bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin E2 induces activation of mTORC1 and mTORC2 in mast cells: selective utilization of mTORC2 for the regulation of chemotaxis and mediator release (151.6)
Authors: Jung Mi-Yeon , Kuehn Hye Sun , Beaven Michael , Metcalfe Dean , Gilfillan Alasdair
Published: January 2023

PGE2 is a potent modifier/regulator of multiple mast cell responses including FcϵRI-mediated degraulation, chemokine production, adhesion to basement membrane proteins, and chemotaxis. Nevertheless, how PGE2 regulates these diverse responses remains unclear. In mouse bone marrow-derived mast cells, we observed that PGE2 induced activation of mTORC1 (mTOR-raptor complex), as indicated by increased p70S6K and 4E-BP1 phosphorylation; and mTORC2 (mTOR-rictor complex), as indicated by increased phosphorylation of AKT at position Ser473. We thus investigated the roles of these signaling complexes in PGE2-mediated mast cell responses. Inhibition of mTORC1, through the use of the selective mTORC1 inhibitor rapamycin or by raptor-targeted shRNA, had minimal impact on PGE2-mediated chemotaxis or chemokine production. In contrast Torin, which inhibited both mTORC1 and mTORC2, and rictor-targeted shRNA, markedly reduced PGE2-mediated chemotaxis and this was associated with down-regulation of PGE2-induced cytoskeletal reorganization. Downregulation of mTORC2 furthermore attenuated PGE2-induced production of the chemokine CCL2, which was linked to a significant reduction in ROS production. Mast cell degranulation and adhesion were unaffected by down-regulation of either mTOR complex. Taken together, these data illustrate that mTOR2, is an important signal for chemotaxis and chemokine production from PGE2-activated mast cells.
Establishing the kinetics of TFH cell development in vivo (152.11)
Authors: Baumjohann Dirk , Okada Takaharu , Ansel K.
Published: January 2023

T follicular helper (TFH) cells have recently emerged as the main regulators of T cell-dependent humoral immune responses. Yet both the developmental pathway and the maintenance of TFH cells are only poorly understood. In addition, it is not clear if TFH cells are a stable T helper cell lineage or if the TFH phenotype represents a transient activation state. The transcriptional repressor BCL6 has been shown to be crucial for the establishment of the TFH phenotype. However, the mechanism by which BCL6 accomplishes this task and the regulation of BCL6 in TFH cells at the pre- and post-transcriptional level remain largely unknown. In order to generate a comprehensive kinetic profile of BCL6 expression in TFH cells on the genomic and protein level, we are taking advantage of a novel murine BCL6-reporter strain and improved intracellular BCL6 staining reagents.
Secreted Frizzled-related protein-1 regulates human peripheral CD4 T cell responses (152.16)
Authors: Lee Yoon-Sook , Lee Kyoo-A , Kang Chang-Yuil
Published: January 2023

Secreted Frizzled-related protein-1 (sFRP1) has been described as a modulator for complex Wnt signaling pathway. In addition to its essential function in T cell development, recent studies have indicated a critical role for Wnt/β-catenin signaling pathway in the biology of mature T cells such as differentiation, polarization, and survival. Here, we investigated the effect of sFRP1 on TCR/CD28 stimulated memory and naive CD4 T cells derived from human peripheral blood in the context of cytokine response of Th subsets. sFRP1 treatment inhibited proliferation and early cytokine secretion of CD3/CD28 activated memory CD4(+)CD45RO(+) T cells, whereas it increased proliferation of CD3/CD28 activated naïve CD4(+)CD25(-)CD45RO(-) T cells. Moreover, sFRP1 influenced lineage choice of naïve CD4 T cells, resulting in enhanced Th1 and Th17 polarization with suppressed Th2 fate. Regulation of Th1/Th2 polarization by sFRP1 was expected from its antagonistic effect on Wnt/β-catenin signaling reported to favor Th2 over Th1 polarization in CD4 T cells. Interestingly, sFRP1 also positively regulated Th17 polarization and sFRP1 treatment of naïve CD4 T cells in Th17-polarizing conditions up-regulated IL-17 expression. Taken together, our observations suggest the function of sFRP1 in the control of activated human peripheral CD4 T cell subsets.
Acute-phase serum amyloid A causes Th1 differentiation in murine CD4+ T lymphocytes (152.18)
Authors: Lui Asona , He Rong
Published: January 2023

Serum Amyloid A (SAA) is an acute-phase protein, released predominantly from the liver during large scale inflammation. High SAA levels, up to 1000 times normal, are also associated with autoimmune conditions like Rheumatoid Arthritis (RA). SAA is found in very high levels in the arthritic joints of patients, along with a high concentration of certain subsets of T lymphocytes, notably helper T cell type 1 (Th1). The marker for Th1 cells is production of the cytokine interferon gamma (IFNγ). The purpose of this study is to determine if SAA stimulation of murine T cells causes Th1 differentiation. CD4+ T-cells isolated from the spleens of wild type mice have been stimulated in vitro with SAA protein. IFNγ production was measured and compared to that of the known inducer of Th1 cells, interleukin-12 (IL-12). Indeed, SAA stimulation induced IFNγ production with levels as high as 22,088 pg/mL, which is comparable to the 20,237 pg/mL induced by IL-12. Our data also shows that SAA and IL-12 can co-stimulate T cells and result in even higher levels of IFNγ secretion. Future experiments will confirm Th1 differentiation by detecting cell surface markers with flow cytometry and will also investigate the intracellular actions of SAA stimulation. These results suggest that SAA and Th1 cells are found together in arthritic joints because SAA induces this T-cell subset’s production, and is perhaps a missing link in our understanding of RA.
E3 ubiquitin ligase Cbl-b negatively regulates T helper 2 cell responses by targeting Stat-6 for ubiquitination (152.19)
Authors: Qiao Guilin , Ying Haiyan , Guo Hui , Chen Bohao , Zhu Xiangdong , Solway Julian , Penninger Josef , Langdon Wallace , Zhang Jian
Published: January 2023

