bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell lineage analysis of KIT D816V and NRAS G12D activating mutations in aggressive systemic mastocytosis (36.25)
Authors: Bai Yun , Simakova Olga , Fu Weiming , Woelfel Jessica , Maric Dragan , Maric Irina , Metcalfe Dean , Wilson Todd
Published: January 2023

Systemic mastocytosis is a clonal disorder characterized by the abnormal proliferation and accumulation of mast cells within tissues. Over 90% of patients with mastocytosis possess a somatic gain of function mutation involving the tyrosine kinase domain of c-kit (KIT D816V). Recently we identified a patient with aggressive systemic mastocytosis who also harbors a somatic NRAS G12D activating mutation. To better understand the clonal evolution of mastocytosis, we evaluated the cell compartments impacted by these two mutations. Bone marrow and peripheral blood cells were collected from the patient. CD34+ progenitors, mast cells, neutrophils, monocytes, eosinophils, basophils, T-lymphocytes and B-lymphocytes were separated by fluorescence-activated cell-sorting. cDNA was prepared and the KIT D816V mutation was tested by nested RT-PCR/RFLP. The NRAS G12D mutation was detected by nested RT-PCR followed by sequencing. The KIT D816V and NRAS G12D mutations were detected in both myeloid and lymphoid lineages. Mast cells harbored both mutations. The KIT D816V mutation was not detected in CD34+ progenitors, whereas presence of the NRAS G12D was observed. These findings suggest that the NRAS G12D mutation occurred prior to the KIT D816V mutation in clonal development. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity.
Extreme GATA-3 dose dependence in early T cell development (36.28)
Authors: Scripture-Adams Deirdre , Arias Alexandra , Elihu Koorosh , Ho I-Cheng , Rothenberg Ellen
Published: January 2023

GATA-3 expression first reaches peak levels during T-lineage commitment, but a specific requirement for GATA-3 at each of the early DN stages in T development has not been proven. Using retroviral expression of shRNA to reduce GATA-3 protein at each of the earliest stages of T-cell differentiation (DN1, DN2, DN3), we identify two dose-dependent effects: a defect in survival or expansion at DN1, and a separable developmental arrest of cells at DN2, which was cell-autonomous, and not relieved by a Bcl2 transgene. The DN2 arrest phenotype was extremely robust; we were able to document this developmental block even in T development assay cultures initiated with a single fetal liver derived precursor. The DN2-stage developmental block was confirmed using transduced Cre to induce deletion of a floxed Gata3 allele. Reduction of GATA-3 also increased PU.1 levels in pro-T cells, despite relatively normal T-lineage gene induction. To test roles of GATA-3 in exclusion of non-T fates, we forced expression of wild type, obligate repressor, and tamoxifen-inducible GATA-3 forms in precursors undergoing B and myeloid development. GATA-3 blocked B cell development via direct repression under conditions which remained permissive for myeloid development. Thus GATA-3 is required for at least two steps of T-cell progression to commitment, and restricts two different alternative fates by distinct mechanisms.
The role of MHC Ib in selecting functional CD8 T cell during acute LCMV infection (36.38)
Authors: He Xiao , Chen Lili , Jay David , Fairbanks Jared , Jensen Peter
Published: January 2023

Conventional MHC Ia & II molecules (H-2K/D/L & I-A/E in mouse) select α/β CD8 & CD4 T cell development, respectively. Although unconventional MHC class Ib, mainly encoded in H-2 Q, T & M regions in mouse, contains more than 20 MHC Ib genes, their functions remain largely unknown. We developed a mouse model, in which 3 genes, Kb, Db & CIITA were deleted, thus called triple knock-out (TKO) mouse. In TKO, CD8 T cells are selected only by MHC Ib. Previously we have shown that α/β CD8 T cells develop well in TKO. Functionally, we observe that TKO mice mount a protective immune response against in vivo acute infections of LCMV. The protection is CD8 T cell mediated & β2m dependent. Rapid production of IFNγ and Granzyme B is detected in expended CD8 cells. Intriguingly, TKO mice do not appear to mount a memory response to a second infection. We report that splenocytes, from in vivo LCMV primed TKO, can be restimulated in vitro, by LCMV infected peritoneal macrophages or bone marrow derived dendritic cells, to produce IFNγ, but little IL-2. This MHC Ib-dependent response appears to be partially mediated by Qa-2. Therefore we have established an in vitro system to analyze MHC Ib-restricted T cell responses to LCMV & we are attempting to map the MHC Ib restriction element(s) and target epitopes. Collectively, our results suggest that MHC Ib can select functional CD8 T cells & these T cells participate in, at least under some circumstances, protective immune responses to pathogens.
Expression and Function of P-selectin and its ligand PSGL1 on hematopoietic progenitors (36.4)
Authors: Sultana Dil , Zlotoff Daniel A , Bhandoola Avinash
Published: January 2023

