bluuebunny/crossref_metadata_2025 huggingface dataset:
TYROSINE PHOSPHORYLATION OF MAL IS ESSENTIAL FOR SIGNAL TRANSDUCTION AND IS BLOCKED IN ENDOTOXIN TOLERANCE (89.8)
Authors: Piao Wenji , Fitzgerald Katherine A. , Medvedev Andrei E.
Published: January 2023

Posttranslational modifications of TLRs, adapter proteins and IRAKs are important in signalosome assembly. This study examined the role of tyrosine phosphorylation for signal-transducing functions of MAL, and analyzed if induction of endotoxin tolerance inhibits LPS-induced MAL tyrosine phosphorylation. Overexpression of wild-type MAL in 293T cells led to its constitutive tyrosine phosphorylation and activation of p38 MAPK, IκB-α degradation, and IL-8 mRNA expression. In contrast, tyrosine-deficient Y86A, Y106A, and Y159A MALs showed impaired abilities to signal upon overexpression in 293T cells, and exerted dominant-negative effects on LPS-driven NF-κB reporter activation in HEK293/TLR4/MD-2 cells. Mutations of the Y187, Y195, and Y196 did not affect MAL signaling and did not confer upon it the ability to act as a dominant-negative inhibitor of LPS signaling. LPS stimulation of HEK293/TLR4/MD-2 cells and human monocytes resulted in tyrosine phosphorylation of endogenous MAL, which was significantly impaired in LPS-tolerant cells. LPS-inducible association between endogenous MAL and Bruton’s tyrosine kinase (Btk) was observed in cell extracts obtained from medium-pretreated human monocytes, whereas LPS failed to induce Btk recruitment to MAL in endotoxin-tolerant cells. Thus, phosphorylation of MAL is required for TLR signaling, and is inhibited in endotoxin tolerance.

This work was supported by NIH grant AI-059524 (to A.E.M.)
Unique processes of antibody assembly and systemic regulation enhance affinity maturation in salmonids (90.15)
Authors: Bromage Erin , Ye Jianmin , Kaattari Stephen
Published: January 2023

The salmonid humoral immune response is mediated primarily by the induction of a single antibody isotype, an 800 kDa tetrameric molecule. However, this antibody molecule possesses considerable structural diversity due to non-uniform disulfide cross-linking of its monomeric subunits. The potential association of antibody affinity with the degree of inter-monomeric disulfide bonding was examined. Three distinctly different routes were taken to examine this relationship; 1) fractionation and analysis of high and low affinity antibodies from individual immune sera by differential immunoadsorption, 2) selective induction of high vs. mixed affinity antibodies in vitro from the same population of cells, and 3) analysis of total serum antibodies undergoing affinity maturation in vivo. All three methods demonstrated a direct relationship between affinity and the degree of disulfide polymerization, with the most cross-linked forms possessing the highest affinity. Utilizing the transfer of labeled immunopurified antibody to naive hosts we have discovered that the persistence of these structural isomers in vivo is also regulated, with the preferential removal of the more reduced forms from circulation. Thus it appears that control of intramolecular disulfide polymerization of the antibody molecule combined with the selective removal of lower affinity antibodies from the circulation, is critical to achieve a mature antibody response in salmonid species.
CM1 ligation induces apoptosis via Fas/FasL interaction in ovarian cancer Caov-3 cells (92.11)
Authors: Hur Dae-Young , Park Ga-Bin , Hur Indo , Kim Hyunjin , Kim Yeong-Seok , Song Hyunkeun , Lee Hyun-Kyung , Kim Yoon Berm , Lee Wang Jae
Published: January 2023

In the previous study, we found that EBV-transformed B cells expressed CM1 (centrocyte/-blast marker 1), which was known as another possible marker of GC B cell and induced apoptosis of GC B cells. In the present study, we found that ovarian cancer cell line (Caov-3 cells) expressed CM1. And CM1 stimulus induced apoptosis in Caov-3 cells. So, we designed to elucidate the requirement of a death receptor, its’ role in subsequent activation of caspase-8 and the role of mitochondria in the induction of apoptosis. CM1 ligation with anti-CM1 mAb induced FasL expression in Caov-3 cells. Furthermore, anti-Fas blocking antibody, ZB4, blocked CM1-mediated apoptosis effectively in Caov-3 cells. Increased mitochondrial membrane permeabilization, which was measured by DiOC6, was observed. Caspase-8 inhibitor, z-IETD effectively blocked CM1-induced cell death. RT-PCR analysis showed up-regulated expression of Bax and Bad and down-regulated expression of Bcl-2. Western blot analysis showed down-regulated expression of procaspase-8 and PARP. The release of cytochrome c and translocation to nucleus of endoG and AIF were showed by confocal micoscopy. Based on these findings, we concluded that CM1 induced apoptosis could be mediated by Fas/FasL interaction in Caov-3 cells and CM1 might act as an effective apoptosis inducer in ovarian cancer cells.
Characterization of peptide binding by heat shock proteins upon antigen induction and under cellular stress (93.4)
Authors: Kelly John B , Binder Robert J , Srivastava Pramod K
Published: January 2023

