bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantifying the magnitude of antigen-specific CD4 T cell responses (39.27)
Authors: McDermott Daniel , Varga Steven
Published: January 2023

CD4 T cells play a vital role in orchestrating adaptive immune responses. Measuring the magnitude of the CD4 T cell response to a given pathogen is currently hindered by the lack of complete knowledge of all of the CD4 T cell epitopes. In cases where most or all of the CD4 T cell epitopes have been mapped, current estimates based on intracellular cytokine production for cytokines such as IFN-γ may also under-represent the frequency of antigen-specific CD4 T cells because of the high heterogeneity in cytokine production exhibited by differentiated effector CD4 T cells. Here we show that following lymphocytic choriomeningitis virus (LCMV) infection, T cell receptor-transgenic CD4 T cells specific to LCMV upregulate CD11a and CD49d and that expression of these adhesion molecules remains stable into memory. Expression of these integrins requires cognate antigen and is not modulated non-specifically by inflammatory signals such as stimulation with LPS or CPG. Additionally, CD11a and CD49d expression is upregulated on endogenous CD4 T cells following acute LCMV infection as well as all LCMV-specific CD4 T cells that respond to previously identified CD4 T cell epitopes. Using these markers we estimate the total magnitude of the virus-specific CD4 T cell response to multiple pathogens and our results indicate that the CD4 T cell response is substantially larger than previously thought.
Development of CD8 T-memory inflation after cytomegalovirus infection determined by initial viral burden (39.28)
Authors: Kincaid Michelle , Thomas Alicia , Zimmerman Peter , Klenerman Paul , Bergdall Valerie , Hickman-Davis Judy , Trgovcich Joanne , Cook Charles
Published: January 2023

Both mice and humans can develop large “inflated” populations of CD8 effector memory T-cells after cytomegalovirus (CMV) infection, and such memory inflation has been associated with immune senescence. Not all humans develop CD8 memory inflation after CMV infection, so we hypothesized that memory inflation might be predicated by the initial viral burden. Because it is impossible to know an individuals CMV inoculum during natural infection, we infected mice with murine CMV (MCMV) titers ranging from 102 to 106 PFU. After infection, peripheral blood was evaluated monthly for MCMV-specific CD8 T-cells and antibody. Using MCMV-specific MHC class I tetramers we confirm that high titer infections induce inflationary T-memory responses. In contrast, low titer infections induce significantly less inflation over time. Serum ELISA for MCMV-antibody responses showed a similar pattern. DNA from lung tissues evaluated 16 weeks after infection confirmed successful infection in all mice. Quantitative PCR showed that latent viral load in lungs correlated well with eventual lung resident T-memory responses (R2=0.96). We therefore conclude that an individual’s initial infectious burden determines their latent viral load and eventual development of memory inflation after cytomegalovirus infection, possibly explaining the wide variability in T-cell responses to CMV observed in humans.
Differential gene expression patterns associated with disease resistance in the MAIDS model (39.29)
Authors: Bakkour Sonia , Kolhatkar Nikita , Karki Sophiya , Herman Nicole , Ursu Oana , Raghavan Vidya , Stranford Sharon
Published: January 2023

Infection of susceptible strains of mice such as C57BL/6 (B6) with the LP-BM5 isolate of murine leukemia virus (MuLV) leads to an immunodeficiency syndrome similar to HIV-induced AIDS, known as MAIDS. However, other MAIDS-resistant strains such as BALB/c develop protective immunity against the virus after infection. Recently, we used DNA microarray analysis to compare lymphoid organ gene expression in these two mouse strains during the first week post infection. We identified a specific set of proteases and certain chemokines that are highly differentially expressed in BALB/c versus B6 mice after infection. This suggested to us that early changes in lymphoid organ architecture and/or cellular recruitment might help shape the early immune response to the virus. In follow-up experiments using real-time PCR and protein-based assays, we now show that neither protease expression nor lymphatic vascular architecture changes appear to be specifically associated with resistance to MAIDS. This despite the fact that these animals very rapidly control the viral load, showing significantly reduced viremia by five days after infection. In B6 mice, the viral load exponentially rises over the same time-course, with strongly divergent patterns apparent by day 5. We do find that certain chemokines are upregulated in the MAIDS-resistant mice at approximately one week after infection. These findings suggest that differences in cell trafficking might influence disease in this AIDS model system.
MyD88 expressing T cells are required for neuropathogenesis of Tacaribe virus. (39.6)
Authors: Tami Cecilia , Pedras-Vasconcelos Joao , Puig Montserrat , Wang Vivian , Butts Cherie , Verthelyi Daniela
Published: January 2023