T cells develop in the thymus from bone marrow-derived hematopoietic progenitors that settle the thymus via the blood. The molecular basis for progenitor entry into the thymus is poorly understood, but p-selectin and P-selectin glycoprotein ligand 1 (PSGL1) are suggested to be involved. We investigated the expression of functional PSGL1 able to bind p-selectin. Our preliminary results suggest that functional PSGL1 is expressed at lower levels on primitive hematopoietic stem cells and downstream multipotential progenitors, but at higher levels on downstream early lymphoid progenitors in the bone marrow. These data indicate that P selectin binding is developmentally regulated. Using competitive thymus reconstitution assays we confirmed that PSGL1 is a functionally important component of the thymic homing process. Our current experiments are focused on understanding the mechanism by which PSGL1 is selectively upregulated on thymus homing progenitors.
Smoking cessation reverses cigarette smoke-induced impairment of B cell development and skewed CD4:CD8 T cell ratios in mice (36.43)
Authors: Swanson Patrick , Fusby Jenny , Kassmeier Michele , Palmer Victoria , Perry Greg , Anderson Dirk , Hackfort Bryan , Alvarez Gwen , Akhter Mohammed , Cullen Diane
Published: January 2023

Cigarette smoking is a risk factor for developing osteoporosis, and previous studies suggest lymphocytes are required for normal bone homeostasis and peak bone mass. We hypothesized that lymphocyte development is adversely affected by smoke exposure. If so, this outcome would provide a mechanism to partly explain how cigarette smoking promotes osteoporosis. To test this possibility, we investigated how the duration and cessation of cigarette smoke exposure affected the development, distribution, and function of lymphocytes normal mice. Consistent with the hypothesis, 3-12 weeks of smoke exposure significantly diminished bone marrow B220+CD43- B cell subsets, but, interestingly, did not significantly affect earlier progenitor B220+CD43+ B cells or thymic T cell subsets. In the spleen, by contrast, we find that individual splenic B cell populations, including transitional, marginal zone, and follicular mature B cell subsets, were largely unaffected by smoke exposure, but smoking did skew splenic CD4+:CD8+ T cell ratio to favor CD8+ T cells compared to sham controls. However, smoke exposure also had little or no effect on lymphocyte function ex vivo. Interestingly, smoking-induced alterations in lymphocyte populations observed at 12 weeks were lost when smoke exposure was stopped for six weeks. These data provide evidence that smoke exposure causes significant changes in lymphocyte populations that can largely be reversed after six weeks without smoking.
Hierarchy of T cell lineage decision is regulated by thymic self-reactive signaling (36.45)
Authors: Marcus Assaf , Friedmann-Morvinski Dinorah , Elinav Eran , Waks Tova , Eshhar Zelig
Published: January 2023

During T cell development, thymic recognition of self antigens leads to negative selection of effector T cells with high TCR avidity. Similar interactions are implicated with emergence of regulatory subsets, including Tregs, NKT, and CD8αα cells. How the degree of self-reactive signaling coordinates the development of the effector and regulatory subpopulations is not fully understood. To elucidate this, we employed transgenic mice, featuring gradually increasing self-reactivity by expression of different levels of a cross-reactive tripartite chimeric receptor targeting 2,4,6-trinitrophenol (TNP). In these mice, increasing cell self-reactivity inversely correlated with effector cell numbers, and led to skewing towards CD4 phenotype, compatible with the kinetic signaling model. In parallel, coreceptor levels of expression were diminished in correlation with gradually increasing self-reactivity, reflecting coreceptor tuning. Higher levels of self-reactive signaling promoted the thymic expansion of CD8αα and NKT cells. T cells with the highest self-reactive TPCR featured defective TCR rearrangement stemming from down regulation of RAG1, RAG2 and PTα. Altogether, we demonstrate a hierarchical order in coping of the immune system with auto-reactivity. In case of weak self-reactivity, coreceptor tuning is implemented, while in stronger self-reactivity, regulatory subsets develop, as possible means of modulating dangerous autoreactive cells into protective guardians of host.
Expanded γδ T cell populations in absence of transcription factor PU.1 (36.49)
Authors: Jabeen Rukhsana , Chang Hua-Chen , Nutt Stephen , Kaplan Mark
Published: January 2023

T cell development is characterized by a succession of developmental stages that lead to the generation of mature αβ and γδ T cells. γδ T cells are considered innate cells that critically contribute to host defense. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi-1) is essential for early stages of T cell development and is down regulated during the pro T-cell stage. PU.1 is expressed in Th2 cells but not Th1 cells. Expression of PU.1 in other T cell subsets has not been studied extensively. In this study, we show that PU.1 is expressed in γδ T cells, and in mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpilck-/-) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild type mice. The increase in γδ T cell numbers in PU.1 deficient mice is reflected in several subsets examined (Vγ2T, Vγ3T and Vδ4T). In mice where PU.1 was deleted using a CD4-Cre transgene, the expansion of γδ T cell numbers was restricted to the intestinal population. PU.1-deficient γδ T cells secrete higher levels of IL-17 than wild type cells, but similar levels of IFN-γ. Thus, our data show that PU.1 is expressed in γδ T cells and that deletion of PU.1 results in γδ T cell expansion. This suggests that PU.1 has a negative influence on γδ T cell development or expansion.
Bcl11b: a novel regulator of T-lineage commitment (36.50)
Authors: Li Long , Rothenberg Ellen
Published: January 2023

