bluuebunny/crossref_metadata_2025 huggingface dataset:
Route of infection determines T helper-1 versus T helper-17 subset differentiation (46.9)
Authors: Linehan Jonathan , Dileepan T. , Cleary Pat , Jenkins Marc
Published: January 2023

How environmental cues distinctly shape CD4+ T cell differentiation in response to specific bacterial infections remains largely unknown. Here we tested whether the route of infection determined which cytokines were produced by specific memory CD4+ T cells. Listeria monocytogenes and Streptococcus pyogenes were engineered to express the 2W1S antigenic peptide (LM-2W1S and SP-2W1S) and used to infect C57BL/6 mice. Endogenous 2W1S-specific CD4+ T cells were tracked using a 2W1S:I-Ab tetramer and magnetic bead enrichment. Infection with either LM-2W1S or SP-2W1S via the intranasal route resulted in splenic 2W1S:I-Ab-specific CD4+ memory cells that produced interleukin-17A (IL-17A) but not interferon-γ (IFN-γ) upon in vivo challenge, whereas infection with LM-2W1S or SP-2W1S via the intravenous route resulted in splenic 2W1S:I-Ab-specific memory cells that produced IFN-γ, but not IL-17A. These results suggest that the lymphoid environment of primary antigen encounter is an important factor in determining the type of CD4+ T cell memory generated and indicate that the route of immunization should be a major consideration when designing vaccines to improve cell-mediated immunity.

This work was funded by the NIH.
c-Maf determines IL-10 regulation during Th17 polarization (47.22)
Authors: Xu Jiangnan , Yang Yu , Lal Girdhari , Ochando Jordi C. , Bromberg Jonathan S. , Ding Yaozhong
Published: January 2023

IL-10 production by Th17 cells is critical for limiting autoimmunity and inflammatory responses. Gene array analysis on Stat6 and T-bet double deficient Th17 cells identified the Th2 transcription factor c-Maf to be synergistically up-regulated by IL-6 plus TGFβ, and associated with Th17 IL-10 production. Both c-Maf and IL-10 induction during Th17 polarization depended on Stat3, but not Stat6 or Stat1, and mechanistically differed from IL-10 regulation by Th2 or IL-27 signals. TGFβ was also synergistic with IL-27 to induce c-Maf, and induced Stat1 independent IL-10 expression in contrast to IL-27 alone. Retroviral transduction of c-Maf induced IL-4/Stat6 independent IL-10 expression, and c-Maf directly transactivated IL-10 gene expression through binding to a MARE motif in the IL-10 promoter. Together, these data reveal a novel role for c-Maf in regulating T effector development, and suggest that TGFβ may antagonize Th17 immunity by IL-10 production through c-Maf induction.
iNKT Cell-development is Orchestrated by Different Branches of TGF-beta Signaling (47.24)
Authors: MARIE Julien C , Doisne Jean-Marc , Garcia Céline , Bartholin Laurent , Losson Régine , Benlagha Kamel
Published: January 2023

iNKT cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although different steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling finely and specifically orchestrates several steps of iNKT cell development. In vivo this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors allowing TGF-β to finely tune the iNKT cell-differentiation program.
Isolated CD39 Expression On CD4+ T Cells Denotes Both Regulatory and Memory Populations (47.26)
Authors: Zhou Qiang , Putheti Prabhakar , Wu Yan , Kassam Nasim , Enjyoji Keiichii , Robson Simon C. , Strom Terry B. , Gao Wenda
Published: January 2023

Foxp3+ regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4+CD39+ T cell pool contains two roughly equal size Foxp3+ and Foxp3- populations. While Foxp3+CD39+ cells are CD73bright and are the bone fide Tregs, Foxp3-CD39+ cells are CD44+CD62L-CD25-CD73dim/-, exhibiting memory cell phenotype. Compared with Foxp3-CD39- naïve T cells, Foxp3-CD39+ cells freshly isolated from non-immunized mice express strikingly higher levels of mRNA for T helper lineage specific cytokines IFN-γ (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines IL-1, IL-6, TNF-α, and MCP-1. Upon stimulation, Foxp3-CD39+ but not Foxp3-CD39- cells rapidly secrete these cytokines. Moreover, the presence of Foxp3-CD39+ cells inhibits TGF-β-induced conversion of Foxp3-CD39- cells into iTregs. Thus, CD39 denotes both Treg and pre-existing memory T cells of Th1-, Th2-, and Th17-types.
TRAF6 is an Intrinsic Negative Regulator of Th17 Differentiation (47.32)
Authors: Cejas Pedro Joel , Walsh Matthew C , Pearce Erika L , Harms Gretchen , Choi Yongwon
Published: January 2023

The newly defined Th17 cell subset of CD4+ T cells is characterized by their ability to secrete IL-17A, IL-17F and IL-22, as well as by the expression of the transcription factor RORγt. Differentiation into the Th17 phenotype requires TGF-β1 and IL-6 both in vivo and in vitro, and is negatively regulated by non-Th17 cytokines such as IL-4, IFN-γ, IL-27 and IL-2. Interestingly, the negative effect of IL-2 is suppressed by IL-1.

