bluuebunny/crossref_metadata_2025 huggingface dataset:
ATPase-driven oligomerization of RIG-I on an RNA ligand allows optimal induction of type I interferon (P1260)
Authors: Patel Jenish , Jain Ankur , Chou Yi-ying , Baum Alina , Ha Taekjip , Garcia-Sastre Adolfo
Published: January 2023

RNA with an exposed 5’-triphosphate (5’-ppp) and terminal double-stranded structures as found in cells during infection with many viruses, constitutes a strong activating ligand for the cytosolic pathogen sensor RIG-I. RNA binding and subsequent ATP hydrolysis by RIG-I are believed to be critical for downstream signaling. During its ligand-mediated activation, RIG-I has been suggested to form multimers but there is insufficient evidence on whether RIG-I is able to form higher-order oligomers on RNA and how multimerization affects signaling. In addition, RIG-I has been shown to translocate on 5’-ppp-dsRNA in an ATP-dependent manner and a precise role for the ATPase and associated RNA translocase activities in RIG-I activation has not been established. Using biochemical analyses of native RIG-I-RNA complexes, we show that RIG-I oligomerizes on this 5’-ppp dsRNA in an ATP hydrolysis-dependent manner. Importantly, there is a drastic reduction in ATP-dependent oligomerization of RIG-I on DI RNA with shorter dsRNA stems, correlating with the level of IFN-I activation by these RNAs. These results demonstrate a clear role for RIG-I ATPase in dsRNA length-dependent oligomer formation and provide a rationale for the ATP-driven translocase activity of RIG-I in stimulation of the IFN response.
Transcription factor ELF4 promotes formation of CD62LLOW effector and memory CD8+ T cells in response to infection (P1422)
Authors: Mamonkin Maksim , Puppi Monica , Lacorazza Daniel
Published: January 2023

At the peak of response majority of pathogen-specific CD8+ T cells are short-lived CD62Llow effectors which eventually die off, revealing CD62Lhigh central and CD62Llow effector memory populations. Intrinsic regulation of CD8+ T cell differentiation to effector and memory lineages still remains poorly understood. We have previously demonstrated that the transcription factor ELF4 regulates homeostatic and antigen-induced proliferation of CD8+ T cells. In this work, we show that ELF4 plays a critical and specific role in the generation of CD62Llow effector and memory CD8+ T cells following Listeria monocytogenes infection. Adoptively transferred Elf4—/— CD8+ T cells displayed impaired formation of CD62Llow effectors, lower magnitude of expansion and reduced numbers of effector memory without affecting development of central memory. Consistent with this finding, we observed decreased levels of Elf4—/— effector and memory CD8+ T cells in non-lymphoid tissues and blood but not in lymph nodes or bone marrow. Impaired expansion and formation of memory was due in part to enhanced cell death of Elf4—/— effector cells and suboptimal priming, evidenced by lower numbers of IL-2Rα positive cells and reduced proliferation 2.5 days after infection. Finally, loss of ELF4 compromised generation of secondary effector cells from central memory in a recall response. Thus, ELF4 emerges as a novel transcriptional regulator of CD8+ T cell differentiation in response to infection.
CD8 T cell metabolism and differentiation in an acute immune response (P1440)
Authors: Verbist Katherine , Wang Ruoning , Green Douglas
Published: January 2023

A popular concept in CD8 T cell biology is that manipulation of T cell metabolism represents a method by which CD8 T cell differentiation, and thus the size and quality of the resultant memory population, is controlled. Indeed, it has long been known that different metabolic programs (i.e. utilization of distinct metabolic pathways to meet the cell’s energetic demands) are associated with different phases of CD8 T cell differentiation during an immune response. Specifically, T cell activation via TCR stimulation results in dramatic increases in the uptake and consumption of glucose and glutamine by effector T cells, whereas naïve and memory CD8 T cells rely primarily on fatty acid oxidation to meet energetic and biosynthetic demands. Interestingly, it has been shown that the initial metabolic programming upon activation is dependent on expression of the transcription factor c-Myc. Here, we utilize c-Myc reporter mice and conditional c-Myc knockout animals to examine the metabolic re-programming that occurs as CD8 T cells transition from the effector to memory phase, and we explore the possibility that c-Myc expression can predict the metabolic profile and eventual fate of CD8 T cells participating in an acute immune response.
Mucosal infection drives rapid development of protective resident memory CD8 T cells (P1444)
Authors: Sheridan Brian , Pham Quynh-Mai , Lee Young-Tae , Cauley Linda , Lefrancois Leo
Published: January 2023

The intestinal mucosa contains unique sites designed for promoting specialized T cell responses. Oral infection with a murinized Listeria monocytogenes (Lm) induced a robust mucosal CD8 T cell response which rapidly converted to a memory population during the peak of the intestinal T cell response. Concomitant with this event, the mice were nearly completely protected against re-infection. Blocking migration with anti-α4β7 from 7 dpi prevented continued migration into the intestinal compartment but did not alter the rapid conversion of early migrating cells into memory suggesting in situ events regulated early memory formation. These early memory cells, but not other effector subsets, preferentially upregulated CD103 identifying them as precursors to mucosal resident memory CD8 T cells. Surprisingly, CD103 expression on infiltrating CD8 T cells was required for early T cell entry into the epithelium but not for long term retention. Moreover, CD103 upregulation on Lm-specific CD8 T cells within the epithelium was predominately independent of TGFβ signals. Thus, the generation of distinct CD8 T cell subsets uniquely tailored to respond to intestinal pathogens may be a hallmark of oral challenge. Indeed, CD8 T cells that infiltrate the intestinal mucosa following intranasal influenza virus infection failed to generate MPECs and were not maintained long-term, demonstrating the importance of intestinal priming for generating robust mucosal memory CD8 T cells.
Temporal dynamics of the primary human T cell response to yellow fever virus 17D as it matures from an effector- to a memory-type response (P1406)
Authors: Blom Kim , Braun Monika , Ivarsson Martin , Gonzalez Veronica , Falconer Karolin , Moll Markus , Ljunggren Hans-Gustaf , Michaelsson Jakob , Sandberg Johan
Published: January 2023

