bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of cardiac myosin-specific CD4 T cells by using MHC II / IAk tetramers in A/J mice (93.12)
Authors: Massilamany Chandirasegaran , Gangaplara Arunakumar , Chapman Nora , Rose Noel , Reddy Jay
Published: January 2023

Myocarditis is one of the predominant causes of heart failure in young adolescents, and Coxsackievirus B strains (CVB1-6) are common suspects. CVB3 isolates from humans induce myocarditis in A/J mice, but the infectious virus cannot be isolated 18-21 days postinoculation. However viral nucleic acids showing deletions in the 5’ non-translated regions persist. The extent to which continuing low level replication of CVB causes inflammation is not known and an autoimmune response is suspected in late disease progression, and cardiac myosin heavy chain-alpha (MYHC-α) has been identified as a dominant autoantigen. To delineate the contribution of autoimmune response to postinfectious myocarditis, it is critical to demonstrate the generation of pathogenic autoreactive T cells as they expand to a significant proportion to cause disease. To this end, we have created major histocompatibility complex class II / IAk tetramers for MYHC-α, 334-352. By using IAk / RNase 43-56 tetramers as controls, we have optimized the conditions to detect MYHC-α 334-352-specific CD4 T cells in lymph node cell cultures derived from A/J mice immunized with MYHC-α 334-352. Preliminary studies suggest that the reagents can also be used to enumerate the frequency of myosin-specific T cells ex vivo in immunized mice. These reagents will now permit us to determine the generation and functions of myosin-reactive T cells as an indicator of autoimmune response to cardiac myosin in mice infected with CVB3.
Activation of C3 promotes experimental autoimmune thyroiditis in C5 deficient female A/J mice. (93.15)
Authors: Njoku Dolores , Talor Monica , Sharma Rajni , Mellerson Jenelle , Burek Carol , Rose Noel , Njoku Dolores
Published: January 2023

Clinical studies suggest that activated C3, an early product of complement activation which can be triggered by infectious or environmental agents, is associated with more severe autoimmune thyroiditis, but few studies have illuminated mechanisms responsible for this observation. Animal models show that the severity of autoimmune thyroiditis is associated with late complement components, such as C5, and genetic susceptibility at the I-Ak sublocus. To elucidate additional complement-mediated effects on autoimmune thyroiditis we induced thyroiditis in I-Ak positive CBA/J and C5-deficient A/J mice with and without cobra venom factor which would activate the alternative complement pathway. A/J mice as a group developed less severe thyroiditis than CBA/J mice (p < 0.001). Surprisingly, female A/J mice developed less thyroiditis than males (p < 0.01) and had lower serum C3 levels (p < 0.05) during thyroiditis induction while thyroiditis and C3 in male A/J was not different from CBA/J mice. Cobra venom factor increased serum C3 levels, TNFα and thyroiditis but not intra-thyroid C3 deposition in A/J females suggesting that systemic complement activation increased thyroiditis in these mice. Our studies suggest that C3 activation promotes experimental thyroiditis in less susceptible A/J females, and may reveal one mechanism by which infections or other environmental agents promote autoimmune thyroiditis in patients.
A BALB/c locus on chromosome 12 confers resistance to lupus in FcγR2-/- mice. (93.23)
Authors: Tarasenko Tatiana , Bolland Silvia
Published: January 2023

FcγR2B-deficient (R2-/-) mice develop autoantibodies and glomerulonephritis with a pathology resembling human lupus when on the C57Bl/6 background. The same mutation on the BALB/c background does not lead to spontaneous disease, suggesting differences in lupus susceptibility between the BALB/c and B6 strains. An F2 genetic analysis from a B6/BALB cross identified region from the B6 chromosome 12 with positive linkage for autoantibodies. We have generated a congenic strain (B6.R2-/-sbb2) that contains the suppressor allele from the BALB/c centromeric region of chromosome 12 in an otherwise B6.R2-/- background. We have observed that the presence of sbb2 renders B6.R2-/- mice resistant to lupus disease by suppressing all antibody autoreactivity and spontaneous immune cell activation. Mixed bone marrow reconstitution experiments indicated that the sbb2 resistant allele is most likely T cell intrinsic. The sbb2 congenic interval contains several candidate genes that are currently being investigated. We generated transgenic lines containing BAC DNA insertions spanning the sbb2 interval. The new BAC transgenic strains were tested in the induced model of lupus by pristane administration. Newly generated BAC transgenic strains were also bred to B6.R2-/- to test for suppression of lupus in spontaneous disease. The goal of our experiments is to identify a new lupus susceptibility gene within the sbb2 interval that is able to suppress lupus disease by altering T cell function.
Activation of innate immunity influences salivary gland function and development of Sjögren’s Syndrome (93.33)
Authors: Deshmukh Umesh , Nandula Seshagiri-Rao , Scindia Yogesh , Bagavant Harini
Published: January 2023

Sjögren’s syndrome (SS) is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the exocrine glands. Loss of salivary gland (SG) function is a major feature of the disease. This study was undertaken to investigate the role of innate immunity activation in pathogenesis of SS. Mice were repeatedly injected with TLR3 agonist poly(I:C), and monitored for SG function and sialadenitis. Poly(I:C) treatment rapidly induced SG hypofunction. However, SG function was restored, once poly(I:C) treatment was terminated. This phenomenon was independent of the genetic background of the mice, and did not require T and B cells. Mice deficient in IFNAR1 and thereby lacking type I IFN signaling, did not loose SG function to the same extent as the wild type mice. In the autoimmune-prone NZB/W F1 mice, poly(I:C) treatment induced severe and accelerated sialadenitis. The SG in these mice showed presence of T-B cell aggregates and germinal center formation. Some of the T cells in these infiltrates were CXCR5+ICOS+ Tfh cells. Significant up-regulation in the expression of IL-21 and Il-21R genes was seen in the SG of poly(I:C) treated mice. This study demonstrates that activation of innate immunity in a genetically susceptible individual modulates the development of SS. This study also shows that type I IFN can adversely affect SG function and that IL-21 pathway and Tfh cells might be important in the pathogenesis of SS.
Mechanisms behind the induction of Ttc7 transcription during B lymphocyte development(93.35)
Authors: Tait Heidi
Published: January 2023