TRAF6 is an intracellular adaptor protein that mediates signalling from the IL-1 receptor, so we reasoned that TRAF6-deficient CD4+ T cells would show impaired Th17 differentiation. To our surprise, TRAF6-deficient CD4+ T cells show preferential differentiation into the Th17 phenotype. In contrast, we have previously shown that in presence of the corresponding stimuli, in vitro differentiation into the Th1 or Th2 subsets is not affected in TRAF6-deficient CD4+ T cells. The observed Th17 bias is also reflected in vivo, as mice with a T cell-specific TRAF6 gene deletion (TRAF6-ΔT) show increased IL-17-producing cells. We have analyzed the effects of increased IL-17 levels in TRAF6-ΔT mice as well as the molecular reasons that explain the negative effect of TRAF6 in Th17 differentiation.
Neither Smad2 or Smad3 is essential for TGF-β to induce Foxp3+ Tregs and Th17 (47.8)
Authors: Zheng Song Guo , Lu Ling , Wang Julie , Brand David , Horwitz David , Shi Wei
Published: January 2023

TGF-ß induces the development of Foxp3+ regulatory T (iTreg) and IL-17-producing (Th17) cells based on the present cytokines. However, the signaling pathways involved in the differentiation of these cells are poorly defined. TGF-ß signals through Smad-dependent and -independent pathways to affect T cell responses. While others reported Smad3 is required for activity of the Foxp3 enhancer, our studies suggest that Smad3 is not essential for TGF-ß to induce Foxp3+ expression. In naïve CD4+CD25- cells isolated from either Smad3 KO or Smad2 conditional KO mice, TGF-ß was still able to induce TCR-activated T cells to express Foxp3 and developed suppressive activity, although the level of Foxp3 was slightly lower than induction in wild type cells. TGF-ß induced naïve CD4+ cells from wild type and Smad3-/- CD4+ to become Th17 cells in the presence of IL-6. Addition of retinoic acid (ATRA) increased iTreg differentiation and controlled Th17 development in both wild type and Smad3-/- mice. These results suggest that although the combined effects of Smad2 and Smad3 may be needed for the induction of iTreg and Th17 cells, Smad-independent signaling pathways are also required for these TGF-ß differentiation effects. (Supported by ACR REF Award and Outstanding youth Scientist Award from NSFC)
Tracking of individual autoreactive and regulatory T cells in Aire deficient TCR "mini" Foxp3-GFP mice (48.16)
Authors: Daniely Danielle Leigh , Ignatowicz Leszek
Published: January 2023

The presentation of tissue specific antigens (TSA) in the thymus is crucial in developing tolerant T cells and the lack thereof, leads to the development of autoimmunity (AI). Peripherally, autoreactive T cells (TA cell) that escape central tolerance can be controlled by FoxP3+ (TRcells). Mice deficient of the Aire gene lack Aire dependent TSA expression and develop organ-specific AI. It has been postulated that in the absence of Aire the diversity of TRcells positively selected is compromised. We propose to examine the effect of Aire on the T cell repertoire at the level of a single T cell clone, using an in vivo model (Aire-/-TCRminiFoxp3GFP mice) where Aire deficient mice express polyclonal, but miniature TCR repertoires. The T cells have a narrow αβ TCR repertoire composed of one transgenic TCR Vβ14+ chain and various TCRα chains produced by the rearrangements of the transgenic Vα2-Jα26,Jα2 mini-locus. The sequence of the CDR3 regions can be used as "tags" to follow the expansion of T cells and GFP driven by the FoxP3 promoter labels all TRcells. We show that irrespective of the mini repertoire, these mice develop multi-organ AI characterized by circulating autoantibodies and organ infiltrates. Analysis of individual TA and TRcell infiltrating organs show non-overlapping repertoires with the expansion of specific T cells. Further research will reveal if these clones are the tissue specific "culprits" responsible for this AI.
NF-κB inhibition leads to increased synthesis and secretion of MIF in human CD4+T cells (48.2)
Authors: Cho Mi La , Moon Young Mee , Heo Yu Jung , Woo Yun Ju , Ju Ji Hyeon , Park Kyung Su , Kim Sung Il , Park Sung Hwan , Kim Ho Youn , Min Jun Ki , Lee Sang Heon , Jung Young Ok , Yoon Bo Young
Published: January 2023

To examine the effects of NF-κB inhibition on the secretion of macrophage migration inhibitory factor (MIF) in CD4+ T cells. Isolated human CD4+ T cells were cultured with NF-κB inhibitors for 24 hrs. MIF levels were evaluated by intracellular FACs, ELISA, and real time PCR. The levels of intracellular O2-, H2O2, and glutathione were evaluated by intracellular FACs. The amounts of phosphorylated Akt and c-Jun were measured by Western blotting. Treatment of CD4+ T cells with NF-κB inhibitors resulted in a significant increase of MIF concentration in culture supernatants, MIF gene expression and O2- production and decrease of intracellular MIF, H2O2, and GSH. Treatment with LY294002, and SP600125 suppressed NF-κB inhibitors induced MIF mRNA expression and MIF secretion and inhibited parthenolide induced phosphorylation of Akt and c-Jun. Treatment with H2O2 also decreased the amounts of intracellular MIF protein and increased MIF level in the culture supernatant. NAC, an antioxidant precursor of GSH, inhibited parthenolide or H2O2 induced secretion of MIF. These results indicate inhibition of NF-κB leads to release of MIF through de novo synthesis of MIF and secretion of preformed MIF in CD4+ T cells through reactive oxygen species production. .
Decreased immunoregulatory activity of B cells derived from patients with Systemic Lupus Erythematosus (SLE) (48.30)
Authors: Tretter Theresa , Gao Nele , Venigalla Ram K.C. , Eckstein Volker , Lorenz Hanns-Martin
Published: January 2023

