bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple effects of Wnt3a on lymphoid progenitors and their environment (83.4)
Authors: Malhotra Sachin , Kincade Paul W
Published: January 2023

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is poorly understood. We over-expressed Wnt3a in stromal cells of osteopetrotic mice and used them in co-culture experiments with highly enriched stem and progenitor cells. The objective was to learn if and how particular stages of B lymphopoiesis are responsive to this canonical Wnt ligand. We found that functional receptors were present on all populations tested, and that Wnt signaling generally inhibited B lineage progression. However, this effect was less in long term cultures. Other results suggest that lineage instability results from Wnt signaling and there was even evidence for loss of differentiation. That is, RAG1+ cells down-regulated expression of this gene and acquired some characteristics of stem cells. At the same time, short term exposure to Wnt3a increased myeloid colony forming potential and retained the ability of progenitors to be transplanted. We also found a surprising effect on expression of cell adhesion molecules thought to retain cells in the bone marrow. VCAM-1 on stromal cells, stem cells or an endothelial cell line was dramatically down-regulated when the cells were exposed to Wnt3a. We conclude that in addition to effects on differentiation, canonical Wnt signaling may control positioning of stem and progenitor cells within the bone marrow.
Regulation of B cell fate determination by EBF (83.7)
Authors: Medina Kay L.
Published: January 2023

B cell fate specification is associated with the expression of genes encoding components of the B cell antigen receptor and the ordered rearrangement of the immunoglobulin heavy-chain locus. Accompanying activation of the early program of B lineage gene expression is the concerted repression of lineage inappropriate genes. EBF is a transcription factor expressed exclusively in the B lineage and evidence is mounting that it is the limiting factor directing the B cell fate choice. To gain insight into mechanisms of EBF directed B cell fate specification, we generated EBF−/− cell lines. The cell surface phenotype, growth factor requirements, gene expression profile, and in vivo functional capabilities of EBF−/− clonal cell lines is consistent with a lymphoid-biased multipotential progenitor. To identify and discriminate novel EBF target genes involved in B cell fate specification, we performed comparative gene expression profiling of EBF−/−, Pax-5−/−, and RAG2−/− cell lines. ANOVA analysis of variance revealed that the EBF−/− lines overexpress several genes associated with early hematopoietic differentiation and stem cell function, notably a subset of HoxA genes. Conversely, the EBF−/− cells are defective in expression of a subset of early B lineage genes including lymphocyte enhancer factor-1, LEF-1. Consistent with the deficiency in LEF-1, EBF−/− lines display elevated levels of fas and increased sensitivity to apoptosis. Taken together, these data suggest novel regulatory circuits involving EBF during B cell fate specification, including inhibition of HoxA genes and activation of the Wnt signaling pathway.
Shnurri-2 controls memory Th1 and Th2 cell numbers in vivo (84.3)
Authors: Nakayama Toshinori , Kimura Motoko Y , Iwamura Chiaki , Hasegawa Akihiro , Yamashita Masakatsu
Published: January 2023

Schnurri-2 (Shn-2) is a large zinc-finger containing protein, and it plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient CD4 T cells, the activation of NF-kB was upregulated and their ability to differentiate into Th2 cells was enhanced. We herein demonstrate that Th1 and Th2 memory cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. Even a week after the transfer of effector Th1/Th2 cells into syngeneic mice, a dramatic decrease in the number of Shn-2-deficient donor T cells was detected particularly in the lymphoid organs. The transferred Shn-2-deficient Th1/Th2 cells express higher levels of the activation marker CD69. No significant defect in the BrdU incorporation in the Shn-2-deficient transferred CD4 T cells was observed. The numbers of apoptotic cells were selectively higher in Shn-2-deficient donor Th1/Th2 cell population. Moreover, Shn-2-deficient effector Th1 and Th2 cells showed an increased susceptibility to cell death in in vitro cultures with increased expression of FasL. Transfer of Th2 effector cells over-expressing the p65 subunit of NF-kB resulted in a decreased number of p65-expressing cells in the lymphoid organs. As expected, T cell-dependent antibody responses after in vivo immunization of Shn-2-deficient mice were significantly reduced. Thus, Shn-2 appears to control the generation of memory Th1/Th2 cells through a change in their susceptibility to cell death.
Combined Photometric and Morphometric Analysis of Apoptosis Using the ImageStream Imaging Flow cytometer (84.8)
Authors: Henery Shannon , George Thaddeus , Hall Brian , Basiji David , Ortyn William , Morrissey Phillip
Published: January 2023

Apoptosis involves multiple biochemical pathways resulting in caspase activation, membrane blebbing and phosphatidylserine (PS) ‘flipping’, nuclear condensation, and nuclear fragmentation. While morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells, a variety of photometric assays are employed to quantify apoptosis by flow cytometry. These assays target hallmarks of apoptosis, such as DNA fragmentation (TUNEL assay), surface PS exposure (AnnexinV assay), and caspase activation (caspase 3 substrates). Here we present a combined method for quantifying apoptosis that directly measures nuclear fragmentation using ImageStream imaging cytometry in addition to photometric parameters.

To investigate both

the temporal relationship between nuclear fragmentation and activation of other apoptotic pathways andpossible false positive and negative events associated with each measurement method, samples were analyzed on the ImageStream using both fluorometric and morphometric classifiers for measuring the apoptotic index.