Changes in Ttc7 (tetratricopeptide repeat domain 7) transcription have been associated with changes in B lymphocyte signaling through changes in both the lymphopoietic environment and homeostasis of B lymphocyte development in mice with the Ttc7fsn/fsn(flaky skin) mutation. We have demonstrated that young Ttc7fsn/fsn mice exhibit decreased naive B lymphocyte populations in the bone marrow and spleen as compared to their wild type littermates, and that this disparity is exaggerated with age. The Ttc7fsn/fsn mutation also leads to excessive production of harmful B1b lymphocytes causing autoimmune disease closely related to SLE. This is significant as both autoimmune and aging human populations share similar B lymphocyte profiles. The evaluation of signaling events upstream of Ttc7 transcription will shed light on stage-specific B cell developmental signaling mechanisms in the bone marrow and spleen that cause immunological dysfunction. We have begun to classify B lymphocyte stages at which Ttc7 transcription rates fluctuate in pursuit of these signaling mechanisms. We have found Ttc7 to be transcribed in the bone marrow at early B lymphocyte stages and have evidence that its transcription is repressed at later stages in the spleen. To further support the hypothesis that induction of Ttc7 transcription during B cell development occurs in early B lymphocytes, we have also found that a 1kb segment of the promoter induces a higher rate of transcription in the pre-B 70Z/3 cell line.
Pre-exposure to uninfected sand fly bites protects rhesus monkeys against cutaneous leishmaniasis (94.14)
Authors: Oliveira Fabiano , Rowton Edgar , Gomes Regis , Lawyer Phillip , Teixeira Clarissa , Aslan Hamide , Valenzuela Jesus , Kamhawi Shaden
Published: January 2023

Leishmaniasis is a vector-borne disease that distresses millions in the poorest regions of the globe. There are no anti-Leishmania vaccines approved for human use. Mice pre-exposed to uninfected sand fly bites (USFB) are protected against cutaneous leishmaniasis (CL). To test if pre-exposure to USFB protects rhesus monkeys against CL, we first developed a model where monkeys challenged with 50 L. major-infected Phlebotomus duboscqi sand flies developed self-healing cutaneous lesions mimicking human CL. In a following experiment, we exposed animals 3 times, every 21 days, to 20 uninfected P. duboscqi. We found that 70% of the monkeys reacted to USFB(DTH reaction and antibodies). Twenty-one days after the last exposure to USFB naïve, pre-exposed reactive (PE-R) and pre-exposed non-reactive monkeys were challenged with 50 L. major-infected P. duboscqi. We observed a reduction in disease burden and healing time in the PE-R group. This protection correlated with a two log reduction in parasite load and the PE-R animals showed a reduced maximum lesion diameter indicative of diminished disease. Skin biopsies two weeks post-infection showed a higher expression of IFN-γ in the PE-R group compared to naïve monkeys. Altogether, these data show that pre-exposure to uninfected P. duboscqi bites can protect monkeys against L. major infection. Identification of the salivary proteins responsible for the observed protection may lead to the discovery of vaccine candidates against human CL.
The link between multiple sclerosis, T regulatory cells and Japanese rice (96.1)
Authors: Wormser Uri , Brodsky Berta , Proscura Elena , Erlanger-Rosengarten Avigail , Shapira Elena
Published: January 2023

The purpose of the present study was to develop a peptide for treatment of multiple sclerosis (MS). We have tested the effect of a novel antiinflammatory peptide (KGHYAERVG, termed IIIM1) on experimental autoimmune encephalitis (EAE), an animal model of MS. Our findings demonstrate significant reduction in neurological score following oral administration of IIIM1, as compared to the control groups received the vehicle (saline). Structural studies revealed that the entire peptide is required for activity. The peptide caused significant reduction in IL17, interferon gamma and IL12 production by isolated splenocytes and concomitant elevation of antiinflammatory cytokines. IIIM1 elevated T regulatory cells (Tregs, CD4+CD25+FoxP3+) in brain and spleen of EAE mice. Similar proliferative effect was observed in isolated human and mouse Tregs in vitro. Stimulation of Tregs by IIIM1 caused production of a new peptide termed RA1 present in Oryza Sativa Japonica group. This Japanese rice peptide ameliorated neurological symptoms in the EAE model. Similar beneficial effect was observed upon oral administration of an extract of Japanese rice. In conclusion, oral treatment with IIIM1 ameliorates EAE symptoms via stimulation of Tregs to proliferate and produce RA1 which reduces EAE symptoms. These findings may explain the relatively low prevalence of MS in Japan and other Japanese rice-eating populations. (This work was supported in part by The Israel Science Foundation grant 747/05).
Therapeutic effect of lithium in EAE: Effects on dendritic cells and encephalitogenic activity of T cells. (96.16)
Authors: Buel Amber , Naves Rodrigo , Mbana Tethia , Raman Chander , De Sarno Patrizia
Published: January 2023

