bluuebunny/crossref_metadata_2025 huggingface dataset:
TIPE2 serves as a molecular switch of phagocytosis during infection (37.12)
Authors: Wang Zhaojun , Johnson Derek , Sun Honghong , Gus Yael , Fayngerts Svetlana , Xue Fan , Ruan Qingguo , Chen Youhai
Published: January 2023

Phagocytosis is essential for host defense against microbial pathogens and clearance of apoptotic cells. TIPE2 (TNF-α-induced protein 8 like-2) is a newly described inhibitor of immune receptor signaling. We report here that TIPE2 knockout (KO) mice were resistant to lethal Listeria monocytogenes infection, cleared the bacteria more rapidly and effectively, and produced lower levels of inflammatory cytokines than wild type (WT) mice. TIPE2 KO macrophages had enhanced phagocytic activity as compared to WT macrophages. TIPE2 regulated phagocytosis at multiple steps, including internalization and phagosome maturation as well as bacteria killing. Importantly, Toll-like receptor activation dramatically downregulated TIPE2 expression while significantly enhanced phagocytic activity of macrophages. These results indicate that TIPE2 may function as a molecular switch of phagocytosis during infection. In response to pathogens, down-regulation of TIPE2 promotes phagocytosis, which in turn eliminates the pathogens.
Mycobacterium tuberculosis and TLR2 agonists inhibit induction of type I IFN and MHC-I antigen cross processing by TLR9 (37.15)
Authors: Simmons Daimon , Canaday David , Liu Yi , Boom W. Henry , Harding Clifford
Published: January 2023

Dendritic cells (DCs) cross process exogenous antigens and present them by MHC-I molecules to CD8+ T cells specific for antigens from viruses and bacteria such as Mycobacterium tuberculosis (Mtb). Unmethylated CpG DNA signals through TLR9 to induce type I interferon (IFN-α/β), which enhances MHC-I antigen cross processing, but lipoproteins that signal through TLR2 do not induce IFN-α/β. We observed that Mtb, which expresses agonists of both TLR9 and TLR2, did not induce IFN-α/β or cross processing by murine DCs. Furthermore, Mtb and TLR2 agonists inhibited induction of IFN-α/β and DC cross processing by CpG DNA. Exogenous IFN-α/β effectively enhanced cross-processing of Mycobacterium bovis BCG expressing OVA, bypassing the inhibition of induction of endogenous IFN-α/β. Inhibition of the response to one TLR by another may affect the ultimate response to pathogens like Mtb that express agonists of multiple TLRs, including TLR2 and TLR9. This mechanism may contribute to immune evasion and explain why IFN-α/β provides little contribution to host immunity to Mtb. On the other hand, downregulation of certain TLR responses may benefit the host by preventing detrimental excessive inflammation that may occur in the presence of persistent infection.
Critical link between intracellular responses and innate autoimmunity in ischemia-reperfusion injury (37.39)
Authors: Lee Haekyung , Ko Eun Hee , Balroop Javi , Richardson Arlan , Zhang Ming
Published: January 2023

Reperfusion of the ischemic tissue may lead to acute ischemia/reperfusion (I/R) injury. Two theories are suggested for the mechanism of I/R injury. One involves intracellular responses to ischemia (e.g. reactive oxygen species, ROS), while the other involves extrinsic acute inflammation manifested as natural IgM-mediated innate autoimmunity. Whether the ROS-mediated mechanism plays a role in natural IgM-mediated mechanism is still unknown. We studied this important question by using transgenic (tg) mice overexpressing antioxidant enzyme, superoxide dismutase (SOD1) in a murine model of intestinal ischemia. If the intracellular response contributes to the extrinsic inflammation, we predict that inhibiting ROS mechanism will protect animals from natural IgM-mediated I/R injury. Our results showed that SOD1 tg have a significant reduction in intestinal I/R injury compared to wild-type littermates. In addition, the IgM mediated inflammatory response is blocked in SOD1 tg. We further explored whether cell membrane damage is the critical link between the generation of intracellular ROS and exposure of self antigens leading to the innate autoimmune response. A cell membrane stabilizer was applied to WT mice prior to I/R surgery, and mice had a significantly reduced I/R injury with blocked IgM-mediated complement activation. Therefore, our current study identified a critical link between the intracellular response to I/R injury and the first step in the innate autoimmune response.
Conjugation of a TLR5 agonist to the Plasmodium falciparum Circumsporozoite (CS) protein enhances murine and human Dendritic cells (DC) interaction in vitro and increases protective immunity following intranasal immunization in mice (37.4)
Authors: Carapau Daniel , Nacer Adela , Mitchell Robert , Meltzer Abby , Shaw Alan , Frevert Ute , Nardin Elizabeth
Published: January 2023