Immune responses play a key role in the pathogenesis of viral encephalitis. Infection of neonatal C57Bl/6 (B6) mice with neurotropic arenavirus Tacaribe (TCRV) results in encephalitis, paralysis and death within 15 days of challenge. Analysis of the expression of 96 immune related genes using a mouse Taqman Low Density Array (TLDA) showed a strong inflammatory response in the brain characterized by high levels of inflammatory chemokines, mainly CXCL10/IP-10, IL1β, Granzyme, IFNγ and TNFα and T cell infiltration. In contrast, CD3ϵ-/- mice which lack T cells (CD3ϵKO) show 100% survival and minimal inflammatory response despite having similar viral loads in the brain. Transfer of either CD4+ or CD8+ T cells from B6 to CD3ϵKO mice resulted in encephalitis and death. Disease was independent of TLR2, 3, 7 or 9, whereas 90% of MyD88-/- mice survived challenge. Brains of MyD88-/- mice showed minimal inflammation and T cell infiltration but had reduced viral titers when compared to B6 or CD3ϵKO mice. MYD88 does not directly affect TCRV replication since infection of B6 and MYD88-/- primary neuro cultures in vitro rendered similar virus titers. However, T cells from MyD88-/- mice were unable to mediate encephalitis when transferred to infected CD3ϵKO. These data show that T cells mediate disease and death in TCRV infected neonates without affecting viral replication and brain tropism and suggest a critical role of MYD88 expression on T cell in the pathology.
Improved T cell restoration by depletion of T regulatory (Treg) cells followed by PD-1 blockade during chronic infection (39.9)
Authors: Penaloza-MacMaster Pablo , Araki Koichi , Ahmed Rafi , West Erin , Freeman Gordon , Rudenski Alexander , Tan Wendy , Jin H. , Aubert Rachael , Omara Leigh , Iyer Smita
Published: January 2023

It remains unclear if chronic infection, and the resulting antigen-specific T cell exhaustion, is a process facilitated by Tregs, cells that are known to prevent autoimmunity and maintain self-tolerance. In a model of chronic lymphocytic choriomeningitis virus (LCMV) infection, we show that depletion of CD4 cells, or specific Treg depletion (in the FoxP3 DTR mouse model), followed by PD-1 blockade results in marked increase of T cell restoration, and enhanced reduction of viral loads compared to mice that received the PD-1 blockade regimen alone. Moreover, in the FoxP3/DTR mouse model, specific depletion of Tregs alone without any further treatment results in dramatic T cell restoration (increased numbers and cytokine expression), which was greater than PD-1 blockade alone. In addition, Treg depletion alone also resulted in re-expression of CD127, and PD-1 downregulation on antigen-specific T cells. These results suggest that brief manipulation of Treg function with or without PD-1 blockade may resuscitate exhausted CD8 T cells during many established chronic infections. Our data also demonstrate an unexpectedly dominant, suppressive role of Tregs during chronic infections, and identify these cells as potential targets for vaccines and prophylactic regimens.
Prolonged Salmonella persistence occurs in the Mesenteric Lymph Nodes of antibiotic treated mice. (40.15)
Authors: Griffin Amanda , McSorley Stephen
Published: January 2023

We have examined a new mouse model where virulent Salmonella infection can be treated using antibiotics. Despite the apparent rapid resolution of infection, withdrawal of antibiotics before 35 days causes recurrent infection. Antibiotic-treated mice resume bacterial shedding, bacteria can be detected in the spleen and liver, and mice eventually suffer fatal recurrent infection. Although the persistent bacteria are undetectable by conventional plating methods, we were able to detect bacterial persistence in the mesenteric lymph nodes (MLN) using a Salmonella-luciferase imaging approach in all tissues of infected mice. Whole organ broth cultures confirmed MLN localization and allowed an estimation of bacterial loads in the tissue. We are currently identifying the specific cell population that harbors these bacteria in infected MLNs. Together, these data demonstrate that Salmonella can evade antibiotic killing via long-term residence in the MLN.
Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells (40.16)
Authors: Suzuki Yasuhiro , Wang Xisheng , Jortner Benard , Payne Laura , Ni Yanyan , Michie Sara , Xu Baohui , Kudo Tomoya , Perkins Sara
Published: January 2023

Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of the cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8+ immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8+ T cells does not require their production of IFN-γ, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8+ T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.
Constructing epitope specific MHC-II tetramers for visualization of CD4 T cells in gram negative bacteria infections (40.18)
Authors: Li Lin-Xi , Lee Seung-Joo , McLachlan James , Moon James , McSorley Stephen
Published: January 2023

MHC class II peptide tetramers can be used to evaluate antigen-specific CD4 T cell responses in models of infectious disease. Here we describe the construction and functional test of epitope specific MHC-II tetramers for use in Salmonella and Chlamydia infection models. Epitope peptide sequences were covalently linked to N-terminal of I-Ab β chain, which also had his-tag for purification, and E.coli BirA signal sequence was attached to C-terminal of I-Ab α chain, which allows for site-specific biotinylation and subsequent tetramerization with fluorochrome-labeled streptavidin. I-Ab α and β chains were co-expressed in Drosophila S2 cells. pMHCII monomers were purified from culture supernatant using a Nickel-charged affinity column, and purified monomer was tetramerized using fluorochrome-labled streptavidin. Functional test of these tetramers was performed by staining peripheral lymph node cells from mice that were immunized with peptide or using spleen cells from infected mice. Therefore, antigen-specific CD4 T cell responses can be efficiently visualized by these epitope-specific MHCII tetramers during Gram Negative bacteria infection.
OX40L mediates susceptibility to chronic Leishmania donovani infection (40.26)
Authors: Snider Heidi , Reville Patrick , Sharpe Arlene , Satoskar Abhay
Published: January 2023