The live attenuated yellow fever virus 17D vaccine provides a well-controlled model to study T cell responses to an acute viral infection in humans. Here, we have studied the temporal dynamics, composition and character of the primary human T cell response to YFV. The acute YFV-specific effector CD8 T cell response was broad and complex, composed both of dominant responses that persisted into the memory population, as well as of transient subdominant responses that were not detected at the memory stage. Furthermore, HLA-A2- and HLA-B7-restricted YFV epitope-specific effector cells predominantly displayed a CD45RA-CCR7-PD- 1+CD27high phenotype, which transitioned into a CD45RA+CCR7-PD-1-CD27low memory population phenotype. The functional profile of the YFV-specific CD8 T cell response changed in composition as it matured from an effector- to a memory-type response, and tended to become less polyfunctional in the course of this transition. Interestingly, activation of CD4 T cells as well as FoxP3+ T regulatory cells in response to YFV vaccination preceded the kinetics of the CD8 T cell response. The present results contribute to our understanding of how immunodominance patterns develop, as well as the phenotypic and functional characteristics of the primary human T cell response to a viral infection as it evolves and matures into memory.
A GATA-3 function distinct from Th2 determination controls basic T cell function (P1410)
Authors: Wan Yisong , Wang Yunqi , Gu Aidi , Curitis Thomas
Published: January 2023

T cells are pivotal to controlling the immune response to fight infection, eradicate tumor and elicit autoimmunity. CD8 T cells are the “cytotoxic” T cells to mediate the killing of target cells. And CD4 T cells differentiate into “helper” T (Th) cell subsets to modulate immune function. To achieve desired T cell response, series of events including cell activation, expansion and differentiation will have to be accomplished following antigenic and mitogenic stimulation. Therefore, identifying the factors critical for T cell activation, expansion and differentiation becomes the central question for T cell immunology. GATA-3 is a master regulator for Th2 differentiation. It is however poorly understood if GATA-3 controls mature T cell function beyond Th2 determination. We found that TCR-signal and cytokine-stimulation promoted GATA-3 expression in CD8 T cells with enhanced proliferation but not Th2-like differentiation. In addition, while GATA-3-deficient CD8 T cells developed normally, their peripheral maintenance was impaired. More strikingly, TCR- and cytokine-promoted expansion of CD8 and CD4 T cells was abrogated in the absence of GATA-3. This study thus reveals an unprecedented GATA-3 function central to basic T cell functions beyond Th2 differentiation.
Notch signalling shapes the CD8 T cell response following Listeria infection (P1416)
Authors: Mathieu Mélissa , Duval Frederic , Labrecque Nathalie
Published: January 2023

Following an infection, naïve CD8 T cells expand and differentiate into effectors able to eliminate the pathogen. At the peak of the response, two populations of effectors are distinguishable: short-lived effector cells (SLECs) meant to die by apoptosis and memory precursor effector cells (MPECs) destined to survive as memory cells that will confer long-term protection. Thus, following activation, the CD8 T cell faces a binary cell fate decision. We postulate that the Notch signalling pathway, known for its role in cellular differentiation and binary cell fate choice, acts as a key player in the MPEC/SLEC choice. To elucidate the role of Notch signalling in CD8 T cell response, we infected mice lacking or not expression of Notch1 and Notch2 in mature CD8 T cells with Listeria monocytogenes expressing OVA. Notch deficiency led to the generation of more OVA-specific effector CD8 T cells but less of these effectors had a SLEC phenotype (CD127loKLRG1hi) at the peak of the response. Surprisingly, Notch did not impair CD8 T cell memory generation following infection. Thus, Notch signalling influences the expansion and the acquisition of a SLEC phenotype following Listeria infection but not the generation of memory cells. Understanding the molecular pathways leading to memory generation is crucial as this knowledge will ultimately contribute to new vaccine development.
Human anti-HIV-1 envelope monoclonal antibodies with a low percentage of mutations have the ability to neutralize sensitive Tier 1 viruses. (P6189)
Authors: Gorny Miroslaw , Li Liuzhe , Wang Xiao-Hong , Williams Constance , Volsky Barbara , Seaman Michael , Zolla-Pazner Susan
Published: January 2023