A needle-free malaria vaccine has been developed based on intranasal (I.N) immunization with a recombinant P. falciparum circumsporozoite (CS) protein conjugated to the TLR5 agonist flagellin. Mice immunized I.N, but not subcutaneously, with flagellin-modified CS developed sporozoite neutralizing antibodies that protect against challenge with a transgenic rodent parasite expressing P. falciparum CS repeats. To better understand the role of innate immune cells in the induction of protective immunity, we analyzed the interaction of flagellin-modified CS with murine bone marrow-derived dendritic cells (DC) and human monocyte-derived DC in vitro. The fusion protein was rapidly taken up by DC and remained detectable in the cytosol as long as 24 hours post antigen pulse. A murine DC line (D1), and a sub-population of human DC, matured and expressed increased levels of CD40 and CD86 following stimulation with flagellin-modified CS. In I.N. immunized mice, the Nasopharyngeal-associated Lymphoid Tissue (NALT) was enlarged 2-fold over naïve NALT, and increased levels of CD11c+ DC were observed by confocal microscopy and FACS analysis. NALT cell subpopulations exhibited a higher B:T cell ratio than those observed in lymph nodes. A better understanding of innate and adaptive immune responses elicited in NALT by TLR ligand-modified CS protein will help identify critical immune parameters required for the induction of sporozoite specific immunity that protects from malaria infection.
Modulation of pro-inflammatory mediators by IL-10 in mouse J774 macrophages stimulated with rMOMP-498 of Chlamydia trachomatis (37.5)
Authors: Yilma Abebayehu , Singh Shree , Taha Murtada , Dennis Evida , Mishra Manoj , Gunn Karyn , Dennis Vida
Published: January 2023

We have recently constructed several recombinant major outer membrane proteins (rMOMP) of Chlamydia trachomatis, as potential vaccine candidates. In vitro characterization of the rMOMP-498 (gene fragment corresponding to amino acids 187-344) revealed that it stimulated the production of IL-6, IL-12p40, IL-1β, TNF-α and IL-10 in mouse J774 macrophages. Since IL-10 is a modulator of macrophage activities, we hypothesized that IL-10 induced by rMOMP-498 in macrophages may serve to control inflammation during the initial phase of a C. trachomatis infection. To address our hypothesis we added exogenous mouse IL-10 to J774 cells stimulated with rMOMP-498. IL-10 decreased the production of IL-6, IL12p40 and TNF-α in these cells. Removal of endogenous IL-10 with neutralizing anti-IL-10 Ab in cultures stimulated with rMOMP-498 in part increased the levels of inflammatory mediators as compared to the control Ab. Analyses of CD80, TLR2, TLR4, and CD86 receptors on cells stimulated with rMOMP-498 alone or combined with IL-10 showed no differences in receptor expression levels between the stimulants, suggesting that these receptors are not involved in the IL-10 down-regulatory activities in rMOMP-498-stimulated macrophages. Overall, our data show that IL-10 may be important in the innate immune response to rMOMP-498 as a vaccine candidate. Current studies are underway to identify the mechanism of IL-10 control of the inflammatory response in macrophages.
A regulatory double negative - NKT and NK cell cross-talk regulates viral-specific CD4+ T cell responses. (38.9)
Authors: Raynor Jana , Lin Adora , Sholl Allyson , Van Kaer Luc , Mattner Jochen , Hoebe Kasper , Hildeman David
Published: January 2023

Natural killer T cells (NKT) express a T cell receptor, restricted to CD1d molecules, as well as natural killer (NK) cell markers. Two subsets of CD1d restricted NKT cells have been described, type I and type II. Although both subsets of NKTs are transiently activated during viral infection, little is known of their role in controlling virus-specific immune responses. Here, we assessed the role of NKT cells in the CD4+ T cell response to viral infection using a lymphocytic choriomeningitis virus (LCMV) central nervous system (CNS) infection model. We found the virus-specific CD4+ T cell response was significantly decreased in LCMV-infected CD1d-/- mice. Further, virally infected CD1d-/- mice had significantly reduced serum type I IFN and increased NK cells. Interestingly, viral-specific CD4+ T cell responses were normal in mice deficient in type I NKT cells, suggesting that type II NKT cells were critical for promoting CD4+ T cell responses. Finally, depletion of NK cells in CD1d-/- mice completely rescued their CD4+ T cell responses. Together, these results suggest a double negative regulatory circuit whereby NKT cells negatively regulate NK cells, which in turn negatively regulate CNS antiviral CD4+ T cell responses. These data also suggest that innate immune cells exert multilayered control over the magnitude of the CD4+ T cell response.
ICAM-1 deficiency results in enhanced maintenance of effector CD8 T cells following infection (39.3)
Authors: Cox Maureen , Bullard Daniel , Zajac Allan
Published: January 2023

CD8 T cell responses are critical for the control of intracellular pathogens, and one hallmark of an effective T cell response is the development of a memory T cell pool which can rapidly respond to reinfection. It is known that the presence of polyfunctional pathogen-specific T cells correlates with more effective protection from persistent infections as well as rechallenge; however the factors that lead to the development of these cells are still poorly understood. Interactions mediated by intercellular adhesion molecule 1 (ICAM-1) have been demonstrated to impact the quality of the T cell response, by enhancing both antigenic and cytokine signals. Utilizing mice deficient for ICAM-1, we investigated the effect of this molecule on T cell priming and the response to acute infections. No pronounced differences were observed in the effector T cell response to acute lymphocytic choriomeningitis virus (LCMV)-Armstrong infection in ICAM-1 deficient animals, however greater numbers of virus-specific cells displaying an effector phenotype (KLRG-1hi CD127lo IFNγ+ IL-2-) were maintained in ICAM-1 deficient mice at memory time points. These ICAM-1 deficient mice exhibited reduced expansion of virus-specific CD8 T cells following secondary challenge when compared to wild type mice, potentially due to already elevated numbers in ICAM-1 null mice. These data indicate a critical role for ICAM-1 in the development of immunological responses to viral pathogens.
Pulmonary Orthopoxvirus infection compromises a murine OVA peptide immune response in a dose-dependent manner (39.8)
Authors: Hrusch Cara , Lipscomb Mary , Wilder Julie , Lyons C.
Published: January 2023