Commitment of HSC to a T-lineage fate is driven by a network of transcription factors and environment cues. Transcription factor Bcl11b is expected to be a crucial component of the network. Bcl11b is an essential regulator for T-cell development. However, little is known about how Bcl11b works in the commitment stage of T-cell development. Using an in vitro growth system, we have found that under certain conditions Bcl11b-deficient pre-T cells fail to pass the DN2/DN3 transition, which is the lineage commitment step in T-cell development. Instead, Bcl11b-deficient cells continuously grow at DN1-like and DN2A-like stages for more than twenty days, while wild-type controls can develop normally to DN3 and DN4 cells. Unlike their wild-type counterparts, the long-term Bcl11b-deficient cells still possess developmental plasticity with an IKDC-like population growing out from the cells. Gene expression profile reveals that Bcl11b-deficient cells express T-cell identity genes including Cd3e and Lck, and continue to silence some important genes associated with major developmental alternatives, such as B-cell factors; however, they fail to turn off stem cell genes and some lineage-regulators, such as Scl, Lyl1, Gfi1b, Id2, Il2rb, Zbtb16 and Bcl11a. The results indicate that Bcl11b plays a major role in T-lineage commitment by controlling the correct exit of T-cell precursors from a progenitor cell state. No other factor has been shown to have this distinctive function.
Membrane localization of the avian chB6 alloantigen (36.62)
Authors: Funk Phillip , Sanderson Bailey , Cheatham Terah
Published: January 2023

Chickens are one of the classic models of vertebrate immunity. We have been interested in the role of the alloantigen chB6 (formerly called BU1) in the development of B cells within the bursa and have presented evidence that chB6 can trigger apoptosis. Furthermore, the pathway that triggers apoptosis is similar to that triggered by FAS, with caspase 8 as an initiator, caspases 9 and 3 as later caspases, and bcl-xl functioning as an inhibitor of apoptosis. We have been unable to demonstrate a steady state association of chB6 and caspase 8 in DT40, so the mechanism of caspase 8 activation remains unclear. Other investigators have shown that FAS segregates into lipid rafts in type I cells and this is correlated with death signaling. In addition, FAS ligation leads to the formation of distinct signaling complexes termed SPOTS. Using immunofluorescence, we observed that chB6, when cross-linked by secondary antibodies, is found in patchy areas on DT40 cells reminiscent of SPOTS. By double labeling with fluorescent cholera toxin B, we show that chB6 co-localizes with lipid raft areas of the DT40 membrane and will be internalized by the cells. This suggests an active distribution of chB6 in the membrane and it may be preferentially loaded into lipid rafts along with BCR in avian cells. This work offers a new insight into the signaling via chB6. This work supported by a grant from the DePaul University Research Council.
Basis for lineage-specific control of PTEN expression by Rpl22 (36.64)
Authors: Stadanlick Jason , Lee Sang-Yun , Wiest David
Published: January 2023

Mutations in a number of ribosomal proteins impair erythropoiesis (e.g., Rps19 mutations in Diamond Blackfan Anemia); however, such mutations have not been reported to disrupt T cell development. We have shown that ablation of ribosomal protein L22 (Rpl22) results in a strikingly specific defect in T cell development, selectively arresting αβ lineage cells through translational de-repression of the tumor suppressor p53 while sparing cells of the γδ-lineage. We performed comparative analysis of Rpl22-/- αβ and γδ precursors to assess the basis for lineage restriction of the blockade. Initial studies revealed a marked decrease in expression of PTEN in mixed-lineage CD4-8- (DN) progenitors from Rpl22-/- mice. This result is paradoxical, as PTEN is a positive regulator of p53 expression, yet its expression is reduced in cells where p53 is elevated. We hypothesized that the reduction in PTEN and the increase in p53 were actually occurring in distinct populations within the DN pool. Indeed, we found that PTEN levels were not reduced in purified αβ-lineage DN3 thymocytes from Rpl22-/- mice, suggesting the loss of PTEN occurs specifically in γδ lineage cells. In agreement, real-time PCR analysis revealed that PTEN mRNA levels are reduced in Rpl22-/- γδ T cells. Taken together, these data indicate that one important factor contributing to the sparing of γδ lineage development in Rpl22-/- mice is the selective reduction in γδ lineage cells of PTEN expression, which destabilizes p53.
Antecedent influenza virus infection enhances the innate immune response to secondary Haemophilus infection in a porcine co-infection model. (37.11)
Authors: Loving Crystal , Brockmeier Susan , Vincent Amy
Published: January 2023

The increasing number of annual influenza (Flu) cases, coupled with the recent Flu pandemic, has amplified concerns about the impact of Flu on human and animal health. It is appreciated that Flu is complicated by secondary bacterial infection. Tracheal epithelial cells (TEC) and pulmonary macrophages (Mac) respond to infection with proinflammatory cytokines for controlling pathogen spread; however, exacerbated responses can lead to immunopathology that may be detrimental to the host. The extent to which antecedent Flu alters TEC and Mac responses to secondary stimulation is poorly understood. Pigs serve as a model for Flu infection, and secondary infection with Haemophilus parasuis (Hps) provides a useful co-infection model for evaluating host responses. Groups of pigs were infected with Flu virus and 5-days later infected with Hps (Flu/Hps). Non-infected, Flu-only and Hps-only groups served as controls. Pigs were necropsied 1 day following Hps inoculation and results show Hps colonization was higher in the nose and lungs of Flu/Hps pigs compared to Hps-only pigs. In Flu/Hps pigs, IL-8, IL-6 and IL-1β protein levels were increased in the lung, and TEC and Mac cytokine mRNA expression levels were significantly increased over Flu-only and Hps-only pigs. Also, TEC and Mac from 5-day post-Flu pigs exhibited dysregulated innate immune responses to secondary Hps exposure in vitro, providing additional evidence that Flu infection alters host responses to secondary stimulation.
New mechanism of innate immunity mediated by human defensin α-1 against Trypanosoma cruzi (37.2)
Authors: Cardenas Tatiana , Kleshchenko Yuliya , Madison M. , Nde Pius , Johnson Candice , Furtak Vyacheslav , Lima Maria , Villalta Fernando
Published: January 2023