Casitas-B-lineage lymphoma protein-b (Cbl-b), an E3 ubiquitin ligase, belongs to the Cbl family. Recent studies indicate that Cbl-b emerges as a gatekeeper that controls the activation threshold of the antigen receptor and maintains the balance between tolerance and autoimmunity. However, the role of Cbl-b in T helper (Th) cell differentiation remains unknown. In this study, we show that Cbl-b deficiency selectively results in a biased Th2 differentiation in vitro. Furthermore, using a mouse model of asthma, we find that the absence of Cbl-b results in severe airway inflammation which associates with stronger Th2 responses including elevated IL-4, IL-5, and IL-13, and accumulation of eosinophils in bronchoalveolar lavage (BAL) fluid and higher titers of IgE in the serum. At the molecular level, Cbl-b selectively associates with Stat-6 upon IL-4 ligation, and targets Stat-6 for K48-linked polyubiquitination and subsequently degradation in the proteasome. These processes are heightened in the presence of TCR/CD28 costimulation. IL-4-induced Cbl-b-Stat6 association requires Cbl-b TKB domain, whereas TCR/CD28-induced Cbl-b-Stat6 interaction is mediated by tyrosine residues of Cbl-b and SH2 domain of Stat6. Therefore, we have discovered a previously uncharacterized function of Cbl-b in Th2 cell differentiation that may serve as an important therapeutic target for allergic diseases including asthma.
IL-10 production by Th2 cells in vivo occurs by sub-differentiation into a novel, suppressive IL-10+CTLA-4hi Th2 subset (152.28)
Authors: Altin John , Goodnow Chris , Cook Matthew
Published: January 2023

IL-10 is a critical regulatory cytokine that can be produced by many cell-types of the immune system, including IL-4-producing Th2 cells where it was first identified. However, current knowledge of Th2 cell IL-10 production comes largely from cultures in vitro, and little is known about how IL-10 is regulated at the single cell level during Th2 cell differentiation in vivo. Here, by directly measuring and manipulating IL-4 and/or IL-10-producing cells formed during inflammatory responses in vivo, we show that Th2 cells acquire the ability to produce IL-10 as part of a regulated differentiation process. This process is characterised by the co-ordinate upregulation of a program of other ‘regulatory’ genes, particularly CTLA-4. Differentiation is exquisitely dependent on Card11-dependent external signals that condition IL-10-CTLA-4lo ‘early’ Th2 cells to become IL-2-responsive IL-10-CTLA-4int Th2 ‘intermediates’. Upon exposure to cytokines such as IL-2, IL-10 and IL-21, these intermediates progress into IL-10+CTLA-4hi ‘end state’ Th2 cells that have potent suppressive function. Our findings define a novel process of external signal-regulated Th2 cell sub-differentiation in vivo, culminating in a suppressive subset that may account for negative feedback during chronic Th2 responses.
Molecular mechanism for pre-B cell loss in mutant rabbits (153.17)
Authors: Robbins Gregory , Knight Katherine
Published: January 2023

VH4 is the most frequently rearranged VH gene during B cell development in mutant ali/ali rabbits; however, most VH4-utilizing cells do not survive the pre-B cell stage. The mechanism(s) by which these pre-B cells are lost remains an enigma. We hypothesize that VH4-utilzing pre-B cells are lost due to inefficient pre-BCR formation and inadequate pre-BCR signaling. Ex-vivo cultures of ali/ali bone marrow cells on OP9 stroma yield fewer blasting pre-B cells and reduced levels of phosphorylated-Syk than cultures of wildtype(wt) controls, suggesting that VH4-utilzing pre-BCRs have impaired signaling. We previously determined that pre-BCRs containing VH4-μ chains are less abundant on the cell surface compared to the pre-BCRs containing VH1-μ chains as are found in wt rabbits. A single amino acid substitution in FR2 of VH4(Y47W)restored the levels of cell surface pre-BCR expression to that of wt pre-BCR, and current studies are testing if this substitution is sufficient to restore pre-BCR cell signaling and pre-B cell development. Loss of VH4-utilizing pre-B cells is not likely due to gross structural defects in VH4-μ chains since the conventional BCRs utilizing VH4 are both functional and diverse. Therefore, studies utilizing the ali/ali rabbit offer a unique model system to identify necessary amino acids for pre-BCR formation and signaling.
Role of antigen in later stages of B cell development (153.20)
Authors: Taham-zadeh Dariush , Wu Yuan , Ratcliffe Michael
Published: January 2023

