bluuebunny/crossref_metadata_2025 huggingface dataset:
Indoleamine 2,3-Dioxygenase is not required for tolerance induction in the airways but is important for allergic airway disease (39.8)
Authors: Xu Hui , Oriss Timothy B , Melgert Barbro N , Henry Adam C , Chen Li , Mellor Andrew L , Munn David H , Irvin Charles G , Ray Prabir , Ray Anuradha
Published: January 2023

Indoleamine 2,3 dioxgenase (IDO) has emerged as an important negative regulator of immune responses. However, the role of IDO in Th2 cell-mediated diseases is not completely clear. We investigated the role of IDO in the induction of inflammation or tolerance in the airways in response to ovalbumin (OVA). The results of these experiments show that IDO−/− mice are susceptible to tolerance induction. However, IDO−/− mice displayed significantly lower airway hyperresponsiveness (AHR), lower serum OVA-specific IgE and lower Th2 type cytokines (IL-5 and IL-13) in comparison to wt mice. In an OVA-induced chronic airway inflammation model, the IDO−/− mice also showed lower AHR and Th2 type cytokines in the lung and less infiltration of inflammatory cells in the airways. To dissect the mechanism underlying IDO-mediated promotion of allergic airway disease, we stimulated lung DC by allergens in vitro. The expression of important co-stimulatory molecules and MHC-II was lower in DCs from IDO−/− mice in comparison to those in DCs from wt control mice suggesting that the reduced airway Th2 type immune response in IDO−/− mice may be due, at least in part, to less effective co-stimulation between DCs and T cells. In summary, our data suggest that IDO is not required for the induction of airway tolerance, but may play a role in promoting Th2-induced allergic airway disease.

Supported by NIH grants HL 77430 (to A.R) and HL60207 (to P.R.)
Role of adaptive immunity in Helicobacter-associated gastric adenocarcinoma (40.1)
Authors: Schmitz Julia Michelle , Lorenz Robin G.
Published: January 2023

The majority of gastric cancer is associated with Helicobacter pylori infection, however the mechanism is unknown. Our laboratory has previously demonstrated that T cells are critical to Helicobacter-induced gastric pathology. This involvement could be through T cell cytokine induced alterations in the protective gastric surface mucous layer. C57BL/6 mice, after infection with H. felis, develop severe gastritis which progresses to a gastric adenocarcinoma that is associated with a decrease in the production of the mucin, muc5ac. C57BL/6.Rag-1−/− mice, which lack both T and B cells, do not develop gastritis, adenocarcinoma, or changes in muc5ac expression after infection. An analysis of gastric cytokine expression by RT-PCR has demonstrated that infected C57B6.RAG-1−/− mice produce less inflammatory cytokines, IL-17, TNFα, and IFNγ, while producing more IL-6 and equal levels of IL-10 and IL-1β. This suggests that B and T cells are only responsible for the secretion of inflammatory cytokines during infection, but not innate or regulatory cytokines. These results also indicate that there is an association between the inflammatory cytokines, IL-17, TNFα, and IFNγ, made by the adaptive immune response and alterations in the protective gastric mucous layer. Further studies will be required to investigate the exact role of each cytokine in the progression to gastric cancer.

This work was supported in part by ACS RSG MBC-103892, NIH R01 DK59911, NIH T32 AI07041, and R24 DK064400.
Promoting dendritic cell migration by mouse airway epithelial cells through IL-17A-induced mouse CCL20 expression (40.10)
Authors: Hung Li-Yin , Wu Reen
Published: January 2023

Dendritic cells are the antigen presenting cells that play a central role in regulating immune response. Their migration on airway epithelium in response to antigen challenge is incompletely understood. In this study, we demonstrated that mouse IL-17A is a potent stimulator for primary mouse airway epithelium to secrete chemotactic factors for the migration of bone marrow-derived dendritic cells (BMDC) along its gradient. Neutralizing antibody study with anti-mCCL20 demonstrated the inhibition of the migration. Real-time quantitative RT-PCR study demonstrated time- and dose-dependent effects of IL-17A on mCCL20 expression, but not the induction of hBD-2 equivalent messages, in mouse airway epithelial cells. The lack of induction of hBD-2 equivalent messages by IL-17A on mouse cell system is in contrast to that was seen in human, suggesting a species difference in response to IL-17A. Using inhibitor, we observed the inhibition of induced CCL20 expression by an NF-κB inhibitor, isohelenin, suggesting an NF-κB mediated transcriptional mechanism in the induction. Based on these observations, we suggest that IL-17A may be responsible for in vivo dendritic cell migration on airway epithelium through the induction of mCCL20 expression.

This study is supported by NIH grant HL077315 and HL077902
Intestinal Bacteria Induce T Cell-Independent IgA2 Class Switching by Linking Epithelial Cells with Lamina Propria B Cells through APRIL (40.3)
Authors: He Bing , Xu Weifeng , Santini Paul A. , Polydorides Alexandros D. , Chiu April , Chadburn Amy , Knowles Daniel M. , Rescigno Maria , Cerutti Andrea
Published: January 2023

Bacteria colonize the intestine shortly after birth and thereafter exert several beneficial functions, including induction of protective IgA antibodies. The distal intestine contains IgA2, which is more resistant to bacterial proteases than IgA1. The mechanism by which B cells undergo IgA2 class switching remains unknown. We found that human intestinal epithelial cells (IECs) triggered IgA2 class switching by activating B cells, including IgA1-expressing effector B cells, through a T cell-independent pathway involving a proliferation-inducing ligand (APRIL). IECs released APRIL after sensing whole bacteria or bacterial products, such as lipopolysaccharide or flagellin, through a Toll-like receptor (TLR)-mediated MyD88-dependent pathway. Bacteria-activated IECs further increased APRIL production by cross talking with dendritic cells via thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine. Our findings indicate that bacteria and their products elicit IgA1-to-IgA2 diversification by linking lamina propria effector B cells arriving from mucosal lymphoid follicles with IECs through a TLR-inducible APRIL-dependent signaling program. Our data also suggest that vaccines should activate IECs to effectively induce protective IgA2 responses in the intestinal mucosa.