The mechanisms of innate immunity against protozoan parasites that cause significant diseases are very limited. Here we show that human defensin α-1 plays a fundamental role in the initiation of innate immune responses to Trypanosoma cruzi. We demonstrate that human epithelial cells respond to early T. cruzi infection by up-regulating the expression and secretion of defensin α-1, which inhibits T. cruzi motility and prevents cellular infection. Human defensin α-1 inhibits trypomastigote motility of T. cruzi and infection at low micromolar concentrations, and this requires direct association of the defensin α-1 with the invasive trypanosome, which causes membrane depolarization and pore formation in the flagellar membrane. Moreover, defensin α-1 alters the flagellar axonemes, breaks both the flagellar membrane that attaches the flagellum to the trypanosome body and the flagellum thereby restricting trypanosome motility, which is required for trypanosome membrane penetration during cell entry. This is the first report showing that defensin α-1 disrupts the T. cruzi flagellum to compromise trypanosome motility and prevents cellular infection. Thus, defensin α-1 possess remarkably distinct modes of activity against protozoan parasites than bacteria and viruses, and their function may provide insights for the development of new antitrypanosomal strategies (supported by NIH grants AI AI080580 and AI007281)
Leishmania mexicana infection induces antibody responses to parasite surface glycolipids that can induce IL-10 and suppress IL-12 from macrophages (37.25)
Authors: Buxbaum Laurence
Published: January 2023

Chronic disease of mice caused by the protozoan parasite Leishmania mexicana requires IL-10 and FcγRIII. Immune complexes consisting of IgG bound to the surface of Leishmania amastigotes can induce IL-10 and suppress IL-12 production from macrophages. However, the targets of these antibodies and the kinetics of their production are not known. Several groups have attempted, without success, to identify surface proteins on the amastigote form of the parasite to which IgG can bind. Using ELISA and thin layer chromatography (TLC) immunoblot methods we now show that parasite glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by IgG1 by 6 weeks of infection with a rapid increase between 14-16 wks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII KO mice. We found that a single prominent spot on TLC is recognized by mouse serum of infected mice and that the glycolipid has a glycosyl phosphatidylinositol (GPI) structure as assessed by GPI-PLC and GPI-PLD susceptibility. Alpha-mannosidase treatment changed the mobility of the recognized glycolipid, indicating that a branched mannose occurs in the structure, but is not required for IgG binding. Further characterization of the target glycolipids will have important implications for vaccine development as well as elucidating the poorly understood role of glycolipids in immunology. Funded by VA Merit, the University of Pennsylvania, and NIH R01-AI081717.
Characterizing mast cell clearance of Yersinia pestis (37.29)
Authors: Lambert Nathaniel , Langefitt Deanna , Nilles Matthew , Bradley David
Published: January 2023

Mast cells have been implicated in protective responses to infectious agents. Yersinia pestis is the causative agent of plague and considered a bioterrorism threat. We have previously demonstrated that B10.T(6R) mice were significantly more resistant to Y. pestis infection compared to Swiss Webster mice. Cromolyn sodium, an inhibitor of mast cell degranulation, abolishes resistance to Y. pestis infection in B10.T(6R) mice, suggesting a protective role for mast cells during Y. pestis infection. The ability of mast cells to internalize Y. pestis was assessed in P815 cells, a murine mast cell-like clone. Both wild-type and type three secretion system mutant strains of Y. pestis were not internalized. There was no visible degranulation of P815 cells cultured with Y. pestis compared to heavily degranulation with compound 48/80 treatment. To determine if Y. pestis may be inducing the release of preformed cytokines without visible degranulation, TNF-α levels were measured in supernatants from P815 cells infected with Y. pestis and S. typhimurium.Y. pestis did not induce any increase in TNF- α (<8 ng/ml), while TNF-α levels were increased with S. typhimurium infection by 30 min and reached 21 ng/ml by 4 h. These findings provide an excellent model to investigate responses by mast cells to various bacteria, and possible functional differences in mast cells from different strains of mice.
Intravital imaging of Borrelia burgdorferi and murine innate immune cells during early cutaneous infection. (37.42)
Authors: Lavik John-Paul , Shukla Vipul , Wooten R. Mark
Published: January 2023

After tick transmission, the spirochete, Borrelia burgdorferi (Bb), remains at the skin inoculation site for up to 48 hours before spreading and causing Lyme disease. Bb surface agonists should elicit potent inflammation and clearance in skin. However, the ID50 is <50 Bb, indicating efficient immunoevasion and suggesting that traditional in vitro models are inadequate for accurately characterizing Bb pathogenesis (i.e. most models display efficient Bb clearance by phagocytes). Thus, we have devised a confocal microscopy method for visualizing infectious fluorescent Bb in situ and in real-time. Here, we inject Bb into ear skin of anesthetized mice and collect four-dimensional data regarding Bb motility during natural infection. This data is complemented by quantitative PCR and histological analyses of the temporal fall and rise of spirochete numbers in vivo. By 4h, the majority of Bb at the injection site are immobile, while Bb adjacent to the site are highly motile. Two movement types are seen: 1) a back and forth shift that may aid Bb in finding an optimal migration path and 2) a directed run that navigates dermal structures. Analysis indicates an average Bb velocity of ≥100 um per minute in skin, which is >10x faster than any immune cell velocities observed in our model. This suggests that Bb motility is a primary mechanism for host cell evasion and indicates our imaging techniques will allow true delineation of host-Bb interactions critical for Lyme disease development.
Mouse Beta-Defensin-1 Plays a Role in Influenza Innate Immunity (37.49)
Authors: Ryan Lisa , Diamond Gill
Published: January 2023