Recently B cells with immunoregulatory properties have been found in mice but evidence for a potential role in humans remains elusive. We have identified a population of highly activated human B cells with regulatory properties in vitro which could induce temporary anergy and apoptosis in Th cells. Now we intend to test the role of such "Breg" cells in human autoimmune SLE. Methods: CD19+B and CD4+Th cells were separated from PBMC of healthy (ND) or SLE donors by MACS-sorting. B cells were prestimulated with SAC for 3d and sorted into highly activated FSChiCD25+ (lgB25+) and small resting FSCloCD25- (smB25-) B cells by FACS. Upon 4d coculture with Th cells and αCD3+IL-2, T cell growth was determined by 3H-TdR incorporation. Results: less inhibition of T cell growth by lgB25+SLE-B (60% of proliferation of T cells alone) was observed compared to lgB25+ND-B (35% of proliferation of T cells alone), independent of T-cell origin: ND-T cell proliferation decreased below 50% of controls in 11 of 12 cases with lgB25+ND-B, in contrast to only 5 of 12 cases with lgB25+SLE-B. No significant difference in cell viability or expression of CD80, CD86, B7-H1, GITR or CD27 could be found between both B-cell populations. We conclude that lgB25+B from SLE patients are less effective in control of Th-cells in vitro. Future experiments deal with the mechanisms, potential correlations with treatment or disease stage, and relevance for pathogenesis of autoimmunity.
Prophylactic or therapeutic administration of anti-GM-CSF-M250 inhibits disease in multiple experimental autoimmune models of multiple sclerosis (48.4)
Authors: Hensley Kelly , MacMaster John , Brewer Avery , Kugler David , Kirchner Jacqueline
Published: January 2023

GM-CSF (granulocyte-macrophage colony stimulating factor) has been implicated in MS by virtue of its elevated expression in resident cells of the target tissue and invading pathogenic T cells, as well as the presence of cell types which require GM-CSF for differentiation and activation. Using a neutralizing antibody to GM-CSF (M250) we evaluated its role in three different EAE models of MS: C57Bl/6/MOG35-55, SJL/PLP139-151 and Adoptive Transfer-EAE (AT-EAE). A single dose of M250 on day of immunization in both the C57Bl/6/ MOG35-55 and SJL/PLP139-151 models prevented disease. Therapeutic administration of a single dose of M250 on day of disease onset reversed disease and reduced relapse in the SJL/PLP139-151 model. In the AT-EAE model, a single dose of M250 on day of cell transfer or disease onset significantly reduced mean clinical score. Histological evaluation of spinal cord tissue in the SJL/PLP139-151 model showed that therapeutic treatment with M250 resulted in a substantial reduction of the inflammatory infiltrate by 24 hours. These data indicate a role for GM-CSF in both the priming and effector phases of EAE, and suggest it may be an important target in MS.
New Roles for LTA4 Hydrolase, LTB4 and BLT1 in Multiple Sclerosis and Experimental Allergic Encephalomyelitis (48.9)
Authors: Parkinson John F , Guilford William J , Mendoza Lisa M , Rosser Mary , Post Joseph , Schaefer Caralee , Halks-Miller Meredith , Kirkland Tom , Cleve Arwed , Lassmann Hans , Reder Anthony T
Published: January 2023

5-lipoxygenase (5-LO), leukotriene A4 hydrolase (LTA4h), leukotriene B4 (LTB4) and its high affinity receptor BLT1 are known drivers of allergy, adaptive immunity and vascular inflammation, but have unknown roles in human multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). We show that LTA4h and BLT1 are markers of inflammatory perivascular cuffs and actively demyelinating plaques in relapsing-remitting and progressive human MS, as well as spinal cord inflammatory foci in rodent EAE. Encephalitogenic murine CD3+ T cells experienced with proteolipid protein peptide antigen (PLP139-151) migrate to LTB4 and express functional BLT1. Peripheral blood immune cell LTB4 synthetic capacity is markedly primed prior to/during onset of rat and mouse EAE and mouse spleen 5-LO/LTA4h/BLT1 mRNAs are dynamically regulated during the immune response after T cell adoptive transfer. Potent, selective LTA4h inhibitors decrease clinical score and cumulative disease burden in rat antigen-induced and murine adoptive T cell transfer EAE. LTA4h inhibitors are also synergistic in suppressing rat EAE when combined with agents having an orthogonal mode of immunomodulatory action, such as the PDE-IV inhibitor mesopram. The results define new roles for the 5-LO / LTA4h / LTB4 / BLT1 pathway in autoimmunity and disease progression in EAE. Human MS may be a therapeutic target for inhibitors of LTB4 synthesis.
Diminished IL-2 responsiveness and FOXP3 expression in T1D subjects is revealed by a disease-associated variant of PTPN2 (49.1)
Authors: Long Sarah Alice , Bollyky Paul , Cerosaletti Karen , Shilling Heather , Pihoker Catherine , Sanda Srinath , Greenbaum Carla , Buckner Jane
Published: January 2023