Purified preparations of selected heat shock proteins (of the hsp90 and hsp70 families) are actually non-covalent complexes of HSPs with peptides that contain the MHC Class I-binding octa/nonamers. Several purified HSP-peptide complexes have been shown to specifically activate the immune system through delivery of the antigenic peptides to antigen-presenting cells (APC) for cross-presentation, but little is known about the formation of intracellular HSP-peptide complexes. The aim of this study is to quantify the relationship between MHC I-binding peptides (and their extended precursors) and chaperone proteins under two immunologically important states: induction of a neoantigen and cellular stress. We present here the development of a novel cell line, PIK23, which employs the modified version of ovalbumin, KOVAK, under the control of a synthetic, inducible promoter system. Using this cell line, we have identified MHC-I binding peptides associated with gp96 and Hsp70, and have begun to quantify these peptides in relation to the translation of the antigenic protein and state of cellular stress.
Monoclonal antibodies to HLA class I restricted influenza epitopes (93.8)
Authors: Gonzalez Melva L. , Wahl Angela R. , Hildebrand William H.
Published: January 2023

The immune response to viruses involves the presentation of peptide epitopes in the context of the human leukocyte antigen (HLA) class I molecule. The HLA class I molecule consists of a heavy chain, a light chain, and a peptide of 9–10 amino acids that is positioned in a groove formed by the class I heavy chain. Identification of HLA class I-restricted peptide epitopes that distinguish a virus-infected cell from a non-infected cell is positioned to facilitate studies of antigen presentation, viral diagnostics, and the design of viral vaccines/therapeutics. Recently, we have directly discovered epitopes presented by the class I HLA of influenza-infected cells. Using HLA tetramers and single chain trimers (SCT) as immunogens, we have produced monoclonal antibodies (mAb) to HLA class I molecules carrying viral peptide epitopes. The peptide/HLA specificity of the mAbs was confirmed by ELISA and by flow cytometry. The results of these experiments demonstrate that mAb specific for HLA/influenza peptide epitopes could be useful for studying the dynamics of viral epitope presentation and may represent a tool for targeting viral infected cells.
The role for DNA methylation in differentially regulating IL-7 receptor alpha expression by human T cells (94.22)
Authors: Kang Insoo , Hwang Kyung-A , Kim Ki-Chan , Kim Hang-Rae
Published: January 2023

IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8+ T cells expressing IL-7Rahigh and IL-7Ralow with different cell survival responses to IL-7. Although these CD8+ T cell subsets have differential IL-7Ra gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and associated with inactivation of gene expression. Thus, we investigated the role for DNA methylation in differentially regulating IL-7Ra gene expression in human CD8+ T cells and Jurkat T cells. IL-7Rahigh CD8+ T cells had decreased methylation in the IL-7Ra gene promoter compared to IL-7Ralow CD8+ T cells and Jurkat T cells with low levels of IL-7Ra. Treating Jurkat T cells with 5-aza-2¡-deoxycytidine (5-aza-dC), which reduced DNA methylation, increased IL-7Ra expression. Plus, the unmethylated IL-7Ra gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that a novel mechanism is involved in differentially regulating IL-7Ra expression in T cells through altering DNA methylation of the IL-7Ra gene promoter, and that methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.
Dissecting the role of miRNAs in Natural Killer (NK) cells (94.27)
Authors: Savan Ram , Owens Garrison , Munroe David J , Young Howard A
Published: January 2023

Natural Killer cells play a vital role in the control of cancer and microbial infections. One of the mechanisms by which these cells act is through the production of interferon (IFN)-gamma in response to many extracellular signals. These extracellular signals have effects on transcriptional and post transcriptional regulation of IFN-gamma gene expression. MicroRNAs may be one of the important agents involved in post-transcriptional regulation of IFN-gamma gene expression. Furthermore, through bioinformatic analysis, putative miRNAs that might target IFN-gamma gene for gene regulation have been identified. Using miRNA arrays, we have screened for the expression of miRNAs in NK-92 cells stimulated for 4h with IL (interleukin) -2, IL-12, IL-15, IL-18, IL-21, IFN-alpha, PMA and a combination of IL-12 and PMA. Our studies by miRNA profiling indicate that miR29a, miR27 and miR181, through bioinformatics are known to target IFN-gamma, are modulated by signals that up-regulate IFN-gamma mRNA. This data forms a base-line to further study the role of microRNAs in NK cell biology. These studies will utilize lentiviruses to overexpress specific microRNAs or disrupt the miRNA pathway in order to elucidate the role of miRNAs in NK cell gene expression.
Temporal regulation of Stat5 activity determines cell differentiation program (94.3)
Authors: Fujii Hodaka , Hoshino Akemi
Published: January 2023

Erythropoietin (Epo) plays essential roles in erythropoiesis including expression of globin genes. It has been shown that Stat5 activation is important for Epo-induced globin gene expression. Although Stat5 is activated by various cytokines including interleukin (IL)-3 and IL-2, only Epo and a small number of cytokines induce erythroid differentiation and globin gene expression. It is not well understood how cytokine-specific gene expression is ensured. Here, by using a reporter gene system to monitor transcriptional activity of Stat5, we showed that sustained activation of Stat5 induces globin gene expression. We also showed that Epo but not IL-3 supports sustained activation of Stat5. These results underscore the importance of temporal regulation of Stat activity in the regulation of cytokine-specific gene expression and cell differentiation. IL-3 or IL-2 stimulation actively inhibits Epo-induced globin gene expression. The acidic region (the A-region) of the IL-2 receptor β chain was essential for this inhibition, suggesting a role of the A-region-coupled signaling molecules in inhibition of globin gene expression. The present study may provide insight into the regulatory mechanism of cytokine-specific gene expression.