This work was supported by NIH grant AI057653 (to A.C.) and NIH T32 training grant AI07621.
αE Integrin Compensates for β7 Deficiency in the Nasal Passages (NP) Following Nasal Immunization with Cholera Toxin (CT) (41.10)
Authors: Maslanka Tomasz , Riccardi Carol , Pascual David W.
Published: January 2023

Nasal immunization stimulates robust Ab responses in the head and neck lymph nodes (HNLN) and nasal-associated lymphoid tissue (NALT). Studies showed that B lymphocyte trafficking in the HNLN and NALT is both L-selectin- and β7-dependent. β7 integrin generally associates with αE or α4 integrins to mediate lymphocyte retention in the epithelium or the mucosa, respectively. To study the contribution of β7 integrin to upper respiratory immunity, β7−/− mice were nasally dosed with CT on days 0, 7, and 14. Mucosal IgA or serum IgG Ab titers were not different from B6 β7+/+ mice at any of the time points examined til day 42. Vaginal IgG responses were slightly enhanced in β7−/− mice, and absent in L-selectin−/− mice. However, the absence of β7 did alter the types of supporting B cells found in the NP. Increased expression of αE was observed on day 42 despite the absence of its co-receptor, β7. This increased αE expression was not observed in B6 β7+/+ mice. αE+ B cells continued to be found in the β7−/− small intestinal lamina propria. Minimal αE+ B cells were found in the HNLN. From these studies, although mucosal Ab responses remain intact in β7−/− mice, it does appear that different homing receptor-dependent mechanisms are employed.

This work is supported by NIH AI-55563.
Using Overlapping Synthetic Peptides from Tumor-Associated Protein Antigens for Breast Cancer Vaccine Development. (41.11)
Authors: Mirshahidi Saied , Whitney James B. , Kramer Victor G. , Anderson Karen S. , Ruprecht Ruth M.
Published: January 2023

An effective vaccine will need to elicit both CD4+ T helper and CD8+ cytotoxic T lymphocytes (CTL) responses in order to maintain effective CTL functions. Peptide vaccines tailored to certain genetic backgrounds are capable of eliciting MHC Class I-restricted (CD8+) and Class II-restricted (CD4+) responses. However, the need to identify specific T-cell epitopes in the context of MHC alleles hampers this approach. We have developed a novel strategy that bypasses the need for epitope mapping for an outbred population: vaccination with overlapping synthetic peptides (OSP), to generate strong antigen-specific cell-mediated immunity (CMI) in individual vaccine recipients with different MHC backgrounds. OSP contain all possible epitopes for the CD4+ as well as CD8+ T cells of a genetically diverse population. BALB/c mice were immunized with OSP derived from a tumor protein, which is overexpressed in 60% in breast cancer specimens. The vaccinated animals developed an in vitro activity of tumor specific- CTL, proliferative response, IFN-g production and CD107 expression. Immunized mice also were challenged with TS/A tumor cells (a murine mammary adenocarcinoma cell) to induce micrometastasis. Vaccination with OSP significantly increased the animal life span. The OSP vaccine strategy provides a novel opportunity to induce cellular immune responses against developing tumors in a genetically diverse population.
Generation and maintenance of antigen-specific CD8 T cells in the vaginal mucosa following systemic versus mucosal routes of immunization (41.6)
Authors: Onami Thandi M. , Suvas Pratima Krishna , Dech Heather M. , Sambira Fleurette
Published: January 2023

In this study we compared the efficacy of two different routes of viral infection on the generation, maintenance, and localization of antigen specific CD8T cells in lymphoid, non-lymphoid, and mucosal tissues during peak effector and memory phases of an immune response. We used an adoptive transfer system of naïve transgenic CD8 T cells, followed by intra-peritoneal (i.p) or intra-nasal (i.n) LCMV infection. Surprisingly, on day 8 the peak of the primary response, i.p. infection resulted in higher numbers of antigen-specific CD8T cells in the vaginal mucosa and ILN only, in addition to 2–3x more antigen specific CD8 T cells that co-expressed both IFNγ and TNFα, when compared to the i.n. route of infection. During memory, equivalent numbers of antigen specific CD8T cells were detected at all sites, irrespective of the route of infection. Following intravaginal re-challenge, both i.p. and i.n generated memory CD8 T cells showed robust secondary responses in the vaginal tract, however the i.n. memory CD8 T cell response was consistently greater. These data suggest that both systemic and mucosal routes of infection can generate a similar quantity of memory CD8 T cells in mucosal sites. However, the initial route of infection appears to influence the programming of the memory CD8T cells generated, and these early differences in priming may impact the quality of protective secondary responses.
Antibody-Mediated Protection against Genital Herpes Simplex Virus type 2 Disease in Mice by Fc gamma Receptor -Dependent and -Independent Mechanisms (41.8)
Authors: Milligan Gregg N , Chu C-F. , Meador M. G. , Young C. G. , Strasser J. E. , Bourne N.
Published: January 2023