Impairment in the IL-2/IL-2R pathway results in a loss of regulatory T cells (Treg) and the development of autoimmunity in animal models. Recent genome-wide association studies (GWAS) have identified several genes associated with Type 1 Diabetes (T1D) which may influence IL-2R signaling including the protein tyrosine phosphatase, PTPN2. We demonstrate that CD4+ T cells of healthy subjects that carry the T1D associated variant of PTPN2rs1893217 exhibit impaired signaling in response to IL-2. This is characterized by decreased phosphorylation of STAT5 and diminished FOXP3 expression in response to IL-2. In T1D subjects, a similar defect in IL-2R signaling is seen, however, in T1D subjects this phenotype is present irrespective of PTPN2 genotype. In comparison to controls, the CD4+ T cells of T1D subjects displayed decreased STAT5 phosphorylation upon exposure to IL-2 and IL-15, decreased FOXP3 expression upon activation in the presence of IL-2 and a loss of FOXP3 persistence in nTreg cultured in IL-2. Together these findings indicate that the PTPN2rs1893217 genotype reveals a phenotype common to T1D subjects. This impairment of IL-2 responsiveness may contribute to the development of T1D through diminished number and function of Treg in the inflamed islet.
Evidence for linkage of Type 1 Diabetes (T1D) resistance and upregulation of eotaxin (CCL11) in pancreatic beta cells to a chromosome 12 locus in the Biobreeding rat (49.2)
Authors: Chao Gary Y.C. , Wallis Robert H , Marandi Leili , Poussier Philippe
Published: January 2023

The Biobreeding (BB) rat spontaneously develops autoimmune Type 1 Diabetes (T1D). We tested the hypothesis that the recently reported upregulation of CCL11 in BB rat βcells prior to insulitis is genetically determined and linked to T1D. This time-dependent upregulation of CCL11 transcription was not observed in T1D resistant ACI rats. Therefore linkage analysis for T1D and pancreatic CCL11 was performed in a conditioned cross/intercross between BB and ACI rats where all animals (n=574) carried T1D susceptibility alleles at the two known BB rat T1D genes. Eight T1D loci (chr 1, 2, 3, 2 in 6, 12, 14 and 17) were mapped establishing the polygenic nature of T1D in the BB rat and at three of these (chr 1, 3 and 12) the BB allele confers T1D resistance. Strikingly, upregulation of pancreatic CCL11 transcription mapped to the T1D locus on chromosome 12 (LOD=3.65) and the peak of linkage (D12rat51) was the same for the two traits. At D12rat51, homozygosity for the BB allele confers high levels of pancreatic eotaxin expression and T1D resistance, raising the possibility that upregulation of the Th2 chemokine CCL11 in the target tissue of the anti-pancreatic autoimmune process is protective. Research supported by Genome Canada and Banting and Best Diabetes Centre.
Copy number variation (CNV) of human FCGRIIIB is concurrent with FCGRIIC and HSP70B: low copy number is a risk factor for and high copy number is a protective factor against systemic lupus erythematosus (SLE) (49.24)
Authors: Wu Yee Ling , Yang Yan , Zhou Bi , Chung Erwin K. , Birmingham Dan J. , Nagaraja Haikady N. , Hebert Lee A. , Rovin Brad H. , Yu Chack-Yung
Published: January 2023

Immunoglobulin Fcγ-receptors FcγRIIA, IIB and FcγRIIIA, IIIB are important effectors of immune response. Fanciulli and colleagues first reported low copy number (0 or 1 copy) of FCGRIIIB-related 5' sequences in 40-45% of SLE patients and ~25% of controls, but their assays did not faithfully reflect the actual CNV of FCGRIIIB. Thus, we investigated the genomic diversity of human FCGR region at chromosome 1q23.3. Through Southern blot analyses of PmeI-digested genomic DNA resolved by pulsed-field gel electrophoresis, we established the concurrent duplications of three contiguous genes FCGRII, heat shock protein HSP70B, and FCGRIII (FRH). We further defined gene specificities of the FRH complex by TaqI-genomic RFLPs in 281 SLE patients and 485 matched controls of European ancestry. One to four FRH modules are frequently detectable in haplotypes with different combinations of FCGRIIIA and FCGRIIIB, polymorphic variants of FCGRIIC, and HSP70B' and HSP70B. Homozygous and heterozygous deficiencies of FCGRIIIB had a combined frequency of 0.125 in SLE and 0.062 in controls. By contrast, high copy numbers of FCGRIIIB (3 or 4 copies) had a lower frequency in SLE (0.082) than in healthy controls (0.120) (p=0.0016). In conclusion, significant increase in low copy number and decrease in high copy number of FCGRIIIB were present in human SLE.
Increased breast cancer-associated metastasis in PyV MT mice induced to develop arthritis (49.3)
Authors: Das Roy Lopamudra , Pathangey Latha , Tinder Teresa , Schettini Jorge , Mukherjee Pinku
Published: January 2023

We determined if inflammation caused by collagen induced arthritis in the PyV MT mice that develop spontaneous mammary gland tumor increases secondary metastasis. The PyV MT mice were injected with 2mgs/ml Type II collagen to induce arthritis at two time points, at 9 and 18 weeks of age for pre-metastatic and metastatic conditions respectively. At both time points, data demonstrated a significantly higher primary tumor burden and a 2-fold increase in lung metastasis in the arthritic versus non-arthritic PyV MT mice. Increased COX2 and CD31 staining in the bones of arthritic PyV MT mice correlated with increase in epithelial metastatic cells in the bone marrow (BM) of these mice compared to non-arthritic PyV MT. We noted increased levels of myeloid suppressor cells in the BM and decreased levels of natural killer cells in the tumor microenvironment of the arthritic versus non-arthritic PyV MT mice. Subsequent increase in circulating levels of proinflammatory cytokines, in particular prostaglandin E2, IL-6, IL-17, vascular endothelial growth factor and tumor necrosis factor-α were found to be the underlying factors contributing to the increased metastasis. The data clearly shows that breast cancer associated metastasis is increased in arthritic conditions and the data have important clinical implications especially in the prevention of secondary metastasis. Supported by DOD BCO63396
Genetic dissection of the effect of Fcgr2b polymorphism on systemic lupus erythematosus (49.30)
Authors: Hirose Sachiko , Lin Qingshun , Ohtsuji Mareki , Tsurui Hiromichi , Nishimura Hiroyuki , Shirai Toshikazu
Published: January 2023

