bluuebunny/crossref_metadata_2025 huggingface dataset:
CD169+ marginal zone macrophages are critical for the clearance and localization of listeria monocytogenes following a systemic infection (INC7P.407)
Authors: Perez Oriana , Maher Leigh , Qiu Zhijuan , Tanaka Masato , Bénéchet Alexandre , Khanna Kamal
Published: January 2023

The spleen is a secondary lymphoid organ that plays an important role in providing protection against blood-borne pathogens. CD169+ marginal zone (MZ) macrophages (MZM) are located along the marginal sinus of the spleen and are positioned to be among the first cells to encounter bacteria. However, the precise role of these cells during infection is not well understood. Thus, we used CD169-diptheria toxin receptor (DTR) mice to selectively deplete CD169+ MZM to study the role of CD169+ MZM after Listeria monocytogenes (Lm) infection. CD169+ MZM depleted mice succumbed to sublethal dose of Lm infection due to inefficient capture and unrestricted growth of LM early after infection. In additon, CD169+ MZM depleted mice fail to localize Lm to T cell zones despite greater numbers of replicating bacteria in the MZ and the red pulp early after infection. This suggests CD169+ MZM play a critical role mediating Lm trafficking to T cell zones. The aberrant Lm localization in CD169-DTR mice also resulted in striking disruption of CD11b+ cell granulomas, further increasing bacterial spread to peripheral tissues. Interestingly, CD8α+ dendritic cells (DC) depletion did not rescue the ability of CD169-DTR mice to clear Lm, suggesting that CD169+ MZMs provide an essential route for Lm clearance that precedes the CD8a+ DC infection niche. Thus, our study revealed a novel role of CD169+ MZM as critical mediators of protection against bacterial infections by direct and indirect mechanisms.
Innate immune correlates of HIV pathogenesis in response to opportunistic mycobacteria (INC8P.428)
Authors: Gasper Melanie , Sherman David , Sodora Donald
Published: January 2023

HIV infection of humans and SIV infection of Rhesus macaques generally results in onset of opportunistic infections (OIs) and AIDS when left untreated. In contrast, SIV infection of African monkeys, such as sooty mangabeys, results in high levels of viral replication, but the immune system remains resilient to dysfunction, OIs and progression to AIDS. Here, we set out to identify innate immune correlates associated with acquisition of OIs in HIV+ humans (and the lack thereof in SIV+ mangabeys), through an ex vivo cytokine and transcriptomic assessment of PBMC and monocyte responses to the opportunistic pathogen, BCG. Our data reveal that HIV+ donors have significantly altered NK cell gene expression profiles and deficient monocyte IL-12 production (p=0.017) in response to BCG stimulation compared to HIV-negative donors. In contrast, SIV+ mangabeys maintain similar gene expression profiles and increase the percentage of monocytes producing proinflammatory cytokines TNFα (p=0.03) and IL-12 (p=0.06) following BCG stimulation compared to uninfected mangabeys. These data suggest that a lack of monocyte-derived IL-12 and deficient NK cell response to opportunistic pathogens may be associated with increased susceptibility to OIs. Further, they raise potential to augment innate immune function through NK cell-targeted IL-12 therapy during pathogenic lentiviral infections.
Monocytes sense hepatitis C virus-replicating cells and induce natural killer cell antiviral activity in an IL-18-mediated manner (INC8P.437)
Authors: Serti Elisavet , Werner Jens , Chattergoon Michael , Cox Andrea , Lohmann Volker , Rehermann Barbara
Published: January 2023

Natural killer (NK) cells are important effectors in the innate immune defense against many viruses. However, NK cell IFN-γ production is attenuated in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is due to an NK cell-intrinsic or extrinsic mechanism. Hepatoma cells expressing luciferase-tagged subgenomic HCV-replicons (Huh7/HCV-replicons) or their HCV-negative counterparts were co-cultured with NK cells in the presence or absence of other PBMC subpopulations. RESULTS: The IFN-γ-mediated antiviral effect of NK cells was reduced when isolated NK cells rather than PBMC were co-cultured with Huh7/HCV-replicons suggesting that other subpopulations contributed to NK cell activation. Increased monokines levels implicated a role of activated monocytes. Indeed, depletion of CD14+ monocytes, siRNA knockdown of the monocyte NALP3 inflammasome and neutralization of the inflammasome product IL-18 decreased the IFN-γ-mediated antiviral activity of NK cells. Interestingly, monocytes from chronic HCV patients were less effective than monocytes from healthy controls in inducing NK cell IFN-γ production. Vice versa, monocytes from healthy controls improved antiviral function and IFN-γ production of NK cells from chronic HCV patients. CONCLUSION: Monocytes sense HCV-replicating cells and induce, via inflammasome-mediated IL-18 production, an NK-cell mediated decrease in HCV replication. Impaired monocyte function in HCV infection attenuates the NK cell IFN-γ response.
Group 2 innate lymphoid cells and IL-33 in infant respiratory syncytial virus infection (INC9P.442)
Authors: Saravia Jordy , You Dahui , DeVincenzo John , Cormier Stephania
Published: January 2023