OX40 is an antigen-specific T cell cosimulatory molecule belonging to the TNF receptor superfamily that is involved in both inflammatory and autoimmune responses. Its ligand, OX40L, is primarily expressed on antigen-presenting cells. The role of OX40-OX40L interactions during visceral leishmaniasis (VL) is currently unclear. Previous studies have reported that OX40-OX40L interactions contribute to increased parasite burdens and pathology of cutaneous leishmanias caused by L. major by increasing Th2 cytokine responses. However, numerous studies have found that during VL, mixed Th1/Th2 cytokine responses are key to disease resolution. In fact, one study has shown that OX40 agonist, in conjunction with anti-CTLA-4 treatment, enhances granuloma formation and parasite clearance during VL. In this study, we examined disease outcome in OX40L-deficient C57BL/6 mice infected with VL caused by L. donovani. We found that while these mice had similar parasite burdens in their livers and spleens early post-infection, they exhibited accelerated parasite clearance during late, chronic stages of disease. Ex vivo antigen stimulation of splenocytes showed that this rapid drop in parasite levels was associated with increased IFNγ production. We therefore propose that OX40-OX40L interactions play a role in the development and pathogenesis of chronic disease caused by visceral leishmaniasis, and their blockade may represent a viable therapeutic target during clinical disease.
PD-L1 is required for optimal protective immunity to Salmonella infection (40.8)
Authors: Lee Seung-Joo , McSorley Stephen
Published: January 2023

Robust antibody and CD4 T cell responses are required for protective immunity against Salmonella infection. Here we examined the role of programmed death ligand 1 (PD-L1) in resolution of primary Salmonella infection. Infected PD-L1-deficient mice had higher bacterial burdens in the spleen and liver at day 21 and day 35 compared to wild-type mice indicating that PD-L1 is required for optimal protective immunity to Salmonella. Although Salmonella specific IgM and IgG2a Ab responses were similar between PD-L1-deificent and wild-type mice, PD-L1-deficient mice displayed a deficiency in CD4 T cells. Specifically, infected PD-L1-deficient mice had a significantly lower percentage of IFNγ-, and TNF-producing CD4 T cells and expressed lower levels of T-bet than wild-type CD4 T cells. These data demonstrate that PD-L1 expression is required for the generation of effective Th1 responses to Salmonella and optimal protective immunity.
Comparison of the use of role-play in both large and small classes to teach the basic concepts of an immune response to undergraduate students (41.3)
Authors: Elliott Samantha
Published: January 2023

Active learning exercises are touted as superior teaching strategies, yet little work has been done to truly evaluate their effectiveness in the science classroom. Role-play is considered an especially strong tool to model concepts and show relationships between entities. The immune response is a particularly difficult topic for students to grasp, due to the myriad of cellular and protein interactions involved. This study looks at the use of role-play as a tool to teach the immune response to undergraduates in both small classes, where all students participate, and large classes where only a subset of students act for their peers. Learning was measured using a standard pre- and post-test format of multiple choice questions, and student perceptions of learning were collected using an anonymous written survey. No significant differences were found in overall learning based upon class size. Role-play participation did not have a significant impact on learning compared to students who did not participate in large classes. No significant differences were seen in learning based upon student learning styles. A full analysis of the efficacy of role-play as a replacement for or an addition to lecture will be presented. This research gives insight into the efficacy of role-play as a learning tool for teaching basic concepts in immunology.
Interleukin (IL)-21, but not IL-2, induces antiviral activity and costimulatory molecules in CD8 T cells without promoting HIV replication (42.15)
Authors: Parmigiani Anita , Pallin Maria , Lichtenheld Mathias , Pahwa Savita
Published: January 2023

Background: Chronic HIV infection is marked by defective cytotoxic T cell (CTL) effector function, skewed terminally-differentiated profile, and reduced expression of the costimulatory molecules CD27 and CD28. Incubation ex vivo of CTL from HIV infected persons with IL-21 augments cytotoxic effector molecules and function. In this study we analyzed IL-21 effects on CTL as compared to IL-2. Methods: Purified CD8 T cells from healthy donors were pre-activated with αCD3+αCD28 and cultured with IL-21 or IL-2 for 6 days. Phenotype and effector function were evaluated by flow cytometry, gene expression by real-time PCR, effect on HIV replication with the reporter cell line TZM-bl. Results: IL-21 induced higher CTL degranulation in response to TCR stimulation, as compared to IL-2. Co-culture of IL-21-treated CTL with autologous HIV infected CD4 T cells reduced HIV replication. Importantly, IL-21 did not induce viral replication when added to HIV infected CD4 T cells. Furthermore, TCR-activated CTL upreglated CD27 and CD28 and acquired a central memory phenotype when incubated with IL-21, but not with IL-2. IL-21 also selectively induced Bcl6, key transcription factor for memory T cells. Conclusions: Since IL-21 enhances CTL antiviral activity, promotes a central memory phenotype and induces costimulatory molecules, this cytokine has an appealing immunomodulatory profile and merits further consideration for cytokine-based therapies, alone or in combination with antiretroviral drugs.
Non-hematopoietic IL12p40-responsiveness is required for tubercular granuloma formation. (42.19)
Authors: Robinson Richard , Cooper Andrea
Published: January 2023