B cell development occurs in the gut associated lymphoid tissue (GALT) of many mammals and birds. In chickens, the early stages of B cell development, including colonization of bursal follicles, B cell proliferation within bursal follicles and repertoire diversification by gene conversion, are supported by surface Ig receptor related constructs that lack antigen binding capacity. Thus early B cell development requires Ig receptor expression but not sIg ligation. In contrast later stages of bursal development, including cortico-medullary redistribution of B cells and their maintenance after hatch, is not supported by those constructs. The later stages of bursal B cell development occur in the presence of gut derived antigens suggesting the possibility that later stage may require antigen mediated sIg receptor ligation as opposed to simply receptor expression. To address this directly, we have introduced Ig receptor related constructs with defined antigen specificity into developing chick embryos. Expression of such constructs is sufficient to support early stages of B cell development, and in the absence of cognate antigen, is not sufficient to support the later stages of B cell development. Preliminary evidence suggests, however, that introduction of cognate antigen results in extended maintenance of receptor expressing B cells after hatch suggesting that ligation of the BCR may act as a survival signal in the later stages of B cell development in the bursa.
Role of CD38 during ontogeny of murine B cells in bone marrow (153.23)
Authors: Romero-Ramirez Hector , Pelayo-Camacho Rosana , Lopez-Santiago Ruben , Rodriguez-Alba Juan Carlos , Santos-Argumedo Leopoldo
Published: January 2023

CD38 is a 45 KD transmembrane protein, expressed by immature and mature lymphocytes. Agonistic antibodies directed to this molecule induce several biological responses: cellular proliferation, protein phosphorylation, intracellular Ca2+ mobilization, rescue from and induction of apoptosis. CD38 ligation with a mAb anti-CD38 (NIM-R5) induces apoptosis of the murine pro-B cell line (Ba/F3); this phenomenon depends on tyrosine kinases activation and the MAP kinase ERK. The expression and function of CD38 during B-cell ontogeny has been poorly analyzed in mice. We analyzed the expression of CD38 during the ontogeny of B cells in murine bone marrow. CD38 is expressed from the preproB stage. ProB, preB and immature B cells from murine bone marrow were all positive for the expression of CD38, but immature B cells had the highest fluorescence intensity, and the proB stage shows a low and high expression of CD38 that suggest two probable populations. The results suggest that CD38 may play a role during the ontogeny of murine B cells. We are analysing whether CD38 ligation on these subpopulations, induces proliferation, maturation or apoptosis.
Molecular interactions of the oncogene NUP98-HOXA9 (153.31)
Authors: Sarma Nayan , Yaseen Nabeel
Published: January 2023

NUP98-HOXA9 is the prototype of the fusion oncoproteins that result from NUP98 gene rearrangements and are associated with acute myeloid leukemia. It consists of an N-terminal FG-rich portion of the nucleoporin NUP98 fused to a C-terminal portion of the homeobox protein HOXA9, and acts as an aberrant transcription factor. Using a yeast two-hybrid assay we have identified several interacting partners of NUP98-HOXA9 including Dynein Light Chain 1 (DYNLT1) and Amino-terminal Enhancer of Split (AES). We showed that the FG repeat region of NUP98 is critical for these interactions. In addition, we found that DYNLT1 localizes to the nuclear periphery, interacts with other nucleoporins and these interactions are dependent on the C-terminal of DYNLT1. Knockdown of DYNLT1 resulted in reduction of the ability of NUP98-HOXA9 to activate its target promoters. AES localizes primarily to the interior of the nucleus and showed a strong interaction with NUP98 but not with other FG repeat nucleoporins. AES augmented the transcriptional activity of NUP98-HOXA9. In the presence of NUP98-HOXA9, AES caused an increase in long-term proliferation of primary human CD34+ cells with an increase in the numbers of primitive cells. AES caused a shift away from the erythroid lineage in primary CD34+ cells, thus counteracting the effects of NUP98-HOXA9 on differentiation. These data establish that DYNLT1 and AES interact and cooperate with NUP98-HOXA9 in transcriptional regulation and cell transformation.
Estrogenic microenvironment influences myeloipoiesis to generate regulatory myeloid cells (153.34)
Authors: VanGundy Zachary , Muth Dillon , White Angela , Taylor Cassandra , Papenfuss Tracey
Published: January 2023

Myeloid cells include macrophages, dendritic cells (DCs), monocytes and their precursors. These cells can promote and regulate inflammation and immunity. Regulatory macrophages, tolerogenic DCs (Tol-DCs) and myeloid derived suppressor cells (MDSCs) are all examples of regulatory myeloid cells. We have found that estriol (E3), a pregnancy specific estrogen, generates Tol-DCs in vivo that protect against the inflammatory autoimmune disease experimental autoimmune encephalomyelitis. To understand the generation of E3-Tol-DCs, we examined E3 effects on myelopoiesis and DC differentiation. Bone marrow (BM) from mice was differentiated for 3-7 days + E3 and phenotypic and regulatory function assessed. Similar to in vivo E3-Tol-DCs, BM-DCs differentiated with E3 showed phenotypic increases in CD80, CD86, PD-L1 and PD-L2 expression and suppressed proliferation of responder cells. E3 exposure also affected myeloid progenitors where E3-BM cells (day 3) suppressed proliferation of responder cells in a dose-dependent manner. These effects were not due to altered differentiation (based on CD11b, Gr-1 or CD11c expression of BM cells). Interestingly, E3-BM cells increased FoxP3+ cells in vitro after 4-5 days in culture with FoxP3+GFP+ reporter splenocytes. These results suggest that E3 promotes Tol-DCs through actions on myelopoiesis and that hormonal modulation during myelopoiesis contributes to the generation of regulatory myeloid cells.
miR-150 differentially regulates the development of NK and NKT cells via targeting the transcription factor c-Myb (153.37)
Authors: Bezman Natalie , Chakraborty Tirtha , Pellerin Alex , Bender Timothy , Lanier Lewis
Published: January 2023

