bluuebunny/crossref_metadata_2025 huggingface dataset:
A molecular mechanism for the increased degradation of E47 mRNA in old murine B cells involves TTP (tristetraprolin) (83.5)
Authors: frasca daniela , landin ana marie , alvarez juan p , riley richard l , blomberg bonnie b
Published: January 2023

We have previously shown that the E2A-encoded transcription factor E47, which regulates class switch recombination (CSR) in splenic B cells, is down-regulated in aged murine B cells, due to increased E47 mRNA decay. At least part of the decreased stability of E47 mRNA seen in aged B cells is mediated by cytoplasmic proteins increased in aged activated B cells. This is the first study to show that TTP is involved in the degradation of E47 mRNA TTP binds AU-rich elements in the 3′-untranslated region (3′-UTR) of mRNAs leading to their rapid degradation. We show herein that TTP mRNA and protein expression are induced by LPS, anti-CD40 and to a lesser extent by anti-IgM, in B cells from young and old mice. The level of TTP in old B cells is always higher than that in young B cells. We found TTP protein expression and function in B cells are dependent upon p38 MAPK and there is less phospho-p38 in old activated B cells. Also there is less phospho-TTP in old than in young splenic activated B cells, which leads to more binding of TTP to the 3′-UTR, therefore decreasing mRNA stability in old B cells. These molecular mechanisms for the regulation of E47 in B cells are important for understanding the control of AID, CSR, and somatic hypermutation of Ig, critical for high affinity Ig and optimal response to pathogens.
HEBAlt: a potent transcriptional regulator of proliferation in developing B cell precursors (83.8)
Authors: Anderson Michele Kay , Wang Duncheng , Claus Carol L.
Published: January 2023

The basic helix-loop-helix transcription (bHLH) factor E2A is essential for B cell development, and can act as a homodimer or as a heterodimer with the closely related bHLH factor HEB. We have identified a short form of HEB called HEBAlt, which differs from the long form of HEB in its structure, function, and regulation, and which is conserved in vertebrates, including humans. Within hematopoiesis, HEBAlt is expressed only in developing T cell and B cell precursors. HEBAlt is strongly upregulated in pre-B cells, but is absent in IgM+ B cells. Retroviral expression of HEBAlt in multipotent hematopoietic precursors partially blocks B cell development. However, committed B cell precursors expressing ectopic HEBAlt undergo rapid expansion and exhibit extended survival in culture. HEBAlt-induced expansion arises from a limited number of precursors, indicating that secondary events collaborate with HEBAlt to cause loss of growth control. IL-7R and cyclin D3 are upregulated in HEBAlt-transduced cells, suggesting that HEBAlt normally functions to promote proliferation of early B cell precursors. Furthermore, downregulation of HEBAlt may be required for quiescence during Ig light chain rearrangement. These results suggest that dysregulation of HEBAlt could play an initiating role in the oncogenic transformation of B cell precursors.
A critical role for DRAK2 in the germinal center reaction and the antibody response (84.1)
Authors: Al-Qahtani Ahmed , Xu Zhenming , Walsh Craig , Casali Paolo
Published: January 2023

DRAK2 (DAP-related apoptotic kinase-2) protein is a DAP family member. DRAK2 is highly expressed in B and T lymphocytes and it is among the few proteins that are highly enriched in the lymphoid system. To determine whether DRAK2 plays a role in B cell activation and differentiation, we analyzed the germinal center development in the spleen of drak2−/− mice immunized with a T cell-dependent (TD) antigen. Immunofluorescence microscopy and FACS analysis showed the number of germinal centers was three to five-folds less in the drak2−/− mice than in their wild type littermates. Impairment of germinal center development was not due to a deficiency in B cell proliferation, as BrdU uptake in vivo and CFSE staining ex vivo of B cells was comparable in DRAK2-deficient and wild type B cells. 7-AAD and TUNEL staining experiments showed that, the proportion of apoptotic germinal center B and T cells in drak2−/− mice was significantly higher than wild type mice. As a result, the generation of high affinity IgG antibodies was impaired in drak2−/− mice. Specific expression of bcl-xl in T cells of drak2−/− mice rescued germinal center formation. Thus, our findings suggest a novel role of DRAK2 in the germinal center reaction and the maturation of the antibody response.