Genetic studies have repeatedly demonstrated that the control of mycobacterial infections, including M. tuberculosis, is tightly coupled with IL12Rβ1-dependent signaling. The explanation historically given for this close association is that IL12p40-dependent cytokines (e.g. IL12p70) are required to generate protective, IFNγ-producing T cells and NK cells; in the absence of IL12Rβ1, these IFNγ-producing cells fail to develop and M. tuberculosis grows unchecked. Here, we present data that suggest an alternative explanation for the association between IL12Rβ1-deficiency and mycobacterial susceptibility. Using a mouse model of tuberculosis we show that in addition to influencing hematopoietic cells, CD11c+-derived IL12p40 also acts on non-hematopoietic cells though IL12Rβ1 to control M. tuberculosis growth. Further examination demonstrated that while mice with il12rb1-deficient stroma accumulate numerous immune cell subsets in the lung following infection, these cells fail to properly organize into protective tubercular granulomas. Granuloma formation was not dependent on IL12Rβ2 or IL23, suggesting that either IL12p40 monomer or IL12(p40)2 was responsible for this effect. Thus, while the absence of hematopoietic IL12Rβ1 does compromise the ability of mice to control M. tuberculosis infection, our data suggests that the influence of IL12p40 on non-hematopoietic cells is equally important.
Interferon-γ controls Th17-mediated immunopathology during mouse hepatitis virus infection (42.2)
Authors: Qin Zhihai
Published: January 2023

Due to its lethality, fulminant hepatitis, the most severe form of viral infection in liver, is a global health concern. Here we examine the function of Th17 cells during the mouse hepatitis virus (MHV) induced hepatitis. We find that IL17 levels in serum are rapidly upregulated and positively correlated to liver damage and death of mice upon viral infection. The unresponsiveness of host cells to IFNγ significantly enhances virus-induced Th17 response. Unrestricted IL17 production accounts for severe liver damage as well as detrimental inflammation in infected IFNγ receptor deficient (IFNγR-/-) mice, since neutralization of IL17 effectively suppresses inflammation and protects mice from liver injury. IFNγ facilitates antigen-induced apoptosis of Th17 cells in vitro and adoptive transfer of IFNγR deficient, but not IFNγR competent CD4+ T cells leads to enhanced liver damage in wild-type mice. The results demonstrate an essential role of Th17 cells in MHV-induced immunopathology and indicate the importance of IFNγ in maintaining immune balance between Th1 and Th17 responses during the acute viral infection.
Innate immune cells are the major determinants of host-related susceptibility differences to experimental spotted fever rickettsiosis (42.24)
Authors: Fang Rong , Ismail Nahed , Shelite Thomas , Walker David
Published: January 2023

Infection of C3H/HeN (C3H) mice with 3 × 105 PFU of R. conorii resulted in 100% mortality, while all C57BL/6 (B6) mice survived the same inoculum. To unravel the strategy by which R. conorii establishes acute fatal disease, we explored its early interaction with the different immune systems in these two mouse strains. Compared to sublethal infection in C3H mice, rickettsiae induced a greater cytotoxic T cell response in infected B6 mice. Lethal infection suppressed antigen-specific proliferation of CD4+ and CD8+ T cells on day 5 post infection, which correlated with upregulated expression levels of PD-1/PD-L1 on T cells and dendritic cells (DCs), respectively. Resistant mice completely cleared the bacteria in four days with greatly expanded cytotoxic NK cells, IFN-γ(+) CD8+ T cells, DCs, but not neutrophils, and enhanced production of MCP-1, MCP-5, RANTES, and IL-12p70. The high resistance of RAG -/- mice on a B6 background to rickettsial infection suggested that the host resistance was determined at the innate immune cell level. Finally, R. conorii induced altered expression of TLR2 and TLR9 in bone marrow derived DCs from C3H and B6 mice, respectively, suggesting that differential TLR signaling pathway was involved in the innate immune responses to rickettsial infection in these two mouse strains. These data suggest that NK cell activation by DCs and their migration to infected sites may play a critical role in innate resistance against Rickettsia.
Alternate delivery systems to improve vaccine performance against foot-and-mouth disease virus (43.15)
Authors: Pandya Mital , Bishop Elizabeth , Pacheco Juan , Scherer Charles , Milward Francis , Doel Timothy , Golde William
Published: January 2023

Foot-and-mouth disease virus (FMDV) is a highly infectious disease of cloven-hoofed animals, with severe agricultural and economic implications. The standard needle inoculation method is not the most efficient system of swiftly vaccinating large numbers of animals at once, as in outbreak situations. Therefore, we conducted studies using a needle free system for vaccination with the killed virus vaccine as a rapid consistent delivery platform. Cattle were vaccinated using a commercially available vaccine formulation at 1x, 1/4x and 1/16x dose using the needle free system and then challenged intradermalingually with FMDV, strain O1 Manisa at 7, 21, or 31 days post vaccination (DPV). All non-vaccinated control cattle exhibited clinical disease, including fever, viremia and vesicle formation. However, cattle vaccinated with the 1/16x and 1/4x doses using the needle free device were partially protected at 7 DPV and fully protected at 21 or 31 DPV. These data suggest that protective vaccination can be achieved with 1/16x vaccine dose when delivered using the needle free intradermal delivery system. This is an indication that the current vaccine stockpile can be extended by many fold using this system.
Determining peptide binding motifs for the SLA-2*0401 Class I MHC and identification of epitopes from foot-and-mouth disease virus (FMDV) P1 protein. (43.16)
Authors: Pedersen Lasse , Patch Jared , Jungersen Gregers , Nielsen Morten , Buus Soren , Golde William
Published: January 2023