Natural killer (NK) and Natural killer T (NKT) cells are important in host defense against pathogens, as well as for the initiation of adaptive immune responses. We and others have recently showed that miRNAs play important roles in NK and NKT cell development, maturation and function, but the roles of specific miRNAs are still lacking. Here we show that modulation of miR-150 expression levels has a differential effect on NK and NKT cell development. Mice with a targeted deletion of miR-150 have an impaired, stem cell-intrinsic defect in their ability to generate mature NK cells. Conversely, a gain-of-function miR-150 transgene promotes the development of NK cells, which display more mature phenotype and produce higher amounts of IFNγ following activation. In contrast, we find that overexpression of miR150 results in a substantial reduction of iNKT cells in the thymus and from the peripheral lymphoid organs. The transcription factor c-Myb has been shown to be a direct target of miR150, and we propose that miR-150 controls the development of NK and NKT cell lineages by targeting c-Myb. Our findings of the increased NK cell and decreased NKT cell frequencies in c-Myb heterozygous bone marrow chimeras are consistent with this hypothesis.
IL-7 receptor expression defines a human common lymphoid progenitor in cord blood lymphopoiesis (153.4)
Authors: Payne Kimberly , Baez Inneavely , Milford Terry , Weldon Abby , Francis Olivia , Sinisa Dovat , Su Rui-jun
Published: January 2023

Expression of the IL-7 receptor alpha (IL-7R) is a distinguishing feature of common lymphoid progenitors (CLP) in mice. Human B cell development has been thought to differ from that in mouse with respect to the requirement for IL-7. Our previous studies show that IL-7 plays a critical role in human B cell production (J Immunol. 2009, 182:4255). Here we use IL-7R expression to identify a human CLP population that can be generated from hematopoietic stem cells (HSCs) in umbilical cord blood (CB). Following two weeks of co-culture with primary human bone marrow (BM) stroma, CB CD34+ cells give rise to a CD34+CD19-IL-7R+ progenitor population that is absent from fresh, uncultured CB. IL-7R+ progenitors make up ~17% of the CD19-CD34+ cells present in culture at 2 weeks. A similar IL-7R+ human progenitor population is generated in the BM of immunodeficient mice following transplant with human CD34+ CB cells. FACS-sorted CD34+CD19-IL-7R+ progenitors produced in co-culture or in the human-mouse xenograft model give rise to CD19+ B cells and CD56+ NK cells in bulk and single cell culture, but lack myeloid potential in CFU assays and when transplanted into immunodeficient mice. Ongoing experiments will evaluate the T lineage potential of IL-7R+ progenitors in the human-mouse xenograft model. These data will be important for developing therapies to accelerate and enhance lymphoid reconstitution from HSCs in CB, a hematopoietic source used increasingly for stem cell transplant.
Over expression of Foxn1 in the bone marrow prevents age-associated perturbations in the expansion and survival of T cell progenitors (153.6)
Authors: Zook Erin , Zhang Shubin , Witte Pamela , Le Phong
Published: January 2023

The thymus requires continuous seeding of progenitors from hematopoietic stem cells (HSC) in bone marrow (BM) for naïve T cell production. HSC give rise to multipotent progenitors (MPP) and their progeny common lymphoid progenitors (CLP); however, MPP are the predominant source of early T cell progenitors (ETP) in the thymus. ETP, HSC, MPP and CLP decline with age, culminating in reduced naïve T cell production. We reported that the age-associated decline in ETP is abrogated in K14-Foxn1 transgenic mice (Foxn1Tg). Here, we show that Foxn1 is expressed in BM and its expression was 98-fold higher in Foxn1Tg. Foxn1 transgene expression increased endogenous Foxn1 mRNA levels by 18- to 24-fold; Foxn1 positive cells were readily detected in the BM. We found that HSC number was 2-fold higher in young Foxn1Tg versus wild type (Wt). HSC and MPP numbers declined in aged Wt but not in Foxn1Tg. A higher percent of HSC was in S,G2/M; further, HSC and MPP in subGo from Foxn1Tg were respectively 3-fold and 4-fold reduced, suggesting inhibition of cell death. In contrast, CLP frequency was 4-fold less in young Foxn1Tg. A higher number of the committed T cell progenitors (CTP) with a 2-fold increase in S,G2/M fraction were found in young Foxn1Tg versus Wt. Our data provide evidence linking Foxn1 to the development of T cell progenitors in the BM and thymus, suggesting that increased expression could limit the decline in naïve T cell production with age.
The anti-viral and signal-3 effects of Type-I interferons exert opposing effects on clonal expansion of CD8 T cells during West Nile Virus infection (154.13)
Authors: Bhaumik Siddhartha , Thomas Sunil , Moguche Albanus , Wijaranakula Michael , Kaja Murali-Krishna
Published: January 2023

West Nile Virus (WNV) is a neurotropic positive stranded RNA virus, which affects central nervous system in a fraction of infected humans in several continents, including North America. In concert with innate responses both antibody and T cell mediated immunity play an important role in conferring protection against WNV. While the importance of type-I IFN-I (IFN-I) signaling on WNV-specific innate immunity is well documented, it remains unclear whether and what effects IFN-I have on WNV-specific adaptive response. Using a murine foot pad infection model of WNV infection here we asked two questions: (1) what happens to virus-specific CD8 T cell responses if the infected host is devoid of IFN-I signaling, (2) what happens to virus-specific CD8 response if only CD8 T cells lack IFN-I signaling. We found that lack of IFN-I signaling in the host results in exaggerated CD8 clonal expansion whereas lack of IFN-I signaling exclusively in CD8 T cells results in diminished clonal expansion. Our results together, suggest INF-I mediated innate viral control and the ability to provide signal-3 exerts opposing effects on CD8 T cell expansion during West Nile Virus infection.
2P imaging reveals that PD-1 blockade promotes clearance of a persistent viral infection by overriding prolonged T cell engagement (154.18)
Authors: Zinselmeyer Bernd , Heydari Sara , Nayak Debasis , McGavern Dorian
Published: January 2023

