bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of T cell development by the transcription factor HEBAlt (82.1)
Authors: Anderson Michele Kay , Wang Duncheng , Claus Carol L. , Braunstein Marsela , Vaccarelli Giovanna
Published: January 2023

The transcription factor HEBAlt is derived from the HEB gene locus, which also encodes the E2A-related factor HEBCan (Can=canonical). HEBAlt is expressed in early lymphocyte precursors. HEBAlt is synergistically upregulated in T cell precursors by Delta-Notch signaling and HEBCan activity, and ectopic expression of HEBAlt in uncommitted precursors enhances entry into the T cell lineage. By contrast, HEBAlt is only expressed in committed B cell precursors, and expression prior to commitment blocks B cell development. A role for HEBAlt in the repression of CD8alpha transcription at the pro-T cell stage of development is suggested by loss of CD8 in the presence of HEBAlt, and upregulation of CD8 in pro-T cells by an HEBAlt-engrailed repressor construct. HEBAlt can also influence the fate choice between the gamma-delta T cell lineage and the alpha-beta T cell lineage. These results suggest that HEBAlt is important in multiple aspects of early T cell development, including locking in the T/B lineage choice downstream of Notch signaling, repressing T cell-specific genes poised for expression past TCRbeta selection, and influencing an early T cell lineage decision.
Developmental-associated differences in membrane cholesterol content account for the differential responses of transitional immature and mature B cells to B cell receptor engagement (83.1)
Authors: Karnell Fredrick G. , Brezski Randall J. , King Leslie B. , Sanborn Keri B. , Monroe John G.
Published: January 2023

Tolerance-sensitive transitional immature B cells maintain significantly lower membrane unesterified cholesterol levels than mature splenic B cells. In addition, the relatively low level of plasma membrane-associated cholesterol in transitional immature B cells impairs compartmentalization of their B cell receptor (BCR) into cholesterol-enriched domains following BCR aggregation and reduces their ability to sustain certain aspects of BCR signaling as compared with mature B cells. By increasing the level of plasma membrane associated cholesterol in transitional immature B to that seen in mature B cells, we observe an decrease in apoptosis post-BCR stimulation as compared to unmanipulated transitional immature B cells. Furthermore, we have generated a stable genetic mouse model in which cholesterol is constitutively produced in B cells, thus increasing the level of plasma membrane associated cholesterol in transitional immature B cells in vivo. These studies establish an unexpected difference in the lipid composition of peripheral transitional immature and mature B cells and point to a determining role for development-associated differences in cholesterol content for the differential responses of these B cells to BCR engagement.

This work was supported by Training Grant AI055428 and Grants AI43620 and AI32592 from the National Institutes of Health.
GALT development in response to Bacillus anthracis (83.10)
Authors: Severson Kari , Mallozzi Michael , Driks Adam , Knight Katherine L.
Published: January 2023

In several species, including rabbit, a large B cell expansion occurs in GALT in response to the intestinal microbiota. To identify which bacterial species induce GALT development, we injected several species into rabbits with a germ-free appendix, a component of GALT. We found that, when injected alone, the spore-forming Bacillus species, Bacillus anthracis, induces large follicles of proliferating B cells. Our goal is to identify bacterial molecules from B. anthracis that promote GALT development, and determine the mechanism by which these molecules stimulate B cell proliferation.

Previous data suggest that the large B cell expansion in GALT occurs in an antigen-independent, polyclonal manner. Additionally, injection of a known B cell superantigen, Staphylococcus aureus Protein A, into germ-free appendices, was sufficient to induce B cell proliferation. We hypothesize that bacterial molecules act as superantigens and polyclonally stimulate B cells directly through the BCR to induce GALT development. To test this, we probed bacterial lysates with rabbit IgM to search for IgM-binding molecules in B. anthracis. By western blot analysis, we identified a 25kD molecule in B. anthracis spores that binds to rabbit IgM. Experiments are underway to determine the identity of this B. anthracis molecule and to determine if it stimulates GALT development through a superantigen mechanism.

Supported by NIH AI50260
Ligation of TLR9 on lymphoid progenitors promotes dendritic cell production at the expense of lymphopoiesis (83.11)
Authors: Pelayo Rosana , Welner Robert S. , Nagai Yoshinori , Garrett Karla P. , Wuest Todd , Carr Daniel J. , Kincade Paul W.
Published: January 2023

Hematopoietic cells express TLR, raising the possibility that bacterial/viral products influence blood cell formation. We now show that common lymphoid progenitors (CLPs) express functional TLR9, and that dendritic cell production was strongly favored by TLR9 ligation with CpG ODN. TLR9 on CLPs rather than indirect cytokine mediated effects accounted for the shift in dendritic cell production, and CLPs became IL-7 unresponsive in culture. Similarly, CLPs from CpG treated mice had little ability to generate CD19+ cells and augmented competence to produce conventional (cDC), plasmacytoid (pDC) and interferon producing killer dendritic cells (IKDC) even in the absence of TNFα. Finally, lymphopoiesis in mice with active, but not latent HSV-1 infection was very similar to that seen in animals given CpG, and the changes were almost entirely mediated by TLR9. The findings bear on long standing controversies concerning the origins of dendritic cells and show how life-threatening infections may boost their production.
Age dependent decline in CD47 expression on circulating mouse erythrocytes and susceptibility to destruction by macrophages (85.11)
Authors: Khandelwal Sanjay , Saxena Rajiv Krishna
Published: January 2023