The requirement for Ab -Fcγ receptor (FcγR) interactions or for virus neutralization in protection against genital HSV-2 challenge was examined. Serum IgG Ab isolated from HSV-immune mice protected normal mice against HSV-2 disease when administered prior to challenge. However, protection was significantly diminished in mice lacking the γ chain subunit utilized in FcγRI, FcγRIII, FcγRIV, and FcεRI and in normal mice depleted of FcγR+, Gr-1+ immune cells suggesting protection was largely mediated by an FcγR-dependent mechanism. To test if FcγR-independent antibody-mediated mechanisms might manifest protection differently, a highly neutralizing, HSV glycoprotein D -specific monoclonal antibody (mAb) was utilized. Administration of IgG1, IgG2a, or IgG2b switch variants of the mAb did not prevent infection of the genital tract but resulted in lower virus loads in the vaginal epithelium and provided significant protection against disease and acute infection of the sensory ganglia independently of host FcγR expression. Together, these data demonstrate two distinct antibody effector mechanisms capable of providing substantial protection against genital HSV-2 disease. The presence of either FcγR -dependent Ab or strongly neutralizing Ab did not completely prevent HSV-2 infection but limited initial infection of genital and neuronal tissues and diminished HSV-2 disease. NIH Grants AI42815 and AI054444.
Oral tolerance defects in congenital T cell-deficient mice (42.1)
Authors: Lee Joon Youb , Oh Nicholas , Surh Charles D
Published: January 2023

Congenital T cell (T)-deficient mice, such as RAG− or SCID mice, succumb to inflammatory bowel disease (IBD) upon adoptive transfer of syngenic naïve T cells - a consequence of chronic inflammation in the intestine in response to commensal bacterial antigens. Currently, the underlying reason for the onset of IBD is believed to be the absence of regulatory T cells (Tregs). However, since donor T cells undergo considerable chronic activation even in the presence of Tregs, abnormalities in the presentation of enteric antigens also appears to be involved. To further explore this idea, we have compared the fate of TCR transgenic T cells adoptively transferred into normal vs. RAG− mice that are fed with the cognate antigen. Strikingly, while the donor TCR transgenic T cells undergo abortive slow cell division in normal hosts and slowly disappear, the same cells undergo rapid proliferation, differentiate into effector cells and persist for prolonged periods. These findings suggest that RAG− mice have an inherent defect in their ability to induce oral tolerance.
Exacerbation of spontaneous colitis in the absence of flora derived TLR4 signals (42.2)
Authors: Mingle Bethany , Afshar Roshi , Prioult Guenolee , Murakami Hidehiro , Nagler Cathryn
Published: January 2023

Complex immunoregulatory mechanisms are required to maintain non-responsiveness to dietary antigens and the commensal flora. We have reported that mice that are unable to signal via TLR4, the receptor for bacterial LPS, are highly susceptible to an allergic response to food. When the composition of the commensal flora is reduced and altered by antibiotic treatment, TLR4 wild-type mice become as susceptible to the induction of allergy as their TLR4 mutant counterparts. More recently we have shown that the mesenteric lymph nodes of TLR4 mutant mice are deficient in the generation of IL-10 secreting regulatory T cells (Tregs) and lack a population of plasmacytoid DC that constitutively makes IL-10. To further examine the contributions of IL-10 secreting Tregs and DC to mucosal non-responsiveness, we prepared TLR4xIL-10 double knockout (DKO) mice. We have found that the Th1 biased colitis previously reported in IL-10 deficient mice is greatly exacerbated in TLR4xIL-10 DKO mice. Our observations stand in contrast to a recent report from Rakoff-Nahoum et al (Immunity 2006 25:319) that shows that MyD88xIL-10 DKO mice are protected from the development of colitis. TLR4 is unique among bacteria responsive TLRs in its ability to signal via both MyD88 dependent and MyD88 independent pathways. Taken together with our observation that food allergy is linked to an inability to signal via TLR4, the exacerbation of spontaneous colitis in TLR4xIL-10 DKO mice supports a unique role for TLR4 signaling in the generation of flora induced immunoregulatory cells that control pathological responses at both ends of the Th1/Th2 spectrum.

Supported by NIH grants DK 55678 and DK43351.
CD8aa-specific CD8ab Effector Memory T Cell Differentiation in the Gut (42.6)
Authors: HUANG YUJUN , Schoenberger Stephen , Cheroutre Hilde
Published: January 2023

CD8 effector memory T cells at the intestinal mucosa are important for rapid containment of the invading pathogens. CD8aa, a homodimer of CD8a, has been found on gut intraepithelial lymphocytes (IELs). It is also transiently expressed on some peripheral CD8ab primary effector T cells that differentiate to memory T cells. In vitro stimulated sorted CD8aa+ and CD8aa− OT-I cells migrate with equal capacity to the gut and spleen of recipient mice. One month later, oral re-challenge with Listeria Monocytogenes-OVA (LM-OVA) leads to an immediate secondary expansion of CD8aa+, but not CD8aa−, donor cells among the IELs, which suggests that only CD8aa+ precursors can differentiate into mucosal memory T cells and respond immediately to an oral pathogen challenge. Later, both CD8aa+ and CD8aa− donor cell-derived secondary responses are detected in spleen and gut. However, 45 days after the secondary response, only CD8aa+donor cells generate memory IELs. It indicates that CD8aa+ primary effector cells are programmed during the initial priming to survive and to become mucosal effector memory T cells. CD8aa expression is transient on primary effector cells and regained after relocation into the gut. The specific role of CD8aa in the survival and differentiation of intestinal CD8 effector memory T cells was investigated.
Influenza specific CD8 T cells require interactions with pulmonary DC in the lungs in order to mount an effective immune response (42.8)
Authors: McGill Jodi , Legge Kevin
Published: January 2023