Antibodies (Abs) specific to the HIV-1 envelope regions, the CD4 binding site (CD4bs), V3 and V2 loops display neutralizing activity and may have protective functions. These Abs are present in almost all HIV-infected subjects and can also be induced by vaccines, as shown in clinical trials, but exhibit rather low neutralizing activities. To determine a threshold of mutations which may represent the maturity needed for cross-neutralizing antibodies, we have studied the percentage of mutations in the variable fragment of the heavy and light chain genes of 63 human monoclonal Abs (mAbs) which are specific to CD4bs, V3 and V2 domains, and tested their neutralizing activities against a panel of 41 pseudoviruses (psVs). All 63 mAbs neutralized, with various frequencies, a panel of sensitive Tier 1 psVs while the more resistant Tier 2 psVs were neutralized sporadically and only by V3 mAbs. Mutation frequencies ranged from 2.4% to 16.7% of the nucleotide sequences were observed for both the heavy and light chain genes of all mAbs. The percentage of mutations was comparable for CD4bs, V3 and V2 mAbs and had no correlation with the breadth of neutralization. The results indicate that mAbs with at least a few percent of mutations can neutralize sensitive Tier 1 viruses, which probably can be achieved during HIV vaccination.
Antiretrovial therapy changes circulating autoantibody profiles in patients chronically infected with human immunodeficiency virus 1 (P6197)
Authors: Meng Zhefeng , Amet Tohti , Byrd Daniel , Lan Jie , Yu Qigui
Published: January 2023

The emergence of severe liver injury in some patients chronically infected with human immunodeficiency virus 1 (HIV-1) during antiretroviral therapy (ART) presents a diagnostic dilemma. It is unclear whether liver injury is a direct result of drug side effects or a consequence of HIV-1-associated autoimmune responses. To investigate the effect of ART on the pattern of autoimmune responses in HIV-1-infected patients, we used a slide-based autoantigen array to profile circulating autoantibodies against a total of 84 autoantigens in HIV-1-infected patients on ART (n=16) and ART-naïve (n=14). Serum samples from 30 uninfected individuals were also included to obtain autoantibody background information. Compared to uninfected individuals, ART-naïve patients had IgG and IgM autoantibodies increased 2 folds or greater to 13 and 8 different autoantigens, respectively. Strikingly, ART profoundly suppressed autoimmune responses in HIV-1-infected patients, as only IgG autoantibodies against 2 autoantigens increased 2 folds or greater than that in uninfected subjects, and no IgM autoantibodies were increased. In addition, the titers of these 2 IgG autoantibodies in patients on ART were significantly lower than that found in ART-naïve patients. Conclusion: HIV-1 infection enhances circulating autoantibodies that can be significantly suppresed by ART, and liver damage in patients on ART is most likely due to drug side effects rather than autoimmune responses.
HIV-1 infection induces a novel B cell response against HERV-K (HML-2) TM protein (P6209)
Authors: Michaud Henri-Alexandre , de Mulder Miguel , SenGupta Devi , Deeks Steven , Martin Jeffrey , Pilcher Christopher , Hecht Frederick , Sacha Jonah , Nixon Douglas
Published: January 2023

The presence of HERV-K (HML-2) mRNA in plasma and a corresponding specific T cell response during HIV-1 infection is well-documented. We hypothesized that HIV-1 also induces a specific HERV-K (HML-2) EnvelopeBcell response due to the expression of envelope protein on the infected cell surface. This led to the concept that HERV-K (HML-2) Env could be used as an HIV-1 Associated Neo Antigen (HANA) to target infected cells. We showed that the humoral response against HERV-K (HML-2) Env is restricted to two sequences on each envelope sub-unit, the surface unit (SU) and transmembrane (TM) proteins. During HIV-1 infection, we observed an increase of the anti-TM antibody titer and a decrease of the anti-SU antibody titer. Interestingly, we found that HIV-1-infected elite controllers had high titers of anti-TM antibodies compared to HIV-1 infected patients on effective HAART or healthy uninfected donors. We showed that TM is preferentially transactivated and expressed on the cell surface in HIV-1 infected cells. Using HA-137, a human anti-TM antibody, we showed that TM protein is preferentially expressed in the myeloid cell subset as well as in a distinct T cell subset. These findings highlight the crucial role of the monocyte/macrophage lineage in the induction of the anti-HERV-K (HML-2) TM Bcell response.This study suggests that the anti-TM humoral response may play a role in HIV-1 pathogenesis and could provide a new tool for immunotherapeutic strategies.
Host correlates distinguish low-risk and high-risk HIV-1 disease progression in HLA-B*5701 individuals (P6169)
Authors: Raposo Andre , Abdel-Mohsen Mohamed , Hecht Frederick , Pilcher Christopher , Pillai Satish , Nixon Douglas
Published: January 2023

HIV-1-infected individuals with HLA-B*57 alleles progress slowly to disease, in part due to restricted CD8+ T cell responses. However, some HLA-B*57 individuals progress faster to disease. We hypothesized that additional host correlates may explain the risk of disease progression. PBMC from six HLA-B*5701 patients were analyzed longitudinally and divided into high-risk and low-risk progressors based on CD4 counts at baseline. We developed a custom made Taqman array to measure mRNA expression of 34 anti-HIV-1 restriction factors. We named the overall gene expression as Cumulative Restriction or CuRe score. T cell activation, viral load, T cell counts and plasma sCD14 were also determined for all patients at all time-points. The CuRe score was significantly increased in high-risk progressors (p<0.0001), with particular increases of APOBEC3F, APOBEC3G, tetherin, SAMHD1 and interferon-inducible genes. CuRe score correlated with viral load only in low-risk progressors (R>0.60, P<0.001). T cell activation was significantly elevated in high-risk progressors (p<0.05), and correlated with a decline in CD4+ T cell counts (Spearman R=-0.92, P<0.0001). Plasma sCD14 was significantly elevated in high-risk progressors, and correlated with CuRe score (R=0.79, P<0.001). In our study, we show that combining a multiple gene expression array of anti-HIV-1 restriction factors and immunological parameters provide important measures of risk for disease progression in HLA-B*5701 individuals.
Human T cell lifespan (P1284)
Authors: Réu Pedro , Mold Jeff , Michaelsson Jakob , Salehpour Mehran , Frisen Jonas
Published: January 2023