Cowpox virus (CPXV), a close relative of variola virus that causes smallpox, can suppress the immune system through a large array of immunosuppressive gene products. We developed a murine model in which DO11.10 T cells specific for an OVA peptide were transferred into BALB/c mice to assess the impact of a pulmonary CPXV infection on DO11.10 T cell proliferation in lung draining lymph nodes following intranasal OVA peptide delivery. High and low-dose CPXV infections were compared. Both doses lead to clinical illness, but the high dose is lethal. A high-dose infection reduced DO11.10 T cell proliferation, but a low-dose infection did not. At the time that proliferation of T cells was assessed (6d),15±1% of lung dendritic cells (DCs) were infected at the high-dose, but only 5±1% of DCs at the low-dose. At both doses, infected and uninfected DCs had decreased expression of co-stimulatory molecules. We speculated that the lung microenvironment created by infection, rather than direct infection of the DCs, suppressed DC immune function. In support, we found that bronchoalveolar lavage fluid and supernatant derived from lung homogenates from infected mice suppressed the function of uninfected lung DCs in vitro. Furthermore, the suppressive activity was more highly concentrated in lungs from high-dose infected mice. These studies strongly suggest that orthopoxvirus infections create an immunosuppressive microenvironment that compromises the host pulmonary immune responses.
How do airborne fungal spores avoid immune recognition? (40.1)
Authors: Bayry Jagadeesh , Aimanianda Vishukumar , Bozza Silvia , Kniemeyer Olaf , Perruccio Katia , Elluru SriRamulu , Clavaud Cecile , Paris Sophie , Brakhage Axel , Kaveri Srini , Romani Luigina , Latgé Jean-Paul
Published: January 2023

Every day we inhale thousands of tiny fungal spores (conidia), originating from many different fungal species such as Aspergillus, Cladosporium, Penicillium and Alternaria. Although these spores are packed with antigens and allergens, yet their inhalation does not continuously activate our innate immune cells or provoke inflammatory responses. By using several fungal inhabitants of the air-borne microflora including the human opportunistic fungal pathogen Aspergillus fumigatus, in vitro assays using human dendritic cells (DC) and alveolar macrophages (AM) and in vivo experiments using a murine model we show that a protein surface-layer called ‘rodlet’ on the dormant conidia masks their recognition by the immune system and hence prevents immune response to conidia. Interestingly, rodlet proteins are found to be immunologically inert and failed to activate either human or mouse DC and AM and did not induce adaptive T responses in vivo upon transfer of rodlet-pulsed DC. Furthermore, dormant conidia that lacked rodlet layer induced strong immune responses in vitro and in vivo despite being dormant in nature. All these results indicate that rodlet layer on dormant conidia masks the immunogenic molecules and prevent immune aggression against inhaled conidia.
Staphylococcus aureus specific memory CD8 T cells in dairy cows (40.12)
Authors: Mullarky Isis , Waithaka Mwangi , Bharathan Mini
Published: January 2023

Staphylococcus aureus is a versatile pathogen that causes disease in both humans and animals. An increase in occurrence of nosocomial infections and chronic mastitis caused by antibiotic resistant strains warrants the need for an effective vaccine. This study assessed the presence of S. aureus specific memory T cells (Tm) in previously infected cows using CD14+ monocyte derived dendritic cells (DC). Results indicate that DC are able to uptake and recognize S. aureus antigens through various pattern recognition receptors (PRRs). Blocking of endocytic pathways using inhibitors confirmed S. aureus uptake by DC. S. aureus stimulated DC showed increased cell surface expression of MHC I and II and cytokine production suggesting activation and antigen presentation. An initial screening study for Tm in cows previously infected with S. aureus showed a significant increase in lymphocyte proliferation compared to control animals. To characterize the proliferating population of Tm, CFSE labeled lymphocytes were co-cultured with irradiated or live S. aureus stimulated autologous DC from previously infected cows identified as CD4, CD8 and WC1+ γδ T cells by flow cytometry. Proliferated CD4 and CD8 T cells were further characterized for memory markers, CD45RO and CD62L. Being the first study to show the presence of S. aureus specific CD8 Tm cells, our next step is to identify the S. aureus antigens that initiate T cell clonal expansion.
Immunobiological Signaling in response to S. aureus Extracellular adherence protein (Eap) (40.27)
Authors: Liubchenko Ganna , Ostapchenko Liudmyla
Published: January 2023

Staphylococcus aureus is an important pathogen that affects a significant part of population and presents a major clinical problem. Extracellular adherence protein (Eap) is expressed on the surface of S. aureus and has been implicated in endothelial adhesion and evasion of host immune mechanisms. This study demonstrates several novel signaling properties elicited by Eap in B lymphocytes. Our results indicate the involvement of ICAM-1-mediated signaling pathway(s) that trigger previously uncharacterized rapid intracellular Ca2+ mobilization and protein phosphorylation (pTyr) events in B lymphocytes upon exposure to Eap. Furthermore, combined application of protein A and Eap to intact murine (C57BL/6) B lymphocytes induced co-clustering of the B cell receptor (sIgM) and ICAM-1 in immune synapse-like structures. This effect may be attributed to the previously described ability of protein A to bind sIgM and act as B cell superantigen. Thus, sIgM/ICAM-1 clusters may form in the absence of antigen and Eap may play arole in this process. We hypothesize that the ability of Eap to interfere with B cell receptor signaling regardless of antigen specificity may play a role antigenic mimicry and premature internalization of sIgM signaling complexes in B cells and thus help S. aureus to evade B cell immune responses.
Mannose lipoarabinomannan from Mycobacterium tuberculosis inhibits T cell migration to sphingosine-1-phosphate and promotes trapping of T cells in lung draining lymph nodes. (40.3)
Authors: Richmond Jillian , Lee Jinhee , Green Daniel , Kornfeld Hardy , Cruikshank William
Published: January 2023