We have recently described a novel technique to tag a cohort of mouse erythrocytes released into blood circulation over a defined time window, and follow age related changes in this population in circulation (Khandelwal S and Saxena RK, Expt. Gerontol. 41, 855, 2006). In the current study, we report the kinetics of age dependent decline in CD47 expression on circulating erythrocytes and its role on destruction of senescent erythrocytes by macrophages. CD47 expression was highest on erythrocytes freshly released into circulation and fell steadily thereafter. Oldest erythrocytes in circulation had 30% lower expression of CD47 than the youngest erythrocytes. Depletion of macrophages by administration of clodronate loaded liposomes resulted in a significant fall in the average expression of CD47 on erythrocytes of young (<15 d), intermediate (15–40 d)and old (>40 d) age groups, but a significant increase was observed only in the numbers of old erythrocytes in blood. A time dependent accumulation of CD47low erythrocytes was noted in blood and spleen though the accumulation was markedly higher in spleen. Taken together our results support the proposition that an age dependent fall in CD47 expression on blood erythrocytes may have a role in regulating the destruction of senescent blood erythrocytes by the macrophages.

This work was supported by a research grant from the Department of Science and Technology, Government of India.
Decreases in E2A, AID, and Ig class switch show intrinsic defects in human B cells with age (85.14)
Authors: blomberg bonnie b , landin ana marie , riley richard l , lechner suzanne c , ryan john g , schwartz robert , frasca daniela
Published: January 2023

Elderly human beings have compromised humoral and cellular immune responses, which lead to reduced protection to infectious agents and to vaccines causing protection of only a small proportion of the elderly population. The capacity to class switch the heavy chain of Ig is critical for an effective humoral immune response in mice and humans. We have shown in mice that the E2A-encoded transcription factor E47, which regulates many B cell functions, is down-regulated in old splenic B cells. This leads to a reduction in activation-induced cytidine deaminase (AID) enzyme, which directly induces class switch recombination (CSR) and Ig somatic hypermutatio necessary for affinity maturation of protective Ig. This reduced AID generates less switched antibodies produced by the activated B cells. We have extended our study to investigate whether aging also affects antibody production, E47 and AID expression in B cells isolated from the peripheral blood (PB) of 50 human subjects (18–86 years). Our results obtained with CD19+ B cells show that the expression of E47, AID and Igf×1 circle transcripts progressively decrease with age. We also show an age-related decline in switch memory B cells (IgG+/IgA+/CD27+) and an increase in naive B cells (IgG−/IgA−/CD27−), consistent with our data on the decrease in CSR. Our results provide a possible molecular mechanism for a B cell intrinsic decrease in the humoral immune response with aging and suggest avenues for improvement of vaccine response in elderly humans.
Effects of T-cell derived leptin on thymic function and immune regulation (85.8)
Authors: vandanmagsar bolormaa , Yang Hyunwon , Carter Arnell , Collins Gary , Pyle Robert , Bertak Dorothy , Taub Dennis
Published: January 2023

The communication between the state of systemic and cellular energy balance to the immune compartment is mediated via a complex array of cytokines, hormones and neuropeptides. Leptin is an adipocyte-derived hormone and belongs structurally to the cytokine family including members such IL-2 and IL-12. Leptin signals via a class I cytokine receptor and represents an important link between energy balance on the one side and immune function on the other. Leptin has recently been shown to be produced in limited quantities by murine T cells. To examine the endogenous role of this hormone in the development and control of the adaptive immune responses and inflammation, we have generated T-cell transgenic mice overexpressing leptin. Leptin overexpression in T cells affects thymic size and cellularity and may have considerable impact on the immune function of aging mice. Preliminary data shows that, leptin differentially affects cytokine production by T cells, increasing IL-1, IL-6, IL-12, G-CSF, and MIP-1a levels in leptin transgenic mice compare to wild-type control. More specifically, IFN-g production was significantly higher in older leptin-TG mice, suggesting that endogenously produced leptin may play an important role in the generation of pro-inflammatory Th1 immune responses and thymic development with age.
Leptin Directly Protects Thymus Tissue from Endotoxin-induced Involution (85.9)
Authors: Gruver Amanda Louise , Ventevogel Melissa S , Sempowski Gregory D
Published: January 2023

Thymus tissue produces new T cells and functions well into adulthood. Thymus tissue is highly susceptible to atrophy induced by stress, infections, and ionizing radiation. Prolonged thymus atrophy can contribute to T cell deficiency or prevent immune recovery after acute T cell depletion. No treatment however is currently available to protect against thymus involution. Leptin is a naturally occurring metabolic hormone currently used therapeutically for obesity. We have previously reported that leptin protects against bacterial endotoxin-induced thymus atrophy in a lipopolysaccharide (LPS) mouse model resulting in increased thymus weight, cellularity, and T cell receptor gene rearrangement. We now report the mechanism by which leptin protects the thymus from acute involution. Leptin directly relieves the LPS-induced block on proliferation in CD4−/CD8− thymocytes and protects against stress-induced CD4+/CD8+ thymocyte apoptosis (9% vs. 34% in control, p < 0.01) in a Bcl-2 independent manner. Taken together, these data demonstrate that leptin directly protects against thymus atrophy by promoting expansion of CD4−/CD8− thymocytes and blocking death of CD4+/CD8+ thymocytes. Together, these data support further investigation of leptin as a therapeutic for stimulating thymic function in settings of stress.