Our studies show that respiratory dendritic cells (rDC) transiently migrate (i.e. within the first 48 hours post infection) from the lungs to the lymph nodes (LN) following influenza virus infections. However, DC are continually recruited into the lungs even after DC migration to the LN has ceased. Since CD8 T cell activation and programming occurs in the LN, the role of these lung-recruited DC in influenza immunity was unclear. To better understand their role, we depleted rDC after the conclusion of normal rDC trafficking to the LN and assessed the immune response. Our results show decreased CD8 T cell responses, resulting in increased viral titers, morbidity and mortality. Initial T cell expansion and activation in the LN, and migration to the lungs appears equivalent in both wildtype and rAPC depleted mice (rAPCneg). However, in rAPCneg mice, these activated T cells fail to continue their expansion once arriving in the lungs. Examination of the DC subtypes present in the lungs revealed decreased numbers of both pulmonary pDC and CD8+ DC in rAPCneg mice through 6 days p.i. Reconstitution of rAPCneg mice with purified pulmonary pDC or CD8+ DC restores influenza-specific CD8 T cell immunity in a MHC I dependent manner. These results suggest that activated influenza-specific CD8 T cells may require additional antigen-specific interactions with recruited DC in the lungs in order to protect against influenza virus infections.
Deterministic linkage between CD4 T cell and B cell specificities to a large virus: vaccinia virus (43.1)
Authors: Crotty Shane , Moutaftsi Magdalini , McCausland Megan , Davies D. Huw , Quiroz Juan Moyron , Johnston Robert , Benhnia Rafii Mohammed , Grey Howard , Hoffmann Julia , Head Steven , Garboczi David , Sette Alessandro
Published: January 2023

Antibody responses are critical components of protective immune responses to many pathogens, but it remains unclear what parameters determine which pathogen proteins are targeted. We have performed large scale studies to map mouse antibody and CD4 T cell responses to the smallpox vaccine virus, vaccinia. Our analysis of this large virus ( > 200 genes) identified 13 viral protein targets of CD4 T cell responses and 23 antibody targets. Unexpectedly, nearly half of the CD4 T cell responses targeted the same viral virion proteins as the antibody responses, suggesting a deterministic linkage between the specificities. Immunizing mice with individual MHC II epitopes revealed that, after viral infection, help between CD4 T cells and B cells of matched viral virion protein specificity is largely non-transferrable to other virion proteins. We used this knowledge to then predict the presence of new MHC II epitopes. In total, 11 of the 18 CD4 T cell responses (61%) we identified were matched by a paired antibody response to the same protein, including all of the top 5 viral protein IgG targets. These data demonstrate that MHC restriction at the protein level is a critical event for anti-virion antibodies to a large virus. This is a powerful predictive principle and is of general relevance for understanding the nature of B cell antigen presentation to CD4 T cells against complex pathogens. NIH-AI-63107
Chlamydia pneumoniae infection modulates cytokine production by human T lymphocytes and monocytes (43.11)
Authors: Hakki Amal , Mamata Yukimitsu , Friedman Herman
Published: January 2023

Continuous cell line cultures of human lymphocytes (MOLT-4) and monocytes (THP-1) were examined for production of immunoregulatory cytokines involved in immune responses to the intracellular opportunistic pathogen Chlamydia pneumoniae (Cp). Infection with Cp is widely prevalent and believed involved in chronic inflammatory diseases such as atherosclerosis and even neurodegenerative diseases like Alzheimer’s disease and multiple sclerosis. The results of this study showed that MOLT-4 and THP-1 cells were readily infected by Cp in vitro assessed by immunofluorescence microscopy for Cp lipopolysaccharide (LPS) and by real time RT-PCR for 16S rRNA expression. Stimulation of MOLT-4 cells with heat killed Cp (KCp) or Escherichia coli LPS (E. coli LPS) induced Th1 cytokines IFNγ and IL-12 and Th2 cytokine IL-10, but infection with viable Cp showed a dominant Th1 cytokine production. Cp infected THP-1 cells mainly produced IL-12 and enhanced TNFα and IL-10 production compared to cultures stimulated with KCp or E. coli LPS as a control. These results suggest that Cp infection modulates the immune response to a Th1 biased cytokine profile by T cells in addition to induction of TNFα by monocytes/macrophages. Such effects are likely involved in antibacterial immunity against Cp infection.
Phenotype and functional properties of Vaccinia specific CD8+ T cells (43.12)
Authors: Ostrout Nicholas D , McHugh Marilyn M , Brustoski Kim , Kazura James
Published: January 2023

Memory CD8+ T cells play a significant role in mediating protective immunity from pathogens, particularly viruses and intracellular bacteria. Inducing the optimal response, from both a quantitative and qualitative perspective, has been a research priority for determining the effectiveness of new vaccines. Depending on the pathogen, central memory (TCM) or effector memory (TEM) may be more important in providing adequate protection. Therefore, when developing new vaccines, it is important to realize which memory CD8+ T cell subset is vital and potentially ‘drive’ the response to elicit a protective level ratio in favor of one subset. Here we describe the phenotype of vaccinia specific memory CD8+ T cells. TEMRA+ memory T cells produce the most IFN-γ of any memory subset and are present in individuals last vaccinated over 30 years ago. Concurrently, we address the development of memory CD8+ T cells upon administration of Dryvax, as well as new smallpox vaccines such as Modified Vaccinia Ankara (MVA) in a murine model. Previous studies have indicated that TEM appear to provide better secondary protection than TCM and the data presented here indicate that these cells are maintained long term (>30 years). Therefore, we propose that new smallpox vaccines, such as MVA, should elicit a memory cell population in favor of TEM similar to that induced by Dryvax or other traditional smallpox vaccines.
cAMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun bind to the IFN-γ proximal promoter in response to M. tuberculosis (TB) stimulation (43.13)
Authors: Samten Buka , Townsend James C. , Weis Stephen E. , Barnes Peter F.
Published: January 2023