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants with an estimated global impact of 64 million cases and 160,000 deaths each year. Severe RSV infections in infants are characterized by airway obstruction and Th2-biased immune responses, including recruitment of Th2 cells and eosinophils to the lung and elevated Th2 effector cytokines (IL4, IL5, IL13). Early elevations in IL13 following RSV infection of neonatal mice suggest a source other than Th2 cells. Group 2 innate lymphoid cells (ILC2s) are a recently identified population of cells that produce high levels of IL13 and IL5 in response to IL33. There are no data available on the involvement of IL33 or ILC2s in Th2 infant RSV responses. In mice, our data demonstrate that RSV causes rapid IL33 expression and an increase in ILC2 numbers in the lung which are elevated in neonates versus adults. Blocking IL33 in the neonate abrogates this increase in ILC2s while treatment with recombinant IL33 in the adult augments ILC2 levels and increases Th2 cell numbers. In humans, we have observed elevated levels of IL33, IL13, and IL5 in nasal washes from RSV-infected infants. Additionally, ILC2s are present in higher numbers in cord blood mononuclear cells compared to adult peripheral blood mononuclear cells. These data indicate an important role for ILC2s and IL33 in the immunopathogenesis of severe infant RSV infection - for which there remains no adequate therapeutic/vaccine.
Pellino-1 and Pellino-3b in endotoxin tolerance and TLR signaling (INM9P.458)
Authors: Murphy Michael , Yanbao Xiong , Medvedev Andrei
Published: January 2023

Endotoxin tolerance (ET) reprograms Toll-like receptor (TLR) 4-inducible responses in monocytes and macrophages by attenuating expression of pro-inflammatory cytokines without suppressing anti-inflammatory cytokines and antimicrobial effectors. We previously demonstrated deficient LPS-mediated TLR4-driven activation of IL-1 receptor-associated kinase (IRAK) 4 and IRAK1 as critical hallmarks of endotoxin tolerance. E3 ubiquitin ligases Pellino regulate TLR signaling pathways via modifications of IRAK kinases. We examined the impact of ET on Pellino-1/3b expression and utilized overexpression and gene ablation approaches to establish their functions in TLR2/4 signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in control THP-1 cells and human monocytes. Endotoxin-tolerant cells showed attenuated expression of Pellino-1 but maintained increased levels of Pellino-3b proteins. Overexpression of Pellino-1 in 293/TLR4/MD2 and 293/TLR2 cells enhanced Pam3Cys- and LPS-mediated NF-κB activation and cytokine gene expression, while Pellino-1 ablation reduced these responses demonstrating Pellino-1 as a positive regulator of TLR4 signaling. Pellino-3b overexpression reduced, whereas Pellino-3b gene knockdown increased TLR2/4-driven NF-kB activation and expression of inflammatory cytokines, establishing it as a negative regulator. These data suggest non-redundant functions for Pellino-1 and Pellino-3b within the TLR4 signaling pathways and implicate their role in ET.
Signaling roles of Dictyostelium discoideum TirA in response to gram-negative bacteria (INM9P.450)
Authors: Snyder Michelle , White Theresa , Berlett Michael , Weichseldorfer Matthew , Shaw Emily , Zapf Ava , Snyder Greg
Published: January 2023

Innate immune Toll-like receptors (TLRs) signal via toll/interleukin-1 receptor (TIR) domains, resulting in initiation of various signaling pathways, including MAPK pathways. The model organism Dictyostelium discoideum, a social amoeba that phagocytizes bacteria for nutrients, lacks full-length TLRs but encodes for several TIR domain-containing proteins. One such protein, TirA, is required for the growth of D. discoideum in the presence of Gram-negative bacteria. Furthermore, addition of LPS increases D. discoideum’s bactericidal activities in a TirA- and ERK- dependent manner. Here we are investigating TirA’s role in the activation of ERK-MAPK pathways in response to bacteria and folic acid. ERK phosphorylation was found to be reduced in TirA-deficient cells as compared to WT cells in response to Gram-negative bacteria, but not in response to Gram-positive bacteria or to folic acid. Furthermore expression of uduB, a MAPK-regulated transcript, has previously been found to be upregulated in TirA-deficient cells during steady-state growth on Gram-negative bacteria. Here we show that upregulation of uduB can be detected within 4 h of exposure of TirA-deficient cells to Gram-negative bacteria. In addition to these procedures we are also working to solve the structure of D. discoideum TIR domains through the use of X-ray crystallography. By obtaining this structural information new insights into the conserved mechanisms of TIR domain signaling in may be revealed.
The imidazoquinoline TLR7 agonist negatively affects virus-induced type I and type III IFN induction pathways in primary human plasmacytoid dendritic cells (INM9P.452)
Authors: Natalia Mahwish , Dai Jihong , Singh Sukhwinder , Cui Chongwei , Fitzgerald-Bocarsly Patricia
Published: January 2023

Human pDC recognize Influenza/Flu and HSV through TLR7 and -9, respectively, resulting in production of type I and III IFN and pro-inflammatory cytokines. We observed that TLR7 is constitutively present in the ER and LAMP1-positive late endosomes in pDC, and upon stimulation with Flu, TLR7 co-localization with late endosomes increased. Synthetic small molecule TLR7 ligands from the imidazoquinoline family are being used for anti-viral therapy and as immune adjuvants. We investigated the mechanisms by which 3M003 induces type I and III IFN production in pDCs. We found that 3M003 induced IFNs much more rapidly than Flu or HSV, but at much lower levels. Surprisingly, neither the TLR7 inhibitor IRS661 nor cysteine endolysosomal protease inhibitor Z-FM-FMK affected 3M003 induced IFN production although both effectively inhibited Flu-induced IFN production. In addition, 3M003 inhibited Flu, HSV, HIV and CpGA induced IFN production in pDC when used together with these TLR7/9 agonists. However, 3M003 did not affect uptake by pDC. Interestingly, 3M003 inhibited virus-induced IRF7 nuclear translocation while enhancing its phosphorylation. Taken together, these results suggest that in pDCs, 3M003 utilizes signaling pathways distinct from HSV or Flu resulting in the different levels of IFN production.
CD4+CD25+ regulatory T cells inhibit NK cell hepatocytotoxicity of hepatitis B virus transgenic mice via membrane-bound TGF-beta and OX40 (IRC10P.464)
Authors: Chen Yongyan , Sun Rui , Wei Haiming , Tian Zhigang
Published: January 2023