Supported by the NIH PO1-HL67314, RO1-AG025150.
TLR agonists induce an alternate pathway for BCR-induced ERK phosphorylation and NF-κB activation (86.9)
Authors: Carter Natalie Annis , Dye John , Palvanov Arkadiy , Rothstein Thomas L
Published: January 2023

The classical pathway for BCR signaling requires signalosome elements including PI-3K, Btk, PLCγ2 and PKCβ. Inhibition or ablation of any of these signaling mediators results in a block in BCR-triggered downstream events such as ERK phosphorylation and NF-κB activation. However, recent work in this lab has demonstrated that exposure to certain molecules such as CD40L and IL-4 produces an alternate pathway such that BCR-induced signaling bypasses the need for signalosome elements. This led us to investigate whether bacterial products that signal “danger” can create an alternate pathway for MAPK activation. We found that BCR-triggered ERK phosphorylation became resistant to the PI-3K inhibitor LY294002 after B cell treatment with LPS, CpG or PAM3CSK. LPS and PAM3CSK were also tested further and found to induce PI-3K independent NF-κB activation recapitulating the alternate pathway induced by CD40L and distinct from that produced by IL-4. Induction of the alternate pathway for BCR signaling by LPS required MyD88. These data demonstrate that TLR agonists induce an alternate pathway for BCR signaling that bypasses essential signalosome elements. This is of special interest considering that high LPS/CpG levels occur during severe gram-negative bacterial infections. This suggests that consideration of the alternate pathway may be prudent when designing treatments for bacterial infections.
Dual Specificity Phosphatase (DUSP) activity regulates ERK activation and positive selection of thymocytes (87.16)
Authors: Bettini Matthew L. , Kersh Gilbert J.
Published: January 2023

Regulation of MAP Kinase phosphorylation is thought to be important for proper translation of TCR signals into thymocyte cell fates. However, the role of phosphatase activity in thymic development has not been extensively characterized. DUSP-6, also known as Map Kinase Phosphatase-3 (MKP-3), is expressed in the thymus and can bind and dephosphorylate ERK. To explore the role of MKP in thymocytes, we expressed MKP-3 or a catalytically inactive form of MKP-3 (DM-MKP3) in an immature thymocyte cell line, 16610 D9. Lower amounts of phosphorylated ERK (p-ERK) were found in D9 cells that over express MKP-3, while D9 cells that over express DM-MKP3 exhibited higher amounts of p-ERK after TCR stimulation in comparison to vector control cells. Thus, over expression of a catalytically inactive ERK phosphatase results in inhibition of all ERK specific MKP activity, and acts as a dominant negative MKP. Utilizing real time PCR, we also observed elevated Egr-1 mRNA levels after TCR stimulation in D9 cells expressing DM-MKP3 compared to control D9 cells. Egr-1 is an ERK dependent immediate early gene known to support positive selection of thymocytes. Finally, we over expressed DM-MKP3 in vivo, and observed enhanced positive selection. Together these results indicate that MKP control of ERK activation after TCR stimulation is critical in regulation of thymocyte cell fate decisions.

This work is funded by grants from the NIH and the ACS.
Generation of Dimers between Itk Molecules, Recruiting Receptors and Substrates (87.23)
Authors: Qi Qian , August Avery
Published: January 2023

Itk is a Tec family of tyrosine kinase that transduces signals from the T cell receptor and other receptors to modulate T cell development and activation. Itk is activated by receptors via a PI3 kinase mediated pathway, which results in recruitment of Itk to the plasma membrane via its PH domain. We show that membrane localization of Itk results in the formation of clusters of at least 2 molecules within 80 angstroms of each other. In addition, only the PH domain, but not the PRR, SH3 or SH2 domain, or kinase activity of Itk, is required for its formation of clusters at the plasma membrane. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane since only receptors that recruit PI3 kinase reside in the same membrane vicinity as the recruited Itk. Itk also forms dimers or clusters when associated with the adaptor protein SLP-76. Our results indicate that Itk forms membrane dimers with receptors in specific membrane regions. Furthermore, Itk forms dimers with adaptor proteins that assemble multiprotein complexes.
In situ visualization of CD8 T cell-DC interactions reveals distinct features of the early and late response to antigen after influenza virus infection (87.27)
Authors: Cauley Linda S , Khanna Kamal M , Lefrancois Leo
Published: January 2023

The anatomical characteristics of T cell activation following infection are poorly described. Using transferred CD8 T cells and confocal microscopy we show that small clusters of virus-specific T cells formed transiently in the lung-draining lymph nodes (DLN) early in the response to influenza virus infection. Several weeks after infection naïve nucleoprotein (NP)-specific CD8 T cells still responded to antigen in the DLN, but remained predominantly in large stable clusters that included CD11c+ APCs. Many activated cells upregulated CD69 and PD-1, but did not lose CD62L-expression and remained clustered with CD11c+ APCs in the paracortex of the DLN until at least 7 days after transfer. Some central/memory cells also entered the DLN after transfer, but did not colocalize with the clustered naïve cells or show any signs of a response to the antigen. These data indicated a change in the response to antigen stimulation in the DLN as the time interval after infection increased and suggested that naïve and memory F5 CD8 T cells used different mechanisms to locate antigen-bearing APCs within the pLN. Some endogenous NP366-374/Db-specific CD8 T cells were also retained in the MLN and showed signs of a continuing response to antigen stimulation, while PA324-332/Db-specific cells were predominantly found in non-lymphoid tissues. Overall all the data revealed a dichotomy in the response to late antigen presentation which could influence epitope selection during reinfection by favoring preferential expansion of the NP366-374/Db-specific cells in the DLN.