The proximal promoter controls transcription of IFN-γ in T cells, which is central for defense against many pathogens. By performing EMSA and supershift assays on nuclear extracts of TB-stimulated PBMC, we found that CREB, cAMP response element modulator (CREM), ATF-1, ATF-2, and c-Jun bound to the IFN-γ proximal promoter. Microaffinity purification with a biotinylated IFN-γ proximal promoter, followed by Western blotting, showed that only CREB, ATF-2 and c-Jun bound to the proximal promoter, and this was confirmed in live T cells by chromatin immunoprecipitation. Coimmunoprecipitation of nuclear extracts of TB-stimulated PBMC indicated interaction of these transcription factors. Introduction of a plasmid expressing a dominant-negative ATF-2 peptide into TB-stimulated T-cells inhibited IFN-γ secretion by >90% and reduced expression of CREB, ATF-2 and c-Jun. Nucleofection of siRNA specific for ATF-2 decreased expression of CREB, c-Jun and ATF-2 in T cells. TB-stimulated PBMC from tuberculosis patients, known to have reduced IFN-γ production, were found to have low levels of CREB, ATF-2 and c-Jun, but not CREM, ATF-1 and c-Fos, compared to PBMC from healthy donors. We conclude that:

CREB, ATF-2 and c-Jun bind to the IFN-γ proximal promoter;ATF-2 contributes to expression of CREB and c-Jun;CREB, ATF-2 and c-Jun contribute to T-cell production of IFN-γ in response to TB.

(NIH AI063514)
CXCL10 is required for the recruitment of leukocytes responsible for controlling herpes simplex virus-1 infection (43.19)
Authors: Wuest Todd , Luster Andrew , Campbell Iain , Farber Joshua , Carr Daniel
Published: January 2023

The chemokine CXCL10 is among the earliest cytokines produced during nervous system inflammation induced by herpes simplex virus-1 (HSV-1) infection. Current evidence suggests CXCL10 is important in the recruitment of a wide range of leukocytes including CD4 and CD8+ T-cells, macrophages, dendritic, and NK cells. Many of these studies have suggested the function of this chemokine extends beyond simple leukocyte recruitment as CXCL10 is also expressed in primary and secondary lymphoid tissues. To elucidate the role CXCL10 has in generating an effective immune response to viral infection of the nervous system, we have analyzed leukocyte recruitment and effector function in CXCL10 deficient (−/−) mice relative to wild type C57BL6/J, CXCL9 deficient, and CXCR3 deficient mice infected with HSV-1. Our results indicate CXCL10 is critical for the recruitment of NK cells and B220+CD11c+ cells, in a CXCR3-dependent manner. Infiltration of HSV-1 specific CD8+ T-cells into the central nervous system of CXCL10 −/− mice is significantly reduced compared to wild type mice, but CXCL10 expression in lymphoid tissues does not contribute in the generation of HSV-specific CD8+ T-cells. Nervous system restricted expression of CXCL10 restored the homing of HSV-1 specific CD8+ T-cells to wild type levels and partially restored homing of natural killer and conventional dendritic (B220-CD11c+) cells.
Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo anti-polysaccharide, but not anti-protein, response to Streptococcus pneumoniae (43.24)
Authors: Chattopadhyay Gouri , Khan Abdul Q. , Dubois Wendy , Potter Michael , Snapper Clifford M.
Published: January 2023

IgG anti-polysaccharide (PS) and anti-protein responses to Streptococcus pneumoniae (Pn) are dependent on CD4+ T cells, B7/CD28, and CD40/CD40-ligand interactions. However, the primary IgG anti-PS response terminates more quickly, requires a shorter period of T cell help, fails to generate memory, and is more dependent on Ig signaling. To determine a role for B cell apoptosis in these differences, we utilized mice expressing an Ig heavy chain enhancer-driven transgene encoding the anti-apoptotic proteins Bcl-xL or Bcl-2. Bcl-xL-Tg and Bcl-2-Tg mice immunized with Pn exhibited a prolongation of the primary IgM and IgG anti-PS, but not anti-protein, response resulting in up to 10-fold enhancements in specific Ig serum titers. Of note, Tg mice still failed to generate PS-specific memory. In contrast, Fas- or Fas-ligand-deficient mice elicited normal humoral immune responses to Pn. In vitro, Bcl-xL and Bcl-2 Tg B cells exhibited higher and more prolonged proliferative responses following multivalent Ig crosslinking, but not after stimulation with LPS or anti-CD40 mAb, relative to WT B cells. In contrast to intact Pn, in vivo anti-PS responses to purified PS or a soluble PS-protein conjugate were similar in Tg and WT mice. These data suggest that apoptosis may selectively limit clonal expansion and subsequent Ig secretory responses of PS-specific B cells during a primary immune response to an intact bacterium.

N.I.H. R01 AI49192
Differential role of CD4 and CD8 T cells in RSV immune pathology (43.28)
Authors: Neupane Rupak , Schaller Matthew , Lukacs Nicholas W
Published: January 2023

CD4 and CD8 T cells produce different cytokines during immune response. Using a model of respiratory syncytial virus (RSV) infection, isolated CD4 T cells were found to produce predominantly Th2, while CD8 produced predominantly Th1 cytokines. To better understand the contribution of CD4 and CD8 T cells in immune pathology during RSV infection, CD4 and CD8 T cells were depleted using specific depleting antibodies and immune response was compared with control mice during RSV infection. Depletion of CD8 T cells showed increased airway hyperresponsiveness (AHR), production of IL-13, as well as increased mucus-associated gene, Gob5, expression. Specific depletion of CD4 induced lower AHR compared to control mice, lower Th2 cytokines and reduced Gob5 production. PAS staining showed both control and CD8 depleted mice with increase mucus production and increased goblet cell hyperplasia whereas mice depleted of CD4 T cell did not produce mucus. These results indicate that CD8 T cells are protective whereas CD4 T cells are responsible for increased pathology observed during RSV infection.