Humans and mice that have been infected with Mycobacterium tuberculosis (MTB) develop lymphadenopathy in lung draining lymph nodes. To determine a possible mechanism for this, we examined human and mouse T cell migration to the lymph node chemoattractant CCL21 and to sphingosine-1-phosphate (S1P) following exposure to various TB components. Mannose lipoarabinomannan (ManLAM), a glycolipid from the MTB cell wall, did not alter T cell migration to CCL21; however, ManLAM did significantly inhibit T cell migration to S1P. As S1P is the primary factor required for egress from lymph nodes, this suggested a potential mechanism. To determine whether this effect could be established in vivo ManLAM was administered intratracheally to C57BL/6J mice. Accumulation of T cells was detected in lung-draining lymph nodes within 24 hr with a four to five fold increase in total T cell numbers. Accumulation patterns were similar to mice that had been infected with Erdman TB for 4 weeks, as both groups exhibited a preferential trapping of Th1 cells. Our data provide evidence that ManLAM may be a primary factor for MTB-associated lymphadenopathy during MTB infection. We hypothesize that alteration in T cell homing patterns induced by ManLAM would affect normal host immune responses and thus potentially facilitate infection by the bacterium.
Mannose lipoarabinomannan from Mycobacterium tuberculosis promotes production of soluble TNF Receptors I & II. (40.4)
Authors: Richmond Jillian , Schuller Elizabeth , Lee Jinhee , Remick Daniel , Kornfeld Hardy , Cruikshank William
Published: January 2023

Serum and BAL fluid from mice that had been infected with Erdman Mycobacterium tuberculosis (MTB) for 6 months were analyzed with an in-house multiplex ELISA system. Out of the panel of cytokines tested, the cytokines that exhibited the largest increase in both serum and BAL as compared with uninfected mice were soluble TNF Receptors I & II. This is consistent with current literature demonstrating that soluble TNF Receptors increase over time during TB infection. To determine the potential source of the TNFRs as well to identify the stimulant, murine T cells were treated ex-vivo with a panel of MTB components. It was determined that the MTB cell wall component Mannose lipoarabinomannan (ManLAM) (10 or 100ng/ml) was able to induce a significant increase in sTNFRI & II following treatment overnight. To confirm the ability of ManLAM alone to induce TNFRs, serum and BAL from mice that had received 25ug purified ManLAM intratracheally exhibited elevated sTNFRI & II 24h following instillation. ManLAM was not able to induce increases in TNF alpha in vitro or in vivo, which is consistent with current literature. These data indicate the ability of ManLAM alone to induce elevated soluble TNFR production from T cells, thereby shifting the balance between TNF alpha and TNF soluble receptors. Based on the role of TNF in granuloma formation and maintenance during MTB infection, this could potentially contribute to the inability of mice to form proper granulomas.
Hiding in Plain Site: The Immune System, Tuberculosis, and Antibiotic Resistance (41.6)
Authors: Stephansky Mark
Published: January 2023

This summer I worked in the Cruikshank lab at the Boston University School of Medicine. The Cruikshank research team determined that mannose lipoarabinomannan, (ManLAM), a cell wall component of M. tuberculosis can decrease S1P sensitivity of activated T cells as well as to alter production of selected cytokines. These alterations likely affect the normal immune response by changing T cell homing patterns and decreasing production of response cytokines thereby increasing the mTB’s chances of survival and optimal infection. This research inspired me to create a high school biology unit. This unit is designed to link the study of the immune system with evolution through a closer examination of mTB, particularly, its two main forms: latent TB infection and active TB disease. The unit includes teacher presentations and demonstrations, an internet activity entitled TB in the Media, and laboratory activities. Students are first introduced to the basic functions of the immune system through its defense against Mycobacterium tuberculosis infection. Then, students work through a simulated ELISA to see its importance in diagnosing disease. Finally, we turn to the emergence of multi-drug resistant mTB as students carry out a laboratory exercise on bacterial transformation. Students present a summary of their learning through the creation of a pamphlet/poster where they investigate a specific disease and describe its symptoms, treatments, suggested causes and immune system interactions.
Interferon-γ and Interferon-γ receptor knockout mice are not created equal: examination of cellular signaling and control of alphavirus infection. (42.12)
Authors: Schultz Kimberly , Lee Eun Young , Griffin Diane
Published: January 2023