This work was supported by the American Lung Association RG1119 (L.S.C.), the Damon Runyon foundation (K.M.K.) and National Institutes of Health grants; R21-AI065895 (L.S.C.) and DK45260, AI41576 and AI56172 (LL).
Negative Regulation of NIP45 Function by Peptidylarginine Deminase 4 (87.28)
Authors: Mowen Kerri A , Hemmers Saskia , Schurter Brandon , Fathman John W. , Glimcher Laurie H. , David Michael
Published: January 2023

IL-4 production in Th2 lymphocytes is regulated by the nuclear factor of activated T-cells (NFAT) and NFAT-interacting protein 45kDa (NIP45). Recently, we showed that arginine methylation of NIP45 modulates its interaction with NFAT and augments IL-4 transcription. Here we report that NIP45 methylation is negatively regulated by peptidylarginine-deiminase 4 (PAD4) via deimination of arginine residues and generation of the atypical amino acid citrulline. NIP45 loses its ability to become methylated after deimination by PAD4. Ectopic expression of PAD4 impairs the association of NIP45 with NFAT and profoundly inhibits IL-4 promoter activity. Whereas, knockdown of PAD4 expression in Th2 cells by a PAD4-directed shRNA-expressing retrovirus leads to elevated IL-4 production. Interestingly, a PAD4 variant is association with rheumatoid arthritis (RA) in a Japanese population. Indeed, many autoantibodies in RA are directed against citrullinated proteins. It has been proposed that PAD4 is linked to RA because citrullination of peptides leads to a breakdown of self-tolerance to self-antigens. Our data suggest that PAD4 may also influence RA development through regulation of the cytokine milieu. Modification of NIP45 by PAD4 provides a novel negative regulatory mechanism whereby NFAT and NIP45 control IL-4 and potentially other cytokines production.
Negative regulation of T cell receptor signaling by NFkappaB2/p100 (87.4)
Authors: Legarda-Addison Diana , Ting Adrian T.
Published: January 2023

The positive regulation of the NFκB signaling pathway in response to T cell receptor (TCR) stimulation has been well studied. However, little is known about the negative regulation of this pathway in T cells. This negative regulation is crucial in controlling the duration of TCR signaling and preventing abnormal lymphocyte activation and proliferation. Therefore, understanding the negative regulation of TCR-mediated NFκB signaling is essential in understanding the mechanisms involved in T cell function and homeostasis. TCR stimulation of human CD4+ T cells resulted in an increase in NFκB2/p100 expression with minimal increase in p52, its cleavage product. Due to the presence of inhibitory ankyrin-repeats in the unprocessed p100, this observation suggests that p100 may function as a negative regulator of the NFκB pathway. Consistent with this hypothesis, ectopic expression of p100 inhibited TCR-mediated NFκB activity and IL-2 production in Jurkat T cells. Conversely, knockdown of p100 expression enhanced NFκB transcriptional activity and IL-2 production upon TCR activation. p100 inhibited the pathway by binding and sequestering Rel transcription factors in the cytoplasm without affecting the activity of the upstream IκB kinase (IKK). Taken together, this study indicates that NFκB2/p100 can act as an inhibitory molecule in the TCR-mediated NFκB signaling pathway.

This work was supported by a grant from the National Institutes of Health to A.T.T.
A biochemical signature for rapid recall of memory CD4 T cells (87.41)
Authors: Chandok Meena R , Okoye Francesca I , Ndejembi Modesta P , Farber Donna L
Published: January 2023

Mechanisms for the rapid effector responses mediated by memory T cells remain unknown. In this study, we used multiparameter analysis of intracellular signaling and function in naive and memory T cells in vitro and in vivo to determine a biochemical basis for immunological recall. We identify here a striking and specific elevation in expression of the ZAP-70 protein tyrosine kinase in resting memory compared to naive CD4 T cells that is stably maintained independent of protein synthesis, whereas low ZAP-70 expression in naive cells increases with sustained antigenic stimulation requiring new protein synthesis. We demonstrate that ZAP-70 levels control effector function, as specific downregulation of ZAP-70 expression in memory CD4 T cells abrogates rapid effector function, and acquisition of effector function occurs exclusively from activated T cells that have upregulated ZAP-70. Moreover, rapid IFN-γ production from ZAP-70hi memory CD4 T cells occurs via a quantitative bypass in distal signaling in vitro and in vivo, contrasting the increased distal phosphorylation events that precede late IFN-γ production from activated naive CD4 T cells. Our findings reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficient functional coupling through increased ZAP-70 expression and reduced downstream signaling events.

This work was supported by NIH AI42092
In vivo and in vitro zinc (Zn) deficiency diminished T lymphocyte activity through the modulation of PKC isoenzymes. (87.46)
Authors: Cremaschi Graciela Alicia , Klecha Alicia Juana , Orqueda Andres , Kaliski María Alejandra , Arcos María Laura Barreiro , Zubillaga Marcela , Wald Miriam , Genaro Ana María
Published: January 2023

Zn is essential to numerous signaling proteins that share Zn-finger structures as a common motif. It is crucial for immunocompetent cells, as its deficiency leads to immune function alterations. The exact mechanisms involved in Zn regulation of lymphocyte activity are still unclear. Here we analyzed both the direct and the in vivo effect of Zn deficiency on normal T cells and the involvement of protein kinase C (PKC) (an enzyme with cysteine-rich domains) and its isoenzyme profile. For this purpose mitogen stimulated normal T cells were cultured in the absence or presence of specific intra- or extracellular Zn chelators. These effects were compared with those obtained in mitogen-stimulated lymphocytes purified from mice fed with Zn deficient diets. Proliferation, PKC activity and the pattern of PKC isoenzymes were evaluated. Zn chelators inhibited normal cell proliferation and their effects were reverted by in vitro Zn addition. Similar effects were observed in lymphocytes from Zn deficient mice. In both experimental conditions, a decrease in PKC activity and in mitogen-induced translocation of the enzyme to cell membranes were observed. These effects were accompanied by a decrease in PKC α and 𝛉 isoforms.