This work was supported in part by NIH RO1AI036302.
Developing MHV-1 infection as an animal model for Severe Acute Respiratory Syndrome (SARS) (43.30)
Authors: Khanolkar Aaruni , Pewe Lecia , Tifrea Delia , Perlman Stanley , Harty John T.
Published: January 2023

SARS is a life threatening infectious disease that acquired epidemic proportions in late 2002 affecting 8000 people worldwide and claiming nearly 800 lives. It is caused by a novel group 2 coronavirus HCoV-SARS. No suitable small animal model currently exists for this disease. Developing a good animal model is crucial for generating effective anti viral therapies. Preliminary data indicate that intranasal infection of A/J mice with a related coronavirus MHV1 closely mimics the disease observed in humans infected with HCoV-SARS. Following infection these mice experience severe weight loss and the lungs show evidence of patchy mononuclear infiltrates and pneumonitis. Significant mortality is observed beginning around day 6–7 post infection suggesting an adaptive immune component to the pathogenesis of the disease. Interestingly, wild type BALB/c and B6 mice are relatively resistant to disease however, resistance is compromised in BALB/c mice lacking both perforin and IFNγ, and B6 mice deficient in receptors to IFNαβ. Our overall goal is to characterize the phenotype and function of the virus-specific T cell response in susceptible strains of mice to ascertain if a dysregulated T cell response contributes to morbidity and mortality. We believe this study will provide useful information regarding the pathogenesis of SARS and aid translational initiatives aimed at infection control and vaccine design
The T cell response to staphylococcal superantigens is modulated by the bacterial cell wall (43.33)
Authors: Madrenas Joaquin , Chau Thu , McCully Michelle L , Britnell Bill , Haeryfar Mansour , McCormick John K , Cairns Ewa , Heinrichs David E
Published: January 2023

Bacterial superantigens (SAgs) are pyrogenic exotoxins that cause oligoclonal T cell activation leading to toxic shock and death. The selective advantage of SAg expression by bacteria remains unknown. We hypothesized that the T cell response to SAg is modulated by the bacteria. If so, one should see intense T cell responses when SAg are introduced into the body alone, but not when the producing bacteria are also present.

We examined the responses of primary T cells to a panel of pure staphylococcal SAg or in the presence of heat-killed bacteria. We found that the IL-2 response to SAg was inhibited by the presence of bacteria, while the TNF-? and IFN-? responses were not affected or were even increased. Such a modulation of T cell responses to SAg was mediated by TLR2/6 agonists but not by agonists of TLR1/2, TLR-9 or NOD proteins, involved activation of the NF-_B pathway, and was prevented by anti-TLR-2 blocking antibodies. At the cellular level, this modulation resulted from death of antigen-presenting cells (APCs) but not of T cells. Thus, we show that the response to SAg is modulated by TLR2/6 agonists from the bacterial cell wall. Such a modulation involves the induction of APC death that then limits the capacity of SAg to cause massive T cell activation. Our findings provide an explanation for the long-standing question of selective advantage of SAg expression, and point to novel therapeutic strategies for SAg-associated diseases.
Role(s) of Pulmonary Macrophage and Dendritic Cells in SARS-CoV-Induced Host Inflammatory Responses: An in vitro Model. (43.39)
Authors: Yoshikawa Tomoki , Wang Nan , Peters Clarence J , Tseng Chien-Te K
Published: January 2023

SARS is a highly contagious respiratory disease with the lungs as a major target. Pulmonary macrophage (MΦ), airway epithelium, and dendritic cells (DC) are three key cells in host innate defenses against respiratory infections. Pulmonary MΦ are situated at the inner epithelial surface, whereas DC reside abundantly underneath the epithelium. Such strategic anatomic locations of these two effector cells within the airway system make it highly relevant and important to investigate their interrelationship with airway epithelial cells in the course of SARS coronavirus (SARS-CoV) infection. In this study, we established highly polarized human bronchial Calu-3 cell cultures in transwell membrane inserts which allowed us to harvest cytokines produced by the apical or the basolateral surfaces of SARS-CoV-infected Calu-3 cells. Here, we report that inflammatory cytokines released by infected Calu-3 cells differ between the two surfaces and are potent in modulating the intrinsic functions of both MΦ and DC. Specifically, they prompt the production of various inflammatory mediators by both MΦ and DC. However, they also significantly compromised the abilities of DC and MΦ in priming naive T cells and phagocytosis, respectively. These results suggest that the interplays among permissive lung epithelial cells, pulmonary MΦ and DC are critically involved in the regulation of inflammatory responses in the course of acute SARS-CoV infection and pathogenesis.
The L Antigen of the LCM Virus is a Target of Dominant CD8+ Responses (43.47)
Authors: Kotturi Maya , Peters Bjoern , Buendia-Laysa Fernando , Sidney John , Oseroff Carla , Botten Jason , Grey Howard , Buchmeier Michael , Sette Alessandro
Published: January 2023

CD8+ T cell responses control lymphocytic choriomeningitis virus (LCMV) infection in H-2b mice. Although antigen-specific responses against LCMV infection are well studied, we found that up to one third of the CD8+CD44hi response to LCMV in H-2b mice was not accounted for by known epitopes. We screened peptides predicted to bind MHC Class I, and overlapping 15-mer peptides spanning the complete LCMV proteome for their capacity to induce interferon-γ (IFNγ) production from CD8+ T cells derived from LCMV-infected H-2b mice. We identified thirteen novel epitopes. These together with the 7 previously known epitopes account for the total CD8+CD44hi response, diminishing the possible role for bystander T cell activation. Strikingly, 9 out of the 13 new epitopes were derived from the viral L polymerase, which, until now, was not recognized as a target of the cellular response induced by LCMV infection. The L epitopes induced significant levels of in vivo cytotoxicity and conferred protection against LCMV challenge. Interestingly, protection from viral challenge was best correlated with the cytolytic potential of CD8+ T cells, whereas IFNγ production and peptide avidity appear to play a lesser role. Taken together, these findings illustrate that the LCMV-specific CD8+T cell response is more complex than previously appreciated.
Bo-lysin: a potential candidate as a biomarker of protection after vaccination against tuberculosis (43.49)
Authors: Scherer Charles Fernando Capinos , Endsley Janice J. , de Aguiar Juliana B. , Waters Ray , Estes D.Mark
Published: January 2023