To date, little is known about the binding specificities for Swine Leukocyte Antigens (SLA) compared to the enormous amount of data available for the Human Leukocyte Antigens (HLA). The increase in immunology and infectious disease research using pigs as an experimental model would benefit from an increased understanding of SLA class I peptide presentation. In this work, we have determined the binding motifs for the SLA-2*0401 molecule, a commonly expressed allele in swine and the PK13 and PK15 cell lines. These peptide motifs were identified using a prediction model developed for the Class I MHC of human and applied to SLA-2*0401. With this, we derived 10 candidate T cell epitopes from the P1 region of the FMDV strain A24 and two more from Ebola virus and V. cholera for comparison. These candidate peptides were tested for binding SLA-2*0401 using an ELISA based protein association assay developed previously for HLA-I molecules. Five of the ten predicted FMDV peptides bound SLA-2*0401 in vitro. These data are now being applied to the analysis of porcine T cell responses to FMDV infection and vaccination.
Comparative analysis of Xenopus immune-related genes (43.22)
Authors: Ota Yuko , Flajnik Martin
Published: January 2023

Evolutionarily, Xenopus species shared a common ancestor with humans ~350 million years ago, and it is one of the high connectivity animals linking mammals to lower vertebrate taxa. Through an in silico approach, we have uncovered many new genes that have important roles in the Xenopus immune system and have analyzed their syntenic relationships relative to other vertebrates. We found that, in contrast to teleost fish, the genomic synteny is remarkably similar between the human and Xenopus, yet in some cases apparent ancestral syntenies can be still found only in the Xenopus genome. We predict that the evolutionarily conserved genes might have vital roles in fundamental immune function, whereas novel genes found only in particular species, especially those within ancient linkage groups, could impart new insights into the immune system. We are specifically interested in the genes that belong to the variable (V) and the constant (C1)-type of the immunoglobulin superfamily (IgSF), including immunoglobulin, T cell receptor, MHC, and B7. In order to understand the phylogenetic relationship and the evolutionary history of these gene families, we used comparative approach; database searches and comparative genomics among different vertebrate classes. Our analysis revealed insights into the architecture of the primordial immune complex that might have played important roles during evolution.
Fishing for CD4 positive cells in the zebrafish. (43.28)
Authors: Mitra Suman , van der Sar Astrid , Alnabulsi Ayham , Secombes Chris , Bird Steve
Published: January 2023

Differentiation of naïve CD4+ T-cells into functionally distinct subsets is essential for an effective immune response against different types of pathogens. The recent discovery of CD4 like molecules in teleost fish and many of the genes important in adaptive immunity, now gives us the opportunity to study the role of T-cells in lower vertebrate immune responses. Despite these discoveries the lack of molecular tools, such as antibodies, in organisms other than mammals has made this difficult to do. Due to this we have begun to develop a number of polyclonal and monoclonal antibodies to immune genes in zebrafish. Zebrafish has become a popular model to study immunity and is being used by several laboratories worldwide to study disease. Using the zebrafish genome we have discovered a locus that contains four CD4 like genes, which is different to the two discovered in other teleost genomes to date. Although related to other vertebrate CD4 molecules, these genes exhibit low identity to each other. Homology modelling of these genes identifies only one of these to be similar to mammalian CD4, as it is the only one predicted to contain 4 immunoglobulin domains. Using a peptide approach we have developed a polyclonal antibody towards this CD4 molecule and have begun using it to study CD4+ cells in the zebrafish using FACS and immunofluorescence analysis. In addition, we have looked by realtime PCR at the expression of all 4 genes in embryos and healthy or infected adult zebrafish.
Chicken IgM interacts with IgY-sensitized cells (43.31)
Authors: Koppenheffer Thomas , Malpede Brian
Published: January 2023

Normal chicken serum agglutinates both homologous RBC and sheep RBC sensitized with specific IgY, but does not agglutinate sheep RBC sensitized with rabbit IgG. This hemagglutination-enhancing (HE) activity indicates recognition of IgY by a component of chicken serum. The HE activity is greatly enhanced in serum that was previously frozen as compared to fresh serum, and when the former is used as a source for isolating macroglobulins with HE activity, gel filtration chromatography (Sephacryl S-300 HR) reveals tow distinct peaks of activity. The first fraction elutes with the column void volume and consists entirely of IgM, as determined by SDS-polyacrylamide gel electrophoresis under non-reducing conditions and Western blotting, while a lower mol. wt. fraction contains both IgM and a smaller macromolecule. It appears, therefore, that chicken IgM has the capacity to bind antigen-bound IgY, and that this activity is enhanced by the tendency of IgM to aggregate during experimental manipulation. While there is as yet no known functional role for this phenomenon, a number of reports have noted that target RBC undergo pronounced agglutination during the course of hemolytic complement tests in which avian serum is used as complement source. This raises the possibility that chicken IgM might modulate effector functions of IgY.
An FcμR expressed by channel catfish, Ictalurus punctatus, clonal CTL mediates inhibitory effector functions and recruits SHIP (43.7)
Authors: Nayak Deepak , Wilson Melanie , Bengtén Eva
Published: January 2023