These results show that Zn plays an important role in T cell growth and that its deficiency would alter the expression of key PKC isoenzymes that are essential signalling molecules involved in lymphocyte activation.
HSF1 is necessary for reactive oxygen species (ROS) homeostasis and proliferation in T cells at fever temperatures. (87.47)
Authors: Woodward Jerold G. , Murapa Patience , Gandhapudi Siva , Skaggs Hollie , Sarge Kevin
Published: January 2023

HSF1 is a major heat and stress inducible transcription factor in eukaryotic cells responsible for the regulation of a variety of genes involved in maintenance of cellular homeostasis. We have previously shown that HSF1 is activated at physiologic fever temperatures (39oC) in T cells vs. much higher, non-physiologic temperatures (42oC) in most other cell types. T cells from HSF1−/− mice proliferate normally at 37oC, but are severely inhibited at 39oC. These T cells are blocked at the G1-S phase transition of the cell cycle. T cells normally increase ROS levels as a result of activation. Antioxidants which block ROS increases also inhibit proliferation. We found that fever temperatures also inhibited the normal activation induced increase of ROS in T cells within 2h. Wild type T cells can overcome this inhibition in ROS generation within 5h and go on to proliferate normally. In contrast, HSF1−/− T cells were unable to overcome this dysregulation and failed to proliferate. Thus, fever temperature causes a dysregulation in ROS homeostasis in T cells and HSF1 is critical in restoring this regulation.
Prolonged FRC-DC interaction negatively affects DC-T cell crosstalk (88.10)
Authors: Kronenberger Konrad , Liwski Robert S. , Trenholm Tara , West Kenneth A.
Published: January 2023

While Fibroblastic Reticulum Cells (FRC) of the lymph node (LN) are known to produce chemokines responsible for the migration of naïve T cells and DC, little is known about their interactions with these cells and their influence on DC and T cell activation. Therefore, we have evaluated the interaction between DC, FRC and T cells in an in vitro model.

For the generation of FRC cells lymph nodes of BALB/c mice were isolated and cultured ex vivo. After co culture with FRC DCs were characterized by FACS analysis concerning their survival rate, maturation state and expression of cell surface molecules as well as for their production of soluble cytokines. The ability of DC to induce T cell proliferation was evaluated using the D011.10 TCR transgenic system and CFSE labeling.

Our in vitro culture method leads to a highly homogeneous FRC population regarding expression of gp38, ER-TR 7, CD44 and V-CAM. Confocal microscopy analysis demonstrated that DC migrated to FRC where they made physical contact leading to stable associations in vitro as well as in vivo in the LN after DC were injected s.c..

Co incubation of DC with FRCs resulted in a complete down regulation of CD40 and partial decrease of co stimulatory molecules. Furthermore there was a strongly diminished ability of these DC to induce a peptide specific proliferation of naïve T cells although a clear CD25 up regulation could be detected. It remains to be investigated whether these CD25 positive T cells show a regulatory phenotype.

Our results demonstrate that LN-FRC provide a milieu that may induce tolerogenic DCs which then drive naïve T cells to a rather regulatory phenotype.
In the tumor microenvironment of head and neck cancer patients (HNSCC), suppression is mediated by a unique subset of CD4+CD25highFoxp3+ T cells secreting IL-10 and TGF-beta1 (88.47)
Authors: Strauss Laura , Bergmann Christoph , Johnson Jonas T. , Whiteside Theresa L.
Published: January 2023

Objective: CD4+CD25highFoxp3+T cells (nTreg) are a characteristic feature of HNSCC. Phenotype and function of nTreg in tumor-infiltrating (TIL) vs. peripheral blood mononuclear (PBMC) T cells remain controversial.

Methods: Flow cytometry was used to study Treg in TIL of 15 HNSCC patients, PBMC of 25 HNSCC patients and PBMC of 15 normal controls (NC). CD4+CD25high T cells were sorted from TIL or PBMC by FACS and tested for function by co-culture with CFSE-labeled CD4+CD25− T responder cells (R). Transwell inserts and neutralizing IL-10 or TGFβ1 Ab were used to evaluate the potential suppressor mechanisms of Treg.

Results: Treg were enriched in TIL (13±3%) relative to PBMC (5±3%) (p ≤ 0.01). The mean % of Treg in NC was 2±1.5% (p ≤ 0.001). Treg in TIL were positive for GITR, IL-10 and TGFβ1, while Treg in PBMC up-regulated CD62L and CCR7 but not GITR, IL-10 or TGFβ1. Treg in TIL mediated stronger suppression (85±7%) of R than Treg in PBMC (20±5%) (p ≤ 0.001). Suppression mediated by Treg in TIL was decreased by transwell inserts (20±3% vs. 80±5%) and anti-IL-10 or TGFβ1 Abs (10±3%). In co-cultures, where the number of R exceeded the number of Treg, suppression was not blocked by the use of transwell inserts (58±1.2% vs. 68±5.13%), although the addition of anti- IL-10 or TGFβ1 Abs abrogated suppression (5±2.51%).