Ramos, JurkatO, and Colo205 cells were incubated with three mechanistically distinct apoptosis inducers (Camptothecin, Staurosporine, and TNFα/IFNγ), and stained for nuclear morphology and AnnexinV, TUNEL, or PhiPhiLux. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms that can exclude artifacts common to these assays and provide a more accurate and complete analysis of apoptosis.
Aberrant Innate Immune Functions of Dendritic Cells in Aged Humans (85.12)
Authors: Agrawal Anshu , Agrawal Sudhanshu , Cao Jia-ning , Su Houfen , Gupta Sudhir
Published: January 2023

The molecular mechanisms underlying the chronic inflammation and reduced immunity observed during aging are not well understood. Comparison of the innate immune functions of monocyte-derived DCs from aging subjects with DCs from young individuals showed that MDDCs from aging individuals display

significantly reduced capacity to capture antigens via macropinocytosis and receptor-mediated endocytosis as determined by flow cytometry;impaired capacity to migrate in vitro in response to the chemokine MIP3-beta; andsignificantly increased LPS induced secretion of TNF-alpha and IL-6.

Investigations of intracellular signaling revealed reduced phosphorylation of AKT in MDDCs from aging, suggesting decreased activation of PI3kinase signaling pathway. Since PI3K signaling pathway plays a positive regulatory role in phagocytosis and migration, and it also functions as a negative regulator of TLR signaling by inducing activation of p38 MAP kinase, this may explain the aberrant innate immune functioning of DCs from aged subjects. Results further revealed an increased expression of PTEN, a negative regulator of PI3Kinase signaling pathway, in MDDCs from aged subjects. Increased PTEN may thus be responsible for the defect in AKT phosphorylation and therefore, altered innate immune response of DCs from aged humans.

This study is supported in part by a grant AG027512 from NIH and partly by new scholar grant from the Ellison Medical Foundation.
Early CD8 T cell proliferative heterogeneity entails diverse memory differentiation programs (85.19)
Authors: Kalia Vandana , Sarkar Surojit , Kaech Susan M. , Ahmed Rafi
Published: January 2023

Initial events governing delineation of the fraction of effector cells that advance into the long-lived memory lineage following antigen clearance remain unclear. We hypothesized that the order of recruitment of naïve CD8 T cells into the immune response regulates the memory potential of an effector cell. To address this hypothesis, we adoptively transferred naïve transgenic P14 CD8 T cells that encode a TCR specific for the GP33-41 epitope of lymphocytic choriomeningitis virus (LCMV) at different days post-infection. As predicted, cells transferred later during an infection demonstrated accelerated acquisition of memory cell phenotype (IL-7RaHi, L-SelectinHi), and proliferated to a greater extent upon secondary challenge compared to cells that were adoptively transferred earlier. Importantly, in a physiologically relevant setting, when cells that had divided < or > 4 divisions (based on CFSE dilution at day 2.5 post-infection) were FACS sorted and their potential to differentiate into long-lived memory cells evaluated, we observed that cells that entered into division later exhibited faster reversion to central memory phenotype, relative to cells that had entered cell division earlier. Memory cells arising from Ag-specific cells that entered proliferation later, also mounted more robust recall proliferative potential compared to those arising from cells that entered proliferation earlier. These data present a model that heterogeneity in early priming events likely dictate heterogeneity in initial proliferation, which in turn regulates their ultimate memory fate.

Supported by Elizabeth Glaser Pediatric AIDS Foundation awards to VK & SS, and NIH & Gates Foundation Grants to RA.
Accelerated T Cell Memory Development in the Peritoneal Cavity (85.24)
Authors: Olson Matthew R. , Varga Steven M.
Published: January 2023

Acute virus infection results in the expansion of the number of virus-specific CD8 T cells, virus clearance, followed by the death (contraction) of 90% of the expanded antigen-specific CD8 T cell population ultimately resulting in a stable memory population. Memory cells are characterized by their enhanced ability to expand in number after secondary infection, enhanced cytokine production (IL-2, TNF-α) and surface expression of the IL-7Rα (CD127), CD27, and the lymph node homing molecule CD62L. Most of what’s known about the kinetics of memory development is from data acquired from the spleen, however it is unclear if other peripheral tissues share those same kinetics. We demonstrate here that CD8 T cells isolated from the peritoneal cavity (PECs) after intraperitoneal or intravenous infection with lymphocytic choriomeningitis virus (LCMV) display enhanced IL-2 and TNF-α production as well as a greater frequency of cells expressing the memory markers CD127 and CD27 compared to their splenic counterparts. Interestingly, PECs demonstrated prolonged expression of the activation marker CD43 and similar levels of CD62L compared to splenic memory cells. PEC cells adoptively transferred into naïve mice maintain their phenotype in all tissues found, whereas splenic memory cells adopt the phenotype of their host tissue. These data suggest that the generation of memory CD8 T cells is controlled by tissue-specific signals.
Ability of aged transgenic B cells to mount both TH1 and TH2 responses (85.25)
Authors: Panwar Akanksha , Porter Katrina , Vogel Dr Laura
Published: January 2023

Humoral immunity has long been known to deteriorate with aging, however the cause is unclear. Previous research has focused on decreased T cell function or changes in microenvironment rather than aging-associated intrinsic alteration in B cells. Our previous in vivo research found that aged B cells can respond to T cell-dependent antigens when appropriate T cell help and a young microenvironment are provided. To examine this interaction in further detail, we investigated if aged B cells had a preference for TH1 or TH2 helper cells. OTII transgenic CD4+ cells were differentiated in vitro into TH1 or TH2 and co-transferred to young normal recipients with aged or young QM B cells. Analysis of expansion and activation phenotype of QM B cells that are induced following immunization was then analyzed using flow cytometry and confocal microscopy. Specific serum antibody amount, isotype and relative affinity were measured by ELISA. The results of the experiment showed that B cells from young and aged QM mice showed characteristic responses to the help provided by both young TH1 and TH2 helper cells in a young microenvironment. These data suggest aged B cells are able to respond to both TH1 and TH2 help.