We have reported that the GSK3 inhibitor, lithium, is therapeutic in acute and relapsing remitting EAE. We have now determined that lithium treatment is effective in attenuating EAE induced by Th1 encephalitogenic T-cells but not Th17 cells. These results were obtained from passive transfer experiments in which encephalitogenic Th1 or Th17 cells were transferred into lithium-treated or untreated naïve recipients. Consistent with this observation, lithium treatment was also ineffective in inhibiting EAE in IFN-γR1-/- mice. Various experiments indicated that the primary target of lithium therapy in vivo were cells of the innate immune system such as, dendritic cells (DC) rather than T-cells. Using primary dendritic cells obtained from spleens and lymph nodes or bone marrow derived DC (BMDC), we have determined that lithium does not affect LPS or CpG-induced maturation of DC. Remarkably, lithium treated DC were as or more efficient than untreated DC in their ability to process and present ovalbumin to OTII T-cells or MOG35-55 peptide to T-cells from 2D2 TCR transgenic mice. These observations raise the possibility that lithium-treatment of DC alters the chemokine and cytokine profile involved in migration and/or Th-subset specific differentiation. Overall, our findings that pharmacological inhibition of GSK3 is effective in Th1 EAE but not Th17 EAE has crucial implications for identifying and treating MS patients.
Deletion of C-reactive protein exacerbates collagen-induced arthritis: A role for CRP in immune tolerance? (96.23)
Authors: Jones Nicholas , Landis Dawn , McCrory Mark , Szalai Alexander
Published: January 2023

Heightened C-reactive protein (CRP) blood level associates with inflammation and tissue damage in rheumatoid arthritis (RA), and so CRP is assumed to have a detrimental effect. However, the exact role of CRP in RA remains unknown. To ascertain the contribution of CRP in RA we subjected wild type mice (WT) and CRP deficient mice (moCRP-/-) to collagen-induced arthritis (CIA). To our surprise, disease developed 14 days earlier in moCRP-/- than WT, achieving a threefold higher clinical score. The beneficial effect of CRP in CIA appears to be linked to the protein’s ability to dampen the activation state of dendritic cells (DCs). For example, incubation of bone marrow-derived CD11c+ DCs (BMDCs) with 10 μg/ml purified human CRP reduced display of MHC class II and CD80/86 compared to BMDCs exposed to media alone (8 ± 4% lower and 5 ± 5.5% lower, respectively). This effect was observed whether BMDCs were derived from WT or moCRP-/- but was lost if cells were from mice lacking expression of FcγRIIB, the latter showing increased activation in response to CRP. Based on these observations we suggest that CRP binding to the inhibitory FcγRIIB conveys a signal leading to reduced display of DC activation markers. Accordingly, in the absence of CRP or FcγRIIB this pathway is disrupted, thereby de-inhibiting DCs and rendering moCRP-/- more prone to CIA. Experiments now underway are investigating whether a CRP→FcγRIIB pathway on DCs contributes to maintenance of immune tolerance.
The therapeutic role of resveratrol in allergic lung inflammation (97.2)
Authors: Chen Jing , Lee Sang-Myeong , Fang Deyu , Atasoy Ulus
Published: January 2023

CD4+ T helper type 2 (Th2) cells are crucial for mediating allergic inflammatory lung disease such as asthma, by producing key cytokines including interleukin (IL)-4, IL-5 and IL-13. The phytoalexin, resveratrol, which is rich in grapes and red wine, can increase lifespan and has been suggested as a potential reagent to treat aging-related diseases. Herein we report that resveratrol prevents mice from developing experimental asthma induced by ovalbumin sensitization and re-challenge. Feeding mice with resveratrol inhibits the chronic allergic inflammation in lungs and reduces the numbers of eosinophil/neutrophils in bronchoalveolar lavage fluid. The mechanism appears to be resveratrol-induced suppression of allergic lung inflammation by inhibition of Th2-cytokine production. OVA-specific IL-5 and IL-13 production by CD4+ T cells in treated mice is reduced. Indeed, resveratrol blocks the in vitro polarization of naïve CD4+ T cells to the Th2 phenotype. Western blotting analysis further shows that resveratrol specifically down-regulates the expression of the Th2 transcription factor, GATA-3. These results suggest that resveratrol has therapeutic potential for altering the course of allergic inflammatory lung diseases such as asthma.
Adenosine promotes de novo generation of Th17 Cells via the dendritic cell A2B adenosine receptor (98.7)
Authors: Wilson Jeffrey , Black Steven , Linden Joel , Ernst Peter
Published: January 2023

Differentiation of naïve CD4+ T lymphocytes into appropriate effector subsets is important for generating protective immunity. Factors controlling the development of IL-17 secreting CD4+ T cells (Th17 cells) remain poorly defined. Adenosine is an endogenous metabolite produced during hypoxia or inflammation. Here we report that the stable adenosine mimetic NECA promotes Th17 responses by acting on dendritic cell (DC) adenosine A2B receptor (A2BAR). Culturing highly purified naïve CD4+ T cells with DCs in the presence of NECA results in the differentiation of IL-17 secreting cells and elevation of mRNA encoding signature Th17 molecules such as IL-22 and ROR-γT. Experiments using selective adenosine receptor (AR) antagonists and cells from A2BAR-/- mice indicate that DC A2BAR is largely responsible for the effect. The Th17 induction requires specific cytokine mediators that are selected by NECA-treated DC including IL-6 and TGF-β. PLC and PKC are important intracellular mediators of the A2BAR-mediated IL-6 response, whereas both adenylyl cyclase and PKA are dispensable as evidenced by studies using pharmacologic inhibitors, thus suggesting that A2BAR promotes the Th17 response via a Gq dependent pathway. Hence, in addition to previously noted direct effects of ARs on Treg development and function, these data suggest that under some circumstances adenosine acts via A2BARs expressed on DCs to favor the Th17 lineage.
CD4+ recent thymic emigrants are biased to the Th2 lineage (99.14)
Authors: Hendricks Deborah , Fink Pamela
Published: January 2023