Conclusions: nTreg in the tumor suppress R by IL-10 and TGFβ1. Cell-to-cell contact is not needed, but optimizes suppression.
Regulation by SHPS-1 of α-GalCer-induced prevention of cancer metastasis (89.14)
Authors: Okajo Jun , Kaneko Yoriaki , Tomizawa Takeshi , Okuzawa Chie , Saito Yasuyuki , Sekigami Tomomi , Matozaki Takashi , Nojima Yoshihisa
Published: January 2023

[Background] α-GalCer activates NKT cells through the interaction between NKT cells and DCs. SHPS-1 is a transmembrane protein predominantly expressed on DCs. The functional roles of SHPS-1 in the regulation of NKT are investigated by the use of mice bearing mutant SHPS-1, which lack most of its cytoplasmic region.

[Methods] B16 melanoma cells were injected into WT or SHPS-1 mutant mice, and α-GalCer or the vehicle was administered. For adoptive transfer of DCs, α-GalCer pulsed DCs were prepared from spleens from WT or SHPS-1 mutant mice, and such cells were injected together with B16 melanoma cells. The mice were sacrificed and the number of tumor colonies in the lung was counted. Splenic or hepatic MNCs were cultured with α-GalCer, and the amounts of IFN-γ and IL-4 in culture supernatants were determined by ELISA.

[Results] The prevention of metastasis by α-GalCer was attenuated in the SHPS-1 mutant mice. The antimetastatic effect was induced in WT recipient mice by adoptive transfer of DCs from WT mice, while such effect by that of DCs from SHPS-1 mutant mice was reduced. The production of either INF-γ or IL-4 in response to α-GalCer was reduced in MNCs from SHPS-1 mutant mice.

[Conclusion] SHPS-1 on DCs is crucial for the α-GalCer-induced prevention of metastasis and Th1-and Th2-responses by NKT cells.
Dendritic Cells from Male and Female Rodents Respond Differentially to Progesterone (90.3)
Authors: Butts Cherie L , Belyavskaya Elena , Sternberg Esther M
Published: January 2023

Females show a 2- to 10-fold higher incidence of autoimmune diseases compared to males, indicating a role for sex hormones. In this study, we determined the role of progesterone (P) in modulating male and female rodent dendritic cell (DC) responses. We found differences both in male and female DC responses to P and differences in DC responses throughout the female estrus cycle. Male LPS-matured bone marrow-derived DCs (BMDCs) secreted higher levels of TNF-? and IL-10 compared to females; however P suppressed BMDC TNF-? and IL-12 secretion in females more than males. Reversal by the PR antagonist RU486 indicated mediation through PR. Females also showed greater progesterone downregulation of BMDC CD80 and MHC Class II expression than males. Female BMDCs differed in P responsiveness depending on the estrus cycle phase, with similar responses to males in proestrus and estrus but greater sensitivity to P during metaestrus and diestrus. This effect is consistent with increased levels of PR in BMDCs during these phases. Thus, depending on estrus cycle phase and gender, P differentially affects mature BMDC function. This could indicate that differences in susceptibility to infection and disease between males and females and at specific estrus cycle phases in females could be related to differential BMDC function.

This work was supported by the NIMH and NIAID Intramural Research Programs.
Regionalized combinatorial diversity provides an evolutionary link between innate and adaptive immunity (90.7)
Authors: Ostrov David A. , Hernández Prada Jose A. , Haire Robert , Cannon John , Litman Gary W.
Published: January 2023

Adaptive immunity emerged in jawed vertebrates exhibiting the ability to express somatically rearranged antigen receptor genes with combinatorial binding sites consisting of residues from each of the paired v-type (variable) Ig domains (i.e., VαVβ). The origins of contemporary antigen receptors have been described by a model in which an ancestral Ig gene diversified to form polymorphic variants consisting of an antigen binding domain linked to an effector module (i.e., VC). The high degree of clustering of polymorphic residues at the combining site between v-type Ig domains throughout evolution of adaptive immunity suggests that this feature confers a powerful advantage in creating a diverse repertoire of antigen receptors. To address the origins of adaptive immunity, we solved the crystal structure of VCBP3 D1 and D2 from the cephalochordate Branchiostoma floridae, which has characteristics of a primordial antigen receptor: a high degree of germline encoded polymorphism and paired v-type Ig domains with polymorphism clustered at the combining site. In addition to finding combinatorial recognition in a jawless invertebrate, we solved the structure of a primordial V domain to 1.15 Å, a level of refinement that permits new insight into the atomic basis for Ig domain organization and stability.
Dendritic cells pulsed with either secretory antigens of Mycobacterium tuberculosis or live mycobacteria show a differential expansion of Th1, Th2 and Th3 type T cells in immune mice (92.10)
Authors: Jagannath Chinnaswamy , Roche Cherie Michelle , Arora Ashish
Published: January 2023

Dendritic cells (DCs) can prime naïve or immune T cells and expand Th1, Th2 and Th3 type of T cells that determine protection against pathogens. While the Th1 immunity has been thought to be protective against tuberculosis, the ability of vaccines to induce Th1-Th3 immunity is unclear. Emerging tuberculosis vaccines include DNA vaccines encoding 85 complex (A, B, C), ESAT-6 and CFP-10 (ES antigens) or live attenuated Mycobacterium tuberculosis (MTB) in comparison with BCG vaccine. Interestingly, MTB H37Rv secretes all three ES antigens while BCG secretes Ag85 but lacks ESAT-6 and CFP-10. We therefore hypothesized that ES antigens and live bacteria may induce different types of T cell responses and the efficacy of vaccines delivering these would vary in mice.

METHODS: Bone marrow derived C57Bl/6 DCs were infected with live H37Rv or BCG or were pulsed with Ag85(ABC), ESAT-6 or CFP-10 antigens. After 24 h, they were cocultured with naïve T cells or immune T cells from heat killed MTB immunized mice. T cells were then stained for intracellular T-bet/IFN-γ, GATA3/IL-4 and Foxp3/IL-10 to determine the expansion of Th1, Th2 and Th3 response respectively.