Funded by NIH grant
Age Related Induction of Functional Transplantation Tolerance to Rat Skin Xenografts in Mice Treated with Co-stimulation Blockade and Donor Antigen (85.28)
Authors: Gordon Ethel J.
Published: January 2023

Our laboratory has developed a protocol to induce functional transplantation tolerance to rat xenografts in mice. Our method requires a single transfusion of donor derived spleen cells (DST) plus four treatments with an antibody to block T lymphocyte activation. Our preliminary analysis of DST donors demonstrated an advantage for using older donors. Median survival time (MST) was longer when older vs. very young DST donors were used. Other variables were constant. We will test the hypothesis that the age of the spleen cell donor has a significant affect on graft survival in xenograft and allograft models. Future studies will analyze cellular and molecular mechanisms responsible for this observation.

This research was supported in part by grant # 2P20RR016477 NCRR/NIH.
CCR2 identifies first responders among human CD4+ memory T cells: long-lived, apoptosis-resistant, antigen-responsive cells with enhanced migration potential, a low threshold for activation, and immediate effector capabilities (85.5)
Authors: Farber Joshua M. , Song Kaimei , Zhang Hongwei H. , Rabin Ronald L. , Hill Brenna J. , Sereti Irini , Prussin Calman , Siegel Richard M. , Douek Daniel C. , Roederer Mario
Published: January 2023

The persistence and roles of memory T cells with effector capability have been questioned. We analyzed human memory CD4+ T cells from peripheral blood defined by their expression of chemokine receptors CCR5 and CCR2, the principal subsets being CCR5−CCR2−, CCR5+CCR2−, and CCR5+CCR2+. CCR2+ cells co-expressed the most chemokine receptor types (up to six) and migrated to the greatest number of chemokines, followed by CCR5+CCR2− cells. Numbers of T cell receptor gene excision circles were CCR5−CCR2− > CCR5+CCR2− > CCR5+CCR2+. The CCR2+ cells were those most readily activated through TCR but showed reduced proliferative potential, were enriched in cells responding to a remote immunogen, secreted high levels of effector cytokines, and were resistant to apoptosis. By contrast, the CCR5+CCR2− population was enriched in recently activated/cycling cells and Treg, and was more susceptible to apoptosis. The data suggest that patterns of CCR5 and CCR2 expression separate effector- vs. memory-cell enriched CD4+ subsets, and that CCR2 marks highly differentiated, long-lived memory cells with effector capabilities. The data also suggest co-ordination among a memory cell’s position on a unidirectional pathway of differentiation, ability to be recruited into tissue, and threshold for activation/effector function, with the most chemokine-responsive cells being best equipped as first responders in a recall response.
Identification of a mouse mutant with highly deficient cytotoxic T cell memory generation using ENU-induced genome-wide mutagenesis (85.7)
Authors: Kung John T.
Published: January 2023

While much has been learned about CTL memory maintenance, much remains to be learned on the early events of CTL activation or induction phase. Through an ENU-induced genome-wide mouse mutagenesis program, we screened G3 mice using CD44 expression on CD8+ T cells as a surrogate indicator of the relative representation of the CTL memory subset. One such identified mutant (P-054) has highly deficient CD44hi subset within CD8+ T cells but normal CD44hi subset in CD4+ T cells. An affected male mouse was bred to normal females to generate F1 mice that all had normal numbers of CTL memory subset based on CD44 expression. Random intercross of F1 mice generated CTL deficienct F2 mice at a ratio of ~25%, indicating heritability of this trait and recessive transmission. Linkage of the mutant gene has been established to chromosome 4 to within a region of ~30 genes. The mutant mouse (H-2b) was immunized by BALB.B spleen cells (H-2b) and CTL memory T cells were identified by H60 (a BALB/c-encoded minor histocompatibility ag)-sepcific CTL by appropriate pentamers and a highly deficient H60-specific CTL response was seen. Adoptive transfer of 2C TCR tg CD8+ T cells activated in the presence of added IL-4, a condition known to promote strong CTL memory generation, into P-054 mice resulted in potent maintenance of 2C memory T cells. Thus, P-054 can provide an environment required to maintain known memory CD8+ T cells and likely represents a novel mutant model that is deficient in the induction phase of CTL memory generation.
B cell receptor signaling governs the subcellular location of Toll-like receptor 9 (86.11)
Authors: Chaturvedi Akanksha , Pierce Susan
Published: January 2023