Telomeric chromosome 1 contains a cluster of potent polymorphic autoimmune-susceptibility genes including Fcgr2b. SLE-prone strains, including BXSB, share deletion polymorphism in Fcgr2b promoter region. Here we established congenic BXSB.IIBB6 that carry normal C57BL/6-type Fcgr2b. While BXSB females do not develop severe disease, BXSB males develop much severe disease because of Y chromosome-linked autoimmune acceleration (Yaa) mutation. Compared with BXSB females, (NZW x BXSB) F1 females develop severe SLE even in absence of Yaa because of the contribution of susceptibility alleles from the NZW strain. The present study addressed the question of whether wild-type Fcgr2b could prevent SLE in mice prone to develop SLE. As compared to BXSB males and (NZW x BXSB) F1 females, all SLE features were markedly suppressed in BXSB.IIBB6 males and (NZW x BXSB.IIBB6) F1 females, respectively. Importantly, activation and proliferation of not only B cells, but also T cells and monocytes/macrophages, were all inhibited in the latter two strains. Thus, the wild-type Fcgr2b appears to have a strong suppressive effect on the susceptibility of both Yaa-dependent and Yaa-independent murine lupus through the mechanisms that inhibit the activation of a wide variety of immune cells. Conversely, due to the loss of such inhibitory effects, autoimmune-type Fcgr2b probably plays a crucial role in SLE susceptibility.
Src-like adaptor protein (SLAP)-deficiency prevents chronic arthritis in SKG mice (49.7)
Authors: Dragone Leonard , Shaw Laura A , Sakaguchi Shimon , Peterson Lisa K
Published: January 2023

Background: Intrinsic defects in lymphocyte signaling have been implicated in the pathogenesis of rheumatoid arthritis (RA) by a mouse model of inflammatory arthritis (SKG). SKG mice have a spontaneous mutation in ZAP-70 that leads to impaired thymocyte development, reduced positive selection and impaired negative selection of thymocytes. These defects result in autoreactive peripheral T cells that cause RA. SLAP is known to influence TCR levels and signaling in developing thymocytes. We hypothesize that SLAP-deficiency can alter TCR avidity and influence the negative selection of SKG autoreactive T cells.

Methods: To test this hypothesis we generated SLAP-deficient SKG expressing mice (DSSKO).

Results: Thymocytes from DSSKO mice have increased TCR complex mediated signaling as evidenced by increased surface expression of CD5 and ERK phosphorylation. DSSKO mice have a 3-fold increase in thymic and peripheral FOXP3+ Tregs compared to SKG controls. Upon zymosan challenge, none of the DSSKO mice develop chronic arthritis. TH17 cells develop normally in DSSKO and SKG mice treated with zymosan suggesting that arthritis protection was not due to lack of their development.

Conclusions: These data support the hypothesis that SLAP deficiency alters thymocyte development possibly by both enhancing the negative selection of autoreactive T cells and converting them to Tregs to prevent the development of arthritis.
Abrogation of established inflammatory arthritis by the GABAA agonist Honokiol. (50.18)
Authors: Munroe Melissa E. , Bishop Gail A.
Published: January 2023

Rheumatoid arthritis is a chronic, systemic autoimmune disease characterized by debilitating stiffness and erosive joint destruction. Honokiol (HNK), a phenolic compound isolated and purified from magnolia, has been found to have anti-inflammatory properties without the toxicity found with current treatments. Whether given i.p. or orally, HNK abrogated established collagen induced arthritis in a dose dependent manner. Treatment with HNK in vivo resulted in decreased antigen-specific pro-inflammatory responses as well as altered T- and B-cell activation and antibody responses. The anti-inflammatory effects of HNK could be reversed using inhibitors of the neurotransmitter GABA(A), a previously reported target for HNK interaction. Preliminary evidence indicates that HNK influences transcriptional regulators of T- and B-cell activation. Thus, the anti-inflammatory properties of HNK show significant promise for blocking the autoimmune response, effectively and without toxicity. These findings also suggest that other agonists of neurotransmitter receptors should be investigated as anti-inflammatory agents. This research was supported by NIH grant 1 R21 AT003998-01.
Diet and DNA methylation in lupus (50.29)
Authors: Ray Donna , Richardson Bruce C
Published: January 2023

T cell DNA demethylation contributes to human lupus. The mechanisms causing demethylation are incompletely understood. DNA methylation depends on DNA methyltransferase activity and S-adenosylmethionine (SAM), and is inhibited by S-adenosylhomocysteine (SAH). SAM levels are determined by Zn, B2, B6, B12, folate, Met and choline, and SAH by homocysteine (Hcy). We hypothesized that decreasing nutrients required for SAM or increasing Hcy increases expression of methylation sensitive T cell genes, and that levels of these nutrients are abnormal in lupus patients. PHA stimulated T cells were cultured in custom media with variable concentrations of each nutrient, and effects on methylation sensitive gene expression (CD70, KIR and perforin) compared by flow cytometry. T cells cultured in media adjusted to median serum levels of each nutrient overexpressed all methylation sensitive genes relative to complete media. Increasing Hcy caused further increases in CD70 while increasing folate or B6 concentrations suppressed perforin overexpression. Lupus patients had lower B6 and Zn levels, and higher Hcy levels than controls. These results suggest that a poor diet may contribute to abnormal T cell gene expression in lupus, and that dietary supplementation may have a beneficial effect.
Probiotics control lupus progression via induction of regulatory cells and IL-10 production (50.30)
Authors: Alard Pascale , Parnell Sarah , Manirarora Jean , Kosiewicz Michele , Atay Safinur
Published: January 2023