RESULTS: ES antigens induced a strong Th1 response with very little Th2 or Th3 responses in immune T cells. Paradoxically, H37Rv and BCG induced a Th1 response as well as significant levels of Th2 and Th3 type of T cells.

CONCLUSIONS: We suggest that live mycobacterial vaccines concurrently induce deleterious Th2 and Th3 T cells during vaccination or infection that may affect the protective function of Th1 T cells.
Motif structure of minor histocompatibility antigen for MHC class II-restricted presentation (93.19)
Authors: Sahara Hiroeki , Imai Akihito , Mori Yoko , Mita Hiroaki , Tamura Yasuaki , Takahashi Nobuaki , Shastri Nilabh , Sato Noriyuki
Published: January 2023

Unlike major histocompatibility complex (MHC) class I, the processing pathway of endogenous MHC class II-restricted antigen has been poorly understood. We previously reported that endogenous MHC class II-restricted minor histocompatibility antigen that encoded murine H46 locus on chromosome 7 was IL-4-inducible gene 1 (IL4i1), which was continually expressed in bone marrow dendritic cells (BMDCs) (J.Exp.Med. 2003, 197: 375). In this study, we investigated the processing pathway of endogenous MHC class II-restricted IL4i1 antigen in BMDCs. IL4i1 antigen localized at RhoB-localized endosome, and MHC class II-restricted presentations were inhibited by endosomal vesicle trafficking inhibitor, such as bafilomycin. Whereas, lysosomal enzyme inhibitors, such as chloroquine, could not influence the presentations, suggesting that seem to be different from autophagy processing pathway. Next, we focused on the IL4i1 gene structure that does arrangement of tri-leucine-based motif in the downstream of tyrosine-based motif, designated YL motif. When YL motif was mutated, the antigen presentations were dramatically inhibited and this mutated-IL4i1 did not colocalize with RhoB-localized vesicle. These data suggested that YL motif would be a sorting signal for it where might occur antigen processing.
In the absence of IGF-1 signaling, IFN-gamma induces T cell apoptosis (94.14)
Authors: Novelli Francesco , Conti Laura , Regis Gabriella , Bernabei Paola , Chiarle Roberto , Giovarelli Mirella
Published: January 2023

IFNg exerts its biological activities by interacting with its cell surface receptor (IFNgR), which consists of two binding chains (R1) and two signal transducing chains (R2).The binding of IFNg to its receptor complex activates the JAK/STAT signal transduction pathway and induces the transcription of IFNg-inducible apoptotic genes. T cells that undergo Th1 polarization or malignant transformation become resistant to the antiproliferative effects of the IFNg/STAT1 pathway.This refractoriness is mainly due to R2 downregulation, due to ligand-independent internalization within clathrin-coated pits. We have previously demonstrated that, by inducing R2 internalization, IGF1 is a critical factors in limiting the IFNg/STAT1 pathway in T cells. Thus, the blockade of its signaling in T cells could hinder their intracellular R2 trafficking and reinstate their sensitivity to IFNg/STAT1 apoptotic signaling. To address this point, a retrovirus-based approach was exploited to stably express a dominant negative form of the IGF1R (IGF1R DN) in ST4 T cell line. Expression of IGF1R DN induced R2 cell surface accumulation and reinstated ST4 cell sensitivity to the IFNg/STAT1 apoptotic pathway in vitro and in vivo. Moreover, the combined exposure to an inhibitor of IGF1R tyrosine kinase activity (picropodophyllin, PPP) and IFNg induced STAT1-dependent apoptosis of three T cell lines (Jurkat ST4 and PF382).These results could be used in the elaboration of new therapeutic approaches based on IGF1 signaling blockade to overcome the resistance of malignant or autoreactive T cells to the apoptotic effect of IFNg.
Interleukin-4-mediated protection of primary B lymphocytes from apoptosis through Stat6-dependent up-regulation of glycolytic metabolism (94.16)
Authors: Gumina Maria , Bleiman Blair , Dufort Fay , Blair Derek , Chiles Thomas
Published: January 2023

Lymphocytes require extrinsic signals to maintain viability and this occurs, in part, through the up-regulation of anti-apoptotic proteins. Several recent studies have implicated cellular energy metabolism as an important component in preventing cell death following growth factor withdrawal. Interleukin 4 (IL-4) prevents death by neglect of naïve B lymphocytes; however, it is not known whether cellular glucose metabolism is crucial for survival by IL-4. We show herein that IL-4 triggers increased glucose uptake, glucose transporter (Glut1) expression, and glycolysis in splenic B lymphocytes. Inhibition of glycolysis results in apoptosis, despite the presence of IL-4. We demonstrate that insulin receptor substrate-2 and phosphatidylinositol 3-kinase activity are dispensable for IL-4-induced glucose utilization. By contrast, Stat6-deficient B cells exhibit impaired induction of Glut1 and glycolysis by IL-4. These findings establish that a key feature of IL-4 prevention of apoptosis in B cells is to promote glucose utilization via a Stat6-dependent pathway.