Supported by N.I.H. grants AR 40908, AI 45011 and AI 60573.
Calcitonin Gene-Related Peptide (CGRP) Inhibits Apoptosis by Differential Phosphorylation of BAD Serines 112, 136 and 155 (84.12)
Authors: Ciornei Radu Tudor , McGillis Joseph P
Published: January 2023

Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that inhibits early B cell differentiation. It enhances B cell survival by upregulating Pim-1, a S/T kinase that regulates Bcl-2 expression, and by shifting expression of Bcl-2 family members to anti-apoptotic members. Studies were done to further characterize CGRP’s effect on Bad phoshorylation. Inactive Bad (a pro-apoptotic Bcl-2 family member) is phosphorylated at ser112, ser136, and ser155 by different mechanisms. Bad ser112is a Pim-1 target. Dephosphorylated Bad forms heterodimers with Bcl-XL and Bcl-2, blocking their anti-apoptotic effects. Studies were done to determine if CGRP influences Bad phosphorylation. 70Z/3 cells were grown in low serum and treated with CGRP. Bad phosphorylation at ser112, ser136 and ser155 was measured by western blot. Serum starvation decreased Bad phosphorylation at 4 hrs and completely depletes phospho-Bad at 24 hours. CGRP upregulated phospho-ser112 and -ser155, peaking at 8–12 hrs and returning to untreated levels at 24 hrs. The results show that CGRP induced Bad phosphorylation at ser112 and ser155 is correlated with increased Pim-1 expression. In contrast, CGRP had no effect on ser136phosphorylation. This study provides further information on cellular and molecular mechanisms underlying CGRP’s protective effect in immature B cells.
A study of the signaling pathway and effector mechanisms of TNF-induced programmed necrosis (84.13)
Authors: Ray Tathagat Dutta , Chan Francis
Published: January 2023

TNF-induced programmed necrosis is an alternate form of cell death that occurs when caspase activation is inhibited. In this study, we have examined the signaling complexes formed during programmed necrosis in jurkat cells. We show that RIP is recruited to the membrane complex with similar kinetics in cells undergoing programmed necrosis and apoptosis. However, ubiquitination of RIP is diminished in programmed necrosis in comparison to apoptosis. We also show that caspase8 dependent cleavage of RIP within the cytosolic complex is inhibited during programmed necrosis. This inhibition of RIP cleavage is crucial for it’s kinase activity, which is essential for the induction of programmed necrosis. We also show that unlike necrosis induced by oxidative stress and macromolecular synthesis inhibitors in fibroblasts, TNF-induced programmed necrosis does not require either ROS or Jnk activation. This shows that the process of programmed necrosis can be mediated by distinct effector mechanisms in different cell types. Our study provides some important insights into the process of TNF-induced programmed necrosis.
Pax5 acts as a negative regulator for p53 (84.2)
Authors: Zhang Zhixin , Park Seayoung , Zhou Jianping , Chen Xinbin , Yu Zhihong
Published: January 2023

Pax5/BSAP is an important regulator for B lineage cell development and function. Pax5 activates many B lineage specific genes expression, suppresses lineage non-appropriate genes expression, and controls B lineage specific immunoglobulin gene VH→DJH recombination. On the other hand, abnormal Pax5 expression has been found in different type of tumors, suggesting that Pax5 might have oncogenic potential. It has been previously shown that Pax5 interferes with p53 function through inhibition p53 gene transcription. Here, we show that Pax5-inhibits p53-meidated induction of different p53-responsive reporter genes without affecting the p53 protein expression level; conversely, forced expression of p53 or activation of p53 by DNA damaging response also suppresses Pax5-mediated transcriptional activation. Co-immunoprecipitation studies provide the evidence that Pax5 interacts with p53, which is essential for the mutual functional interference between these two factors. Moreover, overexpression of Pax5 attenuates DNA damage induced p53 Ser-18 phosphorylation and inhibits p53-medaited induction of downstream target genes, including p21, Bax, Puma, and Gadd45α. Through interfering p53 function, overexpression of Pax5 protects cells from DNA damage induced apoptosis. These results reveal that Pax5 acts as a negative regulator for p53, which might be essential for the dual roles of Pax5 in B lymphopoiesis and tumorigenesis.
The function of the putative tumor suppressor Nit1 in lymphocytes (84.7)
Authors: Zhang Jianke , Zhang Haibing
Published: January 2023