Human healthy HBsAg carriers are susceptible to hepatocyte injury induced by a variety of elements, among which the immune responses and the negative regulation might be critical. It has been reported by us that HBV transgenic mice were oversensitive to liver injury after immunologic triggering and that NK cells played the critical role via NKG2D recognition of HBsAg positive hepatocytes. In this study, using the low dose of Con A-induced oversensitive liver injury model in HBs-Tg mice, it was found that the number of CD4+CD25+Foxp3+Treg cells was significantly increased in the liver after Con A stimulation, which was along with the recovery of liver injury. Adoptive transfer of hepatic Treg cells from HBs-Tg mice treated by Con A could significantly attenuate the oversensitive liver injury in recipient HBs-Tg mice induced by Con A, which was resulted from the inhibited activation of hepatic NK cells. Further, the expression of mTGF-beta was markedly higher on hepatic Treg cells of HBs-Tg mice, which could be further up-regulated by Con A stimulation. Simultaneously, the expression of OX40 on hepatic Treg cells was also up-regulated after Con A stimulation in HBs-Tg mice. Antibody blockade and cell transwell experiments further confirmed that Treg cells significantly inhibited NK cell hepatocytotoxicity of HBs-Tg mice via mTGF-beta and OX40/OX40L interaction. These results would be critical to reveal the mechanisms of immune injury and regulation during human HBV infection.
The immunomodulatory role of glycoprotein nonmelanoma clone B (GPNMB) in macrophage polarization in diabetes (IRC10P.465)
Authors: Yu Bing , Malcuit Christopher , Safadi Fayez , Kim Min-Ho
Published: January 2023

Diabetes is associated with a low-grade chronic inflammation, which has been linked with macrophage dysfunctions manifested by exaggerated pro-inflammatory M1 response and attenuated M2 response. The objective of this study is to validate glycoprotein nonmelanoma clone B (GPNMB) as an immunomodulatory factor that can regulate polarization of macrophages from diabetic mice. In an in vitro study of macrophages polarization, we have observed that GPNMB expression in macrophages from wild-type mice negatively correlates with the gene expression levels of the pro-inflammatory cytokines in which macrophages were induced to polarize into the inflammatory (M1: treatment with 100 ng/mL LPS and 20 ng/mL IFN gamma) or regenerative (M2: treatment with 20 ng/mL interleukin 4) phenotypes. In addition, we have found that polarized macrophages from db/db mice exhibited higher M1 markers and reduced M2 markers as compared with litter mate control mice. More importantly, the exogenous treatment of recombinant GPNMB protein (100ng/mL) could significantly attenuate the level of CD86 expression in M1 macrophages, upregulate CD206 expression in M2 macrophages, and increase the phagocytosis of apoptotic neutrophils by M2 macrophages from db/db mice, with responses that were comparable to those of control mice. Taken together, those results support that GPNMB could act as an immunomodulatory factor that can rescue aberrant functions of macrophages in diabetes.
Restoration of T lymphopoiesis from myeloid progenitors by targeting of C/EBPα (LYM7P.721)
Authors: Welner Robert , Czibere Akos , Yang Henry , Zhang Hong , Ye Min , Tenen Daniel
Published: January 2023

Loss of immune cell function with increased incidence of myeloid skewing and leukemia are well known features of the aging hematopoietic system. As one example, stem and progenitor cells (HSPCs) have greatly reduced potential to generate T lymphocytes. However, the underlying mechanisms are poorly understood. It was previously known that levels of the myeloid transcription factor C/EBPα rise with age and we asked if this might account for some of the previously described phenomena. C/EBPα deficient common myeloid progenitors (CMPs) had a distinct T cell gene expression signature that correlated with an increased potential for differentiation toward the T cell lineage. Moreover, the Notch signaling pathway activation was up-regulated in CMPs that were deficient in this transcription factor. To better understand the mechanisms underlying hematopoietic aging of T cell progenitors, we deleted C/EBPα in aged mice. Remarkably, that increased lymphoid differentiation of HSPCs in both culture and transplantation assays. These findings have implications for our understanding of the transcriptional wiring in lymphoid/myeloid progenitors. Additionally, the data support a model in which age-dependent alterations in gene expression affect lineage choice decisions and could sequentially cause immunosenescence and predispose the aged to myeloid leukemia.
The role of cytotoxic CD4+ T cells in controlling Salmonella. (MPF6P.741)
Authors: Kotov Dmitri , Nelson Ryan , Jenkins Marc
Published: January 2023

Cytotoxic CD4+ T cells (ThCTLs) recognize and lyse cells that express cognate peptide:MHCII ligands. We hypothesized that ThCTLs contribute to the control of chronic Salmonella, which persists in mucosal phagocytic cells that express MHCII. Using an in vivo cytotoxicity assay, we determined that Salmonella-infected mice contain CD4+ T cells capable of killing target cells pulsed with an MHCII-binding Salmonella peptide. In addition, we found that ~15% of Salmonella peptide:MHCII-specific CD4+ T cells express granzyme B and the Eomes transcription factor, which are markers of ThCTLs in other systems. We also discovered that many of the Salmonella specific ThCTLs express α4β7, an integrin important for mucosal homing. We are now poised for future work aimed at understanding the role that these cells play in controlling mucosal infection.
Protective capacity of CD8 T cells targeting a spectrum of Plasmodium-specific epitopes (MPF6P.735)
Authors: Doll Katherine , Pewe Lecia , Harty John
Published: January 2023