In humans, the lifespan and turnover of T cells have been debated for almost half a century. Until now, the analysis of T lymphocyte turnover have been primarily approached through short term labelling studies and nucleotide analogue incorporation. While much has been gained from these endeavours, there is still considerable debate about the half live of the different T lymphocyte subsets and it is apparent that neither approach alone can give the necessary information to reach a conclusive result. Taking advantage of the decreasing concentration of 14C in the atmosphere since the cold war, we have retrospectively assessed the birth date of the different T cell populations in the blood of healthy adult humans.
Transcription factor Bach2 regulates naïve T-cell homeostasis and functional maturation of CD8 T-cell memory (P1309)
Authors: Kim Eui Ho
Published: January 2023

The transcriptional repressor Bach2 is considered as a B cell-specific transcription factor that maintains B cell identity by opposing terminal differentiation to plasma cells. Here, we show that Bach2 is expressed in CD8 T cells and the level of expression depends upon the cellular differentiation state. Bach2 is highly expressed in naïve and central memory T cells (TCM), but the more terminally differentiated effector cells and effector memory T cells (TEM) expressed low levels of Bach2. A larger fraction of CD8 T cells from Bach2-deficient (Bach2-/-) mice displayed the activated CD44HI phenotype and produced cytokines such as IFN-γ. Following an acute viral infection, Bach2-/- CD8 T cells showed normal clonal expansion and effector differentiation. However, without Bach2, effector to memory transition was markedly impaired. A substantially larger fraction of Bach2-/- memory CD8 T cells displayed the senescent KLRG1HI/CD127LO/CD27LO phenotype, with a marked reduction in the numbers of TCM cells. Functionally, Bach2-/- memory CD8 T cells produced lower levels of IFN-γ, TNFα and IL-2 but expressed higher levels of granzyme B, compared to WT. Thus, we have identified novel roles for Bach2 in maintaining naïve CD8 T-cell quiescence, promoting effector to memory transition and steering functional maturation of memory CD8 T cells, and development of TCM cells. These findings have improved our understanding of the transcriptional regulation of naïve and memory T-cell homeostasis.
ITK regulates the dynamics of CD8+ T cell homeostatic proliferation in lymphopenic environment (P1296)
Authors: Huang Weishan , Huang Fei , August Avery
Published: January 2023

Tec family kinase ITK is an essential regulator of T cell development and function. The absence of ITK leads to spontaneous acquisition of innate memory phenotype (IMP) in CD8+ T cells, shown to be induced by bystander-derived IL-4. CD8+ T cells undergone homeostatic proliferation (HP) in lymphopenic conditions share similar memory markers with IMP CD8+ T cells. To examine the role of ITK during CD8+ T cell HP, we compared naïve CD8+ T cells from WT and Itk-/- OTI-Rag-/- transgenic mice. We found that they had similar CD44loCD122- naïve phenotype, but naïve Itk-/- T cells exhibited increased Eomes, KLF2, FoxO3, Bim, Blimp-1 and decreased Bcl-6 mRNA expression. When transferred into lymphopenic Rag-/- recipients, Itk-/- naïve CD8+ T cells exhibited rapid early expansion, giving rise to > 10 fold expansion over that of WT CD8+ T cells by 10 days post-transfer, which was followed by massive retraction with failure to sustain a plateau as seen in WT. The absence of ITK during HP resulted in defects in expression of cytokine receptors and Bcl-2, accompanied by enhanced apoptosis and Fas expression. These data indicate an important role of ITK in regulating the dynamics and maintenance of CD8+ T cells during HP, likely by regulating the expression of cytokine receptors required for sustained survival of these cells. This work suggests that targeting the ITK pathway may allow manipulation of CD8+ T cell number and function in lymphopenic conditions due to infection or irradiation.
Immune mechanisms mediated by Salmonella in ameliorating airway inflammation (P6031)
Authors: Sparwasser Tim , Ganesh Venkateswaran , Baru Mannan
Published: January 2023

Epidemiological data suggest protection from allergic diseases when exposed to infectious agents during early stages of life. As allergies are mainly characterized as an unchecked Th2 response, modulation of the immune responses by pathogens have been considered to be a major factor in mediating the protection. Recent evidences implicate a key role staged by immuno-regulatory mechanisms induced upon infection in ameliorating these allergic disorders. A longitudinal study had demonstrated the reduced frequency and incidence of asthma in children, who had a prior infection from Salmonella typhi. A subsequent investigation demonstrated reduced airway inflammation in mice infected with Salmonella; however the mechanism still remained obscure. In this study, we aimed to delineate the potential mechanisms induced upon Salmonella typhimurium infection which resulted in the amelioration of allergic airway inflammation in mice. We report a significant increase in CD11b+ Gr1+ myeloid cell population in those mice that had been infected with Salmonella. Using in vitro and in vivo analysis we suggest that these myeloid cells could potentially ameliorate airway inflammation by affecting the stability of Th2 cells. In contrast to our initial hypothesis, regulatory T cells were not involved in the protective effect of salmonella infection.
Commensal bacteria induce a barrier protective response to prevent sensitization to food allergens (P6037)
Authors: Feehley Taylor , Stefka Andrew , Tripathi Prabhanshu , McCoy Kathy , Mazmanian Sarkis , Seo Goo-Young , Theriault Betty , Antonopoulos Dionysios , Chang Eugene , Nagler Cathryn
Published: January 2023