Following intra-thymic development, T cells exit the thymus and join the peripheral T cell pool. These newly generated cells, termed recent thymic emigrants (RTEs), undergo phenotypic and functional maturation for the first 3 weeks they reside in the periphery. Using mice transgenic (Tg) for green fluorescent protein (GFP) driven by the Rag2 promoter (RAG2p-GFP Tg), in which GFP reliably distinguishes RTEs from their more mature naïve (MN) counterparts, our lab is able to isolate untouched RTEs for functional and phenotypic analysis. Activated CD4+ RTEs exhibit defects in IL-2Rα upregulation, proliferation and IL-2 production. We now show that CD4+ RTEs are defective in T helper (Th) 0, Th1, Th17 and regulatory T cell lineage commitment, with dampened cytokine production and transcription factor expression. RTE Th lineage commitment is not globally impaired, however, as CD4+ RTES are biased toward the Th2 lineage, with IL-4, IL-5 and IL-13 production that is enhanced compared to that by identically treated mature T cells. This bias in RTEs likely results from dampened negative regulation of the Th2 lineage by diminished levels of T-bet, a key Th1 transcription factor. CD4+ RTEs are thus a transitional population defined by their distinct interpretation of, and response to, immunological cues. These characteristics may be beneficial during the post-thymic maturation period to avoid inappropriate immune responses, particularly in lymphopenic neonates and adults.
Antigen-specific Th9 cell lines exhibit unique features (99.2)
Authors: Tan Cuiyan , Vistica Barbara , Shi Guangpu , Aziz Mehak , Gery Igal
Published: January 2023

Research in recent years has shown that naïve lymphocytes can differentiate into lineages (Th1, Th2, Th17 and Treg cells) with distinct effector functions and signature cytokines. Two recent publications reported the generation of Th cell lines that specifically express high levels of IL-9 and IL-10 and were designated “Th9”. These lines were generated by activation of naïve lymphocytes with antibodies against CD3/CD28, in the presence of the polarizing cytokines IL-4 and TGF-β. Here, we analyzed features of “Th9” cells generated by activation with the specific antigen. T-cells transgenically expressing T-cell receptor (TCR) specific to hen egg lysozyme (HEL) were activated with this antigen in the presence of the polarizing cytokines IL-4 and TGF-β. Main observations: (i) IL-9 and IL-10 were expressed with remarkably different kinetics; (ii) re-activation of “Th9”, following expansion, triggered faster and higher production of both IL-9 and IL-10; (iii) incubating “Th9” cells in polarizing media of other phenotypes stimulated low levels of phenotype switching to Th1 or Th17, but a massive switching to Th2; (iv) “Th9” induced moderate inflammation in HEL expressing recipient eyes, but only when collected at stages at which IL-9 was produced. Thus, “Th9” cells exhibit unique features that differentiate them from other known Th lines.
T Cell Factor-1 Initiates T Helper 2 Fate by Inducing GATA-3 and Repressing IFNgamma (99.8)
Authors: Yu Qing , Sharma Archna , Sen Jyoti
Published: January 2023

Differentiation of activated CD4 T cells into T helper (Th) -1 and -2 cell fate is regulated by cytokines and transcription factors T-bet and GATA-3. Whereas it is clear that IL-12 produced by antigen presenting cells initiate Th1 fate, signals that initiate Th2 fate have not been completely characterized. Here we show that Gata-3, an essential transcription factor for Th2 differentiation, is initially induced by T cell factor-1 (TCF-1) and its cofactor beta-catenin, predominantly from the proximal promoter upstream of exon 1b. This activity is induced in CD4 T cells early after T cell receptor stimulation and is independent of IL-4 receptor signaling via STAT-6. Furthermore, we show that TCF-1 blocks Th1 fate by negatively regulating IFNgamma expression, independently of beta-catenin. Thus, our results indicate that TCF1 initiates Th2 differentiation of activated CD4 T cells by a dual mechanism: by promoting Gata-3 expression and suppressing IFNgamma expression.
Human T follicular helper cells comprise subsets specialized for help of distinct B cell subsets (99.9)
Authors: Bentebibel Salah-Eddine , Schmitt1 Nathalie , Morita Rimpei , Banchereau Jacques , Ueno Hideki
Published: January 2023

T follicular helper cells (Tfh) represent a Th subset specialized for the help of antibody responses. In contrast to recent progress regarding the phenotype and developmental mechanism of Tfh cells, how Tfh cells regulate B cell responses is less well characterized. In particular, whether Tfh cells are involved in the differentiation of naïve B cells into antibody-secreting cells (ASCs) remains largely unknown. Here we show that human tonsillar Tfh cells comprise subsets specialized for the help of distinct B cell subsets. A subset of Tfh cells, here called Pre-Tfh cells, is specialized for the help of naïve B cells. In contrast, Tfh cells at germinal centers (GC-Tfh cells) are the only Tfh subset able to induce germinal center B (GC-B) cells to produce Igs. Despite the capacity to secrete high levels of Interleukin-21 (IL-21), Pre-Tfh cells failed to help GC-B cells, due to the induction of apoptosis of GC-B cells via FAS/FAS ligand interaction. While Pre-Tfh cells were found exclusively at extrafollicular sites, Pre-Tfh cells expressed Bcl6, a master transcription factor of Tfh cells, at equivalent levels with GC-Tfh cells. A comprehensive gene expression profiling strongly suggests that pre-Tfh cells are progenitors of GC-Tfh cells. Thus, the differentiation of functional human Tfh cells already occurs at outside of B cell follicles, and such extrafollicular Tfh cells are engaged in the differentiation of naïve B cells.
Non-covalent interaction of Abacavir with HLA-B*5701 generates an antigenic complex recognized by specific T cells (100.13)
Authors: Adam Jacqueline , Eriksson Klara , Schnyder Benno , Fontana Stefano , Pichler Werner , Yerly Daniel
Published: January 2023

