereby triggers the thymic involution. (Partially supported by grants from NIAID/NIH to DMS).
Correlating expression of syndecan 1, a novel marker for certain thymocytes, with development into unique alpha/beta T cell lineages (86.3)
Authors: Hamad Abdel Rahim , Xiao Zuoxiang , Li Meng , Schneck Jonathan , Mohamood abdiaziz
Published: January 2023

Identification of specific markers of thymocytes that develop into specialized lineages is important for understanding thymic differentiation and lineage commitment. We have recently shown that double-negative (DN) but not conventional T cells express syndecan 1 (sdc1), a major heparan sulphate proteoglycan previously known to be expressed by intestinal epithelia and plasma cells. Here we show a highly restricted expression of sdc1 by a small subset of cells of the αβ lineage during thymic development. Unlike mainstream (sdc1-) thymocytes, sdc1+ thymocytes developed mainly into CD4-CD8-TCRαβ + DN T cells including NKT cells. Sdc1 expression on this small subset was first detected at the DN4 stage, indicating that it was not induced by TCR-MHC interactions. Distribution of sdc1+ thymocytes among the double-positive (DP) thymocyte pool was limited to distinct (TCRβlowCD4lowCD8low) cells. β2m-deficiency blocked maturation of sdc1+TCRαβ + DN cells without affecting the immature subset development. A few sdc1+ thymocytes were selected on MHC class II into CD4 T cells. The existence of phenotypically definable thymocytes such as sdc1-expressing cells within the immature (DN4 and DP) pool suggests that early thymocytes include unique lineage committed precursors that have not been previously identified due to the lack of specific markers.

Funding: NIH DK069279 and DK066039.
Interferon gamma-dependent alterations in the hematopoietic stem cell pool during infection (87.4)
Authors: MacNamara Katherine C , Oduro Kwadwo , Racine Rachael , Choi Kyunghee , Winslow Gary
Published: January 2023

Infection with Ehrlichia muris induces blood cytopenias and a decrease in bone marrow (BM) cellularity and function. Here we addressed whether infection altered hematopoietic stem cells (HSCs) and progenitor cells. We observed changes in the frequency of BM HSCs by day 8 post-infection; lineage-negative (Lin-), c-Kit-, Sca-1+ progenitor cells were absent, and the Lin- population was composed entirely of c-Kit+, Sca-1+ cells (LSK). In addition, common myeloid progenitors (Lin-, Sca-1-, c-Kit+, CD127-) were diminished and common lymphoid progenitors (LSK, CD127+) were increased. Competitive reconstitution experiments revealed that LSK cells isolated from infected mice gave rise to predominantly B lymphocytes, suggesting that infection favored lymphopoiesis. We next stained Lin- cells for expression of other markers for HSCs (CD150, CD48, and CD244) which revealed that the LSK population were likely true long-term reconstituting HSCs (CD150+, CD48-, CD244-) and multipotential progenitors (CD150-, CD244+, CD48-). We examined several knockout strains in our infection model and found that interferon gamma (IFNg)-deficient mice exhibited reduced CLPs after infection, suggesting that IFNg contributes to alterations in the HSC pool. Current research is aimed at understanding the specific requirement of IFNg in processes that govern HSC self-renewal, differentiation, and mobilization.
The concerted action of GM-CSF and Flt3-ligand on in vivo DC homeostasis (87.9)
Authors: Kingston Dior , Schmid Michael A , Onai Nobu , Obata-Onai Aya , Baumjohann Dirk , Manz Markus G
Published: January 2023

Dendritic cell (DC) development is efficiently supported by Flt3-ligand (FL) or GM-CSF in vitro, and lymphoid-organ DC maintenance in vivo is critically dependent on FL. However, the importance of GM-CSF or both cytokines for non-lymphoid organ DC homeostasis is not well defined. Here, we show that Gm-csfr and Flt3 expression in progenitor and myeloid cells correlate with the phenotype seen in the cytokine knock-out (KO) mice. Compared to wild-type, DC progenitor numbers are similar or only slightly decreased in GM-CSF-KO mice, but two-fold decreased in FL-KO mice. Spleen and lymph node cDCs are two- to three-fold reduced in GM-CSF and ten-fold reduced in FL-KO mice, while pDC numbers are only affected by FL deficiency. Epidermal DCs are equally moderately reduced, while dermal DCs are reduced to about two thirds in GM-CSF-KO and half in FL-KO mice. Compared to FL-KO mice, combined lack of GM-CSF and FL leads to a further significant reduction of DC progenitors and dermal DCs. In line with reduction of respective DCs, proliferative T cell and specific IgG responses are reduced progressively from wild-type to GM-CSF- to FL- to double-deficient mice. These data demonstrate the concerted action of GM-CSF and FL on DC maintenance.

This work was supported by the Swiss National Science Foundation and the European Commission FP6 Network of Excellence initiative (DC-THERA and MUGEN).
Tumor antigen cross-presentation by tumor dendritic cells is blocked at a post-processing stage but can be reversed by selected pro-apoptotic agents (88.23)
Authors: Robinson Bruce W S , McDonnell Alison M , Brown Matthew , Prosser Amy C , van Bruggen Ivonne , Currie Andrew J
Published: January 2023

Cross presentation defines the unique capacity of an antigen presenting cell (APC) to present exogenous antigen via MHC class I molecules to CD8+ T cells. This process serves a critical role in host anti-tumor immunity. Dendritic cells (DC), specifically the CD8α+ subset, are specialised cross-presenting cells; however there is little understanding of how different DC subsets contribute to anti-tumour immunity. In this study we have examined the cross-presentation of a marker tumour antigen (transfected HA) expressed by the murine AB1-HA cell line. We have found that tumor antigen is constitutively cross-presented in the draining lymph node throughout disease progression and that both CD8α+ and CD8α - DC subsets are responsible for the observed cross-presentation of HA antigen to CD8+ T cells. Interestingly, tumor-infiltrating DCs fail to cross present, though they are not deficient in tumor antigen uptake or processing. Importantly, a tumor apoptosis-inducing false nucleotide agent, gemcitabine, which primes for anti-tumor immunity, also reverses the defect in antigen cross presentation of tumor DCs. These data suggest that lymph node resident CD8α+ DC are not the sole DC subset capable of cross-priming against tumor antigens and that local cross-presentation within tumors ie. TCR restimulation of tumor-infiltrating CD8 T-cells may be essential for effective anti-tumor immunity. These data have important implications for tumor immunotherapy particularly the use of immunotherapy in conjunction with tumor apoptosis-inducing therapy.