Beta-defensins (BD) are antimicrobial peptides that play a role in bridging innate and adaptive immune responses to infection. The most widely studied is hBD-2, which increases upon stimulation of epithelial cells with lipopolysaccharide (LPS). hBD-1 remains constitutively produced in spite of cytokine or LPS stimulation, but is stimulated by influenza virus in monocytes and plasmacytoid dendritic cells. C57Bl/6 male 8-12 week old mice (WT) and mice with mBD-1 (hBD-1 homologue) deleted (mBD-1-/- KO) were intranasally infected with 300 pfu/50µl of mouse-adapted Hong Kong 1968 influenza A H3N2 virus. Initially, KO mice lost significantly more weight than WT mice until day 5 when KO mice began to die (p ≤ 0.05). Most of the WT deaths occurred on days 8-16. KO mice died sooner, on days 5-10. At day 10, only 15% of KO mice survived compared with 45% of WT mice. After day 16, 10% of KO mice survived versus 18.2% of WT. Mortality of KO mice was significantly greater than that of WT mice (p = 0.028). UV-inactivated virus did not cause body weight loss or mortality in either strain. Although virus titers were equal 24 hr after infection, 3 days following infection the inflammatory cell influx was greater in KO mice compared with WT mice. By day 6, histology in these two strains was comparable, with a large lymphocytic infiltrate and hemorrhage in both strains, suggesting that beta-defensin-1 is important in controlling inflammation early in viral infection.
Combating pulmonary Coxiella burnetii infection: The role of innate immunity. (37.54)
Authors: Calverley Matthew , Read Amanda , Harmsen Allen
Published: January 2023

The innate immune system provides first line host defense. However, within the immune privileged site of the lung, immune response must be closely regulated. As a result, tissue resident alveolar macrophages (AM) are typically immunosuppressed. The intracellular bacteria Coxiella burnetii has been observed within AM during infection and little is known about the role of AM in C. burnetii infection. Therefore, the AM deserves focused study. We hypothesized that, due to the immunosuppressed state of resident AM, these macrophages are particularly susceptible to C. burnetii infection and provide a required niche for infection. Dye labeling techniques were employed to discriminate AM and C. burnetii, allowing for the determination of resident and infected cell populations. These techniques were corroborated with antibody labeling and the various populations were analyzed. We report here that resident AM are infected, even at low infective doses. Interestingly, a heterogeneous population of infected cells is also identifiable. This population is comprised of resident CD11c+ AM expressing CD11b and recruited CD11b+ cells expressing CD11c. These findings suggest CD11c expression serves as a marker of cells susceptible to C. burnetii infection. Identification of this susceptible population may aid vaccine development and prove relevant for treatment of other intracellular bacteria, such as, Mycobacterium tuberculosis and Legionella pneumophila.
CD56+ gamma delta T cells in the peripheral blood during HCV infection (38.15)
Authors: Epie Nicolas , Talwani Rohit , Armstrong Cheryl , Pauza C.David
Published: January 2023

Hepatitis C virus (HCV) is a chronic persistent infection that can cause hepatitis and hepatocellular carcinoma. HCV infection is less responsive to therapy among African Americans compared to Caucasians suggesting that underlying differences may impact disease progression. γδ T cells are present at levels differing by 5-fold among healthy Caucasian and African American, such differences might contribute to the outcomes disparity. We collected blood from African American adults with HCV infection and characterized γδ T cells in this group. Compared to controls, the HCV+ group had > 2-fold higher levels of circulating γδ T cells of the Vδ+ subset, suggesting they were actively involved in the immune response to HCV. Surprisingly, the γδ T cells in HCV+ African Americans were lower than controls for the percentage of cells expressing CD56, a marker associated with cytotoxic effector function in this cell type. The patterns of γδ T cells in African Americans with HCV infection suggest that they participate in an active immune response against virus and that the CD56+ subset may be sequestered in tissue in an effort to block virus replication. Thus, γδ T cells may participate in the complex immune responses to HCV and their lower levels provide one potential explanation for the outcomes disparity among African Americans and Caucasians with HCV infection and liver disease.
Isolation of Cross-Clade HIV-Neutralizing Human Antibodies from Memory B Cell Repertoires Using Short Term Culture and High-Throughput Primary Neutralization Screens (38.17)
Authors: Chan-hui Po-Ying , Wrin Terri , Simek Melissa , Phogat Sanjay , Olsen Ole , Hammond Phil , Poignard Pascal , Walker Laura , Mitcham Jennifer , Fling Steve , Team Protocol G , Burton Dennis , Moyle Matthew
Published: January 2023