Flow cytometric analyses of catfish clonal alloantigen dependent cytotoxic T lymphocyte (CTL) line, TS32.15, grown in the presence of 3% catfish serum revealed that all cells stain positive for IgM and both IgL F and IgL G isotypes, which suggests passive uptake of serum IgM via a putative FcμR. This IgM expression was sensitive to protease treatment, while phosphatidylinositol specific phospholipase C had no effect. Three FcμR or FcμR associated proteins of sizes 52, 40, and 38 kDa were co-immunoprecipitated from biotinylated TS32.15 CTL using anti-catfish IgM. Importantly, FcμR cross-linking led to tyrosine phosphorylation of the 52 kDa protein. Also, Western blot of immunoselect material demonstrated that Src homology 2 domain-containing inositol phosphatase (SHIP) was co-selected with the putative FcμR. However no increase in intracellular calcium was observed. Unlike the previously described catfish IgM bound by catfish NK cells, TS32.15 bound IgM did not mediate antibody dependent cell mediated cytotoxicity. In contrast, IgM cross-linking of TS32.15 cells resulted in reduced cell division and marked decrease in cytotoxicity against allogeneic targets. Cell cycle analyses revealed that cross-linked cells were predominately arrested in the G0/G1 phase and this blockage could be completely reversed by allogeneic stimulation. Together these data indicate this putative FcμR likely functions as an inhibitory Fc-receptor.
Impaired T cell homing and transendothelial migration in CIP4 null mice caused by defective integrin-mediated adhesion (44.1)
Authors: Koduru Suresh , Kumar Lalit , Ozcan Esra , Oyoshi Michiko , Massaad Michel , Le Bras Severine , Ramesh Narayanaswamy , Kaku Mayumi , Fujiwara Yuko , Kremer Lenor , King Sandra , Fuhlbrigge Robert , Sage Peter , Carman Chris , Alcaide Pilar , Luscinskas Francis , Geha Raif
Published: January 2023

The F-BAR domain-containing protein CIP4 interacts with Cdc42 and WASP/N-WASP and is thought to participate in the assembly of filamentous actin. CIP4-/- mice had normal T and B lymphocyte development but impaired T cell-dependent antibody production, IgG antibody affinity maturation, and germinal center formation, despite intact CD40L-CD40 axis. CIP4-/- T cells, but not B cells, homed poorly to lymphoid organs. CIP4-/- mice had impaired contact hypersensitivity (CHS) and their T cells failed to adoptively transfer CHS. CD4+ effector T cells from CIP4-/-/OT-II mice migrated poorly to antigen challenged skin. CIP4-/- T cells exhibited normal rolling on endothelial selectins and chemotaxis to SDF-1α, but impaired adhesion and polarization on immobilized VCAM-1 and ICAM-1, and defective arrest and transmigration across murine endothelial cells under shear flow conditions. These results demonstrate an important role for CIP4 in integrin-mediated T cell adhesive events that are essential for normal recruitment to lymphoid organs and sites of antigen driven immune reactions.
Flotillins and ERM proteins function to promote uropod formation in T cells (44.10)
Authors: Chen Emily , Shaffer Meredith , Niggli Verena , Burkhardt Janis
Published: January 2023

Migrating and adherent T cells form a protruding leading edge and a constricted tail-like structure termed the uropod. The formation of these two structures involves the segregation of specific cytoskeletal elements and cell surface molecules, including proteins that organize cell polarity in other systems. Among the proteins that are segregated to the uropod are ezrin and moesin, ERM family proteins that organize cell membrane domains by linking cytoplasmic and cytosolic proteins to the actin cytoskeleton. Using conditional ezrin-deficient mice in conjunction with siRNA for moesin, we show that ezrin and moesin are required for uropod formation. In addition to ERM proteins, we find that the lipid-raft associated actin-binding proteins flotillins/reggies are also associated with the T cell uropod. Flotillins form a polarized cap even under conditions where a constricted uropod has not formed. Suppression of flotillin expression inhibits uropod formation and the polarization of ERM proteins and their binding partners. These data support a model in which flotillins mark a cell surface domain to which ERM proteins are recruited, and ERM proteins then promote formation of a constricted uropod structure. Since flotillins are not known to interact directly with ERM proteins, intermediary molecules, such as their common binding partner, PSGL-1, may be involved.
Extracellular adenosine triggers lymphocyte entry into the central nervous system during experimental autoimmune encephalomyelitis by regulating chemokine and adhesion molecule expression in the brain (44.12)
Authors: Mills Jeffrey , Alabanza Leah , Mueller Cynthia , Bynoe Margaret
Published: January 2023

Lymphocyte entry into the central nervous system (CNS) is a highly regulated process which serves to promote tissue repair while protecting against potentially fatal inflammation. As the formation of extracellular adenosine (catalyzed from ATP by CD39 and CD73) during inflammation serves as a negative-feedback signal to prevent excessive cell damage, our results suggest that adenosine receptor (AR) signaling also regulates CNS lymphocyte infiltration. We have shown that extracellular adenosine is required for the progression experimental autoimmune encephalomyelitis (EAE), the animal model of autoimmune inflammatory disease Multiple Sclerosis. We now show that AR signaling induces the CNS expression of the chemokine CX3CL1 (fractalkine), which acts as a chemoattractant for monocytes, lymphocytes, and NK cells. In addition, CX3CL1 expression is up-regulated in the CNS immediately prior to EAE onset only in wild type, but not CD73-/- mice, which cannot produce extracellular adenosine, are protected from EAE, and have minimal CNS lymphocyte infiltration following EAE induction. Moreover, CD39, CD73, and ARs are found clustered in the brain only at the choroid plexus, a structure which is known to regulate lymphocyte migration into the CNS. As AR signaling can induce CX3CL1 and ICAM-1 expression and lymphocyte transmigration across an in vitro choroid plexus transwell barrier, we conclude that AR signaling regulates lymphocyte entry into the CNS.
Memory T cells are selectively enriched in lymph nodes of selectin ligand deficient mice (44.14)
Authors: Harp John , Gilchrist Michael , Onami Thandi
Published: January 2023