Despite decades of research, malaria remains a global health crisis. Currently, the most advanced anti-malarial vaccine in human clinical trials (RTS,S) does not provide long-term, sterilizing immunity against Plasmodium infection. Plasmodium-specific CD8 T cells can provide sterilizing immunity to Plasmodium infection in rodent models. However, the numerical requirements for memory CD8 T cell protection against a defined epitope, P. berghei CS252-260 (a CSP epitope) exceeds 1% of peripheral blood lymphocytes - a 103-104 fold higher number than required for protection against some bacterial or viral infections. Recently, three novel H-2b-restricted Plasmodium-specific CD8 T cell epitopes were described from the following antigens: GAP50, S20, and TRAP. Priming with peptide-loaded mature dendritic cells followed by boosting with recombinant L. monocytogenes expressing these epitopes results in antigen-specific CD8 T cell responses constituting 0.3-6% of the PBL in CB6.F1 mice. The capacity of these single CD8 T cell responses to provide protective immunity in a stringent model of liver stage infection with P. berghei will be described. Moreover, we are determining whether combining multiple epitope-specific responses can reduce the total numerical requirement for CD8 T cell-mediated protection. These studies may aid in the preclinical design of a sterilizing CD8 T cell based anti-Plasmodium subunit human vaccine containing multiple CD8 T cell epitopes.
Antigen dependent checkpoints for generating a persisting effector CD8 T cell population during chronic parasite infection (MPF6P.737)
Authors: Chu Hamlet , Robey Ellen
Published: January 2023

We have previously shown that an immunodominant CD8 T cell response to a MHC class I Ld-restricted parasite antigen confers strong resistance against chronic T. gondii infection in mice. Here we show that despite its lower precursor frequency, this immunodominant T cell population is activated earlier, and expands more rapidly during the priming phase compared to the subdominant populations. The number of these T cells is maintained near their peak values throughout chronic infection, with no sign of a contraction phase. Moreover, this population maintained its effector functions and phenotypes without exhaustion. We identified a transient subset within this immunodominant T cell population, which survives, proliferates and gives rise to terminally differentiated effectors only in the presence of persisting antigen in vivo. We propose that the timing and quality of antigen presentation at the acute and chronic phases of infection are critical to the generation and maintenance of this subset, and thus to the overall protective ability of the immunodominant population.
Resident memory CD8 T cells are master orchestrators of protective innate and adaptive immune responses (MUC7P.765)
Authors: Schenkel Jason , Fraser Kathryn , Beura Lalit , Pauken Kristen , Vezys Vaiva , Masopust David
Published: January 2023

CD8 T cells eliminate intracellular infections through direct cytolysis, making them ideal candidates for vaccine design. Recent studies have shown that resident memory CD8 (TRM) are critical in mediating early local pathogen clearance after re-infection. However, given the numerical paucity of CD8 TRM relative to the total number of potential target cells, it is unclear how CD8 TRM could rapidly clear pathogen strictly through direct cytolysis. We therefore hypothesized that T cell activation precipitated local polyfunctional cytokines, which were important mediators of protection after local re-infection. Here, we demonstrate that after local antigen re-encounter, CD8 TRM reactivation within the female reproductive tract (FRT) elicited secretion of multiple cytokines, which induce rapid functional changes in multiple leukocytes. During this recall response, local secretion of IL-2 and IL-15 upregulated granzyme B in local lymphocytes, TNFα matured tissue dendritic cells, and IFNγ recruited circulating memory CD8 T cells and B cells to the FRT. Thus, CD8 TRM cells employ multiple polyfunctional cytokines to orchestrate diverse and parallel responses to infection, modulating both circulating and local leukocytes. Future studies will continue to dissect apart the diverse mechanisms CD8 TRM use to clear pathogen. Vaccines should therefore aim to establish CD8 TRM cells for efficacious adaptive and innate immune responses to pathogen.
Cornea-infiltrating dendritic cells are critical in CD8+ T cell expansion in the draining lymph nodes following ocular herpes simplex virus-1 infection (MUC7P.750)
Authors: Buela Kristine-Ann , Hendricks Robert
Published: January 2023

The cornea is a mucosal surface that has resident dendritic cells (cornea-resident DC) and DC infiltrate the cornea following herpes simplex virus-1 (HSV-1) infection (cornea-infiltrating DC). Both DC populations could theoretically carry viral antigens to the draining lymph nodes (DLN) for direct expansion of HSV-specific CD8+ T cells or for cross presentation by DLN-resident DC. The expanded CD8+ T cells infiltrate the trigeminal ganglion (TG) and prevent HSV-1 transmission to the brain and lethal encephalitis. Local depletion of cornea-resident DC in CD11c-DTR mice did not affect CD8+ T cell expansion (BrdU incorporation) in the DLN. However, continuous depletion of cornea-infiltrating DC abrogated CD8+ T cell expansion at 3 days post infection (dpi) and dramatically reduced their expansion at 7 dpi, leading to reduced numbers in the TG and increased mortality. Cornea-infiltrating DC in DLN showed increased expression of the costimulatory molecules CD70, CD80, and CD86. Blocking CD70 significantly reduced CD8+ T cell numbers in the DLN, by selectively inhibiting proliferation and survival of those reactive to subdominant HSV-1 epitopes, leading to decreased CD8+ T cell numbers in the TG and increased mortality. Our data are consistent with the notion that cornea-infiltrating DCs transport HSV-1 antigens to the DLN for direct presentation to and expansion of HSV-specific CD8+ T cells, and have a critical role in preventing lethal infections.
Neonatal mice failed to induce an effective adaptive immune response to methicillin-resistant Staphylococcus aureus (MUC7P.773)
Authors: You Dahui , Fitzpatrick Elizabeth , Cormier Stephania
Published: January 2023