Tuberculosis (TB) is still a major health problem worldwide. A Th1 type response with release of IFNγ and cytotoxic granules such as granulysin and perforin, play a major role in the disease. Measurements of protection after TB vaccination include IFNγ measures and T cell proliferation assays, which frequently fail to correlate well with protection. The lack of better vaccines than BCG and good correlates of protection demands that new biomarkers are found. A bovine homologue of granulysin (bo-lysin), cloned by our group, may be a good candidate as a biomarker molecule for TB vaccination. In the present study we examined the kinetics of bo-lysin, compared to perforin, IFNγ and Fas-L in vitro, after different stimulation conditions in different bovine T cell populations. Gene expression profile showed bo-lysin and IFNγ expression increase in an equal manner, with IFNγ showing up as early as 4 hours of stimulation and Bo-lysin at 24 hours, in BCG vaccinated animals. No expression was detected in non-vaccinated animals. Detection of IFNγ and perforin by flow cytometry showed increasing percentages of antigen specific perforin+ or IFNγ+ CD4+ and perforin+ or IFNγ+CD8+ T cells in BCG vaccinated animals, but none were observed in non-vaccinated animals. Results show that granulysin is a good candidate as a biomarker of protection after vaccination against TB.

Project funded by grant #USDA-NRICGP-2005-35204-16039
Biomarkers of CD4+ CTL cell mediated immunity to tuberculosis (43.51)
Authors: Endsley Janice J , Hogg Alison , Shell Lis , McAlauy Martin , Scherer Charles Capinos , Coffey Tracey , Howard Chris , Nonnecke Brian , Waters W. Ray , Estes D. Mark
Published: January 2023

The immune responses mediated by interactions between T-lymphocyte subsets and mycobacteria-infected macrophages are critical for control of tuberculosis . In these studies, the bovine model was used to characterize the cytolytic and mycobactericidal CD4+ T cell response induced by BCG vaccination. Antigenic stimulation of CD4+ T-cells from BCG vaccinated cattle induced expression of perforin and IFNgamma in cells expressing a CD45RA−, CD45RO+, and CD62L+ cell surface phenotype. Antigen specific enhancement of granulysin, IFNgamma, perforin, IL-4, IL-13, and IL-21 mRNA expression was detected and not detected for IL-2, IL-6, IL-10, IL-15, TNFα, FasL, and CD40L. Following antigenic stimulation, CD4+ T cells from BCG vaccinated animals contributed to reduction of intracellular BCG in infected macrophages. These results demonstrate that vaccination with BCG induces a subpopulation of mycobacteria-specific CD4+ T cells that are characterized by the expression of a cell-surface memory phenotype, enhanced expression of mycobactericidal molecules, and anti-mycobacterial activity against intracellular M. bovis.

This work was supported by the NIAID Fellowship for Training in Emerging and Re-emerging Infectious Disease and the Sealy Center for Vaccine Development, UTMB, the Texas-United Kingdom Collaborative Research Initiative, and the Institute for Animal Health by DEFRA and the BBSRC, UK.
M. bovis-BCG elicits memory CD8+ T cell responses to mycobacteria in cattle (43.53)
Authors: Hogg Alison , Worth Andrew , Endsley Janice , Howard C.J. , Beverley P. , Villarreal-Ramos Bernardo
Published: January 2023

Tuberculosis (TB) remains one of the leading causes of death due to infectious disease. Current aims to improve vaccines against TB have focused on enhancing immunity conferred by the attenuated M. bovis BCG (BCG) vaccine. The design of a vaccine to specifically boost the protective features of BCG requires a greater understanding of the immune response induced by BCG vaccination. In this work we show that vaccination with BCG induces a population of CD8+CD45RO+ T cells that proliferate, produce IFN-gamma, up-regulate expression of perforin and granulysin, and lyse BCG-infected macrophages, following antigen specific stimulation. To determine whether a similar response could be detected in vivo, vaccinated animals were re-inoculated intradermally with BCG or PBS eight weeks after primary BCG vaccination. Draining lymph nodes were removed 3 days after the second inoculation for analysis by immunofluorescence and confocal microscopy for the presence of activated CD8+ T cells. Clusters of CD8+CD3+perforin+ cells were readily detectable in lymph nodes from animals re-inoculated with BCG but absent in the animals given PBS in the second inoculum. These results suggest that the memory CD8+ T cells induced by BCG vaccination have the capacity to mount secondary responses to mycobacteria in vivo.

Support for these studies was provided by the BBSRC, DEFRA and the Jenner Institute.
Cellular Immune Responses in a Murine Model of Borrelia burgdorferi and Babesia microti coinfection (43.55)
Authors: Vinasco Javier , Braga Walter , Zegarra-Moro Ofelia , Moro Manuel H
Published: January 2023