The mammalian Nit1 gene is homologous to bacterial and plant nitrilases. In the fruit fly D. melanogaster and warms C. elegans, Nit1 is fused to the Fhit gene, whereas in mammals, FHIT and NIT1 are separate genes. FHIT is a tumor suppressor gene located at chromosome region 3p14.2, encompassing the common fragile site of the human genome. Somatic loss of Fhit in humans is associated with cancers in a wide variety of sites, including lung, kidney, stomach, pancreas, cervix, ovary, head and neck, breast, and hematopoetic cells. Proteins that engage in fusion events are expected to jointly participate in a biochemical or cellular pathway and/or to physically interact. Therefore, Nit1 may be functionally related to Fhit. Overexpression of Fhit and Nit1 can induce apoptosis. There was evidence showing that Fhit can induce FADD-mediated cell death. In this study, the in vivo function of Nit1 was analyzed using the Nit1 knockout mice. Various in vitro and in vivo analyses showed that Nit1 plays an important role in regulating cell cycle and proliferation in T lymphocytes.
Aged mice demonstrate altered regulation of distinct B cell developmental pathways (85.10)
Authors: Wolf Sarah , Blomberg Bonnie , Riley Richard
Published: January 2023

Three precursor subsets in bone marrow that generate new B cells were identified in young and aged (20–22 mo.) adult mice. The major subset (c-kit neg) is reduced in aged mice while minor c-kit+ and B1 lineage precursors are better maintained. In vitro, the c-kit+ and B1 precursors more readily generate B cells that undergo spontaneous activation. We suggest that altered levels of these precursors contribute to changes in B cell production and bias to activated phenotypes in aged mice. The “aged phenotype” of few c-kit neg, but maintained c-kit+/B1 precursors, is mimicked in young adult lambda 5 gene knockout mice as are the alterations in new B cell development. Therefore, the preBCR promotes development of c-kit neg B cell precursors, but is not necessary for c-kit+ and B1 precursors. Lambda 5 is decreased in aged B cell precursors, together with E2A. This suggests the hypothesis that, in aged mice, poor preBCR expression interferes with development of c-kit neg B precursors, but not c-kit+/B1 precursors. This, in turn, contributes to changes in the number, specificity, and function of new B cells generated in senescent bone marrow.

Supported by NIH grants AI 064591 and AG 025256 to RLR
Involvement of immunosenescence in the frailty syndrome (85.13)
Authors: morrhaye gabriel , Bruck France , de Saint-Hubert Marie , Weemaels Geoffroy , Swine Christian , Toussaint Olivier , Geenen Vincent , Martens Henri
Published: January 2023

A determinant factor of immunosenescence is the involution of the T-cell compartment, due to a decline in thymic generation of a diverse T-cell repertoire, as well as in peripheral T-cell homeostasis.

Human immunosenescence was investigated through measurement of telomere length by Q-PCR, evaluation of thymic output by Sj and VβTREC methodology, analysis of T-cell phenotype profile, and multiplex cytokine production after polyclonal stimulation. The diseases scrutinized in the elders were pneumonia, heart failure and hip fracture.

TREC value was negatively correlated with age, but no significant relationship could be evidenced with pathological states. A significant rise in pro-inflammatory cytokines IL-6, IFNγ and TNFα was observed in elderly vs. young controls, while IL-10 was significantly decreased. The cytokine profiles as analyzed by protein array showed at least three different patterns: either increased in elderly vs. young and decreased in pathologic states, either increasing or showing no modifications in all cases.

A significant shortening of telomere length in the elderly was observed when compared to young controls. There was a significant decrease between healthy and sick elders.

Significant correlations were evidenced between morbidity and some immune parameters, supporting that immunosenescence contributes to the frailty syndrome.

Supported by the Walloon Region - Senegene project
Recall immune memory in old baboons and mice: implications for vaccine design (85.18)
Authors: Stacy Sue , Dube Peter H. , Kraig Ellen
Published: January 2023

While primary antibody responses are often lower in old individuals, immune memory can survive aging and be vigorously recalled. We assessed whether these long-lived responses might impact vaccine antibody responses in the elderly. Three groups of baboons were tested: young controls, old animals inoculated with tetanus vaccine when young, and old baboons not previously exposed to tetanus. Each group received either a chimeric vaccine, TTFC (tetanus toxin fragment C, the recall antigen) linked to LcrV (a protective plague antigen) or LcrV alone. Among old baboons receiving the chimeric protein, those with previous exposure to tetanus had lower anti-LcrV antibody responses. Unexpectedly, old baboons receiving LcrV only (no tetanus exposure) had higher antibody responses and protective indices than the young; thus, the aging baboon immune system may be atypical. By contrast, aging mice responded as expected; the old mice had lower responses to LcrV compared with young mice. In recall tests, among old mice inoculated LcrV-TTFC, those with previous TTFC exposure had altered anti-LcrV antibody titers; the response was depressed or enhanced depending upon the dose of chimera given. Therefore, caution may be warranted when using subunit vaccines with components that recall previous exposures.