Rationale: Abacavir (ABC) hypersensitivity is mediated by specific CD8+ T cells that interact with ABC in HLA-B*5701+ individuals. To understand how ABC is presented and recognized by T cells, we generated ABC specific T cell clones (ABC-TCC) and analysed the kinetic of ABC recognition. Methods: ABC-TCC from ABC naïve HLA-B*5701+ subjects were generated by limiting dilution. TCC activation by ABC was measured by means of Ca2+ influx kinetic analyses and Annexin V staining. Results: ABC-TCC in presence of autologous antigen-presenting cells (APC) reacted within a short time (3-25 min) to ABC in solution. Interestingly, APC that internalized the drug were able to present it already after 30 min on the cell surface leading to an immediate TCC activation (60 sec). Furthermore, no mRNA for alcoholdehydrogenases (ADH), the enzymes involved in ABC metabolism, was detected in the immune cells of peripheral blood. Conclusions: The rapid kinetic of TCC reactivity to ABC presented by HLA-B*5701 and the lack of ADH activity in peripheral blood mononuclear cells is best explained by a direct, metabolism and processing independent interaction of ABC with the restricting HLA-B*5701 molecule. This interaction either occurs intracellular or on the cell surface, leading to a modification of the HLA-complex able to induce a full T cell response. Our data underline that the immune stimulatory capacity of drugs is not uniquely restricted to the ability to form covalent bonds to proteins.
TAP-independent presentation of the melanoma vaccine candidate epitope gp100209-217 requires no gp100 sequences outside the core peptide and is sensitive to cytosolic TPP2 degradation (100.2)
Authors: Leonhardt Ralf , Vigneron Nathalie , Van den Eynde Benoît , Cresswell Peter
Published: January 2023

gp100209-217 is a promising anti-melanoma peptide vaccine that corresponds to an immunodominant, HLA-A*0201-restricted tumor-epitope. As an immune evasion strategy many tumors display downregulation or even total lack of expression of the TAP transporter, a crucial factor involved in MHC class I-mediated antigen presentation. Surprisingly, we find that HLA-A*0201-mediated presentation of gp100209-217 is remarkably independent of TAP and thus the peptide likely uses an unconventional pathway to access MHC class I molecules. Our data suggest that presentation does not involve proteolysis in one of the diverse protease-rich compartments that gp100 traverses on its way to melanosomes. Neither is melanosomal targeting or ER-export of gp100 required, nor is even co-translational insertion of the protein into the ER membrane necessary. However, presentation of the epitope does depend on proteasome activity. Interestingly, we find that cytosolic tripeptidyl peptidase 2 (TPP2) efficiently counteracts TAP-independent presentation of gp100209-217 suggesting that tumors might use this protease for immune evasion. Strikingly, no sequences outside the core peptide region within gp100 are required for TAP-independent access of the ligand to HLA-A*0201. We conclude that a transporter system besides TAP must exist in the cell, which can partially compensate for loss of TAP function and mediate access of critical tumor-specific antigenic peptides to HLA-A*0201.
An insertion mutant in DQA*0501 restores susceptibility to DM: implications for disease associations (100.22)
Authors: Hou Tieying , Macmillan Henriette , Strohman Michael , Yoon Taejin , Jin Xi , Mellins Elizabeth
Published: January 2023

HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on antigen presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) with autoimmune diseases, like celiac disease, have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. Here, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2/DM interface in order to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on cell surface and the presentation of α-gliadin 57-73 Q65E epitope to T cell hybridomas. We found that the three α chain mutations (α+53G, α+53R and αY22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the α+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that α-gliadin 57-73 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2.
Efficient lipid antigen presentation to NKT cells by targeting CD169/Siglec-1 on macrophages with its glycan ligand. (100.49)
Authors: Kawasaki Norihito , Chen Weihsu , Vela Jose , Nycholat Corwin , Maricic Igor , Vipin Kumar , Crocker Paul , Kronenberg Mitchell , Paulson James
Published: January 2023

Natural killer T (NKT) cells are a subset of T cells expressing an invariant TCR with cross-species conserved specificity to glycolipid antigens in the CD1d restricted presentation. Recent studies have identified CD169/Siglec-1+ macrophages as antigen presenting cells for NKT cells. Here we show that delivery of the lipid antigen through CD169 on macrophages induces NKT cell activation. We developed CD169-targeted liposomes decorated with a specific glycan ligand of CD169 (9-N-biphenylcarboxyl-NeuAcα2-3Galβ1-4GlcNAc) and observed that CD169-targeted liposomes were captured by macrophages in the CD169-dependent manner, and delivered to lysosomes, the known site of lipid antigen loading on CD1d. We next investigated the ability of the CD169-targeted liposomes to promote NKT cell activation by delivery of a lipid antigen α-GalCer to macrophages. We found that macrophages pulsed with α-GalCer containing CD169-targeted liposomes efficiently presented the lipid antigen to NKT cells when transferred into the C57BL/6J mouse. Alternatively, intravenous injection of the α-GalCer containing CD169-targeted liposomes activated NKT cells 100-fold more efficiently than lipid antigen alone in vivo. CD169-targeted liposomes without α-GalCer did not activate NKT cells, demonstrating this activation is through cognate antigen recognition by NKT cells. These data demonstrate that CD169-targeted liposomes dramatically enhance lipid antigen delivery to macrophages for NKT cell activation in vivo.
Essential glycan-dependent interactions optimize MHC class I peptide loading (100.51)
Authors: Wearsch Pamela , Zhang Wei , Cresswell Peter
Published: January 2023