This work is supported by NIH grant R01 AI059315 to H.F.
PLIC-1 is an inhibitor of TLR signaling (94.7)
Authors: Wang Tianyi , Biswas Nabanita
Published: January 2023

Toll-like receptors (TLRs) are key innate immune receptors that recognize non-self pathogens and hence trigger host responses. We have recently identified an ubiquitin-like protein named protein linking integrin associated protein to cytoskeleton 1 (PLIC-1) that interacts with the cytoplasmic domain of TLR4. The interaction between TLR4 and PLIC-1 was verified by co-immunoprecipitation. Further studies suggest that PLIC-1 may be interacting with multiple TIR domain containing proteins. PLIC-1 decreased the production of TRIF in an overexpression system. Consequently, PLIC-1 was found to inhibit the TRIF-mediated activation of both NF-kB and IFN-b pathways in macrophages. Lastly, reduction of endogenous PLIC-1 by shRNAi delivered via retroviral infection enhanced the cellular responses to LPS or poly I:C. Together, our results have implicated PLIC-1 as a negative regulator of TLR pathway.

This work is supported by the University of Pittsburgh faculty start-up fund.
Distinct Effects of IL-12 and IFN-a/b on CD8 T cell Effector Functions (95.18)
Authors: Ramos Hilario Jose , Farrar J. David
Published: January 2023

During viral infections, CD8 T cells act to promote immunity by eliminating virally infected cells. To accomplish this they employ immediate effector functions and promote the generation of memory populations with the capacity to respond to secondary challenge. The innate cytokines IL-12 and type I interferon (IFNa/b) have been shown to participate in both the effector and memory CD8 T cell response. However, the independent and direct pathways they activate have not been well characterized. We have recently examined the direct and independent roles of these cytokines on naïve human CD45RA+ CD27+CD8+ T cells developing in vitro. In these studies, we observed that IL-12 but not IFNa/b promote the generation of CD8 T cells capable of secreting IFN-g upon secondary challenge. Further, this correlates to enhanced CXCR3 expression, a chemokine receptor which homes cells to the site of primary infection. In contrast IFNa/b enhanced the expression of CCR7 which plays an important role in the trafficking of cells to the secondary lymphoid organs. Both IL-12 and IFNa/b augmented the expression of the cytolytic factors perforin and granzyme, however, polarization with both IL-12 and IFNa/b promoted enhanced cytolytic killing than polarization by either cytokine alone. Taken together, these results demonstrate that IL-12 and IFNa/b participate in both overlapping as well as unique pathways in human CD8 T cell development which may function to promote diverse immune responses dependent on the pathogen or cytokine milieu encountered during infection.
Adsorbed Fibrinogen Elicits Cytokine Production by Dendritic Cells (95.27)
Authors: Thacker Robert I. , Retzinger Gregory S.
Published: January 2023

Fibrinogen (fgn) readily coats surfaces, and has been implicated in inflammation, but its role is still ill-defined. Available evidence suggests it is adsorbed fgn that operates during inflammation. To better elucidate the role of adsorbed fgn, the protein was first bound to the surface of microscopic polymeric beads and then placed in the presence of human dendritic cells (hDC’s). Immature DC (iDC’s) produce more IL-6, IL-8 and MIP-1β and mature DC’s (mDC’s) produce more IL-6, MCP-1 and MIP-1β in response to adsorbed fgn than they do in response to solution phase fgn. IL-6 and MIP-1β production by iDC’s are reduced by anti-TLR4, anti-CD18 and anti-CD11c antibodies. IL-8 production by iDC’s is reduced by antibodies against CD11b, CD18 and TLR4. Only anti-TLR4 antibodies reduce IL-6 and MIP-1β production by mDC’s, while MCP-1 production is reduced by antibodies against CD18, CD11c and TLR4. The increased production of these inflammatory mediators by hDC’s in response to adsorbed fgn might enhance immune cell recruitment, thereby potentiating inflammation. These data also suggest that certain fgn receptors play a role in fgn-elicited production of IL-6, IL-8, MCP-1 and MIP-1β by hDC’s.
Altered T lymphocyte response to respiratory syncytial virus in CCR6−/− mice (96.3)
Authors: Kelley Lara , Schaller Matthew , Lukacs Nicholas
Published: January 2023

The CC chemokine receptor 6 is unique in the chemokine family because it binds to only a single ligand, CCL20. CCL20 is expressed in epithelial cells in tissues such as skin, gut mucosa and lungs. Studies using CCR6 deficient mice revealed a defect in dendritic cell positioning in the gut mucosa, suggesting a role for CCL20-CCR6 in leukocyte homeostasis and proper positioning of these cells in mucosal tissues. In a mouse model of asthma, CCR6−/− mice showed reduced airway hyperreactivity and less mucus production. The potential pathological role for CCL20-CCR6 led us to investigate this receptor-ligand pair in a mouse model of viral infection caused by respiratory syncytial virus (RSV), a negative strand RNA virus that can cause severe complications in infants, the elderly and immunocompromised individuals. We found that CCR6−/− mice had lower numbers of activated CD4 and CD8 T cells in the lungs at days 6 and 9 post-RSV infection, as well as reduced Th1 and Th2 cytokines in both lungs and lymph nodes. Interestingly, we also found that these mice had more efficiently cleared virus by day 6 post-infection. This suggests the involvement of a cell type other than T lymphocytes that in the absence of CCR6 is able to impart viral immunity.