Support: the Nathan Shock Center, a Southwest National Primate Research Center pilot grant, a UTHSCSA PREF grant, and NIH [R03 AG 14557 (EK) and R03 AG 22675 (SS)].
Aged Micro-environment Severely Impairs Specific CD8 T Cell Response to Virus Infection (85.21)
Authors: Jiang Jiu , Bennett Andrew J. , Murasko Donna M.
Published: January 2023

Both intrinsinc and extrinsinc factors are considered to affect immunity with aging. However, few studies have reported that the aged micro-environment affects the specific CD8 T cell response to primary virus infection. In this study, we adoptively transferred CFSE-labeled young P14 cells (2-month-old) into young (4-month-old) and aged (24-month-old) mice. The recipients were infected with LCMV (Arm strain) and the Db-GP33–41 specific CD8 T cell response in the spleen was examined for virus specific responses using flow cytometric analysis of Db-GP33–41 tetramer binding and intracellular IFN-γ production at different times after infection. No obvious expansion of the P14 cells was observed on days 1 and 2 after infection in either age group. However, on day 3 post-infection, extensive expansion of P14 cells was observed in young, but not aged, recipients (increased percentage: young vs aged: 275% vs 18%). Coincidentally, 68% percent of CD8 T cells of young recipients were depleted, while no CD8 T cells of aged recipients were depleted on day 3. These results confirm our previous data with E55+MuLV infection, that non-specific depletion of CD8 T cells occurs early after infection in young but not aged mice, and support the hypothesis that this lack of depletion contributes to the hyporesponsiveness of aged mice. While our previous data demonstrated that the aged environment limits the depletion of T cells of young mice, the current data expands these findings by suggesting that the aged environment also limits expansion of virus-specific T cells of young mice.

This work was supported by National Institute of Health Grant AG14913 to D. M. M.
Cytokine adjuvants enhance the cognate helper function of aged CD4 T cells (85.22)
Authors: Eaton Sheri M , Maue Alexander C , Blumerman Seth L , Haynes Laura
Published: January 2023

The elderly exhibit reduced responses to vaccinations, leaving them more susceptible to preventable infectious diseases such as influenza and pneumonia. When the aged are vaccinated, the resulting antibody titers are lower than those seen in younger individuals and the function of the antibodies produced is also diminished. We have examined how reductions in CD4 T cell function contribute to reduced humoral responses, using an adoptive transfer model which allows us to compare identical numbers of antigen-specific naive T cells from young and aged TCR transgenic (Tg) mice. Naive CD4 T cells from aged Tg mice exhibit significantly reduced cognate helper function, leading to reduced B cell expansion and differentiation and reduced antibody production, even in young hosts. The cognate helper function of these aged Tg CD4 T cells can be enhanced significantly by prior effector generation with a cytokine cocktail containing TNFalpha, IL-1 and IL-6 (T16) or by using this cocktail as an adjuvant for immunization. Using this cocktail, enhanced B cell responses, including significantly higher antibody titers, are observed. Gene expression analysis shows that T16 enhances CD40L expression by the aged CD4 T cells, possibly accounting for the enhanced cognate function. Importantly, this cocktail also enhances humoral responses in intact aged animals. These results suggest that the efficacy of vaccines for the elderly can be enhanced by use of appropriate adjuvants.

Supported by NIH grants AG21054 and AG02160 to LH.
The effect of aging on CD4+ Foxp3+ T cells in humans (85.23)
Authors: Kang Insoo , Kim Hang-Rae , Hwang Kyung-A
Published: January 2023

Foxp3 is critical for regulatory-T cell function. Although alterations in immunity occur with aging, little is known how aging affects the number, phenotypes and function of CD4+ Foxp3+ T cells in humans. Here, we report that CD4+ Foxp3+ T cells are largely CD4+ CD25bright T cells in healthy elderly (age¡Ã65) and young individuals (age¡Â40) and that the two groups have comparable frequencies of CD4+ Foxp3+ T cells. Although CD4+ Foxp3+ T cells mostly have a memory phenotype (CD45RA-) in both groups, the frequency of such cells was higher in the elderly. However, CD4+ Foxp3+ T cells from the two groups similarly express CTLA-4 which has immunosuppressive function as well as receptors (R) for chemokines (CCR4, CCR5, CXCR3 and CCR7) and cytokines (IL-15Ra, IL-2/-15Rb and IL-7Ra) which are involved in T cell trafficking and maintenance, respectively. More importantly, the inhibitory effect of CD4+ CD25bright T cells expressing Foxp3+ on CD4+ CD25− T cells was not different between the two groups as measured by cell proliferation, IFN-g and IL-10 production. Our study indicates that aging does not affect CD4+ T cell immune regulation by CD4+ Foxp3+ T cells and that therapeutic strategies targeting the latter cells can be considered regardless of age.
Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune uveoretinitis (EAU) and recruitment of pathogenic ThIL17 and Th1 cells into the retina (85.29)
Authors: Liu Xuebin , Lee Yunsang , Yu Cheng-rong , Silver Phyllis , Caspi Rachel , Igal Gery , Charles Egwuagu E
Published: January 2023