Selectin mediated tethering is one of the earliest steps in T cell entry to lymph nodes (LNs). Selectin binding is dependent on the synthesis of the sialyl Lewis X (sLex) structure, which requires the activity of several glycosyltransferases, including fucosyltransferases -IV and -VII. Mice deficient in expression of both enzymes (FtDKO) reveal profound impairment in T cell trafficking to LNs. We show that T cells isolated from FtDKO LNs were predominantly “memory/effector” (CD44Hi) cells. We analyzed the generation of effector and memory CD8 T cells in FtDKO mice following infection with LCMV. At day 8, the proportion of Ag-specific cells was similar in LNs of FtDKO mice compared to WT. In contrast, at day 60 p.i., we observed ~5-fold increase in the proportion of Ag-specific memory CD8 T cells in LNs of FtDKO. We then examined whether Ag-specific memory CD8 T cells were able to selectively enter LNs of FtDKO recipient mice. Memory CD8 T cells were profoundly impaired in entry to the LNs of FtDKO mice. Additionally, while we measured no differences in exit rate of naïve and memory CD8 T cells out of the LNs, we found more naïve T cells entered the LNs from the blood compared to memory. We hypothesize that an unknown mechanism leads to the selective retention of memory T cells in FtDKO LNs.
Behavior and function of CD11c+ cells within the brain during chronic Toxoplasmosis (44.8)
Authors: John Beena , Harris Tajie , Ricart Brendon , Tait Elia , Hammer Daniel , Weninger Wolfgang , Hunter Christopher
Published: January 2023

Under normal conditions the brain is an immune-privileged site, however during toxoplasmic encephalitis (TE), there is significant increase in the proportion of T cells and antigen presenting cells (APCs) within the brain. Using a combination of real time imaging techniques, transgenic reporter mice, and recombinant parasites, the current studies have characterized the in vivo phenotype and function of CD11c+ cells present within the brain during this infection. These approaches have revealed the presence of an extensive network consisting of microglia and dendritic cells (including myeloid, lymphoid and plasmacytoid subsets) during TE. The CNS resident CD11c+ cells are functional APCs, as revealed by their ability to stimulate naïve antigen specific T cells in vitro. Further, real time imaging studies showed that these cells interacted with antigen specific T cells (for short durations) suggesting in vivo antigen presentation capabilities. The present studies have also identified a distinct chemokine receptor expression pattern on CD11c+ cells isolated from the brain, suggesting the importance of these receptors in trafficking of these cells into the inflamed brain. Using an adoptive transfer system, we show that the migration of DCs into the brain is Pertussis toxin sensitive. This latter model provides an opportunity to define the chemokine receptors responsible for the recruitment and retention of DCs in the brain.
Exhausted CD8 T cells become unresponsive to activation by innate cytokines and bystander bacterial infections (45.12)
Authors: Ingram Jennifer , Zajac Allan
Published: January 2023

During many chronic infections T cell responses initially develop, but succumb to exhaustion, as they lose their ability to produce interferon-γ (IFNγ) in response to antigen. Combinations of interleukins (IL)- 12, 18, and 21 have been shown to induce the antigen-independent production of IFNγ by prototypic memory CD8 T cells; therefore, we evaluated the sensitivity of exhausted lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells to activation with these cytokines. Our findings show that following acute infection, effector CD8 T cells respond synergistically to stimulation by these cytokines, but this responsiveness is abolished in exhausted T cells. To evaluate mechanisms responsible for this loss of responsiveness, we examined the expression of the cognate cytokine receptors. Whereas effector and memory T cells express the IL-18R, exhausted T cells downregulated the IL-18R but expressed higher levels of the IL-21R. To evaluate the biological consequences of this decreased sensitivity, we challenged cohorts of LCMV infected mice with the intracellular bacterial pathogen Listeria. Although LCMV-specific memory CD8 T cells vigorously respond to the bacterial infection, this responsiveness is severely compromised during protracted or chronic LCMV infections. Collectively, our findings show that exhausted cells downregulate IL-18R and become desensitized to activation by innate cytokines, rendering them unresponsive to secondary bacterial infections.
Rheumatoid Arthritis latent tuberculosis infection identification based on the cellular and humoral specific response to rHSP-x, Mycobacterium tuberculosis antigen (45.13)
Authors: Guerreiro Reis Michelle , Tanus Silva Daniela , Silva Bruna , Fouad Rabahi Marcelo , Junqueira-Kipnis Ana Paula
Published: January 2023