In this study we sought to better understand the role of the glycoprotein quality control machinery in the assembly of MHC (Major Histocompatibility Complex) class I molecules with high-affinity peptides. The lectin-like chaperone calreticulin (CRT) and the thiol oxidoreductase ERp57 participate in the final step of this process as part of the PLC (peptide-loading complex). To investigate the mechanism of peptide loading and roles of individual components, we reconstituted a PLC sub-complex, excluding TAP, from purified, recombinant proteins. The minimal components required for PLC assembly and function in vitro were ERp57 disulfide-linked to the class I-specific chaperone tapasin, CRT, and MHC class I complexes bearing monoglucosylated glycans. Mutations disrupting the interaction of CRT with either ERp57 or the class I glycan completely eliminated tapasin-mediated peptide loading. Using the purified system, we also provide direct evidence for a novel role of UGT1 (UDP-glucose: glycoprotein glucosyltransferase 1) in MHC class I assembly. Recombinant UGT1 preferentially recognized and re-glucosylated class I molecules associated with a suboptimal ligand and allowed PLC re-engagement and peptide exchange. Collectively, the data indicate that the interaction of MHC class I molecules with the PLC is regulated by its N-linked glycan, and that UGT1 contributes to the optimization of the MHC class I peptide repertoire in addition to tapasin.
Gastrointestinal tract is the place where encephalitogenic T cells develop in the spontaneous EAE mice (101.11)
Authors: Ito Naoko , Boppana Sridhar , Huang Hui , Dhib-Jalbut Suhayl , Ito Kouichi
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease exhibiting features of demeylination and axonal loss in the central nervous system (CNS). Although CNS-specific Th1 and Th17 cells have been suggested to initiate the disease, it is still unknown how these effector T cells develop in MS patients. To address this question, we have generated transgenic mice that express T cell receptor (TCR) genes specific for myelin basic protein (MBP) and HLA-DR2a genes that were isolated from an MS patient. The humanized MBP-TCR Tg mice developed spontaneous EAE. Interestingly Th1 and Th17 cells developed in the mesenteric lymph nodes and the colon more efficiently than in other lymphoid organs in the spontaneous EAE mice, and their development was much lower in the gastrointestinal (GI) tract of healthy MBP-TCR Tg mice. This data suggests that MBP-specific T cells differentiate into Th1 and Th17 encephalitogenic T cells in the colon and migrate into the CNS from mesenteric lymph nodes. Alternatively, MBP-specific Th1 and Th17 encephalitogenic T cells develop in the other lymphoid organs and preferentially migrate into the gut. Interestingly, CD4 T cells infiltrates in the CNS of spontaneous EAE mice expressed gut-homing receptors in addition to CNS-homing receptors. These data suggest that the GI-tract may be an important site for the differentiation of encephalitogenic T cells in spontaneous EAE mice.
VSV infection ameliorates autoimmune disease in FcγR2B deficient mice (101.14)
Authors: Kole Hemanta , Deane Jonathan , Scott Bethany , Bolland Silvia
Published: January 2023

We have investigated whether infection by pathogens affects the development of autoimmune diseases such as lupus. We tested the effect of infection with vesicular stomatitis virus (VSV) on two mouse strains that spontaneously develop lupus disease. Infection of VSV in lupus prone FcγR2B-/- (R2) mice significantly reduced autoantibody titers and inflammatory cytokines and chemokines in serum, reduced glomerulonephritis, and improved survival. In contrast, VSV infection in R2yaa mice, which develop a more aggressive lupus disease, showed slightly reduced auto-antibody titers in the serum without any major changes in serum cytokines and chemokines, kidney glomreulonephritis, or survival. Characterization of immune cell phenotypes in VSV infected R2 mice showed that B cell development in the bone marrow was not affected whereas germinal center, plasma, and IgG producing mature B cell numbers were reduced. Additionally, VSV infected R2 mice had lower number of follicular helper T cells and CD11c+ cells in the spleen. Compared to uninfected mice, microarray analysis of naïve resting B cells from VSV infected R2 splenocytes showed up-regulation of several candidate genes including a non receptor type protein tyrosine phosphatase, PTPN22 that has been shown to be associated with Lupus and other autoimmune diseases. Studies of the functional implications of Ptpn22 overexpression in VSV-infected R2 B cells are in progress.
Susceptibility to infection in systemic lupus erythematosus: clues from a murine model. (101.3)
Authors: Lieberman Linda , Tsokos George
Published: January 2023