The development of pneumonia in neonates due to infection by methicillin-resistant Staphylococcus aureus (MRSA) is associated with significant morbidity and mortality. Our lab has developed a neonatal mouse model of MRSA-induced pneumonia to investigate the pathogenesis of MRSA in neonates. Neonatal pups or adult mice were intranasally exposed to MRSA (USA300), and the pulmonary immune response was analyzed at various days post infection (dpi). We observed a decrease in the survival rate of neonates and an increase in bacterial load in the lungs of neonates in comparison to adult mice. The increase in bacterial load correlated with a decrease in the total number of cells in the bronchoalveolar lavage fluid, which corresponded to a decrease in macrophages and neutrophils in neonates compared to adults on 1dpi. By 6dpi, neonates failed to develop an effective Th1 response compared to the adults (1.3% CD4+/IFN-γ+ cells vs. 18% CD4+/IFN-γ+ cells). Also unlike the adults, neonates failed to mount a significant Th17 response (0.89% CD4+/IL-17+ vs. 2.6% CD4+/IL-17+). The failure of neonates to mount an efficient adaptive immune response correlated with a decrease in the frequency of DCs in the lung and with less mature DCs as determined by CD86 expression in comparison to adults. These studies suggest that the failure to induce a Th1 response in neonates results in their failure to control bacterial growth.
Hair follicles retain memory CD4+ T cells in skin following infection with herpes simplex virus-1 (MUC7P.774)
Authors: Collins Nicholas , Gebhardt Thomas , Carbone Francis
Published: January 2023

Skin is barrier tissue at the interface of our body and the environment that is exposed to pathogens and therefore must be efficiently protected. Hair follicles are abundant in skin and produce chemokines. However, whether these chemokines regulate skin T cells has not been explored. In the context of a localized viral skin infection with herpes simplex virus-1 (HSV-1), T cell infiltration is necessary for optimal skin protection. Following resolution of infection, memory T cells remain at sites of prior infection. In this study, effector memory CD4+ T cells as well as antigen presenting cells (APC) were found in higher numbers in previously infected ipsilateral skin at memory times compared to contralateral uninfected skin. Histology revealed that the majority of these cells were found in distinct aggregates surrounding hair follicles. The number of CD4+ T cells and APC per aggregate, as well as the frequency of follicular aggregates was increased on the ipsilateral side at memory compared to contralateral skin. Activated CD4+ T cells treated with pertussis toxin did not aggregate, indicating the requirement for chemokine signaling. Thus, hair follicles appear to play a key role in retaining anti-viral memory CD4+ T cells in the memory phase of the response in order to place these cells in an optimal location to respond to secondary viral infection.
Revisiting the role of B cells in Chlamydia muridarum genital tract infection (MUC7P.753)
Authors: Li Lin-Xi , McSorley Stephen
Published: January 2023

B cells can contribute to acquired immunity against intracellular bacteria, but do not usually participate in primary clearance. Using MHC class-II tetramers, we examined the endogenous CD4 T cell response to Chlamydia muridarum genital tract infection. Chlamydia-specific CD4 T cells expanded rapidly and persisted as a stable memory pool for several months after infection. While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract. Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites. However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection. Together, these data reveal heterogeneity in pathogen-specific CD4 T cell responses within the genital tract and an unexpected requirement for B cells in regulating local T cell activation and bacterial dissemination during genital infection. We are currently focusing our efforts on understanding the mechanism underlying the function of B cells during primary Chlamydia infection. Our preliminary data suggest that antibody, but not B cell-dependent antigen presentation, is required for bacterial containment at local genital mucosa.
A HEXIM1-AIRE transcriptional counter-balance in trophoblasts regulates maternal-fetal mucosal immunity in pregnancy (MUC7P.755)
Authors: Wang Zhao-Yuan , Sun Guang , Faucette Azure , Unger Benjamin , Hong Peng , Yao Fayi , Guterman Jacqueline , Mathiarasu Aditya , Deng Jie , Tang Xiangna , Bluth Martin , Armant D. , Lum Lawrence , Siddiqui Maq , Chen Kang
Published: January 2023

Trophoblasts are gatekeepers at maternal-fetal mucosal interface, but trophoblast-derived molecules that initiate and maintain maternal-fetal mucosal immunity are not fully understood. We found that HEXIM1, an inhibitor of P-TEFb essential for eukaryotic RNA Pol II-mediated transcription, is highly expressed in human villous and extravillous trophoblasts. Hexim1-/- mouse placentas exhibit abnormal placental immunity characterized by increased maternal leukocyte and T cell infiltration, a paucity of spiral arteries and reduced placental size. HEXIM1 interacts in trophoblasts with AIRE, a protein critical for central and peripheral immune tolerance, via 7SK snRNA. Such interaction is further dependent on the nuclear localization domains of HEXIM1 and AIRE. Extravillous trophoblasts in human preterm labor with placental inflammation have reduced HEXIM1 and increased AIRE expression. Silencing HEXIM1 in trophoblasts led to increased Treg induction by trophoblasts. Our findings identify trophoblasts as a new type of extrathymic AIRE-expressing immunoregulatory cell, reveal a novel function of epithelial cells in mucosal immune regulation via AIRE-mediated Treg induction, suggest HEXIM1 and AIRE establish a transcriptional counterbalance in trophoblasts to maintain maternal-fetal mucosal immunity in pregnancy by regulating P-TEFb and Pol II, and promote a better understanding of how certain pathogens, such as HIV-1, perturb mucosal homeostasis after hijacking the HEXIM1 machinery.
Chronic exposure to cadmium alters gut immune homeostasis and innate immunity (MUC8P.810)
Authors: Kim Eunsoo , Jee Junbae , Steiner Haley , Cormet-Boyaka Estelle , Boyaka Prosper
Published: January 2023