Interferon-γ (IFN-γ) is a central mediator of the immune response to viral encephalitis. IFN-γ knockout (GKO) and IFN-γ receptor knockout (GRKO) mice have been used interchangeably to examine signaling pathways that control virus infection. GKO mice are unable to produce IFN-γ whereas the alpha chain of the IFN-γ receptor is deleted in GRKO mice. Upon alphavirus infection, both strains of mice succumb to infection with a similar timecourse, however GRKO mice show increased inflammatory cell infiltration and MHC-II-positive cells in the central nervous system compared to GKO mice. Cellular signaling through the mutated receptor was further examined by using mouse embryonic fibroblasts (MEFs). GRKO MEFs were unable to control virus replication in response to IFN-γ treatment. Additionally, signaling through major antiviral IFN-γ receptor pathways (i.e. STAT1) in response to IFN-γ treatment was suppressed in GRKO MEFs as compared to control MEFs. However, inflammatory signaling was intact in GRKO MEFs (i.e. TNFα). These studies indicate that, the mutated IFN-γ receptor can mediate some signaling in response to IFN-γ and therefore, GRKO mice cannot be used interchangeably with GKO mice. This signaling may account for the increased inflammatory cell infiltration in GRKO mice as compared to GKO mice during alphavirus infection.
Type I interferon increases the sensitivity of human immunodeficiency virus (HIV)-specific CD8+ T lymphocytes to CD95/Fas-mediated apoptosis (42.22)
Authors: Fraietta Joseph , Mueller Yvonne , Do Duc , Yang Guibin , Jacobson Jeffrey , Katsikis Peter
Published: January 2023

Innate immune responses elicited during acute and chronic HIV infection are critical to the control of viremia. Based on their well established antiviral effects, the production of type I interferon (IFN-alpha and IFN-beta) has long been considered a protective mechanism in HIV infection. However, it is possible that excessive IFN-alpha and IFN-beta production during HIV infection may impair HIV-specific CD8+ T cell responses and/or contribute to the loss of CD4+ T cells. We show here that CD4+ and CD8+ T lymphocytes from healthy donors and HIV+ patients are sensitized to CD95/Fas-induced death when pre-incubated with type I interferon for 72 hours. Importantly, IFN-alpha increased the sensitivity of HIV-specific CD8+ T cells from HIV+ individuals to CD95/Fas-mediated apoptosis. Apoptosis of CMV-specific and EBV-specific CD8+ T cells from the same patients was not augmented when exposed to IFN-alpha and subsequently stimulated with anti-CD95 antibody. Since CD4+ T cells and HIV-specific CD8+ T cells in HIV infection are known to be sensitive to CD95/Fas-mediated apoptosis, our studies suggest that these lymphocytes may be initially sensitized to CD95/Fas-induced death by type 1 interferon during acute HIV infection. The production of these anti-viral cytokines during chronic infection may further compromise CD4+ and HIV-specific CD8+ T cell survival.
Analysis of the assembly mechanism for multimeric lamprey VLRB antibodies (43.19)
Authors: Li Jianxu , Herrin Brantley , Guo Peng , Cooper Max
Published: January 2023

Instead of the immunoglobulin-type antigen receptors in jawed vertebrates, the surviving jawless vertebrates use variable lymphocyte receptors (VLRs) that are constructed with variable leucine-rich repeat (LRR) sequences to recognize antigens. Two VLR genes in lamprey and hagfish are somatically assembled during lymphopoiesis using a gene conversion-like mechanism in which highly diverse LRR flanking sequences are copied in serial fashion to construct mature VLRs. Lamprey have been shown to secrete polymeric VLRB antibodies that consist of 8-12 identical disulfide-linked subunits. The overall structure of VLRB antibodies resembles that of IgM antibodies in that both are composed of 4-6 dimers. Previous studies have shown that eight cysteines in the C-terminal hydrophobic region are involved in assembly of the multivalent VLRB antibodies through formation of disulfide bonds. We have characterized a panel of VLRB Cys to Ser mutants using site-directed mutagenesis to determine which cysteines are used for disulfide bond formation and VLRB polymerization. Two or more disulfide bonds were found to be essential for VLRB dimerization and polymerization. Our data further suggest that the carboxy-terminal cysteines 5, 6, 7, and 8 form the cornerstone for VLRB dimerization and polymerization, whereas cysteines 1, 2, 3, and 4 contribute to the stability of the polymer structure.
Effects of aging on B and T cell immunity in nonhuman primate (NHP) species (43.20)
Authors: Arteaga-Cortes Lourdes , Mayeux Jessica , Cantwell Angelene , Pasquali Amanda , Stacy Sue , Dube Peter , Rice Karen , Kraig Ellen
Published: January 2023

The ability of humans and rodents to mount effective immune responses declines with age. Yet, when old baboons (19-24 years) were immunized with LcrV, a protective antigen from Y. pestis, antibody titers were significantly higher than in immunized young animals (2½ years) [JI 181:109]. It may be that baboons are unusual, aging without losing immune competence or alternatively, that LcrV is enhancing humoral immunity in older animals. To address this, a second NHP species was tested; young (4 years), middle-age (11 years), and older (18-24 years) rhesus macaques were immunized with LcrV. Significant humoral responses were observed in all age groups, so like baboons, there was no loss of immune reactivity with aging. However, the greatly enhanced response in older baboons was not recapitulated in rhesus. To test the possibility that LcrV is unique, young (6 years), middle-age (12-13), and older (21-23) baboons were immunized with a second plague antigen, F1; a more typical response was seen whereby F1 antibody titers elicited in younger baboons were higher than those seen in the older cohorts. T cell proliferative and cytokine responses to these antigens have been assessed and no definitive age-associated changes were noted. On the other hand, circulating Tregs and some serum cytokines were higher in older NHPs. These experiments provide novel insights into aging effects on primate immunity and suggest a unique function for plague LcrV in enhancing responses in old animals.
Embryo vaccination using novel adjuvant formulation and Eimeria profilin-induced protective immunity against E. maxima infection (43.4)
Authors: Lillehoj H. , Lee S. , Jang S. , Hong Y. , Dominowski D.
Published: January 2023