The recent demonstration of improved anti-viral T cell function following PD-1 blockade is a finding of great clinical importance that can benefit patients with persistent infections and cancer. Presently, it is not clear how blockade of this inhibitory pathway improves the function of exhausted T cells. Infection of mice with an immunosuppressive strain of LCMV is a well described paradigm to study T cell exhaustion. In this study we sought novel insights into the mechanisms of T cell exhaustion and recovery during persistent infection by simultaneously imaging CD8 and CD4 T cell responses using two-photon microscopy in the spleen. To define the motility parameters associated with immune exhaustion, we compared anti-viral T cell dynamics during an acute versus a persistent LCMV infection. Analyses of T cells in these two distinct environments revealed that exhaustion during viral persistence is associated with prolonged TCR signaling and cellular arrest in the splenic marginal zone and red pulp - two regions with abundant LCMV antigen and elevated levels of PD-L1.
Coordinated regulation of PD-1 gene expression and nuclear localization during virus-specific memory CD8+ T cell differentiation (154.20)
Authors: Sacristan Catarina , Youngblood Ben , Xu Jean , Shipper Katelyn , Skok Jane , Boss Jerry , Ahmed Rafi , Dustin Michael
Published: January 2023

Mechanisms that mediate changes in transcriptional regulation during differentiation of long-lived memory T cells during infection remain poorly understood. During chronic viral infection, CD8 T cell differentiation yields a unique transcriptional profile: T-cell exhaustion. The suboptimal effector function of exhausted cells is partly due to sustained expression of the inhibitory receptor PD-1. Using the lymphocytic choriomeningitis virus mouse model of infection, we examined pdcd1 (PD-1) gene regulation at different stages of T cell differentiation. Changes in gene expression are associated with cell-type specific profiles accompanied by distinct patterns of locus conformation and positioning of a gene relative to specific nuclear compartments (e.g. recruitment to nuclear lamina indicating silencing). We hypothesized that pdcd1 epigenetic programming and nuclear localization were coupled to viral persistence and T cell differentiation. Using DNA-FISH, we report a significant decrease in the % of pdcd1 alleles recruited to nuclear lamina in exhausted cells relative to other CD8 T cell subsets from infected mice. Reduced recruitment to nuclear lamina was consistent with upregulated expression of PD-1 mRNA and protein, and with loss of PD-1 DNA methylation in exhausted cells. Our findings contribute to the understanding of the molecular pathways mediating T cell memory and exhaustion, and may provide insight into the use of therapeutic strategies to resolve chronic infections.
CD4 regulatory T cells control CD8 T cell responses to human herpesvirus 8 lytic and latency proteins (154.28)
Authors: Lepone Lauren , Rappocciolo Giovanna , Piazza Paolo , Jais Mariel , Jenkins Frank , Rinaldo Charles
Published: January 2023

Objectives: CD8 T cells likely play an important role in controlling human herpesvirus 8 (HHV-8) infection, the cause of Kaposi's sarcoma (KS). However, these T cell responses are non-robust compared to other herpesviruses, suggesting that they could be under tight control by regulatory T cells (Treg). Methods: HLA A*0201 pentamer complexes for 2 lytic and 3 latency protein epitopes of HHV-8 and polyfunctional flow cytometry were used to detect HHV8-specific CD8 T cells in blood. The effect of Treg was examined by depleting CD4+CD25High cells. Results: Direct staining of PBMC with pentamer complexes showed a frequency of antigen-specific CD8 T cells from 0.02% to 0.2%. This presence of HHV-8-specific CD8 T cells contrasted with low HHV-8 T cell polyfunctional reactivity, suggesting a regulatory suppression. Indeed, removal of Treg enhanced CD8 T cell responses to HHV-8 epitopes. This was not related to proportional increases in number of CD8 T cells after Treg removal. The frequency of T cells specific for HHV-8 lytic antigens was greater than for latent antigens, and this effect was greater when Treg were removed. Conclusions: Treg suppress CD8 T cell responses to HHV-8 lytic and latency antigens. The involvement of CD8 T cells and Treg in control of HHV-8 infection has implications for development of vaccines to prevent KS.
CD11b+Ly6C+ cells, myeloid-derived suppressor cells, activated following Theiler’s virus infection (154.3)
Authors: Olson Julie , Glazer Alex
Published: January 2023

Theiler’s murine encephalomyelitis virus (TMEV) infection of susceptible mice leads to a persistent infection of the microglia/macrophage population in the central nervous system (CNS). The persistent infection leads to the development of a chronic progressive demyelinating disease associated with a myelin- specific CD4+ T cell response. We recently determined that the predominant innate immune cells infiltrating into the CNS following TMEV infection are CD11b+Ly6C+ cells. CD11b+Ly6C+ cells are immature myeloid cells that exit the bone marrow without maturing. Therefore, we wanted to determine the function of these cells during TMEV infection. CD11b+Ly6C+ cells isolated from the CNS during TMEV infection expressed high levels of IL-10 and iNOS and low levels of IL-12, and expressed low levels of costimulatory molecules and MHC class II. The CD11b+Ly6C+ cells were a stable population in the CNS throughout TMEV infection. Most importantly, CD11b+Ly6C+ cells isolated during TMEV infection suppressed both CD4+ T cell and CD8+ T cell responses against virus antigens. Furthermore, depletion of Ly6C+ cells during TMEV infection lead to increased T cell responses associated with increased expression of IFNγ, IL-12, and IL-17 in the CNS and lead to decreased virus load in the CNS. These results suggest that CD11b+Ly6C+ cells generated after TMEV infection are myeloid- derived suppressor cells that suppress T cell responses and may contribute to persistent virus infection in the CNS.
Up-regulation of programmed death ligand 1 expression on human plasmacytoid dendritic cells by viral stimulation (154.31)
Authors: Patel Meher , Dai Jihong , Fitzgerald-Bocarsly Patricia
Published: January 2023