Environmentally-induced alterations in the commensal microbiome have been implicated in the increasing prevalence of food allergy. We found that antibiotic treatment of neonatal mice leads to reduced proportions of regulatory T cells(Tregs) in the colonic lamina propria(LP), impaired production of intestinal IgA, and elevated peanut (PN) specific IgE/IgG1 in response to sensitization. Selective colonization of gnotobiotic mice linked the Treg/IgA inducing capacity to a consortium of bacteria within the Clostridia class. Introduction of either a conventional SPF microbiota or a mixture of Clostridia strains to antibiotic-treated mice restored the Treg and IgA compartments and blocked the induction of a food allergic response. Clostridia colonized gnotobiotic mice displayed increased expression of IL-23 and IL-22 in the colonic LP compartment and the induction of anti-microbial REG3β expression in the epithelium. Collectively, these results suggest that the maintenance of oral tolerance to dietary antigens relies on bacterial populations that induce a barrier protective response, which includes activation of the IL-23/IL22 axis and the expansion of intestinal Tregs and IgA secreting B cells. Our findings hold promise for the development of approaches to prevent or treat food allergy based on modulation of the composition of intestinal microbiota.
Respiratory syncytial virus infection of infant mice disables regulatory T cells with loss of protection from allergic airway disease (P6005)
Authors: Krishnamoorthy Nandini , Khare Anupriya , Oriss Timothy , Raundhal Mahesh , Morse Christina , Moore Martin , Peebles R.Stokes , Ray Anuradha , Ray Prabir
Published: January 2023

Immune tolerance is established early in life beginning in utero. Tregs play a critical role in this process and protect from allergic disease. Although studies have shown a direct correlation between infection by RSV early in life and the risk of development of asthma in adult life, the underlying immune mechanism was not well understood. Towards this goal, we adopted a murine model of maternal transfer of tolerance to the model allergen Ovalbumin (Ova) in infant mice and studied the impact of recurrent RSV infection on tolerance to Ova. We show that infant mice previously tolerized to Ova via their mother’s milk, upon RSV infection, lose tolerance and mount characteristic features of allergic asthma when subsequently challenged with Ova. On the other hand, uninfected tolerized mice did not mount an immune response demonstrating preservation of tolerance. The cause for breach of tolerance in RSV-infected infant mice was found to be the virus’ ability to increase GATA-3 expression and IL-13 production in Foxp3+ Tregs. The Tregs from RSV infected mice had impaired suppressive function. Thus, by promoting Th2 inflammation, RSV interferes with the function of Tregs such that tolerance to an allergen is compromised. Interestingly, treatment of mice with the mucosal adjuvant CpG ODN prevented a switch in Treg phenotype. Our findings highlight a deleterious effect of viral infections in infancy on the immune system, which increases the risk for allergic responses later in life.
The mechanisms of non-depleting YTS177 anti-CD4 monoclonal antibody in controlling ovalbumin-induced asthmatic responses (P6017)
Authors: Kuo Ming-Ling , Wu Cheng-Jang , Lin Chun-Yen
Published: January 2023

Bronchial asthma is an emerged chronic inflammatory disorder of the airway characterized by airway hyperresponsiveness (AHR), pulmonary eosinophilia, elevated serum immunoglobulin (Ig) E, and dysregulated T helper (Th) 2 cytokines. It is becoming urgent to identify target molecules for the preventive and therapeutic purpose. Non-depleting YTS177 anti-CD4 monoclonal antibody (MoAb) has been used to induce immunotolerance in allograft transplantation through antigen-specific Treg cells. This study aims for determining whether YTS177 MoAb is able to inhibit the ovalbumin (OVA)-induced allergic inflammation in an asthmatic murine model. The injection of YTS177 MoAb before and after OVA-sensitization was able to suppress AHR, pulmonary eosinophilia, OVA-specific IgE synthesis, and the production of Th2 cytokines from OVA-activated splenocytes. YTS177 MoAb inhibited the proliferation of DO11.10 CD4+ T cells upon in vivo OVA-stimulation as well. Moreover, YTS177 MoAb-treatment sustained the protein levels of anergy factors, p27kip1 and Cbl-b, and inhibited IL-2 production and cell cycle progression in activated T cells in vitro. The tolerance status might be built on YTS177 MoAb-induced CD4 down regulation and raft dissociation through clathrin-dependent manner. Collectively, YTS177 MoAb binding causes CD4 internalization that further perturbs T cell receptor signaling and ultimately results T cell unresponsiveness.
The role of Eos in T cell function (P1059)
Authors: Rieder Sadiye , Shevach Ethan
Published: January 2023