Toll-like receptor 9 (TLR9), an innate immune system receptor for hypomethylated CpG DNA motifs, has gained substantial attention recently due to its unique ability to promote the activation of autoreactive B cells in response to DNA-containing antigens. Many of the autoantigens targeted in systemic lupus erythematosus (SLE) contain DNA. Moreover, TLR9 has been shown to play a critical role in regulating anti-DNA autoantibody production in mouse models of lupus by mechanisms that involve the simultaneous engagement of TLR9 and the B cell receptor (BCR). However, the cellular and molecular basis of the interaction between TLR9 and the BCR is not known and given the spatial segregation of the BCR on the plasma membrane and TLR9 in early endosomal compartments, it is not obvious how synergy is achieved. Here we show that when activated independently the BCR and TLR9 signal from discrete subcellular compartments, the BCR initially from the plasma membrane and later from large multivesicular bodies (MVB) and TLR9 from multiple endosomes. Remarkably, stimulation of B cells simultaneously through the BCR and TLR9 triggers the recruitment of the TLR9-containing endosomes to the MVB by a Src-family kinase-dependent, MyD88-independent mechanism resulting in a synergistic response. Thus, the BCR triggers the recruitment of TLR9- containing vesicles to the MVB presumably to allow TLR9 to survey the BCR bound antigen for its DNA ligand, which if present, results in TLR9 activation and enhanced B cell responses. Our study adds a new spatial dimension to our view of TLR9 signaling in the B cells, potentially providing a new arena for therapeutic drug design.
CS1 (CRACC) induces Proliferation and Autocrine Cytokine expression on Activated human B Lymphocytes (86.3)
Authors: LEE JAE KYUNG , Vaidya Swapnil V , Kumaresan Pappanaicken R , Mathew Porunelloor A
Published: January 2023

CS1 (CRACC, CD319), a member of the CD2 family of cell surface receptors is implicated in the activation of NK cell- mediated cytotoxicity. Previous studies showed that CS1 is also expressed on activated B cells. However, the functional role of CS1 in human B-lymphocytes is not known. Two isoforms of CS1, CS1-L and CS1-S are expressed in human NK cells that differentially regulate NK cell function. CS1-L contains immunoreceptor tyrosine-based switch motifs (ITSMs) in its cytoplasmic domain whereas CS1-S lacks ITSM. Here we show that human B lymphocytes express only CS1-L isoform and its expression is upregulated upon B cell activation with various stimulators. Moreover, anti-CS1 mAb strongly enhanced proliferation of both freshly isolated as well as activated B cells. The enhanced proliferation effects of CS1 were most prominent on B cells activated by anti-CD40 mAbs and/or hrIL-4. Human cytokine microarray and quantitative real-time PCR results indicated that CS1 activation enhanced mRNA transcripts of flt3 ligand, lymphotoxin A, TNF, and IL-14. These results suggest that activation of B lymphocytes through surface CS1 may be mediated through secretion of autocrine cytokines and CS1 may play a role in the regulation of B lymphocytes proliferation during immune responses.
Intracellular signaling pathways triggered by activation of the Epstein-Barr virus receptor (gp140, CR2, CD21, EBV/C3dR) on cell surface of human B lymphocytes (86.5)
Authors: FRADE Raymond , LOTTIN Severine
Published: January 2023

We originally isolated gp140, the receptor of human C3 (the third component of complement) of human B lymphocytes (1981). gp140 is the C3d receptor (1985a) and also the Epstein-Barr virus receptor (1985b). EBV/C3dR regulates proliferation (1985c) and transformation (1985b) of human B lymphocytes. Thus, we analyzed intracellular events triggered by activated EBV/C3dR. EBV/C3dR activation triggered its intracellular interactions with a p120 nuclear RNP (1987) and the p53 oncoproteine (1995). Then, we first identified two signaling pathways specifically triggered by activated EBV/C3dR. The first pathway is characterized by PI-3 kinase activation:

upstream PI-3 kinase, pp60src kinase is activated to tyrosine phosphorylate nucleolin. Then, tyrosine phosphorylated nucleolin interacts with SH2 domains of the p85 subunit of PI-3 kinase, activating this latter;downstream PI 3-kinase, AKT-GSK3 pathway is activated. This pathway is independent of BCR and CD19 activations (1999, 2001, 2003).

The second pathway is characterized by:

Cbl phosphorylation;Cbl interactions with Crk-L, Syk and the p85 subunit of PI-3-kinase;dissociation of the Cbl-Vav complex (2006).

This dissociation, in opposite to Cbl-Vav association triggered by BCR and CD19, emphasizes the specificity of EBV/C3dR signaling. We herein identified the functional relationship between both pathways.

This work is supported by INSERM
T Cell Receptor-induced NF-KB Activation Is Negatively Regulated by E3 Ubiquitin Ligase Cbl-b via Akt- and PKC-theta-dependent Pathways (87.10)
Authors: Qiao Guilin , Li zhenping , Alegre Maria-Luisa , Ying Haiyan , Zhang Jian
Published: January 2023

Previously, we demonstrated that Cbl-b tunes the threshold for T cell activation mediated by CD28 and CTLA-4. Cbl-b negatively regulates Vav-1 via a PI3-K-dependent mechanism. However, it is unknown whether Cbl-b also targets other signaling pathway(s). In this study, we found that loss of Cbl-b selectively results in aberrant activation of TCR-induced NF-κB which is mediated by PI3-K/Akt and PKC-𝛉. The absence of Cbl-b also results in enhanced TCR-induced activation of PLC-γ1 which may selectively induce activation of PKCs including PKC-𝛉 but not Ca2+ signaling pathway. Cbl-b associates with PKC-𝛉 upon TCR stimulation and regulates TCR-induced PKC-𝛉 activation through Vav-1 which couples PKC-𝛉 to PI3-K and allows it to be phosphorylated possibly by PDK-1. PKC-𝛉 then couples IKKs to CBM complex, resulting in activation of IKK complex. Therefore, our data first demonstrate that down-regulation of TCR-induced NF-κB activation by Cbl-b is coordinately mediated by both Akt- and PKC-𝛉dependent signaling pathways in primary T cells.
Measurement of nuclear translocation in primary cells using correlation analysis of images obtained on the ImageStream imaging flow cytometer (87.13)
Authors: George Thaddeus , Harmon Ian , Peterson Erik J , Burbach Brandon J , Shimizu Yoji , Matsuda Jennifer L , Gapin Laurent , Connors Mark , Dowdell Kennichi C , Farrar J David , Hall Brian E , Morrissey Philip J
Published: January 2023