ThIL17 cells are implicated in etiology of several inflammatory and autoimmune diseases. Although recent reports have emphasized essential roles of IL-6 and TGF-β1 in promoting commitment of naive CD4+ cells into ThIL17 lineage, their exact roles or contributions to development of ThIL17 cells is not well understood. IL-6 is a pleiotropic cytokine that mediates some of its biological activities through STAT3. In this study we have used a mouse with targeted deletion of STAT3 in CD4+ cells to investigate whether ThIL17 development or their involvement in pathogenic mechanisms of experimental autoimmune uveoretinitis (EAU) requires STAT3. In wild type (WT) mice, EAU is characterized by infiltration into the retina of ThIL17 and Th1 cells that express high levels of VLA-4 and LFA-1. In contrast, STAT3 conditional knockout (STAT3KO) mice do not develop EAU, fail to differentiate into ThIL17 cells, express low levels of VLA-4/LFA-1 integrins and do not enter the eye. Moreover, TGF-β1 and IL-6 or IL-23 are unable to induce naive STAT3KO T cells to differentiate into ThIL17 cells, providing further evidence that STAT3 is indispensable for commitment to the ThIL17 lineage. The requirement of STAT3 for integrin expression and the defective recruitment of IRBP-primed STAT3KO Th1 and ThIL17 cells into the eye during EAU, suggest that STAT3 is a potential target for therapeutic modulation of uveitis and other CNS inflammatory diseases.
Functional Significance of PD-1 expression on aging T cells (85.4)
Authors: Suvas Susmit , Joo Hye Mee , Sehrawat Sharvan , Suvas Pratima Krishna , Sangster Mark Y , Yagita Hideo , Azuma Miyuki , Rouse Barry T
Published: January 2023

Increasing age is associated with the lesser ability of T and B cells to mount an effective immune response against the microbial antigens. Numerous reports have documented the malfunctioning of the different components of immune system with aging but a more detailed mechanistic understanding of the age associated immunological changes in the elderly individuals is needed to develop better vaccines specific for this population. In this study, we observed that PD-1, a key inhibitory receptor in chronic viral infections, was highly expressed on T cells from uninfected aged but not young mice. Next, we determined if PD-1 expression on T cells regulate adaptive immune responses in aged animals. Young and old groups of mice were treated with anti-PD1 or anti-PDL1 mAbs prior to HSV-1 or influenza A virus infection. Our results showed a significant increase in the number of HSV-1 or Influenza specific CD8+ T cells during acute and secondary expansion of memory CD8+ T cells in the aged mice group when compared with control infected aged animals. Furthermore, a significant increase in the number of flu-specific ASCs (antibody secreting cells) and memory B cells was clearly evident in the spleen of antibody treated aged animals in comparison to the control infected aged mice group.

Supported by NIAID-AI-063365 grant
Endogenous naive CD8 T cell precursor frequency and infection type control CD62L gene expression (85.6)
Authors: Obar Joshua , Khanna Kamal M. , Pham Quynh-Mai , Lefrancois Leo
Published: January 2023

CD62L is essential for homing of lymphocytes to lymph nodes. Moreover, CD62L expression identifies distinct lineages of memory CD8 T cells. However, the factors regulating memory cell lineage differentiation are ill defined. Our data show that CD62L gene regulation is controlled by the input number of transferred antigen-specific CD8 T cells. To enable analysis of endogenous antigen-specific CD8 T cells, we have now utilized a method to enrich naïve antigen-specific CD8 T cells using MHC class I tetramers in conjunction with magnetic bead separation. Of three specificities tested thus far, naïve precursor frequencies ranged from ~100–1000 cells/mouse. Interestingly, following infection with recombinant vesicular stomatitis viruses, the naïve precursor frequency inversely correlated with the time required for the resulting memory population to shift to CD62L+ cells. Remarkably, in the case of ova-specific CD8 T cells, the shift toward CD62L+ ova-specific memory cells occurred substantially faster following infection with Listeria monocytogenes expressing ovalbumin than after VSV-ova infection. This system will allow us to examine parameters of the endogenous CD8 T cell response to infection from initial activation to memory development.