Eos, an Ikaros family transcription factor, is preferentially expressed in T regulatory cells (Tregs). Although previous studies have demonstrated that the elimination of Eos by siRNA in Tregs resulted in the loss of their suppressive function both in vitro and in vivo, Tregs from mice with a global deletion of Eos suppress normally in vitro and in the IBD model in vivo. While non-activated T conventional (Tconv) cells fail to express Eos, Eos expression is rapidly upregulated following TCR activation in vitro. TCR activated Tconv cells from Eos deficient (-/-) mice proliferate poorly, but make normal amounts of IL-2. The diminished proliferative response is secondary to a defect in the upregulation of CD25 that is accompanied by a decrease in p-STAT5 and p-S6 levels. Surprisingly, Eos-/- mice develop more severe EAE compared to wild type (WT) mice. Mixed chimeras between WT and EOS-/- bone marrow donors also develop very severe EAE. A greater number of Eos-/- CD4+ T cells than WT CD4+ T cells can be detected in the CNS and they produce higher levels of IL-17 than WT CD4+ T cells. Increased IL-17 production by Eos-/- CD4+ T cells correlates with their defect in IL-2 signaling. While WT Tregs can be detected in the CNS of the chimeric mice, Eos-/- Tregs are not detectable. These studies raise the possibility that Eos negatively regulates the production of IL-17 in Tconv, and may also play an important role in the migration of Tconvs and Tregs.
Exosomes represent a novel mechanism of regulatory T cell suppression (P1079)
Authors: Steinkamp Heidi , Delgoffe Greg , Frase Sharon , Castellaw Ashley , Vignali Dario
Published: January 2023

Regulatory T cells suppress via a variety of mechanisms, both contact-dependent and -independent mechanisms. These is some controversy as to which mechanisms play more dominant roles during various disease states as well as maintenance of immune homeostasis. Our laboratory has found that regulatory T cells secrete exosome-like vesicles that are 20 to 50 nm in diameter. Membrane vesicles have been shown to mediate communication amongst immune cells, but they also mediate communication between immune cells and cancer cells and between and immune cells and pathogens. Membrane vesicles mediate physiologic changes in recipient cells by initiating cellular signaling events following receptor binding, or via uptake and integration of vesicular cargo, which can include receptors, enzymes, cytokines, chemokines and genetic material. We have found that functional regulatory T cell membrane vesicles mediate suppression of conventional CD4+ T cells, whereas resting regulatory T cell and conventional T cell vesicles do not. Furthermore, we have found that 293F cells package overexpressed immunosuppressive cytokines into exosomes following transfection. Taken together these data suggest that exosome-like vesicles represent a bridge between contact dependent and independent regulatory T cell suppression.
Dual regulation of CD8+ T cell and NK cells following systemic cancer immunotherapy (P1087)
Authors: Alvarez Maite , Sckisel Gail , Bouchlacka Myriam , Murphy William
Published: January 2023

NK-CD8 T cell regulation has been demonstrated in viral infection models. It is believed that NK cells regulate CD8 T cells to prevent exacerbated activation resulting in pathological effects. Current immunotherapy regimens use immunostimulation with blockade of negative regulators to achieve optimum antitumor effects. We observe that NK and CD8 T cells play dual roles in their concurrent regulation following systemic administration of IL2 and αTGFβ as the depletion of either population resulted in a higher expansion of the other. This data indicates a possible cytokine competition between both NK and CD8 T cell compartments. However, antigen nonspecific bystander CD8 T cells were particularly enhanced after NK depletion whereas CD4 T cells were not affected. Therefore, this data suggests a possible elimination of CD8 T cells after NK activation which is FasL-dependent as Fas expression is upregulated on CD8 T cells after NK depletion. This mechanism is not mediated by perforin because the IL2 treatment of perforin-deficient mice resulted in a higher CD8 T cell expansion after NK cell depletion. In conclusion, this study illustrates the existence of an NK-CD8 T cell regulation in an antigen-nonspecific stimulation model which demonstrates a tight regulation between adaptive and innate immune responses. Further, the study demonstrates the potential efficacy of increasing antitumor responses by concurrent application of immunostimulation with blockade of negative regulators.
TLR2 signaling in epithelial cells initiates sterile tissue inflammation through recruitment of NK cells (P1089)
Authors: Kim Hye Jeong , Lee Jong Soo , Kim Ahra , Koo Sumi , Cha Hee Jeong , Kwon Byoung S. , Mittler Robert , Cho Hong R. , Kwon Byungsuk
Published: January 2023

It is well recognized that damage-associated molecular patterns (DAMPs) released from damaged kidney cells initiate post-ischemic inflammation, an essential step in the progression of kidney ischemia-reperfusion injury. However, the mechanism that coordinates this highly specific process in the ischemic kidney remains to be clarified. We report here that endogenous TLR2 ligands released from ischemic TECs induce expression of CCR5 chemokines in an autocrine manner, thereby promoting recruitment of NK cells. By implantation of CD137L-/- TECs into the kidney capsule of TLR2-/- mice and vice versa, we further show that TLR2-mediated recruitment of NK cells is an uncoupled event occurring independently of CD137L signaling in TECs and sufficient to result in kidney ischemia-reperfusion injury (IRI) by inducing subsequent recruitment of neutrophils that is dependent upon CD137L signaling in TECs. Therefore, our findings identify TECs as not only a target for kidney damage but also a master regulator that plays a central role in initiation and amplification of acute sterile inflammation inflicting kidney IRI. Clinically important, the signaling pathway of innate receptors in epithelial cells may therefore represent a good target for blocking acute sterile inflammation occurring as a result of tissue damage, including kidney IRI.
Detecting genuine regulatory relationships among transcription factors, miRNAs and target genes post influenza virus infection (P1091)
Authors: Miao Hongyu
Published: January 2023