This research is supported by NIH grant AI36302
Integrin alpha-1 deficiency does not affect resistance to pulmonary Mycobacterium tuberculosis infection in mice but leads to necrosis within granulomatous lesions (97.12)
Authors: Taylor Jennifer Lynn , Bielefeldt-Ohmann Helle , Pozzi Ambra , Izzo Angelo
Published: January 2023

The hallmark of Mycobacterium tuberculosis infection is the granuloma, a highly dynamic immune structure that ‘walls off’ the bacilli during chronic infection. Here, we examined if α1β1 integrin is required in the development and maintenance of the granulomatous structure during infection using an α1-integrin knockout mouse (α1-null). The α1β1 integrin is expressed on activated macrophages and T cells, and interacts with collagen molecules in the extracellular matrix (ECM). To determine if the α1-integrin molecule is necessary for trafficking of activated T cell within the lungs, cells from the lungs of infected α1-null mice were stained for CD4, CD44 and CD62L. The numbers of activated and IFNγ-producing CD4-positive T cells were increased in the α1-null strain during chronic infection. While there were no differences in bacterial load between the α1-null and wild type mice, histology revealed striking differences in granuloma formation and structure. MMP-9 and TNFα were decreased in the α1-null mice and alveolar septae were distinct and markedly thickened. By day 180, there were regions of necrosis within the granulomas, characterized by cellular and nuclear debris. These results reveal that while the α1β1 integrin is not a factor required for resistance to M. tuberculosis, it does play a role in granuloma formation and integrity of the structure.

Funding: NIH AI52040 and Colorado State University CRC
CRUCIAL ROLE OF CD44 IN LIVER INFLAMMATION (97.6)
Authors: kimura kiminori , nagaki Masahito , Kakimi Kazuhiro , Nakayama Toshinori , Taniguchi Masaru , Moriwaki Hisataka
Published: January 2023

CD44 on inflammatory cells is well known as an adhesion molecule and blocking CD44 is thought to be one of the therapeutic strategies for inflammatory diseases. Here, we showed that CD44 knock-out (KO) mice exhibit suppressed liver inflammation during the early phase after carbon tetrachloride (CCL4) injection due to reduced infiltration of inflammatory cells and reduced cytokine production. In contrast, these mice showed severe liver inflammation with increased numbers of apoptotic hepatocytes at the late phase. This induction of hepatocyte apoptosis was triggered by reduced NF-kB activity, which was induced by low concentrations of inflammatory cytokines in the liver. Through this inflammatory response, the kinetics of NKT cells were distinctive, with increased infiltration into the liver by greater proliferation and migration, which was independent of the CD44 pathway. Furthermore, MIP-2 derived from NKT cells contributed to exacerbation of liver injury, since neutralization of MIP-2 significantly protected against hepatocyte apoptosis in CD44KO mice. These findings suggest that blocking of CD44 as a therapeutic approach for inflammatory liver diseases might be specific for particular immune systems in the liver.
The Role of LFA-1 in T cell Trafficking and Functions in Demyelinating Disease (99.5)
Authors: Dugger Kari J , Zinn Kurt R , Bullard Daniel C , Barnum Scott R
Published: January 2023

Multiple sclerosis (MS) is characterized by autoimmune damage to central nervous system (CNS) myelin, resulting in impaired nerve function. Integrins such as lymphocyte function-associated antigen-1 (LFA-1) are thought to be important in leukocyte trafficking to the CNS and disease development. Studies using monoclonal antibodies to LFA-1 in experimental autoimmune encephalomyelitis (EAE), the animal model for MS, have provided conflicting results regarding the role of LFA-1 in EAE. To overcome the limitations of the antibody-based approach, we used LFA-1-deficient mice and a transgenic mouse (T-lux) with a hCD2 promoter that drives expression of the luciferase gene specifically in T cells thereby providing a novel method of a real-time depiction of T cell trafficking patterns. We found that both LFA-1-deficient and LFA-1 hemizygous mice do not develop clinically significant EAE and have markedly reduced leukocyte infiltration into the CNS. Encephalogenic LFA-1-deficient T-lux cells adoptively transferred into wild type recipients do not localize to the CNS, whereas MOG-specific wildtype T-lux cells transferred to wildtype or LFA-1-deficient recipients, readily migrate to the CNS and draining lymph nodes and proliferate. These data demonstrate that LFA-1 on T cells is necessary for myelin-specific T cells to enter into the CNS and initiate disease.

Supported by NIH grant NS46032.
Developmental Control of Integrin Expression Regulates T Helper-2 Effector Homing (99.6)
Authors: Lee Seung-Hyo , Kheradmand Farrah , Corry David
Published: January 2023

Integrin CD18, a component of the lymphocyte function-associated antigen-1 (LFA-1) complex that also includes CD11a, is essential for Th2, but not Th1, cell homing, but the explanation for this phenomenon remains obscure. Here, we investigate the mechanism by which Th2 effector responses require the LFA-1 complex. CD11a-deficient T cells showed normal in vitro differentiation and function. However, Th2 cell-dependent allergic lung disease was markedly reduced in CD11a null mice and wild type mice given LFA-1 inhibitors, whereas control of infection with Leishmania major, a Th1-dependent response, was enhanced. In both disease models, recruitment of IL-4−, but not IFN-g, secreting cells to relevant organs was impaired, as was adhesion of Th2 cells in vitro. These diverse findings were explained by the markedly reduced expression of CD29, an alternate homing integrin, on Th2, but not Th1, cells, which precludes Th2 homing in the absence of CD11a. Thus, murine Th1 and Th2 cells utilize distinct integrins for homing, suggesting novel opportunities for integrin-based therapeutic intervention in diverse human ailments.
CXCR6 promotes atherosclerosis by supporting T cell homing, interferon gamma production and macrophage accumulation in the aortic wall (99.8)
Authors: Galkina Elena , Harry Brian , Ley Klaus
Published: January 2023