The function of exhausted CD8 T cells in chronic HIV-1 infection is negatively regulated by the PD-L1/PD-1 pathway resulting in suppression of specific anti-viral T cell responses. Blockade of this pathway reconstitutes T cell function and enhances antiviral immune responses. In HIV-infected individuals, increased PD-L1 (B7-H1) expression on APCs correlates directly with viral load, indicating a potential effect of the virus on PD-L1 expression. However, the mechanisms of PD-L1 up-regulation in these settings are not well understood. In this study, we investigated how PD-L1 expression is regulated in human pDCs. We found PD-L1 expression on pDC was up-regulated with different viral stimulations including HSV, Sendai virus, Influenza virus and both live and AT-2 inactivated HIVMN. PD-L1 levels were differentially regulated by stimulation with different viruses. In comparison with these virus stimulations, TLR9 ligands CpGA and-B were less efficient at inducing PD-L1 expression. TNF-α and IL-6 had no influence on PD-L1 although IFN-α had a moderate effect on it, indicating that up-regulation of PD-L1 on pDC was specifically a primary response to viral stimulation. Furthermore, we observed that LPS enhanced virus-induced PD-L1 expression on pDC, suggesting potential involvement of LPS in the up-regulation of PD-L1 in chronic HIV-1 infection. Taken together, our results suggest a potential role for up-regulation of PD-L1 expression on pDC in HIV-induced immune exhaustion.
Novel approaches to improve influenza and cytomegalovirus vaccines (155.25)
Authors: Fischer David , Sundick Roy
Published: January 2023

Cytokines have been extensively used as adjuvants in vaccines. However, several factors limit their use including diffusion from antigen, short half-lives and production costs. To address this, we developed a technology that efficiently produces inactivated, whole-virus influenza vaccine bearing membrane-bound cytokines. Tethering the cytokine to the antigen of interest keeps the adjuvant in close contact with the antigen ensuring that immune cells recruited by the adjuvant react with the antigen as well. Influenza can be used to test this model since it has two surface proteins with conserved signal sequences that direct these proteins to the infected cell surface where they are picked up budding virions. Cytokines can be fused to these signal sequences producing viral particles that will then contain an attached cytokine adjuvant. Trials using IL2 and GMCSF have shown this method may be effective at increasing seroconversion rates. IL4 and complement factor C3d are also being tested as adjuvants in this system. This platform can also be used to deliver and enhance immunogenicity of antigens from other viruses. The cytomegalovirus surface protein gB can be anchored to influenza virions using conserved signal sequences. We hypothesize that delivery of gB via an influenza virion, with or without a bound adjuvant will result in enhanced immune responses.
Modification of VRC01 for enhancing half-life and ADCC activity (155.39)
Authors: Ko Sung-Youl , Kuo Timothy , Blumberg Richard , Nabel Gary
Published: January 2023

Several studies have demonstrated that increased binding of IgG to hFcRn extended the half-life of antibody in serum. In addition, the Fc region of IgG binds to FcγRIIIa, mediating ADCC. Modification for hFcRn and FcγRIIIa may negatively affect their functions respective to the other. Here, to increase the potency and duration of VRC01, a broadly neutralizing antibody to HIV gp120, we have combined hFcRn-binding enhancing mutations (QL, LS, A, AAA and YTE) with ADCC enhancing mutations (DE and DLE) to improve in vivo activity. We demonstrated that all of the mutants have Ag binding affinities similar to VRC01 wild type (WT), indicating that the neutralizing activities are likely maintained with these mutants. All mutants except mutant group A have increased binding to hFcRn at both pH6.0 and pH7.4 compared to VRC01 WT. Using a transcytosis assay, we showed that DE-QL and DE-LS mutants had increased transport across epithelial cells compared to others in the DE mutant groups. ADCC enhancing mutations recovered the loss of ADCC acitivities and FcγRIIIa binding affinity associated with hFcRn-binding enhancing mutations. Unlike other mutations tested, a single LS mutant has FcγRIIIa binding and ADCC activity similar to VRC01 WT. The combined DE-LS and DLE-LS mutants showed increased functional activity in transcytosis and ADCC assays. Taken together, the DE-LS or DLE-LS mutants were able to enhance both half-life and effector functions in vitro.
Synthetic LbL nanoparticle vaccines containing the chemokine mimic epitope of RSV-G protein and a CD8 epitope of RSV-M2 protein elicit broad-based cellular and humoral responses (155.7)
Authors: Powell Thomas , Boyd James , Jacobs Andrea , Palath Naveen , DeRome Mary , Tang Jie , Cardenas Edwin , Harcourt Jennifer , Haynes Lia , Anderson Larry , Tripp Ralph
Published: January 2023