Signal transduction pathways regulate T cell activation and differentiation and have a critical influence on the outcome of adaptive immune responses. It has recently been shown that nuclear translocation events within non-adherent tumor cells can be quantified by correlating transnscription factor and nuclear images collected on the ImageStream multispectral imaging flow cytometer. Here we extend the quantitative technique to the study of nuclear localization within primary human or murine T cells in the following systems:

translocation of T-bet in CD4 cells derived from murine spleen and lymph node cellstranslocation of NFAT in tetramer positive CD8 cells from human peripheral blood,translocation of NFκB in CD4+CD8+ thymocytes involved in conjugates with APC presenting cognate peptide, andlocalization of phosphorylated STAT4 in human CD4 cells following cytokine stimulation.

These data demonstrate image-based nuclear localzation within primary cells with large nuclear to cytoplasmic area ratios using ImageStream technology.
Ets1-dependent IP3R3 expression in naïve CD4+ T cells is required for cytokine gene expression (87.22)
Authors: NAGALEEKAR VISWAS K , DIEHL SEAN , Juncadella Ignacio , Charland Colette , Garrett-Sinha Lee Ann , Muthusamy Natarajan , Anguita Juan , Rincón Mercedes
Published: January 2023

IP3 receptors (IP3Rs) are critical for the release of Ca++ from intracellular stores in response to IP3 generated upon T cell receptor (TCR) ligation. However, little is known about the expression of the different IP3Rs in CD4+ T cells and their contribution to cytokine gene expression during antigen stimulation. Here, we show for the first time that prior to activation, naïve CD4+ T cells only express IP3R3, but not IP3R1 and IP3R2. IP3R3-mediated Ca++ flux for cytokine gene expression is required for an extended period of time on the order of hours that varies for specific cytokine. IP3R3 gene expression in CD4+ T cells is dependent on the transcription factor Ets1 and is downregulated during the activation due to the loss of this transcription factor. The downregulation of IP3R3 in activated cells correlates with the failure of TCR ligation to trigger intracellular Ca++ mobilization in these cells. Thus, IP3R3 plays an important role in cytokine gene expression during early activation of naïve CD4+ T cells.
Endogenously produced IL-4 strongly enhances CD8+ T cell proliferation and accumulation in homeostatic conditions and during Th2 responses (87.24)
Authors: Morris Suzanne C. , Heidorn Stephanie M. , Yang Jun-Qi , Herbert De’Broski R. , Khodoun Marat V. , Finkelman Fred D.
Published: January 2023

IL-7 and IL-15 have important stimulatory roles in CD8+ T cell homeostasis. We evaluated whether another γc-related cytokine, IL-4, also stimulates CD8+ T cell proliferation and accumulation in vivo. Wild-type (WT) mice have twice as many splenic CD8+ T cells as IL-4- or IL-4Rα-deficient mice, with similar numbers of naïve cells but ~5-fold more memory phenotype cells. WT CD8+ T cells outcompete IL-4Rα-deficient CD8+ T cells following cell transfer and SCID mice reconstituted with equal numbers of WT and IL-4Rα-deficient CD8+ T cells have twice as many splenic WT as IL-4Rα-deficient CD8+ T cells 2 weeks later. WT, but not IL-4Rα-deficient CD8+ T cells proliferate when transferred into IL-4Rα-deficient mice that have been immunized with goat anti-mouse IgD antibody or infected with Nippostrongylus brasiliensis to stimulate a Th2 response. Consistent with this, splenic CD8+ T cell accumulation is entirely IL-4- and IL-4Rα-dependent and proliferation is predominantly IL-4- and IL-4Rα-dependent in mice infected w132th Schistosoma mansoni and pulmonary CD8+ T cell proliferation is rapidly suppressed by anti-IL-4R□ mAb in a mouse model of asthma. Furthermore, even ng quantities of IL-4 stimulate CD8+ T cell proliferation and accumulation in vivo. Thus, IL-4 is a potent, biologically important stimulator of CD8+ T cell proliferation and accumulation in vivo.

Supported by the US Dept of Veterans Affairs and NIH.
Enhanced development of memory like CD8 T cells with innate immune function in the absence of Tec kinase signals (87.26)
Authors: Hu Jianfang , August Avery
Published: January 2023