T cells are important in atherosclerosis, but very little is known about the mechanisms behind lymphocyte recruitment into atherosclerosis-prone aortas. We tested the hypothesis that CXCR6, a chemokine receptor that is expressed on a subset of T cells and natural killer T cells, is involved in lymphocyte homing into the aortas and modulates atherosclerosis. To investigate the role of CXCR6 in atherosclerosis, we bred CXCR6-deficient (CXCR6GFP/GFP) mice with apolipoprotein E-deficient (ApoE−/−) mice. CXCR6GFP/GFP/ApoE−/− mice fed a western diet for 17 weeks or a chow diet for 56 weeks had decreased atherosclerosis compared with ApoE−/−controls. Flow cytometry analysis of the aortas from CXCR6GFP/GFP/ApoE−/− mice showed that the reduction of atherosclerosis was accompanied by a decreased percentage of CXCR6+ T cells within the aortas. Short–term homing experiments demonstrated that CXCR6 is involved in the recruitment of CXCR6+ leukocytes into the atherosclerosis-prone aortic wall. The reduced percentage of CXCR6+ T cells within the aortas was associated with diminished production of IFNγ and reduced accumulation of CD11b+/CD68+ macrophages in the aortas. These data provide evidence for a functional role of CXCR6 in atherosclerosis by altering of the recruitment of CXCR6+ leukocytes and modulating of the local immune response within the aorta.

This work is supported by NHI HL 58108, 55798 and AHA 0525532U.
NK Cell Mediated Contact Sensitivity is Elicitable 1 Hour After Immunization and Depends on IFN-gamma and IL-12 Production (B101)
Authors: Askenase Philip W , Szczepanik Marian , Majewska Monika
Published: January 2023

NK cell mediated contact sensitivity (CS) has recently been described. We confirmed CS in SCID and RAG1−/− mice but not in SCID-beige mice without NK cell function. NK mediated CS was transferred with liver mononuclear cells and unlike conventional CS not with mixed lymph node and spleen cells. Positive selection with anti-DX5 beads confirmed the NK nature of the transferring cells. Participation of NKT cells and B-1 cells was ruled out with Jα18−/− and JH−/− mice. Remarkably, NK CS was elicitable 1-hr after immunization and with an early 2-hr component 1 day after LMNC transfer, and lasted at least 7 days. Adoptive cell transfer required MHC matching and IL-12 and IFN-γ−/− were involved in the mechanism of CS.

In conclusion, NK CS is a distinct entity. IL-12 and IFN-γ are involved in the effector mechanism of NK-mediated CS, similar to classical CS mediated by Th1 cells. The requirement for MHC matching may involve the specificity of the NK receptor, which in this case may be hapten altered self. In conclusion, NK cells provide a very rapid and long lasting, Ag-specific mechanism to elicit cell-mediated immunity, with features of innate and acquired immunity.

Supported by NIH grants AI-59801 and AI-07174 to PWA; and the Polish Committee of Scientific Research to MS.
Streptococcus pyogenes activates human plasmacytoid and myeloid dendritic cells (B123)
Authors: Veckman Ville , Julkunen Ilkka
Published: January 2023

Human peripheral blood contains two major dendritic cell populations, namely CD11c−CD123+ plasmacytoid DCs (PDCs) and CD11c+CD123− myeloid DCs (MDCs). Here we have compared PDC and MDC activation in response to Gram-positive Streptococcus pyogenes (group A streptococci) and influenza A virus. S. pyogenes stimulation induced the maturation of both DC types as evidenced by enhanced expression of co-stimulatory molecules and production of pro-inflammatory cytokines and chemokines. Furthermore, S. pyogenes-stimulated PDCs and MDCs activated naive CD4+ T cells and enhanced their Th1 type cytokine production. Influenza A virus infection induced rapid PDC activation whereas MDCs were extremely sensitive to influenza A virus-induced cell death. The most significant differences between DC types were seen in the production of IL-10 and IL-12, which were produced only by S. pyogenes-stimulated MDCs. Although S. pyogenes was able to induce PDC activation, only influenza A virus infection resulted in detectable IFN-α production. Our results show that depending on the infecting microbe the functions of PDCs and MDCs may be partially overlapping suggesting a considerable flexibility of the human DC system.
Generation of a protective T cell response following viral infection of the CNS is independent of either IL-12 or IL-23 (B141)
Authors: Held Katherine , Glass William , Orlovsky Yevgeniya , Shamberger Kimberly , Petley Ted , Branigan Patrick , Cunningham Mark , Benson Jacqueline , Lane Thomas
Published: January 2023

The functional role of IL-12 and IL-23 in generating effector T cells following viral infection of the central nervous system (CNS) was determined. Instillation of mouse hepatitis virus (MHV) into the CNS of C57BL/6 mice results in an acute encephalitis followed by a chronic demyelinating disease. MHV-infected mice express mRNA transcripts specific for IL-12p35 and IL-23p19 subunits within the brains. Antibody-mediated blocking of IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) did not reduce numbers of virus-specific T cells and treated mice cleared virus from the CNS demonstrating that blocking IL-23 does not affect the generation of a Th1 response. Therapeutic administration of anti-IL-23p19 to infected mice with established demyelination did not attenuate disease indicating IL-23 does not contribute to viral-induced demyelination. Treatment with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition of EAE, an autoimmune model of demyelination highlighting the importance of IL-23 in CNS autoimmune inflammation and disease. These data indicate that i) IL-12 and IL-23 signaling are dispensable in generating a protective response following CNS infection with MHV and ii) IL-23 amplifies disease in a model of autoimmune demyelination. Therefore, targeting of IL-23 for the treatment of autoimmune diseases may offer unique advantages by reducing disease severity without muting protective responses following viral infection.
Modulation of Adaptive Immune Responses in Chickens by Low Pathogenicity Avian Influenza Virus H9N2 (B142)
Authors: Xing Zheng , Cardona Carol J , Li Jinling , Tran Tu , Andrada Jason
Published: January 2023