Microorganisms can evade the immune response via induction of regulatory cells, and can prevent the development of various autoimmune diseases. Probiotics, including Lactobacilli, are beneficial bacteria that play an important role in the immune system development. The goal of this study was to test whether gavaging (NZBxNZW)F1 (BWF1) lupus-prone mice with Lactobacilli weekly could control lupus development and identify the mechanisms mediating protection. BWF1 mice fed with Lactobacilli starting before disease onset exhibited a delay in lupus onset indicated by decreased severity and increased survival. This protection was associated with an increase in the percentage of CD4+Foxp3+ regulatory cells and IL-10 production. Moreover, T cells from Lactobacilli-fed BWF1 mice transferred some protection when injected into young BWF1 mice. More importantly, mice that were fed after disease onset remained stable for several months and exhibited a delay in disease progression and an increase in survival. Regular feeding of Lactobacilli appears, therefore, to delay lupus progression via regulatory cell induction and possibly IL-10 production.

Altogether, these data indicate that the use of probiotics may be a viable strategy for controlling disease development and progression in patients with lupus, i.e., extending the length of remission and reducing flare frequency. Funded by the Lupus Research Institute.
Immunotherapy of experimental autoimmune encephalomyelitis by adoptive transfer of B cells stimulated with MOG (50.4)
Authors: Do Jeong-Su , Song Eun-Jung , Seo Hyo-Jung , Hwang Jin-Ki , Kim Jun-Hee , Shin Sung-Hye , Hwang Pyung Han , Yi Ho Keun , Nam Sang-Yun
Published: January 2023

The efficacy of adoptive transfer of activated B cells in an experimental autoimmune encephalomyelitis (EAE) model was investigated. B cells were prepared from C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein (MOG 323-339) in CFA. Primary (1o) or secondary (2 o) stimulation of purified B cells was performed with MOG, LPS, and anti-CD40. EAE was induced by immunization with MOG plus M. tuberculosis and pertussis toxin. Adoptive transfer of 2 o B cells on day-1(D-1) of MOG immunization significantly prevented EAE development in a dose-dependent manner, but not 1o B cells. Adoptive transfer on D13 of immunization also suppressed EAE progress. At day 50, controls and treated mice were reimmunized with MOG and the results showed that adoptive transfer of activated 2o B cells induced long-term resistance to disease. 2o B cell treatment enhanced MOG-specific IgG1 but suppressed IgG2a antibody secretion. Cytokine profiles of splenic CD4+ T cells obtained on D21 of immunization clearly indicated that adoptively transferred 2o B cells drive Ag-specific TH2 responses. The fail of transfer of EAE with T cells from treated mice, but not control mice, supported that T cells polarized toward TH2 are responsible for the efficacy of 2o B cells. Neither regulatory T cells nor TH17 were likely to be involved in 2o B-mediated suppression of EAE. In conclusion, adoptive transfer of 2o B cells may be a promising strategy for immunotherapy of TH1-dominant autoimmune disorders.
A novel flow cytometric based in vivo ADCC assay for screening lytic murine antibodies (50.42)
Authors: Evans Lawrence S , Butz Eric A
Published: January 2023

CD70 is a TNF superfamily member that is not normally expressed on resting cells, but is induced on lymphocytes and dendritic cells upon activation. Aberrant upregulation of CD70 is observed in several human autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and graft versus host disease (GVHD). Therefore, depletion of CD70+ cells could have a significant impact in these diseases and in animal models where CD70+ cells play a prominent role. Though in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays perform well with human cells they are difficult to do using mouse effectors and targets. We have developed an in vivo ADCC assay using carboxyfluorescein succinimidyl ester (CFSE) labeled target cells that allows us to measure in vivo target depletion in the presence of anti-CD70 antibodies. The antibodies tested displayed differential lytic capacities in vivo that were not evident from in vitro ADCC assays. The relative roles of NK cells, FcγIII/II Receptor (CD16/CD32), and complement on in vivo depletion have also been examined. We believe that this assay is a useful tool in examining the lytic activities of mouse Ab that have been difficult to determine using in vitro assays.
Transgenic inhibition of astroglial NF-κB improves functional recovery in EAE by suppressing chronic neuroinflammation. (50.7)
Authors: Bethea John R , Brambilla Roberta , Shestopalov Valery I , Barnum Scott R
Published: January 2023

In the CNS the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. Here we show that transgenic inactivation of astroglial NF-κB (GFAP-IκBα-dn mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis (EAE). At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared to WT, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, while the loss of neuronal-specific molecules essential for synaptic transmission was limited compared to WT mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following EAE, directly implicating astrocytes in the pathophysiology of this disease.
Revisiting the effect of anti-VLA-4 monoclonal antibody treatment in experimental autoimmune encephalomyelitis (50.9)
Authors: Ji Niannian Eva , Forsthuber Thomas G.
Published: January 2023