Cadmium is a toxic heavy metal that can be ingested due to its presence in contaminated water and its accumulation in leafy vegetables, fish and grains. Since cadmium can compete with iron for intestinal absorption, long-term ingestion of cadmium can change composition of gut-commensal bacteria by inhibiting the growth of selected bacteria. Gut-commensal bacteria are critical to maintain intestinal homeostasis since they limit attachment of infectious agents, regulate pro-and anti-inflammatory milieu and secretory IgA levels. However, consequences of chronic exposure to cadmium for gut immune homeostasis, and mucosal innate immunity are poorly understood. To investigate the effect of cadmium on gut-homeostasis, C57BL/6 mice were treated with cadmium-contaminated water for 28 days. Mice exposed to cadmium exhibited a significant reduction of secretory IgA levels, which correlated with reduced expression of polymeric Ig receptor mRNA. These mice also showed increased intestinal permeability to macromolecules when compared to control untreated mice. The chronic exposure to cadmium also render these mice more responsive to oral administration of cholera toxin since the frequency of Paneth and goblet cells, as well as the levels of mucus production and anti-microbial mRNA responses seen 16 hours after ingestion of cholera toxin were higher than those of control mice. In summary, environmental pollutant cadmium is a major regulator of gut immune homeostasis and innate immunity.
Intestinal IL-25 signal and ILC2 regulates the susceptibility to IgE-mediated experimental food allergy (MUC8P.813)
Authors: Lee Jee-Boong , Chen Chun-Yu , Liu Bo , Burwinkel Karen , Hogan Simon P , Finkelman Fred , Wang Yui-Hsi
Published: January 2023

Food allergy is an IgE-mediated disorder manifested by uncontrolled type-2 immune response in the gastrointestinal tract. Little is known about the underlying mechanisms that drive the propensity to develop food-induced intestinal hypersensitivity. IL-25 is a distinct member of IL-17 inflammatory cytokine family, functioning at evoking type-2 inflammatory response. We aimed at investigating the involvements of IL-25 and its responding cells in the development of IgE-mediated experimental food allergy. We observed that IL-25 expression was rapidly elevated in the epithelium of inflamed duodenum before the onset of anaphylactic reaction to ingested allergens. Mice overexpressing intestinal IL-25 or deficient of IL-17RB displayed exacerbated or attenuated symptomatic features of IgE-mediated experimental food allergy, respectively. Subsequent analysis revealed that Lin-IL-17RB+ type-2 innate lymphoid cells (ILC2) that reside in the laminar propria of small intestine prior to the development of IgE-mediated experimental food allergy are the primary producers of Th2 cytokine, IL-5 and IL-13, in response to IL-25 stimulation. Furthermore, irradiated mice reconstituted with bone marrow cells that are deficient of RoRα or IL-17RB became resistant to developing IgE-mediated experimental food allergy. Our results suggest that intestinal ILC2 residents are poised to respond to induced IL-25 and function in promoting the development of IgE-mediated experimental food allergy.
Neonatal immunization with insulinoma-released exosomes induces humoral immune response that protects islets from autoimmune pathogenesis (THER5P.835)
Authors: Dai Yang , Rahman Muhammad , Bashratyan Roman , Regn Danielle
Published: January 2023

Exosomes (EXOs) are immunostimulatory microvesicles that can activate both innate and adaptive immunity. We have demonstrated that autoreactive B and T cells in prediabetic non-obese diabetic (NOD) mice can recognize EXOs released by insulinoma. In this study, we examined EXO-specific humoral immune response and its contribution to the islet-specific autoimmunity. We immunized NOD neonates with EXOs, in which mature T cells are rare, and then monitored antibody and B cell responses to EXOs in 4- or 8-week-old mice. At 4 weeks, the immunized mice showed increased B cells in pancreatic lymph nodes but not in spleen as compared with untreated mice. EXO-specific serum Ig titers remained higher than the controls. At 8 weeks, the immunized group had higher IgG1 but lower IgG2a and increased expression of FoxP3 and PD1 on Th cells, suggesting a Th1/Th2 shift and induction of regulatory T cells. As a result, the neonatal-immunized mice remained insulitis-free till 8-week old, whereas, age/gender-matched control mice developed insulitis. This effect can be partially explained by the induction of protective, EXO-specific antibodies, since the immunized sera were inhibitory for CD86 expression on activated B cells, more efficient than control sera and in vivo, the sera protected islets from insulitis caused by diabetogenic T cells. Thus, EXOs may contain unique antigen(s) that could be used as neonatal vaccine to prevent autoimmunity.
A targeted peptide controls and reverses type 1 diabetes. (THER5P.838)
Authors: Vaitaitis Gisela , Olmstead Michael , Waid Dan , Wagner David
Published: January 2023

CD40-CD154 interaction is decisive in directing autoimmune inflammation; therefore, controlling this interaction and hence destructive inflammation is a major goal in a number of autoimmune diseases including Type 1 Diabetes (T1D). Initial attempts to block CD40-CD154 interaction used antibody, random peptides, and small organic molecules, which all were problematic. Therefore an efficacious treatment remains a major goal. Here we reveal a unique targeted peptide, KGYY15, based on the domain of CD154 that binds to the CD40 receptor. Immunoprecipitation utilizing the peptide demonstrates that it binds directly to the CD40 receptor protein which in turn alters the CD40 signaling outcome. KGYY15 prevents T1D in 87% of non-obese diabetic (NOD) mice and histology reveals significant decreases in β-islet infiltration. Interestingly, KGYY15 treatment reverses overt hyperglycemia in 80% of new onset diabetic mice with significant improvement in β-islet insulin granule score. Structure-activity relationship studies demonstrate that the 15-mer peptide is optimal for efficacy and that five specific amino acids are critical in its activity. KGYY15 is well tolerated and functions to control the culprit Th40 effector T cells that are causative in T1D. The peptide sequence is 80% homologous to the human sequence suggesting an important candidate for translational studies.
Eradication of surface MHC class Ia expression in an insulinoma cell clone by TALEN-mediated gene deletion prevents recognition by diabetogenic CD8+ T cells (THER5P.843)
Authors: Hess Paul , Gojanovich Gregory , Holmes Jennifer
Published: January 2023