Most low pathogenicity avian influenza (LPAI) viruses cause no or mild diseases in avian species. The H9N2 subtype are commonly detected LPAI viruses that have crossed the species barrier to infect humans, unlike most other LPAI viruses. We report in this study that chicken macrophages are susceptible to infection with H9N2 and H6N2 viruses and infection led to apoptosis. Additionally, in H9N2 virus-infected macrophages, major histocompatibility complex (MHC) antigens, class II in particular, were downregulated, which also occurred in the lungs of H9N2 infected chickens. Pro-inflammatory cytokines and chemokines were differentially regulated in both infected macrophages and lungs. Among the regulated genes, IL-4, IL-4R, and CD74 were downregulated, all of which are pivotal in the activation of CD4+ helper T cells and humoral immunity. Remarkably, in H9N2 virus-infected chickens the antibody response was largely suppressed. This is in contrast to what was observed in those infected with H6N2 virus. This finding suggests that the pathogenesis of H9N2 infection in chickens may involve humoral immune suppression. Modulation of MHC antigens and the subsequent impact on the immune responses in mammals infected with avian influenza viruses is largely unclear. These findings raise questions about how some LPAI viruses regulate avian immune responses and if they have similar effects on mammalian immune function.
Pgp (MDR1)-dependent resisrance of malignant cells towards natural killer (NK) cells is reversed by IL-2 and by the Pgp inhibitor KT-5720: implications on graft versus tumor (GVT) effects in adoptive immunotherapy (B144)
Authors: Nagler Arnon , Bar Iris , Ostrovsky Olga , Galski Hanan
Published: January 2023

P-glycoprotein (Pgp) encoded by the MDR1 gene, may render tumor resistant to NK and CTLs mediated immunotherapy (DLI). We assessed Pgp expression on tumors and their response to DLI & tried to reverse tumor resistance towards NK cells.

The KB-V1, Pgp over-expressing cell line was found significantly less sensitive to resting & IL-2 activated NK cells than its parental KB-3-1, Pgp non expressing cell line. NK-resistant Colo205 cell line expressed high level of Pgp RNA, while low expression level was found in NK-sensitive K562 cell line. The allele polymorphism C3435T of MDR1 is associated with phenotypic variations in Pgp expression, with high-, intermediate- or low-expression level of the CC, CT and TT genotypes, respectively. Both KB-V1 & Colo205 (MDR1 over-expressing and NK-resistant) cell lines carried the CC genotype while the K562 cell line (MDR1 low-expressing, and NK-sensitive) carried the TT genotype. Incubation of resting NK cells with IL-2 increases significantly the killing of both KB-3-1 & KB-V1 cell lines. The Pgp inhibitor KT-5720 increases the killing of KB-V1 cells (but not of KB-3-1 cells) by resting NK cells. In summary:

Low cytolytic response of tumor cells to NK-cells is associated with high Pgp expression and the CC genotype of the MDR1 geneIL-2 & the Pgp inhibitor, KT-5720, reverse Pgp-dependent resistance of tumors towards NK cellsExposure of NK-cells to IL-2 increases their cytolytic activity. The effect of IL-2 is larger on Pgp-high expressing- than on Pgp-non expressing-target cellsSince KT-5720 is a novel Pgp inhibitor this study may lead to future clinical application of KT-5720, to increase GVT effects in adoptive immunotherapy.
Memory T cells in the tumor-infiltrating lymphocytes for adoptive cell transfer therapy (B145)
Authors: Zhou Juhua , Nagarkatti Prakash , Robbins Paul F , Rosenberg Steven A , Nagarkatti Mitzi
Published: January 2023

It has been documented that immune memory has a critical role in mediating protection against infections as well as potentiating allergic and autoimmune diseases. Memory T cell populations in human anti-tumor tumor-infiltrating lymphocytes (TILs) for adoptive cell transfer have not fully been characterized. FACS analysis showed that T cells in the initial TIL microcultures generated from the tumor tissues of melanoma patients did not express CCR7, however, 67¡À5 % of T cells expressed CD27, 70¡À6 % of T cells expressed CD28 and 35¡À5 % of T cells expressed CD62L. As compared with the TIL microcultures before large-scale expansion, T cells in the administered TIL samples had significantly decreased levels of CD62L and CD27 expression. CD28 expression, however, did not change dramatically after large-scale expansion. These memory phenotypic T cells also expressed high levels of CD44. Furthermore, T cell expansion significantly decreased the percentage of CD8+CD44+ T cells. These results indicated that effector memory T cells were present in TIL samples and the maintenance of memory T cells in TILs may represent an important factor in effective adoptive cell transfer therapy.

(Supported by NIH grants R01AI053703, R01ES09098, R01 AI058300, R01DA016545, R01HL058641 and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.)±±
OX40 signals preferentially mediate the generation of effector memory but not central memory CD4+ T cells (B15)
Authors: Soroosh Pejman , Ine Shouji , Sugamura Kazuo , Ishii Naoto
Published: January 2023