Systemic lupus erythematosus (SLE) patients invariably display defects in both innate and the adaptive branches of the immune system, contributing to inflammation and organ damage. Nevertheless, infection is a leading cause of death in patients suffering from SLE. Though SLE patients are treated with immunosuppressive drugs, the high rates of infection suggest that inherent defects in host immunity may contribute to their increased susceptibility. The mechanisms responsible for the increased susceptibility of SLE patients to infection are unclear, but the observed defects in cytokine production and T cell subsets in these patients would intimate that they have a less robust immune response to infection. We have developed a murine model to study the relationship between infection and SLE pathogenesis and preliminary data supports an increased susceptibility of SLE-prone mice to infection. B6.MRL/lpr mice infected intraperitoneally with the obligate intracellular parasite Toxoplasma gondii succumbed to infection acutely. The mechanism behind this susceptibility is not clear. There was no increase in parasite burden or decrease in serum cytokines in the infected SLE-prone mice as compared to the wild type controls. The activation status of various immune cells was normal in the infected SLE mice, but a defect was observed in antigen-specific IFN-γ production. Further studies will elucidate the mechanism responsible for the increased susceptibility of SLE-prone mice to infection.
Development of autoreactive CD8+ T cells in Aire-deficient mice (101.38)
Authors: Ji Niannian , Forsthuber Thomas
Published: January 2023

Autoimmune regulator (Aire) regulates the transcription of a myriad of self antigens in the thymus, which is important for the process of central tolerance to eliminate autoreactive T cells and to prevent autoimmune pathology. Aire-deficient mice (aire-/-) have previously been shown to develop multi-organ autoimmune disorder. We were interested in investigating the role of Aire for autoreactive CD8+ T cells using aire-/-Ia-/- mice. We found that CD8+ T cells were increased in aire-/-Ia-/- mice as compare with their aire+/+ or heterozygous littermates. Furthermore, aire-/-Ia-/- mice showed significantly lower body weight as compared with their littermates. Immune infiltrates mainly composed of CD11c+ cells and CD8+ T cells were found in various tissues of aire-/-Ia-/- mice, especially in liver, lung, pancreas and stomach. Autoantibody was detected in the serum of aire-/-Ia-/- mice directed against gastric tissue and was determined to be predominantly IgM. Collectively, our study shows that autoreactive CD8+ T cells arise on the background of Aire-deficiency are capable of initiating and promoting autoimmunity in the absence of CD4+ T cells.
Mapping the epitope in Epstein Barr Virus nuclear antigen-1 that elicits cross-reactivity with dsDNA (101.43)
Authors: Yadav Pragya , Ebegbe Roland , Anam Adiba , Mumbey-Wafula Alice , Gottlieb Paul , Spatz Linda
Published: January 2023

Several studies have shown an association between Systemic Lupus Erythematosus (SLE) and the Epstein Barr Virus (EBV). Our laboratory recently demonstrated that BALB/c mice injected with the major EBV nuclear antigen, EBNA-1, developed antibodies to EBNA-1 that cross-react with double stranded DNA (dsDNA). We generated monoclonal antibodies (MAbs) to EBNA-1 from EBNA-1 injected mice and demonstrated by anti-dsDNA ELISA, dsDNA affinity columns, and Crithidia luciliae assays, that these antibodies cross-react with dsDNA. Studies are underway to determine the pathogenicity of these MAbs. We have been mapping the epitope in EBNA-1 that serves as a peptide mimotope for dsDNA. We now demonstrate that the mimotope is contained within the carboxyl region of the EBNA-1 protein, between amino acids 459 and 607. This region contains well-defined secondary structure, which suggests that the basis for the cross-reactivity with dsDNA could be due to a conformational epitope in EBNA-1. Studies are in progress to further map this cross-reactive epitope. Identification of this epitope may help in designing diagnostic and therapeutic strategies that can mask the epitope from the immune system following EBV infection and thereby prevent the development of antibodies that cross-react with dsDNA.
Susceptibility to autoimmune myocarditis is associated with strain-dependent differences in IL6 receptor expression on naive CD4+ T cells (101.5)
Authors: Chen Ping , Baldeviano G. , Ligons Davinna , Talor Monica , Barin Jobert , Rose Noel , Daniela Cihakova
Published: January 2023

The goal of this study is to identify non-H-2 loci controlling differential susceptibility to experimental autoimmune myocarditis (EAM). A.SW and B10.S mice share the same MHC (H-2s) haplotype, yet A.SW are susceptible to EAM, while B10.S are resistant. We have identified a locus on Chromosome 1 (termed Eam1) that is important in determining susceptibility to myocarditis. In this study we have found that naive A.SW mice had a higher percentage of CD4+ T cells in their spleens, and CD4+ T cells from naive A.SW had great potential to differentiate towards the Th17 pathway than that from naive B10.S mice. To examine why T cells from A.SW mice more readily develop a Th17 phenotype, we assessed the cytokines and related receptors needed for Th17 induction. Level of IL-6 did not differ in the hearts of A.SW and B10.S mice. However, IL6Rα expression was greater on naive A.SW CD4+ T cells compared to B10.S mice, no difference was found for TGF-βRI expression. B10.A.Eam1 congenic mice were generated where Eam1 was derived from A.SW and the rest of the genome was of B10.S origin. The percentages of CD4+ T cells and IL-6Rα expression were intermediate on B10.A.Eam1 congenic mice, highest on A.SW and lowest on B10.S mice. Our data suggest that a higher percentage of CD4+ T cells in A.SW mice together with increased expression of IL6Rα and the tendency of CD4+ T cells to differentiate towards a Th17 phenotype contribute to heightened susceptibility to autoimmune myocarditis in A.SW mice.
Deletion of IL-6 in a murine model of primary biliary cirrhosis leads to exacerbated autoimmune cholangitis and lymphomas (101.6)
Authors: Zhang Weici , Tsuda Masanobu , Yang Guo-Xiang , Tsuneyama Koichi , Rong Guanghua , Ridgway William , Ansari Aftab , Flavell Richard , Coppel Ross , Lian Zhe-Xiong , Gershwin M. Eric
Published: January 2023