T cells with a memory phenotype and innate immune function have been reported, but the signals regulating these cells are still unclear. The Tec family kinases are highly expressed in T cells and involved in T cell activation and differentiation. We show here that Itk null mice have a consistent increase in the proportions and absolute numbers of CD8+ T cells that have memory phenotype (CD8+ CD44highCD122high). This elevation is observed in mice carrying an Itk mutant lacking the kinase domain, but not in mice carrying a transgene of the related kinase Rlk/Txk, which indicates that active Tec kinase signaling suppresses their presence. These cells carry preformed message for IFN-gamma and are able to produce IFN-gamma directly ex vivo. They also carry abundant transcripts for Granzyme B and eomesodermin. We also show that memory phenotype CD8+ T cells rapidly secrete IFN-gamma after infection with Listeria monocytogenes (LM) and Itk null mice have the ability to effectively response to infection. Antibody-mediated depletion of CD8+ T cells resulted in significantly reduced ability to clear LM infection in both WT and Itk null mice. Furthermore, transfer of small number of memory phenotype CD8+ T cells can reduce bacterial counts in IFN-gamma deficient mice 3 days after LM infection. These results indicate that these cells are functional CD8+CD44+ cells with innate like function and that the active signals from Tec kinases regulate their development.
Stomatin-Like Protein 2 Sustains Signalling from T Cell Receptor Signalosomes (87.36)
Authors: Kirchhof Mark G , Chau Luan A , Lemke Caitlin D , Vardhana Santosh , Darlington Peter J , Márquez Maria E. , Taylor Roy , Rizkalla Kamilia , Blanca Isaac , Dustin Michael L , Madrenas Joaquín
Published: January 2023

T cell activation involves the formation of an organized interface between the T cell and the antigen-presenting cell known as the immunological synapse (IS). Sustained T cell signalling at the IS emanates from peripheral supramolecular activating complexes (pSMACs). We hypothesized that a molecular machinery would anchor the TCR microclusters in the periphery of the synapse to the cytoskeleton allowing the recruitment of signalosome components and providing sustained TCR signalling. Here, we report that stomatin-like protein-2 (SLP-2) is a key element of this machinery by linking the components of the pSMACs to the actin cytoskeleton. SLP-2 is expressed at low levels in resting human lymphocytes but is rapidly up-regulated upon activation. In T cells, SLP-2 polarizes to the IS and actively accumulates in the pSMACs, within lipid rafts. SLP-2 sequentially associates with CD3-ε, Lck, ZAP-70, phospho-LAT and phospho-PLC-γ1, as well as with polymerized actin. In vivo, SLP-2 expression can be detected in areas of lymphocyte activation in human lymph nodes and thymi. Consistent with these data, up-regulation of SLP-2 in T cells correlates with enhanced activation, whereas knocking-down SLP-2 expression in human effector T cells correlates with loss of sustained TCR signalling and down-regulation of effector responses.
Bcl-3 is required for adjuvant mediated T cell survival and sustained inhibition of GSK3ß (87.38)
Authors: Jayaraman Padmini , Chilton Paula M , Sengupta Sadhak , Mitchell Thomas C
Published: January 2023

Increased transcription of bcl3 correlates with adjuvant-mediated survival of activated T cells. To determine whether Bcl3 is required for adjuvant-dependent survival of activated T cells, OT-II.Bcl3 knockout (KO) mice were challenged with ova peptide (Ova) in the presence or absence of LPS and T cell survival was assessed after 20 hr culture. Ova-treated CD4 T cells from the Bcl3 KO mice activated with or without LPS failed to survive whereas wild type (WT) OTII cells survived when activated with LPS. Using low-number adoptive transfer of WT or KO OTIIs, we show that Bcl3 is required for effective adjuvant-dependent increase in clonal expansion in response to antigen. At peak expansion, 10-fold fewer KO OTIIs were recovered after LPS treatment, compared to the WT OTIIs. Similar defects were seen in adjuvant treated OT-I.Bcl3 KO cells. GSK3ß is a target molecule whose activity is affected by adjuvants during T cell expansion. Bcl3 KO T cells do not maintain the inhibitory p(ser9) on GSK3ß as the WT OTII cells although the initial increase after T cell activation is not affected. This indicates increased phosphatase activity in Bcl3 KO T cells. Studies are underway to address unchecked phosphatase activity as a mechanism of death in the KO cells. These data establish the importance of Bcl-3 in adjuvant- mediated survival of in vivo activated T cells.

This work was supported by USPHS grants AI51377 and AI059023 and the Commonwealth of Kentucky Research Challenge Trust Fund.
Synergistic control of T cell development by Diacylglycerol kinase alpha and zeta (87.44)
Authors: Zhong Xiao-Ping , Guo Rishu
Published: January 2023

Signaling from the T-cell receptor (TCR) plays a critical role in thymic selections and lineage differentiation. Abnormal TCR signaling can lead to aberrant T cell development and autoimmune diseases. Thus, it is critical to understand how TCR signaling is regulated during T cell maturation. Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that catalyze phosphorylation of DAG and convert it to phosphatidic acid (PA). An important feature of DGKs mediated reaction is that both the substrate, DAG, and the product, PA, can be important second messengers. Both DAG and PA associate with and regulate the activitys of many signaling molecules. It has been hypothesized that DGKs may act as crucial regulators of receptor signaling and cellular function by modulating DAG and PA concentrations. Previous studies have demonstrated that DGKalpha and zeta are important negative regulators for peripheral T cell activation by negatively controlling DAG mediated signaling. We show here that DGKapha and zeta synergistically regulate multiple signaling pathways downstream of the TCR in developing thymocytes and that DGK activity plays a crucial role in thymic selection and CD4/CD8 lineage commitment.
A non-apoptotic FADD/caspase-8 dependent mechanism essential for T cell clonal expansion (87.48)
Authors: Walsh Craig Michael , Leverrier Sabrina , Bell Bryan D. , Arechiga Adrian F. , Weist Brian M.
Published: January 2023