Active coinfection of Borrelia burgdorferi and Babesia microti has been suggested to result in an increased disease severity due to changes in immune response patterns. However, the immunological mechanisms involved in this processes are still unknown. The aim of this study is to characterize the cell immune responses during the progression of B. burgdorferi and B. microti coinfection. To address this issue, C3H.HeN and BALB/c mice cohorts were coinfected or singly infected and monitored for 1, 7, 10, 14, 21 and 60 days post-infection. Spleens and lymph nodes from infected and non-infected mice were removed for tissue culture experiments. Mononuclear cells were analyzed by flow cytometry assays using monoclonal antibodies. Interestingly, we found minimum changes among CD4+/CD3+, CD8+/CD3+, gamma-delta and NKT cells in C3H and BALB/c strains. In contrast, B cell populations were significantly augmented in BALB/c than C3H mice after infection. Furthermore, significant increased levels of B cells were also detected in coinfected BALB/c mice at days 14, 21 and 60. Spleen macrophage populations reached a peak at day 7 in both C3H and BALB/c strains for singly infected mice, in contrast with coinfected mice that showed significant decreased population of these cells. The reduction of spleen macrophages in coinfected mice may suggest an impair immune response during B.burgdorferi and B. microti coinfection resulting in an increased disease severity and persistence of Lyme disease. NIH P20RR016443-04.
Chronic Psychogenic Stress (predator exposure) Improved Survival and Altered the Cytokine/Chemokine Profile in Two Sepsis Models (44.11)
Authors: Dugan Amy , Gregerson Karen , Gardner Jason , Neely Alice , Buckley Donna , Noel Greg , Ogle Cora , Horseman Nelson
Published: January 2023

We determined the impact of chronic psychogenic stress (predator exposure) on sepsis-induced mortality and serum cytokine alterations. Male 7-week-old C57Bl/6 mice, individually housed, were exposed to either an unoccupied rat cage (non-exposed) or a rat cage containing 1 male Long Evans rat (predator-exposed), but with no physical contact between the mouse and rat. Exposure sessions lasted 1 hour and occurred on 9 consecutive days. Immediately before the 3rd exposure, non-exposed and predator-exposed mice received either saline, LPS (from E. coli, 20 mg/kg), or live E. coli (1 X 108 CFU) i.p. and were monitored for survival over the remaining 7 days. Predator exposure markedly improved survival rates after either LPS or bacteria injection. Mortality in the non-exposed mice (LPS) was 58%, but only 8% among the predator-exposed mice (p = 0.030). Following E. coli injection, mortality was 67% in the non-exposed mice, but only 17% in predator-exposed mice (p = 0.038). In a separate experiment mice were killed 24 hours after injection and serum cytokines were measured. Predator exposure attenuated the sepsis-induced increase of TNFα while enhancing IL-6. We hypothesize that predator-induced alterations in cytokine profiles during sepsis may be responsible for the observed survival advantage.

This work was funded by the Shriners of North America
Differential secretion of IL-12 and IL-18 based on intracellular localization of Listeria monocytogenes (44.2)
Authors: Meeks Karen D. , Sieve Amy N. , Balch Joshua B. , Berg Rance E.
Published: January 2023

Listeria monocytogenes (LM) is a gram-positive, intracellular bacterium that is commonly used to investigate the immune response to infection. Pro-inflammatory cytokines, such as IL-12 and IL-18, are secreted during LM infection to promote clearance of the bacteria through the induction of IFN-γ. LM secretes listeriolysin O (LLO) which allows the bacterium to invade the cytoplasm. Neither heat-killed LM (HKLM) nor LLO deficient LM (LLO−LM) actively produce LLO. When bone marrow derived macrophages (macs) and dendritic cells (DCs) are infected with LM, HKLM, or LLO−LM and co-cultured with naïve splenocytes, only macs or DCs infected with LM are able to induce IFN-γ secretion. ELISA data demonstrates that IL-12 is secreted from LM, HKLM, or LLO−LM infected macs and DCs, but IL-18 is only secreted from macs and DCs infected with LM. Therefore, IL-12 can be secreted by macs and DCs regardless of cytoplasmic invasion of LM whereas the secretion of IL-18 requires LM to gain access to the cytoplasm. When mice are infected with HKLM, there is no detectable IFN-γ secretion and this can be attributed to a lack of both IL-12 and IL-18. This implies that IL-12 is induced differently in response to HKLM in vivo versus in vitro. Further studies are ongoing to determine which pattern recognition receptors and cell types are responsible for recognizing and secreting IL-12 and IL-18 during in vivo LM infection.

Supported by NIH AI064592.
Listeria induces NKDC to produce IFN-gamma via IL-12, IL-18 and TLR9 ligation (44.24)
Authors: Plitas George , Chaudhry Umer I. , Kingham T. Peter , Stableford Jennifer , DeMatteo Ronald P.
Published: January 2023

Introduction: Natural killer dendritic cells (NKDC, NK1.1+CD11c+) are innate effector cells that secrete IFN-γ when stimulated. Initial control of Listeria monocytogenes (LM) infection depends on IFN-γ. The mechanism of IFN-γ secretion by NKDC in response to LM is unknown.

Methods: NKDC and NK (NK1.1+CD3−) cells were isolated by fluorescence activated cell sorting from the spleens of C57BL/6 mice and cultured with LM strain 10403s at a multiplicity of infection of 1 for 3 days. IL-18 (20ng/ml), IL-12 blocking antibody (10μg/ml), or an inhibitory CpG (iCpG) sequence (50μg/ml) was added. Supernatant IFN-γ was analyzed by cytometric bead array.

Results: LM infection or IL-18 alone did not induce significant IFN-γ production by NK cells or NKDC, however the combination of IL-18 and LM induced substantial amounts of IFN-γ from NKDC (19.3±2.7 ng/ml), and to a lesser extent, NK cells (2.0±0.6 ng/ml). Addition of anti-IL-12 antibody abrogated the IL-18/LM induced IFN-γ production of both NK cells and NKDC (<0.4 ng/ml). The addition of iCpG led to an 85% reduction of IL-18/LM induced IFN-γ production by NKDC and a 20% reduction by NK cells.

Conclusion: IL-18, IL-12, and TLR9 ligation regulate NKDC IFN-γ production in response to LM.

This work was supported by grant DK068346.