Due to increasing concerns on the use of antibiotics in animal production system, development of alternative strategies for disease control in livestock and poultry is becoming important. Avian coccidiosis is caused by several distinct species of Eimeria which undergo intracellular development in different parts of gut reducing significant nutrient absorption. Our previous studies have shown significant protection against coccidiosis using recombinant Eimeria vaccines in laboratory models. This study was carried out to investigate the effect of immunizing 18-day-old embryos with Eimeria profilin protein in novel adjuvant formulation which is composed of Quil A, cholesterol, DDA, and carbopol (QCDC). Chickens which were immunized in ovo with Eimeria profilin/QCDC vaccine produced significantly higher antibody and cytokine responses, and showed better protection against live challenge infection with E. maxima compared to the control chickens which were immunized with Eimeria profilin protein alone. This study demonstrates the first immunological evidence to show immunoenhancing role of QCDC in poultry.
Titanium used in the prosthetic devices may act as an adjuvant driving Th2 cell differentiation (44.15)
Authors: Mishra Pankaj , Rozo Christina , Joseph Benevenia , Gause William
Published: January 2023

Inflammation contributing to periprosthetic osteolysis following joint replacement is a potential complication of arthroplastic surgery. The components of the prosthetic device that elicit inflammation and the characteristics of the innate and adaptive immune response that contribute to inflammation are as yet little studied. The prosthetic material Titanium (Ti) was examined for adjuvant properties. Mice were immunized with the Ti particle (12.5 mg) + LPS-free OVA protein (50 µg) and found to elicit elevated levels of serum IgE, IgG1 and IgG2a compared to OVA alone. CFSE labeled transgenic OVA-specific DO11.10 CD4+ cells were transferred to recipient BALB/c mice and inoculated i.p two days later with Ti plus OVA323-339 peptide or with OVA323-339 peptide alone. Five days after inoculation, CFSE fluorescence analysis showed that donor CD4+, KJ1.26+ DO11.10 T cells had undergone markedly more cell cycling in recipient mice receiving Ti+OVA compared to recipient mice administered OVA alone. OVA-specific ELISPOT assays of cell suspensions from draining lymph nodes showed marked increases in DO11.10 T cell IL-4 and lesser elevations in IFN-γ expression from mice inoculated with Ti + OVA compared to mice immunized with OVA peptide alone. These studies suggest that Ti can act as an adjuvant in vivo to induce naïve T cell differentiation to cytokine producing effector T cells indicating that this inert nanoparticle has potent inflammatory characteristics.
TNF-α Induces Endothelial Cell Expression of Intercellular Adhesion Molecule-1 (ICAM-1) Required for Recruitment of Antigen-Primed CD8 T Cells to Mediate Responses in the Skin (44.2)
Authors: Kish Danielle , Baldwin William , Fairchild Robert
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten sensitization and challenge of the skin. Elicitation of CHS is initiated when hapten-primed CD8 T cells producing IFN-γ and IL-17 traffic to the challenge site and simulate endothelial cells (EC) to produce CXCL1/KC. KC is required for the neutrophil recruitment directing CD8 T cell infiltration through the EC where they are activated to induce the edema of CHS. Factors required for this KC production were tested. Hapten-immune CD8 T cells stimulated hapten-labeled EC to produce KC in vitro and this was blocked by antibodies to IFN-γ, IL-17, and ICAM-1 but not to TNFa. Immunohistochemical staining and qRT-PCR analyses indicated that hapten challenge induced ICAM-1 expression on skin challenge site EC and TNFa expression in both naïve and sensitized mice within 4 hrs of hapten challenge. Transfer of hapten-primed T cells to naïve wild-type but not ICAM-1 knockout mice induced KC production at 6 hrs after hapten challenge. In vivo treatment with antibodies to TNF-α or to ICAM-1 at the time of hapten challenge eliminated KC production, neutrophil infiltration, and CHS. Anti-TNF-α mAb significantly reduced ICAM-1 as well as IFN-γ, and IL-17 mRNA expression in the challenged skin of sensitized mice. Thus, hapten challenge stimulates TNFα production that induces the EC expression of ICAM-1 required for hapten-primed T cells activation to produce the IFN-γ and IL-17 that induces the EC to produce KC.
Non-T cells are required for a poised Th17 phenotype and elevated IL-17 secretion by T cells from type 2 diabetes patients. (44.3)
Authors: Jagannathan Madhumita , McDonnell Marie , Shin Hyunjin , Hasturk Hatice , Nikolajczyk Barbara
Published: January 2023