Nanoparticle vaccines synthesized via layer-by-layer (LbL) fabrication were loaded with designed peptides representing epitopes of the attachment (G) and matrix (M2) proteins of respiratory syncytial virus. The CX3C chemokine mimic epitope of RSV-G has been proposed to contribute to inflammatory responses post-challenge while CD8+ T-cell responses against RSV M2 have been shown to limit the severity of infection and inflammatory pathology. Both monovalent designs (G or M2) and multivalent designs (G+M2) were tested. Mice immunized with RSV-G nanoparticles produced antibody responses that recognized the CX3C epitope only in its folded conformation and not in a linearized state. The same sera also bound native RSV-G protein, inhibited binding of RSV-G protein to the CX3CR1 chemokine receptor, and inhibited migration of human leukocytes toward RSV-G protein. Mice immunized with RSV M2 nanoparticles generated CD8+ T-cell responses and in vitro CTL activity against M2-labeled target cells. The multivalent vaccines containing both G and M2 elicited higher antibody responses to RSV-G and, surprisingly, more potent cellular responses against RSV-M2. These novel nanoparticle vaccines are currently being tested for the induction of neutralizing antibody responses and protection from viral challenge. If successful, the LbL nanoparticle fabrication strategy will provide an innovative approach to formulating safe and effective subunit vaccines for respiratory pathogens including RSV.
Programming of antigen-specific CTLs from induced pluripotent stem cells for tumor protection (156.1)
Authors: Lei Fengyang , Haque Rizwanul , Song Jianxun
Published: January 2023

Generation of highly reactive antigen(Ag)-specific CTLs derived from induced pluripotent stem(iPS) cells can provide an unlimited source of functional CTLs that can be used for adoptive immunotherapy. The iPS cell-derived T cells have the advantages of averting potential immune rejection, as well as circumventing ethical and practical problems associated with other types of stem cells. Previously we demonstrated that iPS cells are able to differentiate into progenitor T cells in vitro by the stimulation of Notch ligand Delta-like 1 (DL1) over-expressed on OP9 bone marrow stromal cells. Full maturation of iPS cells was observed upon adoptive transfer into RAG1-deficient mice. Here we show that iPS cells differentiate into functional CTLs in vivo after over-expressing MHC I-restricted Ag-specific T cell receptors (TCRs). In this study, murine iPS cells are genetically modified with OVA specific, MHC-I restricted TCR (OT-I) by retroviral transduction. We demonstrate that after adoptive transfer into recipient mice, the majority of OT-I/iPS cells undergo differentiation into CD8+ CTL. In vivo developed TCR-transduced iPS cells are able to respond to ex vivo peptide stimulation and secrete IL-2 and IFN-γ. Moreover, adoptive transfer of TCR-transduced iPS cells results in the infiltration of OVA-reactive CTLs into tumor tissues and protection of animals from tumor challenge. Our results provide a novel approach for generating Ag-specific T lymphocytes for T cell-based therapies.
Modulating therapeutic antitumor T cell immunity with MicroRNAs (156.21)
Authors: Rutkowski Melanie , Nesbeth Yolanda , Toraya Seiko , Scarlett Uciane , Jacobs Ana , Conejo-Garcia Jose
Published: January 2023

MicroRNAs are attractive candidates for enhancing the therapeutic effectiveness of adoptive T cell therapy, although overcoming tumor-induced immunosuppression in the tolerogenic ovarian cancer microenvironment remains a formidable barrier. We hypothesized that over-expression of miR181a in adoptively transferred tumor-reactive T cells would result in sustained anti-tumor immunity within the tolerogenic ovarian tumor microenvironment. To test this, congenic tumor-reactive T cells were intraperitoneally injected into mice bearing established transplantable ovarian tumors. Twelve and 26 days post-transfer, RNA was isolated from sorted peritoneal host or transferred CD4 and CD8 T cells. Contrary to our initial hypothesis, miR181a expression was significantly and exponentially greater in transferred CD4 and CD8 T cells, compared to host-derived T cells at these temporal points during which the function of transferred T cells was impeded by the tolerogenic microenvironment. This suggested an unexpected negative role for miR181a in the context of T cell immunotherapy. To confirm these findings, T cells over-expressing miR181a were adoptively transferred into tumor-bearing mice and survival was monitored. Compared to mice receiving either non-transduced or control-vector transduced T cells, mice treated with T cells over-expressing miR181a had significantly reduced survival, supporting the novel finding that miR181a may have a negative role in adoptive T cell immunotherapy.
Primary medulloblastomas express functional CD1d and can be targeted for immunotherapy with NKT cells (156.25)
Authors: Liu Daofeng , Song Liping , Salsman Vita , Gao Xiuhua , Wei Jie , Teo Wanyee , Lau Ching , Ahmed Nabil , Metelitsa Leonid
Published: January 2023

Medulloblastoma (MB) is the most common brain tumor of childhood. Current therapies are not curable for a third of patients and have debilitating long-term toxicity. In the initial screen of potential targets for immunotherapy, we performed gene expression analysis of twenty primary MB tumors and found that nine of them expressed high levels of CD1d RNA. Nearly all tumors cells in the corresponding specimens expressed CD1d protein on the cell surface. Two of five analyzed MB cell lines (DAOY and MHH-MED-8A) also were CD1d-positive. Functional experiments demonstrated that both cell lines effectively presented αGalactosylceramide (αGalCer) to activate NKT-cell cytokine production, proliferation, and cytotoxicity. The effector to target ratio as low as 2:1 was sufficient to kill nearly 100% of MB cells in vitro. The cytotoxicity was CD1d-restricted as it was inhibited by anti-CD1d blocking mAb. A single intracranial injection of 2X106 ex-vivo expanded human NKT cells and αGalCer in NOD/SCID/β2m mice with 6-day established xenografts of luciferase-transduced DAOY cells resulted in rapid tumor regression with 30% cure rate. The therapy was well-tolerated. Therefore, CD1d-positive MB could be targeted for immunotherapy with NKTs that has a curative potential.
Regulatory B lymphocyte production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice (156.27)
Authors: Horikawa Mayuka , Colin-Minard Veronique , Tedder Thomas
Published: January 2023

Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of malignancies that represents ~4% of all cancers. Rituximab, a chimeric CD20 monoclonal antibody (mAb) that effectively treats half of NHL patients variably depletes non-malignant tissue B cells. Whether endogenous or remnant B cells affect CD20 mAb-induced lymphoma depletion is unknown. To determine whether endogenous B cells can influence in vivo lymphoma therapy in mice, the therapeutic effectiveness of a potent mouse anti-mouse CD20 mAb was compared in wild type and CD20-deficient (CD20-/-) mice given homologous CD20+ primary lymphoma cells. CD20 mAb efficiently depleted endogenous mature B cells and lymphoma cells in wild type mice and prolonged survival significantly. By contrast, endogenous B cells are not depleted in CD20-/- mice, including a rare IL-10-producing regulatory B cell subset (B10 cells). Furthermore, the persistence of endogenous B cells significantly inhibited the anti-tumor effects of CD20 mAb in CD20-/- mice. Even small numbers of adoptively transferred CD20-/- B10 cells dramatically suppressed lymphoma depletion in wild type mice by inhibiting monocyte activation and effector function through IL-10-dependent mechanisms. Thus, endogenous B10 cells are potent negative regulators of innate immunity, with even small numbers of residual B10 cells inhibiting lymphoma depletion by CD20 mAb. This may provide a new explanation for treatment resistance in some lymphoma patients.
Combination of DTIC and peptide-vaccination elicit a renewal of high-avidity late-differentiated T-cell clones in melanoma patients (156.6)
Authors: Palermo Belinda , Del Bello Duilia , Franzese Ornella , Di Donna Cosmo , Gualtieri Novella , Nuzzo Carmen , Proietti Enrico , Catricalà Caterina , Ferraresi Virginia , Nistico Paola
Published: January 2023

Combination of chemo/immunotherapy to increase the effectiveness of the antitumor immune responses associated with a clinical efficacy is an attractive antitumor strategy. We have recently reported that the administration of dacarbazine (DTIC) one day before peptide (Melan-A and gp100)-vaccination in disease-free melanoma patients increases the number of peptide-specific effector-memory CD8+ lymphocytes. Notably, patients treated with DTIC before vaccination, showing a longer survival, display a progressive enhancement of TCR repertoire diversity of Melan-A+ CD8+ clones, accompanied by the maintenance of high-avidity, whereas patients treated with vaccine alone showed a reduction of TCR repertoire diversity and a decline of their tumor lytic activity. To study the phenotype and the functional lytic activity of CD8+ T-cell clones in association with their renewal rate we have analyzed the expression of CD28 and CD27 co-stimulatory molecules, in both Melan-A- and gp100-specific T-cell clones, isolated before and at different times after the treatments. We found newly recruited clones with high-lytic activity and highly-differentiated phenotype only after the combined therapy. Studies are in progress to define the role of replicative senescence in the continuous renewal induced by the combined therapy, by studying activity and regulation of telomerase in endogenous-induced or vaccine-driven clones, in correlation with their different phenotype and anti-tumor lytic capability.
Presence of NLRP3 in myeloid cells increases sensitivity to membrane damage (157.3)
Authors: Heid Michelle , Chu Jessica , Sun Chengqun , Thomas L. , Keyel Peter , Salter Russell
Published: January 2023

Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. We and others have shown that NLRP3 inflammasome induced processing and secretion of mature IL-1 beta is dependent on the activity of caspase-1 as well as potassium efflux. Unexpectedly, we now observe that NLRP3 knockout macrophages are more resistant to membrane damage compared to wild type macrophages exposed to nigericin or TLO, as assessed by a dye uptake assay. We also were able to increase sensitivity to membrane damage in myeloid cells deficient in ASC, an adaptor protein required for formation of the NLRP3 inflammasome, by over-expression of ASC using retroviral transduction. Through the use of both pharmacological inhibitors and caspase-1 knockout mice, we observed this NLRP3 and ASC dependent effect on membrane integrity is independent of the activity of caspase-1, unlike the secretion of mature IL-1 beta. However, the loss of membrane integrity is dependent on potassium efflux, as is mature IL-1 beta secretion. These data suggest a mechanism through which the NLRP3 inflammasome is able to influence the membrane integrity of myeloid cells in a caspase-1 independent manner, distinguishing the loss of membrane integrity from IL-1 beta processing and secretion.
Pyrin-only protein 3: a pseudogene product in the human genome regulating NF-kB activity (157.4)
Authors: Porter Kristen , Nyland Patricia , Harton Jonathan
Published: January 2023

Pyrin-only proteins influence NF-kB and inflammasome signaling pathways. In viruses, POPs act as pathogenic factors by reducing innate immune responses and favoring viral survival within the host. In humans, two known POPs are thought to have a role in regulating innate immunity and inflammation. POP1 interferes with IKKα/β activation and prevents the downstream activation of NF-kB with no known effect on ASC-dependent inflammasomes. POP2 however, inhibits NF-kB signaling at the level of the p65 (RelA) subunit and down-modulates the formation and activation of a variety of pyrin domain/ASC-dependent inflammasomes. Here we describe a third human POP, POP3, the product of an NLRP pseudogene encoded on chromosome X that is expressed in a wide variety of tissues. Like its close genetic relative POP2, POP3 inhibits p65 activation downstream of IKK, but lacks the capacity of inhibit the NLRP3 inflammasome. Our data suggest that the human POP family has three members, all of which may play distinct biological roles during inflammation.