We previously showed that recent latently tuberculosis infected individuals could be identified by their antibody response against rHspx, an antigen related to latent/early tuberculosis. Here, using different tests we tried to identify latency among individuals with Rheumatoid Arthritis (RA). Twenty-two RA patients were classified by tuberculin skin test (TST) and tested using IgM ELISA to rHspx, specific rHspx TCD4+IFNγ+ quantified by flow cytometry(FC) and Elispot TB (TSPOT.TB). A cut off was established for each test and those who had a positive response were considered possible latently infected individuals. Sixteen healthy controls (HC) were also classified and tested. TST, here considered as a golden standard test for latency, identified 18% of the RA individuals latently infected, while specific TCD4+IFNγ+ recognized 72,7%, ELISA 63,6% and Elispot 27,2%. These results show incongruent identification of TB latency by several tests among RA patients. When combining two tests, the identification of latent tuberculosis among RA individuals changed: FC/ELISA identified 50%, FC/Elispot 18%, ELISA/Elispot 13,6%. The last two associations work the same way as TST. However, FC and ELISA were better than TST in identifying latently infected among RA patients. These results suggest that it may be useful an association of tests (FC and ELISA) to discriminate latently infected individuals among RA patients from an endemic area.
Seoul virus alters proinflammatory and antiviral responses of macrophages from Norway rats (45.14)
Authors: Li Wei , Au Rebecca , Klein Sabra
Published: January 2023

Hantaviruses cause persistent infection, upregulation of regulatory responses, and downregulation of proinflammatory responses in reservoir hosts. To determine whether hantaviruses suppress the inflammatory activity of antigen presenting cells, we infected bone marrow-derived macrophages (BMDMs) from Norway rats with Seoul virus (SEOV). The expression of MHCII and CD80, production of IL-6, IL-10, and TNF-α, and expression of Ifnβ were attenuated after infection. Stimulation of BMDMs with poly I:C prior to SEOV infection increased the expression of activation markers and production of inflammatory cytokines and suppressed SEOV replication. Infection with SEOV also suppressed LPS-induced activation of BMDMs, including NF-κB signaling and production of proinflammatory cytokines. Because inhalation is a major route of hantavirus transmission between rodents, with alveolar macrophages providing the first line of defense against inhaled pathogens and playing a pivotal role in the initiation of infection-induced inflammatory response, we are currently investigating whether rat alveolar macrophages response to SEOV infection in a similar manner as BMDMs and whether SEOV infection of alveolar macrophages enhances regulatory responses, including TGF-β production. Hantavirus-induced inhibition of inflammatory responses of macrophages may be one mechanism contributing to the persistence of virus in reservoir hosts.
Quantitative bioimaging of WRvFire and of IHDJ-Luc vaccinia virus dissemination in mice and the effects of prophylactic and therapeutic treatments with IgG and antiviral. (45.18)
Authors: Kapnick Senta , Zaitseva Marina , Scott John , Meseda Clement , Nielsen Henriette , Merchlinsky Michael , Weir Jerry , Golding Hana
Published: January 2023

Whole body bioluminescence imaging was employed to test the effects of IgG-based therapies and Cidofovir on lethality and virus dissemination in BALB/c mice challenged intranasally with 10^5 pfu of recombinant vaccinia viruses (VV) expressing luciferase, WRvFire or IHDJ-Luc. Both viruses induced lethality within 6-8 days, with slightly different kinetics of replication in internal organs. Using statistical analysis of 80 animals/group we determined that replication of IHDJ-Luc (increased Extracellular Enveloped Virion compared to WR) rose faster and peaked earlier than WRvFire (predominantly Intracellular Mature Virion) in the liver and spleen. In the nasal cavity, WRvFire persisted at significantly higher levels than IHDJ-Luc. Vaccinia immunoglobulin for intravenous injection (VIGIV) administered at day -2 protected 100% of animals from WRvFire and IHDJ-Luc infections. Sym002 (26 anti-vaccinia human mAbs) completely protected mice from both challenges at 300-fold lower dose than VIGIV. A single treatment of Cidofovir (100 mg/kg) at day 1, 2, or 3 p.i. fully protected mice from WRvFire challenge. Complete protection conferred by treatments correlated with significant reductions in bioluminescence in nasal cavity or lungs and in both the liver and spleen between days 1-5. Our data demonstrated that despite some differences observed in the kinetics of VV dissemination, infections with these viruses were similarly susceptible to prophylactic and therapeutic treatments.
Development of a sensitive serodiagnostic assay for human Lyme disease based on immunodominant epitopes of an in vivo expressed microbial surface antigen (45.19)
Authors: Coleman Adam , Pal Utpal
Published: January 2023

Lyme disease (LD) is a common bacterial infection transmitted by the ticks belonging to Ixodes scapularis complex. The diagnosis of LD is complicated by several factors, including unnoticed exposure and variation in patient presentation. Serology is currently the primary laboratory means of diagnosis in suspected LD cases, and the development of new diagnostics is an important goal of LD research. Central to this effort is the identification and characterization of new immunodominant antigens to serve as serodiagnostic markers. Borrelia burgdorferi, the causative agent of LD, encodes BBK07, an outer membrane lipoprotein thought to be an important antigen expressed during infection. We show that BBK07 gene expression is selectively induced during mammalian infection when compared to B. burgdorferi grown in vitro or in ticks. We show that BBK07 is surface-exposed on the bacterium, and that an antibody response to BBK07 is detectable throughout the murine infection. We use recombinant BBK07 protein to dissect the early (IgM) and late (IgG) immune response over the course of infection. A synthetic overlapping peptide library of BBK07 was used to identify the most immunogenic epitopes of the antigen using a human LD serum panel. We used a combination of immunodominant peptides to develop a specific and sensitive assay for the diagnosis of LD. These data suggest possible development of a BBK07 peptide-based serodiagnostic kit for the detection of human LD.