The development of Systems Biology approaches has provided unprecedented opportunities for researchers to gain novel and deeper insights into biological mechanisms. In particular, Systems Biology methods have been proposed to identify gene regulatory relationships based on high-throughput omics data in the infectious disease setting. However, successful applications of the Systems Biology approaches in experimental studies have been hampered by two problems: first, post-transcriptional regulation is usually missing in previous modeling work; second, many approaches are purely data-driven and thus could have a high false positive rate in predicting regulatory relationships. In this study, we propose a new framework for detecting genuine regulatory relationships by exploiting both available expert knowledge and time-course expression data of transcription factors, miRNAs and target genes. The regulatory relationships are explicitly anchored to a set of potential interactions and then selected by rigorous quantitative methods such as DBN and LASSO. We apply our method to influenza virus infection in human and predict the target genes regulated by each transcription factor and miRNA with the highest potential.
Essential role of E3 ubiquitin ligase activity of GRAIL in T cell functions (P1111)
Authors: Nurieva Roza , Wang Junmei , Alekseev Andrei
Published: January 2023

T cells are the master regulators of adaptive immune responses, and many autoimmune diseases arise due to a breakdown of self-tolerance in T cells. Understanding of the molecular mechanisms underlying T cell tolerance will lead to development of pharmacological approaches either to promote the tolerance state in terms of autoimmunity or to break tolerance in cancer. E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. We as well as other groups have reported that T cells activated in the absence of both CD28 and ICOS costimulation developed into tolerant T cells, associated with markedly upregulated expression of the E3 ubiquitin ligase GRAIL. In order to understand the physiological function of GRAIL, we generated mice deficient in Grail by replacing region that encompassing most of the RING domain and responsible for E3 ubiquitin activity. Remarkably, genetic inactivation of E3 ubiquitin ligase function of GRAIL led to T cell hyper-responsiveness to TCR/CD3z signaling and their independency to costimulation for activation. As a result, GRAIL-deficient mice were more predisposing to autoimmune diseases. On the other hand, modulation of GRAIL function helped to boost T cell immune responses to cancer, and, therefore, mediate tumor rejection. Thus, modulation of the E3 ligase activity of GRAIL might be an important approach to control T cell functions in autoimmunity or cancer.
CD43 activates moonlight functions of pyruvate kinase (P1113)
Authors: Bravo-Adame Maria Elena , Vera-Estrella Rosario , Barkla Bronwyn , Cruz-Muñoz Mario , Rosenstein Yvonne
Published: January 2023

CD43 is the most abundant costimulatory molecule on the T cell surface; it transduces activation signals through its cytoplasmic domain, modulating the outcome of T cell responses. CD43 KO mice revealed a positive role for CD43 in T cell migration and in the regulation of cell number and homeostasis. The aim of this study was to identify new signaling pathways modulated by CD43. Protein expression profiles of normal human CD4+ T cells stimulated through TCR and CD43 identified changes in 189 proteins. Bioinformatic analysis revealed that glucose metabolism was significantly represented. We evaluated pyruvate kinase (PK) and lactate dehydrogenase (LDH) activities, as well as glucose uptake. TCR+CD43 stimulated cells showed a 1.5 fold increase in PK activity, but only a moderate increase in LDH activity and glucose uptake. Interestingly, tyrosine phosphorylation of PK triggers its ability to phosphorylate Stat3. Phosphorylation of Y105 of PK and of Y705 of Stat3 was induced in response to TCR+CD43 and TCR+CD28. Since phosphorylation of Y705 of Stat3 by PK induces MEK5 transcription, we evaluated the MEK5/ERK5 pathway activation by measuring the levels of pCREB at S133, an ERK5 target. CREB pS133 was increased in TCR+CD43 and TCR+CD28 stimulated T cells. Altogether, our results show that CD43 regulates monosaccharide metabolism, as well as moonlight functions of metabolic enzymes.
The direct impact of key cytokines on memory effectors from older adult human CD8+ T cells to live influenza virus (P4297)
Authors: Zhou Xin , Swain Susan , McElhaney Janet
Published: January 2023

An age-related decline in cytolytic activity has been described in CD8+ T cells and we have shown that the poor CD8+ effector T cell response to influenza A/H3N2 challenge is due to a decline in the proportion and function of these cytolytic T lymphocytes (CTL). Here we report that there is a significant decline in the levels of many cytokines and chemokines including IFN-γ, IL-2, IL-10 and IL-17A, but increased IL-4 and IL-6, following influenza-stimulation of PBMCs from vaccinated older compared to young adults. We find that addition of exogenous IL-2 acts to maintain naïve CD8+ T cells survival and strongly enhances the proliferation of existing memory effector CD8+ T cells in response to live influenza virus challenge. In addition exogenous IL-6 plays a similar but lesser role in the proliferative response when compared to IL-2. Our data show that only exogenous IL-4 enhances the proliferative capacity of the CD8+ T cells and particularly the promotion of naïve CD8+ T cell differentiation. However, IL-4 treatment has no effect on the accumulation of CD8+ effector T cells in response to influenza challenge. These results suggest mechanisms that might be exploited to develop influenza vaccines with improved efficacy in older adults.
Attenuated dendritic cell function in BALB/c mice following HPAI vaccination results from failed C-type lectin receptor signaling (P4309)
Authors: Vaughan Sarah , Stout-Delgado Heather , Pollock Zemmie , Plourde Jennifer , Pyles John , Harrod Kevin
Published: January 2023