Blocking of adhesion molecule VLA-4 has been shown to ameliorate disease in human MS patients and in experimental autoimmune encephalomyelitis (EAE). We were interested in determining long-term effects of anti-VLA-4 monoclonal antibody (mAb) treatment on persistence and function of myelin-reactive T cells in EAE. Unexpectedly, we found that anti-VLA-4 mAb (PS/2) treated mice succumbed to toxic shock-like death after repeated injection of the mAb. Investigating the underlying mechanism we found that treatment of mice with PS/2 in combination with Pertussis toxin (PTX) which is commonly used to facilitate induction of disease in mouse EAE models resulted in death of the animals. Our results show that CD4+ T cells are critical for this effect and that systemic release of cytokines including IL-1 β and TNF-α may play a role. The results reveal a previously not appreciated effect of VLA-4 antibody treatment in mouse EAE model and could be relevant for the treatment of MS patients.
The lipid environment regulates DM-dependent loading of foreign antigens onto MHC class II in dendritic cells (78.17)
Authors: Berger Adam C. , Roche Paul A.
Published: January 2023

MHCII resides on intraluminal vesicles contained within cholesterol-rich lysosomal compartments in immature DCs. This localization is proposed to allow efficient interaction with the MHCII-like chaperone protein DM (localized to the lysosomal limiting membrane). In this study, we investigated the role of the lipid environment in regulating DM-dependent loading of foreign antigens onto MHCII through the use of U18666A, an inhibitor of lysosomal cholesterol egress. U18666A treated DCs upregulate CD40 and CD86 upon stimulation, have similar cellular MHCII levels, and efficiently internalize and degrade foreign protein antigens. Nevertheless, these DCs have a diminished ability to stimulate naïve antigen-specific T cells. We demonstrate that U18666A pretreatment reduces MHCII-peptide surface expression by 60% and completely inhibits the LPS-maturation induced increase in cellular expression of complexes. Microscopic analysis of U18666A treated DCs shows that MHCII molecules are retained on the limiting membrane of cholesterol engorged lysosomal structures and that these molecules are largely devoid of peptide. MHCII-CLIP levels are also significantly decreased in both immature and mature DCs. We conclude that when MHCII and DM are retained on the same membrane, DM retains the ability to elicit CLIP removal from MHCII, but not to allow efficient peptide loading.

This work is supported by the NCI.
The membrane distribution of MHC-II-peptide complexes and its role in T cell activation (78.18)
Authors: Bosch Pages Berta , Roche Paul A.
Published: January 2023

The efficiency of the interaction between MHC-II-peptide complexes (pMHC-II) and T cell receptor depends on the amount of antigen present at the surface of DCs. MHC-II molecules are constitutively present in plasma membrane lipid rafts, membrane microdomains that are important to induce T cell activation at low antigen doses. In this work we investigated if DCs can possess a specific pMHC-II distribution at the surface that can enhance T cell activation. In murine bone marrow derived mature DCs (mDCs), MHC-II molecules displayed a clustured phenotype. Using an antibody that recognizes specific IAk-HEL pMHC-II complexes, we observed that pMHC-II also showed a clustered distribution on the surface of antigen-loaded mDCs. While DCs treated with LPS for only 12 h had 50% less IAk-HEL pMHC-II complexes at the cell surface than DCs treated for 30 h, both cell types showed a clustured distribution of these complexes. Despite the fact that 12 h DCs expressed less IAk-HEL pMHC-II complexes and less CD86/CD40 molecules at the cell surface, T cell activation was similar using 12 h- or 30 h-mature DCs. We are currently investigating the relationship between pMHC-II complex number and cluster distribution over time with the antigen presenting cell function to elucidate the role of microdomain association of pMHC-II in T cell activation.

This work is supported by NCI.
Inherent plasticity of a T-cell receptor binding interface prior to pMHC recognition (78.2)
Authors: Faraldo-Gómez José D , Dash Banaja
Published: January 2023

Structural and biophysical studies have established that the recognition of antigenic peptide-MHC complexes (pMHC) by T-cell receptors (TCR) involves conformational changes in the so-called CDR loops, which define the binding interface of the TCR. This plasticity is believed to underlie the receptors' cross-reactivity, a key feature given the vast diversity of antigens and the limited T-cell repertoire. It remains unclear, however, whether this plasticity is induced upon recognition of specific pMHC, or whether it is an intrinsic characteristic of the CDR loops. To gain insights into this question, we present results from large-scale, atomically-detailed computer simulations of the molecular dynamics of a TCR in its unliganded form and in complex with two distinct pMHC. Our results indicate that the CDR loops can populate bound-like conformational states prior to the engagement of the pMHC, and thus, that the structural plasticity of the binding interface is indeed inherent. It follows that cross-reactivity of this TCR may not be solely determined by their potential interactions with specific pMHC complexes, but also by the nature of this pre-existing conformational equilibrium.
H. influenzae and M. catarrhalis trigger a potent immune response through activation of dendritic cells (78.23)
Authors: Dew Kristin E , Henry Curtis J. , Mitchell Latoya M. , Hiltbolt Elizabeth M. , Swords W. Edward
Published: January 2023