Background. We have recently isolated 2 broadly neutralizing monoclonal antibodies (bNAbs), PG9 and PG16, from an HIV-infected subject in an African Protocol G cohort that are more potent than the bNAbs discovered in the past decade. The discovery highlights our human antibody (Ab) discovery approach based on short-term memory B cell culture, high-throughput primary microneutralization screening, and recombinant monoclonal antibody (mAb) reconstitution (Walker LM, et al, 2009, Science, 326:285-9). The epitope specificities of PG9 and PG16 reveal a novel target for designing vaccine immunogens that may elicit broad anti-HIV protection. To identify additional epitopes for vaccine design, we extended the bNAb discovery to more HIV-infected subjects. Method. We selected several HIV-infected subjects with broad neutralization activity in serum. Memory B cells from these elite neutralizers were activated and the IgG-containing culture media were screened for neutralizing activity against multiple pseudotyped viruses. mAbs were rescued from selected B cell culture wells by RT-PCR and subcloning. Results. From an HIV-infected elite neutralizer, we have identified in the primary screen multiple human Abs that cross-neutralize MGRM-C-26 of clade C, and 93BR020 and JR-CSF of clade B. Antibodies that neutralize either MGRM-C-26 alone or 94UG103 of clade A alone were also detected. Recombinant mAbs are currently being rescued for further characterization. (Supported by IAVI and USAID)
Pronounced T Cell Receptor-dependent Activation Drives Exhaustion but Sustains Interferon-γ Transcript Levels (38.6)
Authors: Zajac Allan , Mackerness Kathryn , Cox Maureen , Lilly Lauren , Weaver Casey , Harrington Laurie
Published: January 2023

The ontogeny of exhausted CD8 T cells as well as the underlying mechanisms that account for the functional inactivation of these cells remains ill-defined. We have utilized cytokine reporter mice, which mark the synthesis of interferon-γ by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that, during chronic lymphocytic choriomeningitis virus infection, the precursors of exhausted T cells all upregulate interferon-γ mRNA and become susceptible to depletion with anti-Thy1.1 antibodies. This potent phase of hyperactivation which precedes exhaustion is antigen-dependent and is dictated by viral levels and T cell precursor frequencies. Unlike acute infections, which result in massive expansion of the responding T cells, during chronic infections further expansion of the initial response becomes rapidly attenuated. The exhausted T cells which subsequently emerge in chronically infected hosts attain a distinct CD127lo, KLRG-1lo phenotype but do not silence interferon-γ transcription even though protein expression is abolished. Thus ablation of interferon-γ production by exhausted cells is not due to transcriptional silencing, implicating post-transcription regulatory mechanisms in disabling this effector module.
Virus-specific CD4+ T cells are recruited de novo during persistent polyomavirus infection (39.11)
Authors: Lin Eugene , Kemball Christopher , Hadley Annette , Wilson Jarad , Lukacher Aron
Published: January 2023

Virus-specific CD4+ T cells optimize anti-viral responses by providing help for anti-viral humoral responses and CD8+ T cell memory differentiation, as well as operating as direct anti-viral effectors. While CD4+ T cell responses to viral infections that undergo complete clearance have been extensively studied, comparatively less is known about virus-specific CD4+ T cell responses to viruses that persistently infect their hosts. Using a mouse polyomavirus (MPyV) infection model, we previously demonstrated that CD4+ T cells are essential for recruiting naïve MPyV-specific CD8+ T cells in persistently infected mice. In this study, we defined two dominant MPyV-specific CD4+ T cell populations, one directed toward an epitope derived from the nonstructural LT antigen and the other from VP1, the major viral capsid protein. These MPyV-specific CD4+ T cells vary in terms of their magnitude, functional competence, and phenotype during acute and persistent phases of infection. Employing a minimally myeloablative mixed bone marrow chimerism approach, we further show that naïve virus-specific CD4+ T cells, like anti-MPyV CD8+ T cells, are primed de novo during persistent virus infection. In summary, these findings reveal quantitative and qualitative differences in the CD4+ T cell response to a persistent virus infection and demonstrate that naïve antiviral CD4+ T cells are recruited during chronic polyomavirus infection.
Murine LP-BM5 viral infection induced peripheral neuropathy in C57BL/6 mice (39.2)
Authors: Cao Ling , Green Kathy , Li Wen , Tan Leonard Tan , Green William
Published: January 2023

Distal symmetrical polyneuropathy (DSP) is the most common form of HIV infection-associated peripheral neuropathy and is often associated with pain. C57BL/6 (B6) mice infected with LP-BM5, a murine retroviral isolate, develop a severe acquired immunodeficiency syndrome similar to humans infected with HIV, hence the term murine AIDS (MAIDS). We investigated the induction of peripheral neuropathy post- LP-BM5 infection (p.i.) in B6 mice. First, LP-BM5 infected mice displayed significant symmetrical hind paw mechanical hypersensitivity (behavioral sign of peripheral neuropathy) starting from week 6 p.i. up to the time of sacrifice at week 12. The density of PGP9.5+ (a pan-neuronal marker) intraepidermal nerve fibers (IENFs) was found to be significantly reduced at week 2 p.i. and this loss was gradually recovered with time. There were no significant differences between left and right paws. Also, significantly increased levels of viral gag RNA in all segments of spinal cord tissue were detected in infected mice via quantitative real-time PCR. Further, FACS analysis showed an early increase of lumbar spinal cord microglia p.i.. Altogether, LP-BM5 induced the development of peripheral neuropathy that occurred concurrently with the development of MAIDS and was correlated with viral infection of the spinal cord. The potential role of spinal cord microglia in LP-BM5-induced painful neuropathy will be further investigated.
Exhaustion of intra-hepatic CD8+ T cells targeting intact and escaped MHC class I epitopes in chronic phase of hepatitis C virus infection (39.20)
Authors: Callendret Benoit , Bowen David , Fuller Michael , Eccleston Heather , Rutckiewitz Jennifer , Hasselschwert Dana , Freeman Gordon , Walker Christopher
Published: January 2023