FADD constitutes an essential component of TNF receptor-induced apoptotic signaling. Paradoxically, FADD has also been shown to be crucial for lymphocyte development and activation. Using mice expressing a T cell specific transgene encoding the death domain of FADD (FADDdd), we have found that FADD participates in T cell clonal expansion. Based on BrdU uptake assays, we have found that FADD signaling is required for transition between the late G1 and S-phase of cell cycle. The defective progression through cycle is marked by a defect in cyclin E expression and CDK2 activation. We have also identified a defect in the maintenance of S6K1 activity and ribosomal S6 phosphorylation, two essential intermediaries of proliferative signaling in T cells. We have found that caspase-8 is essential in this FADD dependent process, suggesting that FADD promotes the assembly of a non-apoptotic caspase-8 complex that serves to relieve inhibition of cell cycle progression during the G1/S transition. In line with this hypothesis, we have found that caspase-8 activity is induced in T cells in a cell-cycle dependent manner in normal T cells. The potential that this signaling axis may modulate autophagy is presently under investigation. Since TNF receptor family members play key roles in regulating adaptive immunity, these results suggest that FADD and caspase-8 serve multiple functions to maintain immune homeostasis.
From a single viral peptide epitope to effective lysis of target cells by CTL (87.50)
Authors: Sykulev Yuri , Anikeeva Nadia , Beal Allison , Vasiliver Gaia , Varma Rajat , Norris Philip J , Mattoussi Hedi , Dustin Michael L
Published: January 2023

Cytotoxic T lymphocytes (CTL) possess a unique ability to destroy virus-infected cells by releasing cytolytic granules that upon delivery to target cells induce apoptosis. It has been proved that perhaps even a single peptide presented on a target cell renders this cell susceptible for killing by CTL. Using a novel biosensor containing quantum dots (QD) carrying controlled number of cognate and non-cognate peptide-MHC (pMHC) proteins, we have shown that pMHC attached to QD cooperate and that the presence of a single cognate pMHC with all others being non-cognate is sufficient to induce a robust CTL response.

To understand how triggering of CTL response leads to delivery of effective lethal hit to target cells, we examined granule polarization and release as well as segregation of immune receptors at interface between CTL and planar bilayer containing cognate pMHC and ICAM-1. We show that exceptionally tight ring junction formed at CTL-bilayer interface functions as a gasket precluding granule escape, which are concentrated in the secretory domain of immunological synapse. We propose that efficient target cell lysis requires 2 essential manipulations of cytolytic granules: release and effective targeting.
Regulation of B cell differentiation by the adaptor protein HSH2 (87.53)
Authors: Justement Louis B. , Herrin Brantley R. , King R. Glenn
Published: January 2023

The adaptor protein HSH2 is selectively expressed at low basal levels in cells of the B lineage. However, stimulation of B cells via CD40, BAFF-R, TLR4 and TLR9 upregulates HSH2 expression within 6–12 h in a NF-kB-dependent manner. Thus, it was hypothesized that HSH2 may regulate B cell survival and/or differentiation. HSH2 transgenic (Tg) mice were generated that constitutively express HSH2 in cells of the B lineage to determine if HSH2 causes gross changes in B cell subpopulations or functional responses. No changes were observed in developing B cell subpopulations in the bone marrow and the peripheral B cell compartment appeared normal with the exception of a 50% decrease in marginal zone B cells. Analysis of serum Ig levels in HSH2 Tg mice revealed a 40–50% decrease in IgM, a 20% decrease in IgA and a 70–85% decrease in other class switched isotypes compared to control animals. HSH2 Tg mice exhibited a significant impairment in their response to immunization with T-independent type 1 and 2 antigens and exhibited a similar defect in their 1° and 2° response to T-dependent antigen. Constitutive HSH2 expression was not observed to alter BCR-mediated signaling. In contrast, HSH2 Tg B cells exhibited defective responses to CD40 and BAFF-R signaling in vitro, but responded normally to TLR agonists. Thus, HSH2 appears to be important for selectively regulating the B cell functional response to CD40 and BAFF-R signaling.
Ubiquitination of RIP1 regulates an NF-κB-independent cell death switch in TNF signaling. (87.8)
Authors: O’Donnell Marie Anne , Addison Diana Legarda , Ting Adrian
Published: January 2023

TNF receptor 1 (TNFR1) can trigger opposing responses within the same cell: one leads to a pro-survival response whereas the other leads to cell death. The pro-survival response is mediated primarily by activation of the NF-κB signaling pathway, which leads to the expression of pro-survival genes such as c-FLIP, c-IAPs, TRAFs and BCL-2 family members. On the other hand, TNF can also trigger activation of CASPASE 8 and 3 leading eventually to apoptosis. In most cell types, stimulation with TNF does not lead to apoptosis unless NF-κB signaling is blocked. Hence, NF-κB-mediated transcription of pro-survival genes acts as a cell death switch during TNF signaling. This current study demonstrates the existence of another cell death switch elsewhere in the pathway that is independent of NF-κB. Our results show that lysine 63-linked ubiquitination of RIP1 on lysine 377 inhibits TNF-induced apoptosis first through an NF-κB-independent mechanism, and subsequently, through an NF-κB-dependent mechanism. In contrast, in the absence of ubiquitination, RIP1 serves as a pro-apoptotic signaling molecule. Therefore, RIP1 is a dual-function molecule that can be either pro-survival or pro-death depending on its ubiquitination state and this serves as an NF-κB-independent cell death switch early in TNF signaling. These results provide an explanation for the conflicting reports on the role of RIP1 in cell death, which was previously implicated to be both pro-survival and pro-death. Since TRAF2 is the E3 ligase for RIP1, these observations also provide an explanation for the NF-κB-independent anti-apoptotic function of TRAF2 previously described.
New differentiation pathway for double-negative regulatory T-cells that regulates the magnitude of immune responses (87.9)
Authors: Zhang Dong , Yang Wei , Degauque Nicolas , Tian Yan , Mikita Allison , Zheng Xin Xaio
Published: January 2023

CD4−CD8− (double-negative, DN) T-cells are capable of down-regulating the immune response. However, the origin and developmental pathway of DN T-cells are still unclear.