Otitis media is one of the most common pediatric public health problems worldwide. It is caused by bacterial opportunists that normally reside in the nasopharynx such as non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat). Once established, these infections are largely resistant to clearance by host immune effectors. Much remains to be learned regarding how these pathogens persist in the middle ear. Dendritic cells (DC) are the major antigen presenting cell at mucosal surfaces and are the link between the innate and adaptive immune responses. The goal of this study was to determine if NTHi and Mcat activate DC in vitro, and if these activated DC are able to stimulate effector T cell responses. Mouse bone marrow derived DC were stimulated with NTHi strain 86-028NP or 2019, Mcat strain 7169, or lipooligosaccharide (LOS) and the level of co-stimulatory molecule expression (CD86, CD80, CD44 and I-A[b]) was measured using flow cytometry. Bacteria activated DC were then used to stimulate CD8+ and CD4+ T cells, and their proliferation and cytokine production were measured. The results indicate that both NTHi and Mcat stimulate DC maturation in vitro. Based on this data, it appears that the mechanism of persistence of these agents in otitis media does not involve evasion of antigen presenting cells. Ongoing work in our laboratory will focus on defining why immune effectors fail to resolve these infections.
Statins impair CD1d-mediated antigen presentation (78.5)
Authors: Khan Masood A , Gallo Richard , Gourpura Renukaradhya , Brutkiewicz Randy
Published: January 2023

ABSTRACTStatins are widely used as cholesterol-lowering agents that also decrease inflammation, and target enzymes essential for prenylation, an important process in the activation and intracellular transport of proteins vital for a wide variety of cellular functions. Here, we report that statins impair a critical component of the innate immune response, CD1d-mediated antigen presentation. The addition of specific intermediates in the isoprenylation pathway reversed this effect, whereas specific targeting of enzymes responsible for prenylation mimicked the inhibitory effects of statins on antigen presentation by CD1d as well as MHC class II molecules. This study demonstrates the importance of isoprenylation in the regulation of antigen presentation and suggests a mechanism by which statins reduce inflammatory responses.
The absence of ER trimming alters peptides presented by non-classical MHC class I molecules (78.8)
Authors: Nagarajan Niranjana A , Gonzalez Federico , Shastri Nilabh
Published: January 2023

The nature, function and pathways used to produce peptides for presentation by non-classical MHC class I molecules is poorly understood. Here we show that cells from mice lacking the ER aminopeptidase associated with antigen processing (ERAAP) present an immunologically distinct peptide-MHC class I repertoire. Wild-type mice immunized with ERAAP-deficient cells mount robust CD8 T cell responses specific for peptides presented by classical as well as non-classical MHC I molecules. These CD8 T cells produce pro-inflammatory cytokines and effectively kill ERAAP-deficient target cells in vitro as well as in vivo. Further, peptides presented by non-classical MHC class I molecules are expressed in a tissue-specific manner and elicit CD8 T cell responses both by direct and cross-priming. Thus, ERAAP trims the final peptides presented by classical as well as non-classical MHC class I molecules, and may be involved in trimming peptides for cross-presentation.

This research was supported by grants from the NIH (AI060040) to N.S.; N.A.N. is supported by the Irvington Institute Fellowship Program of the Cancer Research Institute.
CD8 T cell depletion in a chronic asthma model inhibits resolution of lung inflammation (79.12)
Authors: McNamara Jeffrey Thomas , Singh Anurag , Secor, Jr Eric , Guernsey Linda , Lefrancois Leo , Thrall Roger
Published: January 2023

Although the pro-inflammatory role of CD4 T cells in the development of allergic airway disease (AAD) has been extensively studied the role of CD8+ T cells remains controversial. CD8 T cells have been shown to be pro-inflammatory in certain models and suppressive in others. We have previously demonstrated that acute daily OVA aerosol exposure (3-14days) results in AAD while continued daily aerosol (42 days) results in resolution of inflammation and the development of local inhalational tolerance (LIT). This biphasic model uniquely enables the investigation of CD8 T cells in the resolution of AAD and development of LIT. CD8 T cells were depleted after AAD (but prior to LIT) using rat IgG anti mouse CD8α (clone YTS 169AG 101HL). This protocol depleted CD8 T cells by > 95% in the perripheral blood and lymph nodes. Control mice received non-specific rat IgG. Bronchoalveolar lavage (BAL) and lung histology were used to assess lung injury at LIT. CD8 T cell depletion resulted in enhanced lymphocytic perivascular /peribronchial inflammation (H&E) and increased collagen deposition (Trichrome) as compared to the control group. There was no evidence of increased mucus production (PAS), lung tissue eosinophilia or increased eosinophils in the BAL. This suggests CD8 T cells may play a role in development and/or maintenance of tolerance in this model.
Early life chlamydial infection enhances allergic airways disease through age-dependent differences in immunopathology (79.20)
Authors: Hansbro Phil M , Starkey Malcolm R , Horvat Jay C , Kim Richard Y , Phipps Simon , Gibson Peter G , Foster Paul S
Published: January 2023

Chlamydial respiratory tract infections are associated with the development and exacerbation of asthma in both children and adults, but the mechanisms underpinning this association remain unknown. We investigated the effect of the age of chlamydial infection on key features of asthma in later life using mouse models of Ovalbumin-induced allergic airways disease (AAD). Neonatal and infant, but not adult infection resulted in differential and permanent alterations in airway eosinophils, T-cell cytokines and dendritic cells (DCs). Infection of neonates suppressed eosinophil influx into the airways and reduced T-cell cytokine release which correlated with a significant reduction in the number and activation of DCs. In stark contrast, infant infection augmented eosinophil influx and increased T-cell cytokine release which correlated with increased DC numbers and activation. Importantly, both neonatal and infant infections enhanced physiological (mucus secreting cell hyperplasia and airways hyper-responsiveness) responses in AAD in later life, which correlated with enhanced IL-13 expression in the lung. Furthermore, neonatal infection induced substantial alterations in lung structure which was linked with increased TGF-β in lung tissue. Thus, early life infection enhances pivotal features of AAD through age dependent, differential affects on immune responses and lung structure.