Autoimmune liver diseases are characterized by elevated serum levels of IL-6. We have previously reported the development of a PBC-like model in dnTGF-βRII mice engineered to express a dominant negative form of transforming growth factor β receptor (dnTGF-βRII, dnR) under the control of CD4 promoter. By producing a double construct, namely IL-6 deficient mice on the dnTGF-βRII background (dnTGFβRII IL-6-/- mice), we previously reported that such animals manifest improvement in their underlying inflammatory bowel disease but worsening of their biliary infiltrates. To further understand this model, we have studied dnTGFβRII IL-6-/- mice and, in particular, focused on the pathology over a longer period of observation of up to 9 months. Importantly, loss of the IL-6 gene, although it does improve underlying inflammatory bowel disease, dramatically increases lymphoid aggregation, elevates inflammatory liver cytokines and promotes the appearance of activated T cells in liver. Indeed, loss of the IL-6 gene results in lymphomatous lesions in liver, spleen and lung at 6-9 months of age in 7 of 22 male dnTGFβRII IL-6-/- mice. No lesions were found in control dnTGFβRII mice. These data further highlight the promiscuous role of IL-6 and suggest caution in the therapeutic use of anti-IL6R antibody in the treatment of patients with autoimmune disease.
Neutrophil migration to the lung is enhanced in selectin-ligand deficient mice (102.11)
Authors: Onami Thandi , Harp John , Zeng Junwei
Published: January 2023

The first step in leukocyte trafficking into tissues and peripheral organs is mediated by selectin binding. Mice deficient in α1,3 fucosyltransferases-VII and -IV (FtDKO) are unable to synthesize sLex, which is required for E-, P-, and L-selectin binding. We recently reported that due to an inability to enter peripheral lymph nodes, FtDKO mice show increased numbers of T cells in peripheral blood and increased T cell accumulation in the lung. We now report that FtDKO mice show substantial increases in granulocyte populations in peripheral blood, with an ~20-fold increase in Ly6G+Mac-1+ neutrophil populations in the lung parenchyma. To determine whether increased neutrophil localization in the lung influences disease processes, we infected FtDKO mice with influenza. As previously reported, FtDKO mice show impaired neutrophil trafficking to the peritoneal cavity following inflammation due to loss of selectin-mediated binding. However, FtDKO mice show rapid and enhanced neutrophil trafficking to broncho-alveolar regions of the lung following influenza infection. These data suggest that instead of reducing inflammatory cell populations, loss of selectin-mediated binding may paradoxically enhance accumulation of neutrophils in the lung parenchyma, which may in turn facilitate the influx of these cells into broncho-alveolar regions of the lung following inflammation or injury.
Renal dysfunction increases aortic macrophage and lymphocyte accumulation during atherosclerosis development (102.2)
Authors: von Vietinghoff Sibylle , Hertel Barbara , Ley Klaus , Haller Hermann
Published: January 2023

Atherosclerosis is a chronic inflammatory disease of the artery wall with innate and adaptive immune components. Decreased renal function is a major risk factor for cardiovascular events and mortality. While altered hemodynamics and mineral metabolism have been investigated in detail, the atherosclerotic vascular leukocyte infiltrate in renal impairment has not been systematically explored. This study tested whether moderate renal impairment alters leukocyte composition in the aorta of atherosclerotic mice. Apolipoprotein E deficient (Apoe-/-) mice underwent nephrectomy or sham surgery and were maintained on high-fat diet for 12 weeks. Aortic leukocyte infiltration was determined by immunofluorescence staining and confocal microscopy and by flow cytometry of aortic single cell suspensions. Unilateral nephrectomy significantly increased serum creatinine levels as a measure of renal function but did not alter serum calcium and phosphorous levels. While total blood leukocyte counts were un-altered, the number of aortic leukocytes was significantly increased after nephrectomy. Specifically, aortic B cells and macrophages were increased. Proliferation of aortic macrophages was increased. Aortic en face and root atherosclerotic lesion size were significantly larger in mononephric mice. We conclude that mild renal failure is sufficient to alter inflammatory aortic leukocyte accumulation in atherosclerosis.
Age-associated changes in Non-muscle Myosin IIA and CXCR4 regulates increased migration induced by SDF1 in CD4+ T lymphocytes from elderly human donors. (102.8)
Authors: Cane Stefania , Ponnappan Subramaniam , Sullivan Dennis , Ponnappan Usha
Published: January 2023

Non-muscle Myosin-IIA has varied functions in T cell contractility, migration and cytokinesis. Evaluation of non-muscle Myosin-IIA in CD4+ T cells from the elderly, demonstrated not only altered association of the Myosin heavy chain with the chaperone HSP90, but also increased basal levels of protein expression, and constitutive phosphorylation of the myosin light chains. These alterations manifested as increased actomyosin complex formation in cells from the elderly, as opposed to those from young donors. Additionally, chemotactic migration in response to agonist SDF1, demonstrated increased migration index in CD4+ T cells from the elderly, when compared to those from young donors. As SDF1-mediated migration is clearly dependent on the expression of CXCR4 on T cell surface, we assessed CXCR4 levels in CD4+ T cells from young and elderly human donors. Our results for the first time demonstrate a significant increase in CXCR4 levels on the surface of T cells from the elderly, when compared to those from young donors. As Myosin-IIA regulates both the motility and endocytosis of CXCR4, we hypothesize that alterations in Myosin-IIA may underlie endocytosis of CXCR4 by SDF1 in the elderly. Thus, combined alteration in Myosin-IIA and CXCR4 levels in T lymphocytes may underlie increased motility observed during aging and may impact the strength and duration of interaction with antigen-presenting cells.