Hepatitis C virus (HCV)-specific CD8+ T cells persist in the chronically infected liver but provide no apparent control of virus replication. Epitopes targeted by some of these T cells contain escape mutations while others remain intact. Studies in acutely infected humans suggest that circulating CD8+ T cells targeting the intact epitopes express more PD-1, a molecule associated with loss of effector function. Whether this is true in the persistently infected liver is unknown. CD8+ T cells targeting intact or mutated epitopes were visualized using tetramers in the liver of chronically infected chimpanzees. Unexpectedly, there was no obvious relationship between expression of PD-1 on the HCV-specific CD8+ T cells and the nature of the targeted epitope. More importantly all HCV-specific CD8+ T cells we examined displayed functional incapacity regardless of the nature of the targeted epitope and anergy could not be reversed by blockade of the PD-1/PD-L1 pathway. We conclude that decreased PD-1 expression previously observed on acute phase CD8+ T cells after escape of the targeted epitope is not necessarily maintained in the liver during chronic hepatitis C. Rather, our data suggest that mechanisms other than continuous TCR triggering might be responsible for PD-1 upregulation and exhaustion of CD8+ T cells, or that escaped epitopes are still able to induce TCR engagement without T cell activation resulting in exhaustion of viral specific CD8+ T cells.
A molecular characterization of how the immunomodulatory E3-19K protein from adenovirus binds to and retains MHC class I molecules in the endoplasmic reticulum (39.23)
Authors: Bouvier Marlene , Fu Jie , Li Lenong
Published: January 2023

Adenoviruses (Ads) cause persistent infections. The E3-19K protein from Ads retains MHC class I molecules in the endoplasmic reticulum (ER), suppressing antigen presentation to T cells. The ER-lumenal domain of E3-19K is subdivided into a variable (residues 1-81), conserved (residues 82-100), and linker (residues 101-107) regions. We show that proteolysis of serotype 2 E3-19K (residues 1-100) generates a large N-terminal 1-88 fragment and a small C-terminal 94-100 fragment that span the variable and conserved regions, respectively. Neither of these fragments associates with HLA-A*1101. However, the N-terminal 1-93 residues bind with high-affinity to HLA-A*1101; Tyr93 is critical for this interaction. The data suggest that a delicate structural interplay between the variable and conserved regions enables E3-19K to exert its MHC binding function. Interestingly, Ad37 E3-19K displays weaker binding affinities for class I molecules relative to Ad2 E3-19K but retains the same locus specificity; both proteins associate with HLA-A and -B molecules but not HLA-C molecules. We provide evidence that the class I locus specificity of E3-19K is defined by MHC residue 177. Finally, we present a model which MHC residues 56 and 177 represent “hotspots” for the variable and conserved regions, respectively, of E3-19K. In conclusion, the knowledge derived from our studies helps not only to better understand host/Ad interaction, but also to design novel antiviral therapies against Ad infections.
The activating receptor CD94-NKG2E is required for protective NK cell responses to a lethal viral infection (39.26)
Authors: Fang Min , Shamsedeen Ahamed , Orr Mark , Lanier Lewis , Sigal Luis
Published: January 2023

The CD94/NKG2 receptors of NK cells are C-type lectin-like heterodimers formed by CD94 and inhibitory receptor NKG2A, or activating receptor NKG2C or NKG2E. However, the physiological functions of those receptors in the NK cell responses to viral infections are not clear. Ectromelia virus (ECTV) is an orthopoxvirus (OPV) that causes mousepox, the mouse equivalent of human smallpox. Previous studies have shown that NK cell responses are essential for the natural resistance of C57BL/6 (B6) mice to mousepox. Here, we show that B6 mice deficient in CD94 (CD94 KO) and B6D2-D6 mice (a B6 congenic mouse strain harboring NK complex from the DBA2/J, also deficient in CD94) succumbed to ECTV infection due to defective NK cell cytolytic activity. In addition, [B6-CD94 KOxB6D2-D6] F1 mice remained susceptible to mousepox whereas CD94 KO or B6D2-D6 mice expressing transgenic CD94 on NK cells were resistant. Together, these data demonstrated an essential role for CD94 in resistance to mousepox. To determine the specific CD94/NKG2 receptor required for resistance, we co-incubated reporter cells expressing CD94 and different NKG2 molecules with ECTV infected cells. We found that this resulted in the activation of only the reporters co-expressing CD94 and NKG2E. These results suggest that the activating receptor CD94-NKG2E mediates the protective NK cell response to ECTV. This is the first report showing the functional involvement of CD94/NKG2 receptors in an anti-viral NK cell response.
Cytomegalovirus reactivation occurs in burn patients and correlates with increased central memory and regulatory T cell populations. (39.32)
Authors: Maile Rob , Peppercorn Amanda , Mackinnon Karen , Schmitz John , Barnes Carie , Serody Jonathon , Cairns Bruce
Published: January 2023