Type 2 Diabetes (T2D) has recently been identified as a chronic inflammatory disease with changes in immune cell function. Th17 cells and their major product, IL-17, have been implicated in multiple chronic inflammatory diseases. However, the relationship between Th17s and T2D has not been reported. Pro-inflammatory cytokines hyper-secreted by T2D patient monocytes promote Th17 activation. These data suggest a functional link between Th17s and monocytes in T2D. To identify such a link, we first showed that fresh ex vivo T cells from T2D patients have molecular signatures of Th17 cells. T2D T cells stimulated in the context of peripheral blood mononuclear cells (PBMCs) activated Th17 signature genes and elevated IL-17 secretion. In contrast, LPS stimulation of PBMCs from T2D patients failed to elevate IL-17 secretion, indicating that increased monocyte cytokine production, characteristic of T2D, does not further stimulate Th17 cells. Unexpectedly, continuous co-culture of T cells with the non-T cell fraction was required to maintain elevated Th17 function. These data indicate a crucial role for non-T cells in maintaining the Th17 phenotype and elevating IL-17 production in T2D. We conclude the interaction between T cells and non-T cells, most likely monocytes, promotes inflammation in T2D through previously unappreciated mechanisms.
Clonotypic structure of human memory alpha-T cell repertoires to influenza in elderly individuals (45.20)
Authors: Gil Anna , Yassai Maryam , Naumov Yuri
Published: January 2023

The age-associated thymic atrophy and reduced repertoires of naïve T cells could be confounding factors of high morbidity and mortality of older populations due to the seasonal influenza infections. Given that influenza A viral epitope M158-66 induces robust CD8 T cell responses among middle-aged populations, we examined clonotypical α-TCR repertoires among older blood donors. Previously we reported that α-TCR repertoires in middle-age individuals are polyclonal where M158-66 specific T cells were selected as memory cells due to expression JA42 α -chains with poly-G/S/A runs in the CDR3. In the present study we compare α-TCR repertoires between middle-aged and older individuals in respect to Vα family usage and clonotypical diversities. Using CDR3α- spectratyping and sequencing, we found that middle-aged adults possess stable polyclonal response to influenza where diverse JA42 CD8 T cells are distributed among eleven Vα families and VA27 family was dominated in vitro response. In contrary, with aging such dominance was still present in some but not all individuals. Two subjects responses to M158-66 was mediated by VA12.1 family, which represents minor component in the middle-aged cohort. In addition to reduced diversity of VA families, the older subjects posses reduced diversity of JA42 clonotypes. Therefore, we concluded that influenza re-exposure during lifetime induces clonotypical exhaustion of T cells leading to reshaping of T cell repertoire.
Protective effects of monomeric and dimeric forms of the 2G12 neutralizing antibody against HIV-1 infection in a modified humanized mouse model (45.28)
Authors: Luo Xin , Lei Margarida , Feidi Rana , West Anthony , Balazs Alex , Yang Lili , Baltimore David
Published: January 2023

Broadly neutralizing anti-HIV antibodies are rare and have proved hard to elicit with any immunogen. Designer antibodies, on the other hand, are unnatural and have to be introduced through the engineering immunity approach. Yet, it is important to evaluate both types of anti-HIV antibodies in vivo to identify potential HIV vaccine candidates. In this study, we have used a modified humanized mouse model to investigate the protective effects of 2G12 versus a mutant of the antibody that favors dimer formation. Our results showed that while 2G12 was able to prevent CD4 loss and decrease HIV viral load at concentrations higher than 70 μg/mL upon HIV infection, the dimer mutant showed a similar potency at concentrations between 10~15 μg/mL, possibly due to an increased avidity and/or stability of the dimer versus the monomer. We also found that an initial concentration of 2G12 dimer at around 40 μg/mL through one single injection was more effective than a sustained dimer concentration of 10 μg/mL to prevent CD4 loss and to delay the increase of viral load. HIV escape mutants were detected in the presence of either monomeric or dimeric forms of 2G12, indicating that both antibodies have pressured HIV to mutate and the addition of other antibodies such as b12 may be required to provide a sterilization effect against HIV. Collectively, we have identified a more potent variant of 2G12 in vivo, which can be used as part of an antibody cocktail to prevent HIV infection.
Nanogel antigen delivery system for adjuvant-free intranasal vaccines (46.16)
Authors: Nochi Tomonori , Yuki Yoshikazu , Takahashi Haruko , Sawada Shinichi , Mejima Mio , Kohda Tomoko , Harada Norihiro , Kataoka Nobuhiro , Kong Il Gyu , Sato Ayuko , Tokuhara Daisuke , Kurokawa Shiho , Takahashi Yuko , Tsukada Hideo , Kozaki Shunji , Akiyoshi Kazunari , Kiyono Hiroshi
Published: January 2023

Nanotechnology is an innovative method of freely controlling nanometer-sized materials. The recent outbreak of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce antigen-specific both mucosal and systemic immune responses. However because of lacking the effective antigen delivery system to aero-digestive mucosa, co-administration of mucosal adjuvant mediating protective but also undesired immunity is continuously needed. Here we developed a novel intranasal vaccine-delivery system with a nanometer-sized hydrogel (“nanogel”) consisting of a cationic type of cholesteryl group-bearing pullulan (cCHP). A nontoxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells (DCs) after its release from the cCHP nanogel. Vigorous botulinum neurotoxin A (BoNT/A)-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid (TT) with cCHP nanogel induced strong TT-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.
Comparison of antibody responses induced by a virus-like particle-based vaccine upon intramuscular, pulmonary, and vaginal delivery. (48.14)
Authors: Hunter Zoe , Smyth Hugh , Durfee Paul , Chackerian Bryce
Published: January 2023