This work was supported by NIH grants AI41576 and DK45260 (LL) and a Damon Runyon Foundation fellowship (KMK).
Memory B Cells Assort by Phenotype, BCR Repertoire & Genotype (86.1)
Authors: McHeyzer-Williams Louise , O’Connor Adam P , McHeyzer-Williams Michael G
Published: January 2023

Effective vaccination requires the generation of high-affinity B cell memory. After immunization with T-dependent antigens, multiple cellular subsets of antigen-specific memory B cells develop. Specifically, following the germinal center (GC) reaction, three B cell subsets emerge: two non-secreting distinct subsets of memory B cells are apparent (6B2hi post-GC and 6B2lo pre-plasma memory cells) as well as secreting CD138pos plasma cells. All three B cell compartments express isotype switch, affinity matured antigen-binding antibody. In the current study, we focus on the IgG1+ memory B cell response to antigen recall in vivo to deepen the molecular and genetic analysis of the two memory subsets and their short-lived plasma cell progeny. Single cell RT-PCR repertoire analysis indicates a majority of VH186.2 variable region gene usage among all three NP-specific memory responders with distinguisable groups of clonotypes based on junctional and somatic variations in the BCR. Notably, BCR associated with the highest affinity for antigen preferentially assort into the 6B2lo pre-plasma memory compartment. Based on gene expression profile and Q-PCR analysis the two memory B cell subsets express unique developmental programs that control subsequent cell fate and impact memory B cell function upon antigen recall.
The role of c-Rel in EBV-mediated B cell survival (86.10)
Authors: Valentin Anibal Jose , Lu Kristina T. , Covey Lori R.
Published: January 2023

LMP1 is an Epstein-Barr virus encoded protein that is expressed on the surface of latently infected cells and is known to promote cell survival through activation of the NFκB pathway. Previously our lab developed an immortalized B cell line from a patient with non-X-linked HIGM in which LMP1 is conditionally expressed under the control of a tetracycline-inducible promoter (pt1-LCLtet cells). Analysis of these cells revealed an increased level of apoptosis even in the continuous presence of LMP1 stimulation. In addition, analysis of NFκB/Rel subunits showed a markedly lower expression of c-Rel (c-Rel75) and the presence of a novel 25 kD form (c-Rel25) in pt1but not control-LCLtetcells. Western analysis of immunoprecipitated c-Rel revealed several higher mw forms in pt1- vs. control-LCLtet cells. To investigate the phosphorylation status of c-Rel75 in the pt1-LCLtet cells, in-vivo labeling was carried out and immunoprecipitation of extracts with anti-c-Rel antibodies revealed that c-Rel75 is hyperphosphorylated in these cells. Therefore, we hypothesize that hyperphosphorylation of c-Rel75 targets a post-translational processing event that results in both the reduced level of c-Rel75 and the appearance of the 25 kD form observed in pt1-LCLtet cells. Importantly, the low abundance of c-Rel75 appears to be closely linked to ongoing apoptosis which is not inhibited by LMP1 signaling.

(Supported by AI37081)
The Role of STAT-3 and LIF in regulating IgM secretion in B-1 cells: CD25+ B-1 cells and their characteristics (86.12)
Authors: Tumang Joseph R , Holodick Nichol , Kaku Hiroaki , Rothstein Thomas L
Published: January 2023

B-1 cells spontaneously secrete natural Ig that acts as a primary line of defense against infection. A major shortfall in our understanding of this key process centers on the molecular mechanisms regulating natural Ab secretion by B-1 cells. Herein, we identify a novel subset of B-1 cells that express the high affinity IL-2 receptor (IL-2Ra/CD25) and preferentially have the activated form of the signal transducer and activator of transcription-3 (STAT3). We show that upon inhibition of pSTAT3, Ig secretion is elevated preferentially in the CD25+ subset. Consistent with the induction of Ig secretion, we detected increases in the expression of key regulators of the plasmacytic program, B lymphocyte inducer of maturation program 1 (BLIMP-1) and X-box binding protein 1 (XBP-1). In contrast to the CD25- subset of B-1, we also found that the CD25+ subset differentially expresses a number of genes including the STAT3 target SOCS3, the cytokine receptor GP130 and the leukemia inhibitory factor receptor (LIF-R). We demonstrate that treatment of B-1 cells with LIF induces Ig secretion preferentially in the CD25+ subset. Together, these data identify STAT3 and LIF as possible regulators of Ig secretion in B-1 cells. Furthermore, these results implicate a role for cytokines in regulating B-1 Ig secretion.
Differential regulation of NF-κB survival signaling in transitional and mature Fo B cells (86.13)
Authors: Castro Iris , Hoek Kristen L. , Shinners Nicholas P. , Khan Wasif N.
Published: January 2023