Memory T cells can be divided into effector memory (TEM) and central memory (TCM) subsets based on their effector function and homing characteristics. Although previous studies have demonstrated that TCR and cytokine signals mediate the generation of memory T cells, it is unclear how the generation of the two memory subsets is regulated. We found that OX40-deficient mice showed a marked reduction in the number of CD4+ TEM cells, while the number of CD4+ TCM cells was normal. Adoptive transfer experiments using Ag-specific CD4+ T cells revealed that OX40 signals during the priming phase were indispensable for the optimal generation of the CD4+ TEM, but not the CD4+ TCM population. Furthermore, CD4+TEM cells that were generated in the absence of OX40 signals showed impaired cytokine production. Collectively, the present results indicate that the generation of functional CD4+TEM cells, but not TCM cells, is critically regulated by OX40 signals.
Defective CCR7 function in aging: implications for dendritic cell migration (B156)
Authors: Grolleau-Julius Annabelle , Harning Erin , Yung Raymond
Published: January 2023

We recently reported that bone marrow-derived dendritic cells (DCs) from old C57BL/6 mice are less effective than their young counterpart in inducing the regression of pre-established B16-OVA melanomas. However, the mechanisms for this are unclear. DCs migration to lymphoid organs is essential to effective DC anti-tumor activity and is regulated by interactions between CCR7 and its ligands CCL21 and CCL19. We therefore examined the effect of aging on DC migration, CCR7 expression and function. Young (3–6 months) and old (18–20 months) DCs were labeled with CFSE and CMPTX, and their in vivo migration quantified by fluorescent microscopy. Half as many aged DCs were present in the popliteal lymph nodes (p<0.025). DCs from aged mice express 50% less CCR7 compared to young DCs (p< 0.0005). Surprisingly, the reduced gene expression did not result in any significant change in CCR7 surface expression on DCs. Nevertheless, the reduced CCR7 gene expression correlated with the impaired in vitro migration of aged DCs in response to CCL21 (p< 0.05). Finally, we showed that aged DCs have impaired signaling following exposure to CCL21. In conclusion, our data showed that old DCs have defective in vivo migration that correlates with reduced CCR7 gene expression, as well as impaired in vitro CCL21 migratory response and signaling. These findings may contribute to the observed impaired DC tumor immunotherapeutic response in aging.
Ex vivo expansion of PR1-CTL elicited by autologous dendritic cells pulsed with PR1 peptide for adoptive immunotherapy of leukemia (B157)
Authors: Wang Changqing , Ma Qing , Wieder Eric , Lu Sijie , Molldrem Jeffrey J.
Published: January 2023

Cytotoxic T lymphocytes (CTL) specific for PR1, a HLA-A*0201-restricted peptide derived from proteinase 3, are capable of killing leukemia cells and contribute to the elimination of CML. Cytogenetic remission of CML after treatment with IFNα correlates with an expansion of PR1-CTL in patients. INFα is also an abundant cytokine produced by plasmacytoid DC which is critical for priming adaptive immune responses and MHC class I antigen cross-presentation. To determine whether a clinical trial with adoptively transferred PR1-CTL will benefit leukemia patients, we used INFα in combination with GM-CSF for ex vivo expansion of PR1-CTL and examined whether theses PR1-CTL can eliminate AML in the established NOD/SCID model. PBMC from HLA-A*0201+health donors were stimulated weekly with autologous DC pulsed with PR1 peptide in the presence of INFα and GM-CSF. By 3 weeks, there were 38.7% of PR1-CTL in the culture compared to 1.83% detected in the original sample using PR1-tetramer staining. We found that PR1-CTL generated in vitro can migrate to sites of disease and maintain their capacity to eliminate the human primary AML cells in NOD/SCID mice. Mice received AML alone had 72–88% blasts in a hypercellular marrow, whereas mice that received AML plus PR1-CTL had normal hematopoietic elements and only 10–11% blasts in a hypocellular marrow. We conclude that ex vivo expansion of PR1-CTL using this method is suitable for adoptive immunotherapy of AML.
Differential Type I Interferon Induction by Respiratory Viruses (B179)
Authors: Durbin Joan E. , Jewell Nancy A. , Vaghefi Negin , Mertz Sara E. , Akter Parvis , Peebles R. Stokes , Bakaletz Lauren O , Durbin Russell K. , Flano Emilio
Published: January 2023

Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the TLRs expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs, and it is not clear how this property impacts IFN-alpha/beta induction by this virus in vivo. In this study we compared type I IFN production by RSV and influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-alpha/beta production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-alpha/beta in response to either virus; but in pDC-depleted animals only type I IFN induction by influenza virus infection was impaired.

This work was supported by grants R01AI47226 and R01DC006468 to JED from the National Institutes of Health.
MKP-1, a critical regulator of the innate immune response to Gram-negative bacterial infection (B180)
Authors: Frazier Joshua , Wang Xianxi , Kuhlman Joshua , Liu Yusen
Published: January 2023

Mitogen-activated protein (MAP) kinases play a critical role in the innate immune response by inducing the transcription of pro-inflammatory cytokines. MAP kinase phosphatase (MKP)-1 is an archetypal member of the MKP family which inactivates p38 and JNK MAP kinases. We have previously shown that deficiency of MKP-1 predisposes experimental animals to mortality upon challenge with lipopolysaccharide, a Gram-negative bacterial endotoxin. However, the impact of MKP-1 deficiency in the setting of true Gram-negative bacterial infection has not been elucidated. We hypothesize that MKP-1 deficient animals will experience increased mortality from live bacterial infection compared to wild type animals. Age- and sex-matched wild type and MKP-1 deficient mice were injected intravenously with Escherichia coli at a dose of 2.5 x 107cfu/g body weight. After treatment, the animals were monitored for 48 h and mortality data collected for analysis. The mortality of the MKP-1 deficient animals was significantly higher than that of similarly treated wild type mice (p = 0.032). Our results indicate that MKP-1 deficiency confers a disadvantage to animals facing live Gram-negative infection. Thus, MKP-1 appears to play a critical role in down-regulating the inflammatory response to Gram-negative bacterial pathogens.
Activation of C5 via the lectin pathway of complement (B181)
Authors: Salvi Veena P. , Rajagopalan Rema , Kunkalla Kranthi , Rawal Nenoo
Published: January 2023