In type 1 diabetes, islet transplantation to replace β cells killed by pathogenic T cells is a promising means of attaining insulin independence; however, graft durability is limited by auto- & alloreactive responses. While autologous stem cells ultimately may circumvent the latter obstacle, the inciting CD8+ T-cell effectors are a threat to long-term engraftment. Islet cell manipulation to prevent expression of class Ia molecules could render them “invisible” to diabetogenic CTL, & we hypothesized that transcription activator-like effector nucleases (TALENs) could be used to engineer this modification. TALENs designed to flank a Bgl1 site in exon 1 of H2-Kd/Db were transfected into the NOD insulinoma line NIT-1, & cells with high nuclease activity were sorted using a GFP reporter plasmid encoding the target sequence. A clone isolated by limiting dilution (KG) was negative for H2-Kd/Db surface expression, & PCR amplicons were resistant to Bgl1 digestion. Genomic DNA sequencing showed biallelic modifications of both genes, with one Db sequence containing a 4 bp insertion. The remaining 3 alleles appeared to be repaired by gene conversion, causing frameshift mutations. When pulsed with cognate peptide, KG failed to stimulate diabetogenic CD8+ T cells purified from TCR-transgenic NOD.8.3 mice, as measured by CFSE dilution & IFN-γ production. These data demonstrate that TALEN-mediated genomic modification of insulin-producing cells can prevent recognition by autoreactive CTL.
CD354 in lupus nephritis (THER5P.825)
Authors: Du Yong , Wu Tianfu , Agac Didem , Zhou XinJin , Davis Laurie , Mohan Chandra
Published: January 2023

CD354, Triggering Receptor of Myeloid Cells -1 (TREM-1), is a potent amplifier of myeloid immune responses. Our goal is to determine the expression and function of TREM-1 in lupus nephritis. We used an anti-glomerular basement membrane antibody-induced nephritis (anti-GBM) model, which shares pathologic features with lupus nephritis (LN). 5 days after mice were sensitized with rabbit IgG in adjuvant, the mice received anti-GBM serum i.v. to induce disease. Anti-GBM treated 129x1/svJ mice developed severe nephritis and B6 mice did not. Anti-GBM disease induced elevated urine sTREM-1 levels in 129x1/svJ (dys 7-21) compared to B6 mice. TREM -1 blockade with an inhibitory peptide ameliorated renal inflammation compared to control peptide. TREM-1 blockade inhibited proteinuria and renal disease as measured by GN class, severity of tubulo-interstitial disease, crescent formation, and inflammatory cell infiltrates. Elevated sTREM-1 levels were detected in serum of spontaneous LN models and in their renal biopsies. Elevated TREM-1 levels were detected in serum of lupus nephritis patients and in SLE renal biopsies. We found that elevated TREM-1 expression in 129x1/svJ mice has importance pathologic consequences. TREM-1 is amenable to therapeutic targeting for nephritis. These studies could be important in understanding of pathogenic mechanisms driving SLE renal disease
Massive monoclonal expansion of functionally stable human regulatory T cells (THER5P.826)
Authors: Nowatzky Johannes , Manches Olivier , Bhardwaj Nina , Lafaille Juan
Published: January 2023

Adoptive Treg transfer is a promising therapeutic strategy for autoimmune diseases. Most current approaches rely on the enrichment and in vitro expansion of non-clonal, presumably thymus-derived Treg cells. Limitations include low expansion rates, contaminating effector cells, and unstable suppressive activity. Here, we hypothesized that cloning of human CD4+CD127lowCD25highFoxP3+HELIOS+ cells yields a highly pure cellular product with stable suppressor function throughout repeated re-expansion cycles. Cloning was performed through selection for CD4+CD127lowCD25+ cells by magnetic bead isolation and expansion in limiting dilution conditions. Suppressor function was tested in suppression assays of CD3/CD28-stimulated allogeneic Teff cells. Clonality was assessed by Vbeta staining. To demonstrate validity of our approach for a human autoimmune disease we also cloned Treg from a patient with Behcet's disease, a multisystem vasculitis. Cloning efficiency was 2-5/100 seeded cells. Treg clones were homogenously CD4+CD127lowCD25highFoxP3+HELIOS+, and clonality was confirmed by staining for a unique TCR Vbeta chain. Clones maintained at least 80% suppression at a 1:4 Treg:Teff ratio over 8 weeks in in vitro culture. Expansion rate was up to 9.8 billion-fold. Our results indicate that massive expansion of human antigen-specific Treg clones is a feasible approach for the generation of large numbers of functionally stable regulatory T cells for preclinical and clinical testing.
Impact of myeloid-derived suppressor cells on adoptive immunity in autoimmune arthritis (THER5P.827)
Authors: Liu Peng , Crook Kristen , Weeks Michael
Published: January 2023