The earliest transitional (T1) B cells are significantly more sensitive to cell death than the late transitional (T2) and mature follicular (Fo) B cells and aberrant survival of T1 B cells can lead to autoimmune disease. While it is known that survival of splenic B cells is predominantly regulated by B cell antigen receptor (BCR) and B cell activation factor receptor (BAFF-R), the mechanisms by which these receptors mediate survival is less clear. We have investigated the developmental stage-specific regulation of anti-apoptotic genes and the transcription factor NF-κB, which is a major modulator of these genes. We found that in vivo, T1 B cells express the highest levels of Mcl-1 and Bcl-xL, whereas mature Fo B cells predominantly express Bcl-2. Consistent with T1 cell sensitivity to cell death following BCR crosslinking, T1 B cells only weakly induced the expression of anti-apoptotic genes (Mcl-1 and Bcl-xL) relative to robust induction in T2 and mature Fo B cells following BCR stimulation. BCR-induced expression of NF-κB (NF-κB1, RelA, cRel, NF-κB2 and RelB) coincides with that of anti-apoptotic genes; lowest in T1 and highest in mature Fo B cells. These findings suggest that T2 and mature Fo B cell survival may be linked to sustained expression of NF-κB and anti-apoptotic genes.

This study is supported by NIH RO1 A1060729
Gene expression in Blimp-1-transduced fibroblasts (86.16)
Authors: Buldak Gerald L , Frank Matthew , Jackowski Suzanne , Brewer Joseph W.
Published: January 2023

B-lymphocyte-induced maturation protein-1 (Blimp-1) is an important regulator in the development of several immune cell lineages, as well as embryogenesis, where it is expressed in all three primordial germ layers. The best understanding of mechanistic regulation by Blimp-1 comes from B cells, in which it is a master regulator of terminal differentiation into professional secretory plasma cells (PC). Blimp-1 expression results in the induction of another master regulator called X-box-binding protein 1, or XBP-1. Both genes are critical for PC differentiation; it is difficult, however, to identify genes regulated by Blimp-1 independently of XBP-1. In other cell types, knowledge is limited to either the requirement, or temporal and spatial localization, of Blimp-1 expression.

To better understand the genes and pathways regulated by Blimp-1, we have developed a reductionist system to investigate its effects in NIH-3T3 fibroblasts. Blimp-1-expressing fibroblasts are viable to at least 48 hours, exhibit morphological changes, and altered gene expression profiles, without inducing XBP-1. Therefore, this system can be used to analyze XBP-1-independent events downstream of Blimp

J.W.B. is supported by NIH-GM61970, S.J by NIH-GM45737, and G.L.B by NIH-T32 AI007508.
SOCS3 regulates B lymphopoiesis at the immature B cell stage (86.4)
Authors: Le Yi , Zhu Bing-Mei , Hennighausen Lothar , Silberstein Leslie E
Published: January 2023

CXCL12-mediated FAK phosporylation and sustained pro-adhesive responses occur uniquely in progenitor bone marrow (BM) B cells, but not in mature peripheral B cells. Since CXCR4 receptor expression is similar at all B cell stages, we hypothesized that progenitor and mature B cells possess different mechanisms to control FAK phosphorylation. Here we demonstrate that Suppressor of Cytokine Signaling 3 (SOCS3) regulates CXCL12-induced FAK phosphorylation through the ubiquitin-proteosome pathway. CXCL12 triggers increased FAK ubiquitination in peripheral B cells and spleen B cells, but not in BM B cells. In accordance with this finding, SOCS3 expression is relatively low in progenitor BM B cells and high in peripheral blood and spleen B cells. Over-expression of SOCS3 results in decreased CXCL12-induced FAK phosphorylation and impaired adhesion to VCAM1. Next, we examined B lineage cells from MMTV-Cre SOCS3fl/flmice in which SOCS3 protein expression was reduced greater than 95%. SOCS3 deficient splenic B cells have increased and prolonged FAK phosphorylation and enhanced sustained adhesion to VCAM-1, similar to progenitor B cells. MMTV-Cre SOCS3fl/flmice have a two-fold increase in the absolute number of IgM+,IgD−, immature B cells in BM compared to wild type mice (n=8, p<0.01). IL-7 dependent pre-B cell proliferation was unaffected by SOCS3 deficiency. However, withdrawal of IL-7 induced the generation of immature B cells that survived longer in culture compared with wild type cells. Taken together, our results suggest that SOCS3 may be an important negative regulator of immature B cells by affecting their survival and pro-adhesive responses in the bone marrow microenvironment.
THE HEAVY METAL MERCURY MODULATES p-ERK1/2 IN ACTIVATED B-CELLS (86.7)
Authors: Laiosa Michael , Rosenspire Allen , McCabe Michael
Published: January 2023