Activation of C5 via the lectin pathway of complement was analyzed by examining C5 cleaving properties of lectin pathway C3/C5 convertases. C4b-dependent C3/C5 convertase, ZymM1,C4b,C2a and EManM1C4b,C2a (M1 represents MBL-MASPs) assembled on zymosan or sheep erythrocytes coated with mannan (EMan) exhibited a weak affinity for C5 (Km = 2.7–5.3 μM). Very high affinity C5 convertases (ZymM1,C3bC4b,C2a and EManM1,C3bC4b,C2a) were generated when low affinity C4b-dependent C3/C5 convertases were allowed to deposit C3b by cleaving native C3. ZymM1,C3bC4b,C2a and EManM1,C3bC4b,C2a exhibited Km (4.9–8.3 nM) well below the normal concentration of C5 in blood (0.37 μM). Although, the Km, Vmax, and kcat of C3/C5 convertases of lectin pathway were similar to those reported for classical pathway, the ability of C4b to bind C2 indicated that every C4b deposited on zymosan/EMan was capable of forming a convertase. The findings differ from those reported for classical pathway C3/C5 convertase where one out of four C4b molecules deposited formed an enzyme. The four times greater potential of the lectin pathway than the classical pathway in generating C3/C5 convertases and producing proinflammatory products, such as C3a, C4a, and C5a implies that activation of complement via the lectin pathway might be a more prominent contributor to the pathology of inflammatory reactions.

Research supported by NIH grant HL073804
Differential regulation of cytotoxicity and cytokine production via surface receptors in CD45-deficient natural killer cells (B185)
Authors: Hesslein David G.T. , Takaki Rayna , Hermiston Michelle L. , Weiss Arthur , Lanier Lewis L.
Published: January 2023

Little is known about how CD45 regulates NK cell receptor signaling and the resulting effector functions. We find that NK cells from CD45-deficient mice are competent for immunoreceptor tyrosine-based activation motif (ITAM)-dependent receptor-induced cell-mediated cytotoxicity. In contrast, CD45−/− NK cells are completely deficient for cytokine secretion after stimulation with ligands to or antibodies against the ITAM-associated receptors, NK1.1, CD16, Ly49H, Ly49D, and NKG2D. This deficiency in cytokine/chemokine production occurs at the level of mRNA expression. After receptor engagement, the extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation was markedly perturbed, whereas p38 activation was not substantially affected. Further more, we have uncovered other cytotoxicity pathways that are defective in CD45−/− NK cells. These findings indicate that CD45 is not required for ITAM-dependent cytolytic activity, but is required for cytolytic activity through other receptor pathways and for ITAM-dependent cytokine production.
Dry powder formulations of plague F1-V vaccine provide protective immunity in mice (B195)
Authors: Huang Joanne , Ferriter Matthew S. , Alarcon Jason B. , Mikszta John A. , Evans Michelle , D’Souza Ajit , Ford Brandi , Stewart Todd , Amemiya Kei , Ulrich Robert G , Sullivan Vince J.
Published: January 2023

The potential use of Y. pestis as a bioterror agent is a great concern. Development of a more stable powder vaccine against Y. pestis and using non- or minimally- invasive administration methods could provide an effective alternative to traditional intramuscular (IM) injection of liquid formulations.

We evaluated two powder formulations of a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted F1-V powders via IM, microneedle-based intradermal (ID) or non-invasive intranasal (IN) administration. By standard IM injection, the reconstituted powders induced serum antibody responses and provided protection against 1000 LD50 Y. pestis lethal subcutaneous challenge at levels equivalent to those elicited by unprocessed liquid vaccine (70 – 90% protection). Overall, ID delivery showed similar efficacy as IM injection, while IN delivery required an extra dose of vaccine to achieve a level of protection similar to that provided by injection.

In summary, we demonstrate the feasibility of using reconstituted F1-V powders and microneedle-based ID delivery to provide protection against plague in a mouse model. Nasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and powder vaccine formulations may result in substantial health and economic benefits.
Abrogation of potent humoral vaccine responses in patients on long-term anti-CD25 therapy (B196)
Authors: Ragheb Jack A , Buggage Ronald , Reed George , Levy-Clarke Grace , Nussenblatt Robert
Published: January 2023

Daclizumab, a monoclonal antibody that binds CD25, has been effective in the treatment of a number of autoimmune diseases. Despite continuous IL-2 receptor blockade for extended periods in these patients, there have been no instances of opportunistic infection and the rate of community-acquired infections is no greater than that of the general populace. This suggests that daclizumab dosing is balanced between prevention of autoimmunity and susceptibility to infection. To test this hypothesis, as a surrogate for infection we measured hepatitis vaccine responses in five patients with autoimmune uveitis who had been on daclizumab therapy for as long as 92 months. Surprisingly, only one equivocal vaccine response was observed. Given the high frequency of seroconversion (>95%) induced by these vaccines in healthy adults, this study is sufficiently powered to demonstrate statistically significant differences from the expected hepatitis vaccine response rate (p=0.0005). These results show that daclizumab can block the response to a potent immunogen in vivo. Given that alum based vaccine responses are highly dependent on IL-4, the known inhibition of Th2 cytokine production by daclizumab is likely to have contributed to the vaccine failures we observed. Our results indicate that, where possible, antibiotic/antiviral drug prophylaxis may be preferable to vaccination in patients on daclizumab therapy.