Abnormal B- and T-cell recognition of self-antigens and autoantibody production result in autoimmunity. We previously showed that myeloid-derived suppressor cells (MDSCs) suppress T cell proliferation in autoimmune arthritis. To further explore the impact of MDSCs on B cells, we examined B cell functions in vitro and in vivo in collagen-induced arthritis. We found the proliferation of collagen-primed B cells was inhibited by autologous monocytic MDSCs, but not by Ly6G+ neutrophils. The production of both IgG and IgM was reduced in the supernatant of B cell and monocytic MDSC co-cultures as compared to the supernatant from B cells alone or the supernatant from B cell-Ly6G+ cell co-cultures. Mechanistically, the levels of iNOS and PGE2 increased in the B cell-MDSC co-cultures, and addition of either iNOS or PGE2 inhibitors abolished the MDSC suppression of B cell proliferation. While neutralizing IL-12 also reestablished B cell proliferation, blockade of IL-6 and TNFa did not show similar effect. Finally, adoptive transfer of monocytic MDSCs to collagen-immunized mice resulted in reduced autoantibody production and mitigated arthritis severity. In summary our data show that monocytic MDSCs suppress autoreactive B cell proliferation and antibody production. The suppressive function requires iNOS, PGE2 and IL-12, but is independent of IL-6 and TNFa. These MDSCs can potentially be used in cell-based therapy to treat autoimmune arthritis by limiting B cell functions.
Antigen-specific tolerance of diabetogenic T cells inhibits mTOR expression and function (THER6P.854)
Authors: Chen Weirong , Khairallah Marie-Therese , Miller Mindy , Ellis Jason , Ukah Tobechukwu , Zaghouani Habib
Published: January 2023

In prior studies we have shown that antigen-induced tolerance of diabetogenic Th1 cells leads to diminished expression of the transcription factor T-bet and downregulation of the chemokine receptor CXCR3. Consequently, the cells are retained in the spleen, can not traffic to the pancreas, and the mice are resistant to type 1 diabetes (T1D). Investigation of the molecular events underlying this form of Th1 tolerance indicates that expression of the mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase involved in regulation of cell metabolism, is inhibited and its function is impaired. Specifically, phosphorylation of the ribosomal protein S6 kinase, a target of mTOR complex 1 (mTORC1) is decreased whereas mTORC2-dependent phosphorylation of AKTS473 is unaffected. In contrast, overexpression of mTOR or its positive regulator, Akt, by viral transduction nullifies antigen-induced tolerance and accelerates progression of T1D. Thus, mTOR plays a major role in the regulation of T cell activation and provides a useful target for modulation self-reactive T cells and suppression of autoimmune or type 1 diabetes.
The potent immunosuppressive role of Interleukin (IL)-37 in the pathogenesis of psoriasis (THER6P.846)
Authors: Li Jiong , Teng Xiu , Hu Zhonglan , Wang Zhen , Wei Xiaoqiong
Published: January 2023

IL-37 has emerged as a fundamental inhibitor of innate immune response. Psoriasis is thought to be initiated by abnormal interactions between cutaneous keratinocytes and systemic immune cells, triggering epidermal hyperplasia, increased inflammatory cells infiltration and high inflammatory mediator production. To research the presumed unique “cooling fires of inflammation” properties of IL-37 in psoriasis, we firstly found that IL-37 was abundantly distributed in human psoriatic plaques (mainly localized in effector memory T cells and macrophages not regulatory T cells). Further, we explored the possible immunomodulatory mechanism of IL-37 in psoriasis models in vitro and in vivo. IL-37 was effective in suppressing the production of chemokines, proinflammatory cytokines and antimicrobial peptides in human keratinocytes which recapitulated numerous features of psoriasis in vitro. IL-37 relieved the psoriatic symptoms of keratin14-VEGF Tg mice (widely-used psoriasis model) including thinner epidermis and diminished lymphocyte infiltration, and greatly dampened Th1 type cytokines production and mast cells recruitment in psoriatic lesions. Our results support the idea that the presence of IL-37 attenuates the inflammatory process in psoriasis by disrupting the linkage between innate immunity and adaptive immune response, indicating a hopeful therapeutic approach for immune-mediated disorders.
Targeting IL-6/STAT3 pathway with small-molecule compounds for multiple sclerosis therapy (THER6P.849)
Authors: Yang Yuhong , Li Chenglong , Nuro-Gyina Patrick , Lovett-Racke Amy , Racke Michael
Published: January 2023

Multiple Sclerosis is an immune-mediated chronic CNS disease where IL-6/STAT3 pathway plays a critical role in disease pathogenesis. Dysregulated IL-6/STAT3 signaling skews Teff/Treg balance toward an enhanced T effector response, favoring the development of CNS autoimmunity. Specifically, we previously showed IL-6 induces the development of highly encephalitogenic Th17 cells, suggesting IL-6/STAT3 pathway may serve as an innovative target. In support of this strategy, both IL-6-/- and STAT3-/- mice are completely resistant to EAE. Therefore, we designed four small-molecule lead compounds targeting IL-6/STAT3 pathway. MDL-5 and MDL-16 bind to the D1 domain of GP130, preventing the IL-6/GP130 interaction; while LLL-12 and LY-5 bind to the SH2 domain of STAT3, preventing STAT3 phosphorylation. All four lead compounds significantly inhibit IL-6 induced IL-17 production in myelin-specific CD4 T cells without significant toxicity. Furthermore, adoptive transfer of small-molecule treated myelin-specific CD4 T cells into naïve recipient mice show a significant reduction in EAE severity compared to control group, suggesting these compounds suppress the encephalitogenicity of CD4 T cells. More importantly, immunized small-molecule treated mice demonstrate delayed onset and reduced severity compared to control mice, suggesting in vivo treatment suppresses EAE development. Together, our data suggest that these four lead compounds have great potential to serve as an innovative therapy.