Cytomegalovirus reactivation (CMV-R) is an important cause of morbidity and mortality in immunocompromised patients. CMV-R has been recognized in critically ill patients. Severe burn injury leads to early and prolonged immune and adaptive immune dysfunction. We hypothesized that CMV-R would occur in burn patients correlating with specific changes in T cell phenotype. We prospectively evaluated incidence of CMV-R longitudinally among patients with >10% total body surface area (TBSA) injury using serum PCR. We determined changes in CMV specific CD8+ T cells by utilizing CMV-specific MHC Class I tetramers, functional responsiveness to CMV peptide and the generation of Tregs using qPCR for FoxP3. 24 burn patients enrolled, with CMV-R occurring in 36.8% of patients. We detected no difference in frequency of CMV-reactive CD8+ T cells nor in vitro anti-CMV CD8+ T cell response by IFN-γ staining between R and non-R patients. CMV-R patients had a significantly decreased proportion of CMV-specific CD8+ T cells that were central-memory (CD45RO+CD62L+CD28+) and increased effector-memory phenotype (CD28-) during the first week following injury. Elevated FoxP3 expression in the 10 days prior to R correlated with R in 4/6 patients tested. CMV-R occurred following burn injury and may be related to remodeling of the CD8+ T cell population after burn. Increased T cell regulatory activity after burn may also predict for CMV-R in a subset of patients.
A Novel L-Ficolin-Mannose Binding Lectin Chimeric Molecule With Enhanced Activity Against Lethal Viral Infections (39.33)
Authors: Lear Calli , Zeitler Corinne , Karpell Marshall , Spear Gregory , Olinger Gene
Published: January 2023

Ebola viruses are potentially lethal enveloped RNA viruses for which no effective therapy exists. Human mannose-binding lectin (MBL) and L-ficolin (FCN) bind to conserved epitopes on pathogens and eradicate microbes by opsonophagocytosis, complement activation, and/or regulation of adaptive immune responses. Three FCN-MBL chimeras containing the carbohydrate-recognition domains of hMBL and varying lengths of the collagen stalks of FCN were stably expressed in HEK293F cells. rhMBL (Enzon, NJ) formed octadecameric and larger oligomers vs. smaller chimera multimers. Compared with rhMBL, chimeras bound a similar range of carbohydrate ligands but FCN-MBL76 had significantly improved function as shown by: 1) greater affinity to mannan (KD, 47 vs 73 pM, p<0.004); 2) greater capacity to activate complement in the range of 7-30 ng/mL, p<0.001; and 3) greater binding to human placental calreticulin, a putative cellular receptor for MBL (p<0.001). All chimeras and rhMBL inhibited Ebola glycoprotein-pseudotyped lentivirus and native Ebola virus infection of HepG2 cells although FCN-MBL76 was the most effective protein against native virus. Tapping-force atomic force microscopy revealed that FCN-MBL76 was less tall than the other proteins suggesting enhanced flexibility. Compared with rhMBL, a novel FCN-MBL chimeric protein exhibited superior binding to select carbohydrates, activation of complement, binding to calreticulin, and greater inhibition of Ebola virus infection in vitro.
Molecular profiling of memory CD8 T cells after repeated antigen stimulations (39.5)
Authors: Badovinac Vladimir , Wirth Thomas , Xue Hai-Hui , Rai Deepa , Harty John
Published: January 2023

Repetitive antigen stimulation by prime-boost vaccination or pathogen re-encounter increases memory CD8 T cell numbers, however the impact on memory T cell differentiation is unknown. Here we show that repetitive antigen stimulations induce the selective accumulation of CD8 T cells with a uniform effector memory phenotype. In contrast to this, genome-wide microarray analysis reveals that each additional antigen challenge results in the differential regulation of several hundred genes in memory CD8 T cells and therefore in stepwise diversification of T cell transcriptomes. As a consequence, repeatedly stimulated memory CD8 T cells and primary memory CD8 T cells differ significantly in their molecular signature and share expression of only a small but preserved group of memory signature genes. Our results provide new insight into the complex regulation of memory CD8 T cell differentiation and identify potential new molecular targets to dissect and manipulate the function of memory T cells in human diseases.
The role of Programmed Death-1 (PD-1) in pulmonary M. tuberculosis infection: friend or foe? (40.10)
Authors: Lazar-Molnar Eszter , Chen Bing , Almo Steven , Jacobs William , Nathenson Stanley
Published: January 2023

The programmed death-1 (PD-1) co-inhibitory receptor attenuates T and B cell responses and plays a crucial role in peripheral tolerance. Recently, PD-1 has been shown to inhibit T cell responses during chronic viral infections such as LCMV and HIV, and blockade of PD-1 restored anti-viral effector T cell responses. In this study, we examined the role of PD-1 in infection with Mycobacterium tuberculosis, a common co-infection with HIV. Surprisingly and paradoxically, PD-1 deficient mice showed dramatically reduced survival compared to wild-type mice. Moreover, lungs of the PD-1-/- mice showed uncontrolled bacterial proliferation, and focal necrotic areas with predominantly neutrophilic infiltrates, but a lower number of infiltrating T and B cells. Pro-inflammatory cytokines, such as TNF-α, IL-1, and especially IL-6 were significantly increased in the lung and sera of infected PD-1-/- mice, consistent with an excessive inflammation. Microarray analysis of the lungs infected with M. tuberculosis showed dramatic differences between PD-1-/- and control mice: 297 genes were differentially expressed, resulting in profoundly altered inflammatory responses, with implications in both innate and adaptive immunity. Our studies demonstrate that M. tuberculosis infection in the absence of PD-1 results in fatal inflammation with increased bacterial load; therefore we suggest that the PD-1 pathway is involved in controlling inflammatory responses after M. tuberculosis infection in the lungs.