Highly pathogenic avian influenza (HPAI) has an approximate 60% fatality rate in humans. In the United States, a vaccine for HPAI has been manufactured and stockpiled using FDA approved methods for seasonal vaccines. Compared to seasonal vaccines, the HPAI vaccine is less immunogenic in humans despite similar production methods. We developed a mouse model of influenza vaccination to examine differences in the host immune response to these vaccines. Mice that received the seasonal vaccine produced a robust neutralizing antibody response whereas no neutralizing antibodies were detected following HPAI vaccination. Dendritic cells (DCs) cultured with HPAI vaccine had decreased expression of activation markers CD40 and CD86, and produced significantly lower levels of cytokines IL-6, IL-12, and TNF-α compared to those treated with seasonal vaccine. C-type lectin receptors have been implicated as a class of pattern recognition receptors in innate immunity and immunization. Pretreatment with mannan diminished cytokine induction by DCs in a dose dependent manner following seasonal but not HPAI vaccine treatment, suggesting a role for C-type lectin receptors in DC activation by influenza vaccines. These findings implicate a potential mechanism for attenuated DC function following HPAI vaccination and may explain the lack of immunogenicity of the currently approved HPAI vaccine.
Recombinant Ebolavirus antigens from insect cells are potent immunogens inducing cellular and humoral immunity in rodents and non-human primates and provide protection against virus challenge (P4325)
Authors: Lieberman Michael , Lehrer Axel , Humphreys Tom , Johns Lisa , Wong Teri-Ann , Olinger Gene , Dye John , Carrion Ricardo , Patterson Jean , Marzi Andrea , Feldmann Heinz
Published: January 2023

Soluble recombinant Filovirus surface glycoproteins (GP) and matrix proteins (VP24 and VP40) were generated in the Drosophila S2 cell expression system and purified by immunoaffinity chromatography. The immunogenicity of individual recombinant Zaire ebolavirus (ZEBOV) subunits and admixtures with or without adjuvants was evaluated in mice, guinea pigs and macaques. Strong antigen-specific IgG responses were observed, includng Ebola virus neutralization responses. In mice and macaques subunit proteins were shown to elicit cell mediated immunity, as significant B- and T-cell stimulation was observed in immune lymphocytes after antigen re-stimulation. Analysis of secreted cytokines in batch-cultured, antigen-stimulated splenocytes or PBMC’s demonstrated Th1 and Th2 type responses. Vaccine candidates were tested in mice and guinea pigs for direct protection against challenge with species-adapted ZEBOV. All vaccine formulations containing ZEBOV GP were protective in mice and serum transfer from such animals into naïve mice demonstrated that humoral immunity alone can be fully protective. Furthermore, the transfer of immune splenocytes into naïve mice showed that recombinant GP and VP24 subunits elicit functional T cell responses that lead to protection against live virus challenge. In guinea pigs, lead vaccine formulations consistently produced high antibody responses and demonstrated 100% protective efficacy in the ZEBOV challenge model.
Heterologous prime-boost vaccination with DNA vaccine and recombinant subunit containing four serotypes of dengue virus envelope protein domain III elicits neutralizing antibodies and specific T-cell responses (P4327)
Authors: Pan Chien-Hsiung , Hu Hui-mei , Hsiao Yu-Ju , Wu Sze-Hsien
Published: January 2023

Dengue is still an important public health problem and no dengue vaccine is available now. In this study, we characterized prime-boost vaccine regimens using heterologous and homologous dengue DNA vaccine and recombinant subunit vaccine consisted of envelope (E) protein domain III (ED III) from four serotypes of dengue virus (DENV). A broader and balanced IFN-gamma and IL-4 responses was observed in DNA-prime and subunit-boost immunized mice, compared to a narrower but strong IFN-gamma production after homologous DNA vaccination and an IL-4 only response in homologous subunit vaccine immunized mice. In addition, a significant ED III specific antibody response was detected in all immunized mice except vector control. The highest IgG titer was appeared in homologous subunit vaccine immunized mice, followed by heterologous prime-boost regimen and homologous DNA vaccine. However, that heterologous prime-boost immunized mice generated a highest neutralizing antibody than the others, suggested a better protection provided by this DNA-prime and subunit vaccine-boost regimen.
SeVRSV, a PIV and RSV vaccine, proves efficacious in a cotton rat maternal antibody model (P4279)
Authors: Hurwitz Julia , Portner Allen , Russell Charles , Surman Sherri , Jones Bart , Sealy Robert , Rudraraju Rajeev , DeVincenzo John
Published: January 2023

Respiratory syncytial virus (RSV) and the human parainfluenza viruses (PIV) are causes of significant morbidity and mortality among infants each year. We are now advancing SeVRSV, a Sendai virus based Jennerian vaccine for human PIV-1 and a recombinant vaccine for RSV. SeVRSV expresses RSV F and induces immunity that protects against RSV in small animals and non-human primates. Immune responses persist systemically and in the respiratory tract, which ensures that pathogens can be blocked at their point-of-entry. One concern surrounding pediatric vaccines is that maternal antibodies may obstruct vaccine efficacy. We therefore designed a cotton rat maternal antibody model to test SeVRSV. First, to define the levels of maternal antibodies typical of the vaccine's intended target population, we titered antibodies from approximately 100 human infants. Then, by passive transfer techniques, we matched these titers in animals. Intranasal vaccination in test animals was followed by the measurement of de novo PIV-specific and RSV-specific antibodies, and cotton rat challenge with RSV to measure protection. In models using polyclonal or monoclonal antibody products with specificities for PIV and/or RSV, the SeVRSV vaccine protected against RSV challenge. Data suggest that SeVRSV will not be inhibited by maternal antibodies in the clinical setting, and encourage rapid development of the vaccine for protection of human infants from PIV and RSV.