This work was supported by USPHS grant AI-49994.
Cholinergic agonists regulate monocyte chemoattractant protein-1 (MCP-1) production by activated endothelial cells: Involvement of the JAK/STAT3 pathway (94.29)
Authors: Chatterjee Prodyot , Sherry Barbara A , Metz Christine N
Published: January 2023

Numerous studies demonstrate the anti-inflammatory effects of cholinergic agonists, including nicotine. Investigations by our laboratory reveal that cholinergic stimulation inhibits endothelial cell activation, in part, through the NF-κB pathway. Monocyte chemoattractant protein-1 (MCP-1) is produced by endothelial cells and is a target of the JAK/STAT3 pathway. Human umbilical vein endothelial cells (HuVECs) were isolated and used to investigate the potential role of the JAK/STAT3 pathway in regulating MCP-1 production by activated endothelial cells following cholinergic stimulation. LPS induced MCP-1 expression in a dose-dependent manner. Soluble IL-6 receptor (IL-6sR), but not IL-6 itself induced significant MCP-1 expression. MCP-1 production following LPS or IL-6sR stimulation was mediated, in part, through the JAK/STAT3 pathway. Cholinergic agonists, nicotine and GTS-21, significantly reduced both LPS and IL-6sR-induced MCP-1 production. Cholinergic agonists also attenuated STAT3 phosphorylation following LPS and IL-6 stimulation. Finally, IL-6 blockade partially inhibits LPS-stimulated MCP-1 production and STAT3 phosphorylation. Together, our observations show that both LPS and IL-6sR induce MCP-1 production in endothelial cells, in part, through the JAK/STAT3 pathway that is inhibited by cholinergic mediators.

This work was supported by NIH NIGMS R01GM070727-02.
IL-2 inhibits its own production through a Blimp-1-dependent autoregulatory loop (94.4)
Authors: Gong Dapeng , Malek Thomas R.
Published: January 2023

IL-2 is essential for T cell expansion and effector cell development in vitro and for the development and maintenance of Treg cells in vivo. In our attempts to identify differentially expressed molecules dependent on IL-2 in activated T cells, DNA microarray analysis identified Blimp-1 as one such target. Blimp-1 is minimally expressed by naïve T cells, but is highly expressed by activated T cells. This expression mainly depends on IL-2, with lower Blimp-1 levels induced by IL-4 and IL-12. For activated T cells that expressed lower IL-2, Blimp-1 expression was higher, correlating Blimp-1 with the inability to properly produce lL-2. Activated T cells that were transduced with Blimp-1 produced lower IL-2 and inhibited an IL-2 reporter construct consisting of 8.4-kb upstream DNA sequence of the IL-2 gene, while granzyme B and CD25 expression were somewhat enhanced. These findings indicate that IL-2 inhibits its own production through a Blimp-1-dependent negative feedback loop while promoting a T effector cell phenotype. As Blimp-1-deficient T cells in vivo exhibit dysregulated T cell homeostasis and autoimmunity, our findings raise the possibility that improper regulation of IL-2 production may be a contributing factor to these pathological abnormalities.
Human Interferon Lambda-1 (IFN-λ1 / IL-29) modulates the Th1/Th2 response. (95.20)
Authors: Gallagher Grant , Jordan William , Eskdale Joyce , Srinivas Shekar , Pekarek Vera
Published: January 2023

Interferon lambda one ((IFN-λ1, IL-29) is a member of the Type-III interferon family, which contains three ligands: IFN-λ1, 2 and 3. These ligands use the same hererodimeric receptor, comprised of the CRF2-12 (IFN-λ1/IL-28Rα) chain and the CRF2-4 (IL-10Rβ) chains. Like their close relatives the Type-I interferons, IFN-λ1, 2 and 3 promote the phosphorylation of STATs 1&2, induce the ISRE3 complex, elevate OAS and MxA expression and exhibit anti-viral activity in vitro. Their use of the IL-10Rβ chain and their ability to phospohrylate STATs 3, 4 & 5 suggested that they might also exhibit immunoregulatory activity; their antiviral activity led us to hypothesize that this might be directed towards the Th1/Th2 system. Here, we demonstrate that IFN-λ1 altered the activity of human Th cells in three experimental systems: mitogen stimulation, MLR and the stimulation of naive T-cells by monocyte-derived dendritic cells(mDC). In general the inclusion of IFN-λ1 consistently led to a marked diminution of secreted IL-13 (15/16 donors) with occasional (5/16 donors) coincident, modest elevation of secreted IFN-γ. IL-13 secretion was 100-fold more sensitive to IFN-λ1 than was IFN-γ secretion. IFN-λ1 was able to alter Th-biasing and when naive T-cells were exposed to allogeneic mDC that had been developed in the presence of IFN-λ1, secreted IL-13 was again markedly and consistently reduced. These functions were independent of IL-10. Our data support a hitherto unsuspected role for IFN-λ1 in modulating the development of Th1 and Th2 cells, with an apparent emphasis on the diminution of IL-13 secretion in human in vitro cell culture.
Augmenting the biological activity of IL-15 with soluble IL-15Ralpha (95.21)
Authors: Rubinstein Mark P , Surh Charlie D , Sprent Jonathan , Goldrath Ananda W
Published: January 2023

IL-15 is thought to be normally presented in vivo as a cell-associated cytokine bound to IL-15Rα. We show here that the biological activity of soluble IL-15 is much improved after interaction with recombinant soluble IL-15Rα; after injection, soluble IL-15/IL-15Rα complexes rapidly induce strong and selective expansion of memory CD8+ cells and natural killer cells. Surprisingly, we also find that sIL-15/IL-15Rα complexes can drive conversion of naive T cells to memory T cells in vivo, without administration of antigen. This conversion is associated with an acquisition of effector function. The enhancing effect of IL-15Rα binding may explain why IL-15 normally functions as a cell-associated cytokine. Our results suggest the biological activity of IL-15 as a therapeutic reagent could be considerably enhanced by administration of preformed soluble IL-15/IL-15Rα complexes.