In this study, we showed that proliferated CD4+ T-cells could convert to DN T-cells after 4–5 rounds of either allogeneic or syngeneic DC triggered proliferation, IL-2 and IL-15 enhanced the conversion. The disappearance of CD4 molecular on converted DN T-cells was a result of CD4 gene silence. Converted DN T-cells were resistant to AICD and expressed unique cell markers and gene profile. Moreover, DN T-cells retained a stable phenotype after re-stimulation. DN T-cells converted from CD4+ T-cells exerted powerful inhibition on same, but not third party, alloantigen triggered proliferation of naïve T cells. Perforin, a cytotoxic lymphocyte related cytokine, was highly expressed by DN T-cells, played a role in DN T-cell mediated suppression. Transferring of DN T-cells alone could prevent allograft rejection and the onset of autoimmune disease in vivo.

This previously unknown pathway of differentiating DN regulatory T-cells represents a negative feedback mechanism of immune response. The results of using ex vivo CD4+ T-cells converted DN T-cells in transplantation and autoimmune disease models support the concept and the feasibility of potentially utilizing this novel cell-based therapy.
T Regulatory Cells Control the Numbers of NK Cells and Thereby Protect CD8alpha+ Immature Dendritic Cells Presence in the Lymph Node Paracortex (88.13)
Authors: Giroux Martin , Yurchenko Ekaterina , St-Pierre Jessica , Piccirillo Ciriaco A , Perreault Claude
Published: January 2023

In the absence of infection, the spleen contains numerous natural killer (NK) cells located mainly in the red pulp and whose differentiation profile is characterized by a preponderance of mature elements. In contrast, lymph nodes (LNs) contain few NK cells that are sited mostly in T-cell zones and that are phenotypically skewed toward immature developmental stages. We present evidence that CD4+CD25+ T regulatory (Treg) cells hamper generation of mature NK cells in the LNs through short-range interactions with NK precursors. Moreover, we show that Treg cells are both necessary and sufficient to repress accumulation of NK cells in resting LNs and an absence of Treg totally alleviates the differentiation blockade and leads to an accumulation of mature NK cells in the LN paracortex. In turn, mature NK cells specifically regulate the amount of CD8α+ phenotypically immature dendritic cells (iDCs) present in LN T-cell zones. We propose that the dominant influence of Treg cells on NK cell precursors and consequently on CD8α+ iDCs explains why “quiescent” LNs, in the absence of infection, function as privileged sites for induction and maintenance of tolerance to peripheral self antigens.
CD101 surface expression discriminates potency among murine FoxP3+ regulatory T cells (88.18)
Authors: Fernandez Irina , Zeiser Robert , Karsunky Holger , Kambham Neeraja , Beilhack Andreas , Sodestrom Kalle , Negrin Robert , Engleman Edgar
Published: January 2023

CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-versus-host disease (GVHD). However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit monoclonal antibody against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25–30% of CD4+CD25+ Treg and a small population of conventional memory T cells. CD101+ Treg displayed greater in vitro suppression of alloantigen driven T cell proliferation as compared to CD101 negative (CD101-) Treg. In a model of GVHD induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101+ Treg, compared to CD101− Treg. Moreover, treatment with CD101+ Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.
Gamma irradiation alters the phenotype and function of CD4+CD25+ regulatory T cells (88.2)
Authors: Cao Mengde , Xu Yiling , Cabrera Roniel , Liu Chen , Nelson David
Published: January 2023

Naturally occurring regulatory T cells (Tregs) are importantmodulators of immune responses to pathogens, tumors, and transplantationantigens. Gamma irradiation of stimulator cells is commonly used to achieve unidirectionality of response in the mixed lymphocyte reaction (MLR), but little is known about effects of gamma irradiation on phenotype and suppression function of Tregs. To elucidate the effects of gamma irradiation on Tregs, we analyzed changes in phenotype and suppression function in Tregs treated with or without gamma ray. We investigated and found that proliferation of Tregs in response to gamma irradiation was reduced in a dose-dependent way ( 0Gy: 44007 ±6785cpm; 1.875Gy: 15038 ±5542cpm; 7.5Gy: 6618 ±1069cpm; 30Gy: 439 ±100cpm; p<0.05), and the apoptosis induction of Tregs was increased compared with untreated cells (0Gy: 6.88%; 1.875Gy: 17.71%; 7.5Gy: 21.70%; 30Gy: 54.49%). Gamma irradiation down-regulated the expression of CD62L, FOXP3, membrane TGF-β, CD127, but up-regulated PD-1, CD25, caspase3, Bax and GITR expression in Tregs. High dose gamma irradiation (30 Gy) significantly abolished the suppression of Tregs on CD4+CD25− T cells proliferation. The neutralization of anti-TGF-β has the same function as high dose gamma irradiation to block the suppressive ability of Tregs. It was concluded that gamma irradiation of Tregs not only influences the phenotype of Tregs but also alters their capacity to suppress the proliferation of autologous T cells. Our findingssuggest that Tregs could be potential targetsfor improving the efficacy of radiotherapy for cancer.