Virus-like particles (VLPs) can be used as platforms to increase the immunogenicity of antigens that are normally poorly immunogenic, such as self-antigens. We have developed a bacteriophage VLP-based vaccine that targets the HIV coreceptor CCR5 and shown that, following intramuscular administration, it induces high-titer serum antibodies against CCR5 that can inhibit HIV/SIV infection. We have also assessed the ability of this vaccine to induce mucosal immune responses, which is desirable as many pathogens, including HIV, infect at mucosal surfaces. In rats, both intramuscular and pulmonary immunization induced high titer IgG and IgA against the vaccine in the serum, but only the aerosolized vaccine induced CCR5-specific IgA locally in the lung and remotely in the genital tract. We will also present data comparing the magnitude of this "secondary" response in the genital tract to the IgA response invoked following direct immunization of the genital mucosa with different VLP platforms, including VLPs from a virus (human papillomavirus) that normally infects in the genital tract and bacteriophage Qβ VLPs. Our results provide a general method for inducing broad systemic and mucosal antibody responses using VLP-based immunogens.
The effect of altering the homeostatic regulation of alveolar macrophages (48.7)
Authors: Lyonga Daphne , Wissinger Erika , Hussell Tracy
Published: January 2023

The lungs are constantly bombarded by inhaled antigen, both innocuous and harmful. In order to avoid a detrimental over-exuberant response to the myriad of harmless particles inhaled daily, the lung micro-environment is highly regulated. Alveolar macrophages are held in a state of tight suppression by CD200R, TGF-B and IL-10 signalling, and by the nuclear macrophage regulator PPAR-gamma. An absence of any of these immune modulators can severely impact on this tight macrophage regulation. When challenged with LPS in vitro, CD200R-/- alveolar macrophages produce higher levels of IL-6 and TNF compared to wild type controls; and an absence of PPAR-gamma in alveolar macrophages leads to Th1-associated pulmonary inflammation. Here we block IL-10R signalling at homeostasis using an IL-10R mAb and aim to investigate how this impacts on the phenotype of murine alveolar macrophages, whether they become more or less regulatory, classically or alternatively activated. Preliminary data suggests that an absence of IL-10R signalling affects the phenotype of alveolar macrophages at homeostasis. During a subsequent influenza infection and importantly, after IL-10R blockade has ceased, we see a heightened immune response in the IL-10R mAb-treated animals relative to controls. This indicates that even temporarily altering lung homeostasis can affect the outcome of a subsequent infection.
Cell Contact Facilitated Delivery of IL-2 from Dendritic to T Regulatory Cells (49.14)
Authors: Kulhankova Katarina , Rouse Todd , Nasr Mohamed , Field Elizabeth
Published: January 2023

CD4+CD25+ T regulatory lymphocytes (Tregs) play an important role in maintaining immune homeostasis and require IL-2 for their development, expansion, maintenance, and function. Because Tregs do not produce IL-2, they must obtain it from a paracrine source. The identity of a cell providing IL-2 to Tregs is controversial, and the manner in which IL-2 is delivered to Tregs is not well understood. We found that Treg in vitro inhibitory function depends on IL-2 produced by dendritic cells (DCs). By confocal microscopy of in vitro cultures we documented that CD25 (IL-2Rα) concentrated at the JAWSII (DC hybridoma) - RD6 (Treg hybridoma) contact sites, internalized DC material co-localized with CD25 in the RD6, and Tregs internalized IL-2 from transfected DCs. Tregs cultured with WT, but not IL-2 KO DCs, up-regulated CD25 and Foxp3 in contact-dependent manner. To examine IL-2 trafficking between DCs and T cells we examined co-cultures of IL-2mCherry transfected DCs and Treg or T effector cells. DC-Treg contact significantly enhanced IL-2 uptake by Tregs and resulted in up-regulation of CD25 on Tregs. In contrast, Teffs did not acquire IL-2 from DCs in culture. Altogether, these data suggest that 1) DCs are an important source of IL-2 for Tregs, and 2) delivery of IL-2 from DCs to Tregs is Treg-specific and facilitated by close cell-cell contact. These novel findings may further our understanding of requirements for Treg biology during the homeostatic maintenance and immune response.
T cell-intrinsic IL-10 signaling negatively regulates Th1 immunity by restraining IL-12 responsiveness of effector T cells. (49.20)
Authors: Yap George , Wilson Douglas , Marple Andrew , Kamanaka Masahito , Flavell Richard
Published: January 2023

The principal mechanism by which IL-10 mediates downregulation of T cell immunity is through its action on antigen presenting cells, including dendritic cells and macrophages. Whether or not T cells themselves are important physiologic targets of IL-10 regulation has remained unclear. Here we report that transgenic mice expressing a dominant-negative acting, truncated form of the IL-10 R alpha chain in T cells (CD4-IL-10DNR transgenic mice) exhibit enhanced Th1 responses to the parasite, Toxoplasma gondii. CD4-IL-10DNR transgenic mice were considerably more susceptible to peroral T. gondii infection and exhibited enhanced production of IFN-gamma, but not IL-17 or IL-12, indicating that T cells are important targets of IL-10 downregulation in a Th1 response in vivo. To investigate the mechanism of IL-10’s negative effect on Th1 cytokine, we interrogated the ability of Th1 effector cells from the peritoneal cavity of T. gondii-primed mice to respond to IL-12. Our analysis showed that normally, effector cells are highly responsive to IL-12, but rapidly lose signaling competence 12 hrs following the challenge with parasites in vivo. Remarkably, effectors from CD4-IL-10DNR transgenic mice retain their IL-12 responsiveness under the same circumstances. Our results clearly establish the importance of T-cell intrinsic IL-10 signaling for the control of Th1 responses in vivo and shed light on a new mechanism for the antagonistic interactions between IL-10 and IL-12.