The heavy metal mercury (Hg) is known to have immunomodulatory properties affecting lymphocyte signal transduction, death receptor signaling and autoimmunity. We tested the hypothesis that Hg exposure modulates B-cell activation in response to BCR stimulation. To test this hypothesis; WEHI-231 immature B-cells, were stimulated with Goat-anti-IgM in the presence or absence of 5 μM HgCl2. We found a significant reduction in p-ERK1/2 in the presence of Hg by Western blot. However, it is unclear whether the reduction of BCR dependent pERK is a consequence of Hg modulating the amount of p-ERK1/2 in each cell, or whether Hg is reducing the number of responding cells in the population. Single cell proteomics were employed to address whether pERK1/2 is modulated on a per-cell-basis using a ERK phosphospecific fluorochrome labeled antibody. We found nearly 80% of the WEHI cells, possess pERK1/2 by 5 minutes after stimulation. Interestingly, pre-incubation with HgCl2 causes a 25% decrease in the percentage of responding cells after IgM stimulation. Moreover, the MFI of pERK1/2, is reduced by more than 30% following exposure to HgCl2. Additional studies in primary mouse B-cells have found Hg also modulates phosphorylation of the proximal BCR kinase Syk. Taken together these data suggest Hg modulates BCR signal strength which may have profound implications for establishment of self-tolerance within the B-cell repertoire.
FcγRIIB1 regulates plasma cell apoptosis through a c-Abl-dependent pathway (86.8)
Authors: Tzeng Shiang-Jong , Bolland Silvia , Bonvini Ezio , Pierce Susan
Published: January 2023

In B cells the FcγRIIB1 blocks BCR signaling when both receptors are coengaged by binding to immune complexes. Inhibition is mediated by the recruitment of SHIP to the FcγRIIB1’s ITIM motif that has been phosphorylated by Lyn. In contrast, FcγRIIB1 regulates B cell apoptosis when clustering without BCR engagement. We show that the FcγRIIB1 triggers apoptosis by a mechanism that is distinct from the mechanism by which the FcγRIIB1 inhibits BCR signaling. Following clustering the FcγRIIB1 is tyrosine phosphorylated in an ITIM- and SHIP-independent fashion. The phosphorylation of the FcγRIIB1 and the apoptosis mediated by FcγRIIB1 are abolished in c-Abl and Arg double knockout cells. We then determined that long lived plasma cells that express FcγRIIB1 but not BCR and memory B cells when stimulated by a BCR-independent, TLR-dependent pathway are likely the targets of FcγRIIB1-mediated apoptosis. In both naïve and immunized mice, FcγRIIB1 clustering on bone marrow CD138+ plasma cells supported by TLR stimuli, e.g. CpG, resulted in a decrease in the number of Ig-secreting cells and an increase in the number of cells undergoing apoptosis. In human CD27+ memory B cells we found that FcγRIIB1 clustering blocked the CpG-induced differentiation to plasma cells. Taken together, our findings provide strong evidence that FcγRIIB1 serves as a peripheral checkpoint to maintain tolerance by regulating both apoptosis and differentiation of plasma cells.
Differential Regulation of SOCS Gene Expression by IL-27 in Astrocytes and CD4+ T Cells (87.11)
Authors: Qin Hongwei , Cong Yingzi , Roberts kevin L. , Baker Brandi J. , Elson Charles O. , Benveniste Etty N.
Published: January 2023

Suppressor Of Cytokine Signaling (SOCS) proteins are critical feedback inhibitors of the JAK/STAT signaling pathway. In addition, several studies have demonstrated that SOCS-1 and SOCS-3 have neuroprotective effects. Recently, several reports have shown that the cytokine IL-27 functions as an inhibitory regulator, and represses the development of TH17 cells associated with autoimmune brain inflammation. Stimulation with IL-27 results in the activation of members of the STAT family, predominantly STAT-1 and STAT-3. Astrocytes, the major glial cell in the CNS, have a number of important physiological properties related to CNS homeostasis, and have also been shown to produce IL-27. In this study, we describe that IL-27 induces SOCS-3 mRNA expression in primary astrocytes, but not in the macrophage cell line RAW264.7, the microglial cell line BV2 and primary microglia. IL-27 induces STAT-1α and STAT-3 activation as assessed by tyrosine phosphorylation in astrocytes, which is correlated with the induction of SOCS-3 gene expression. There was no induction of SOCS-1 mRNA after IL-27 treatment in primary astrocytes. Furthermore, we demonstrate that IL-27 induces STAT-1α and STAT-3 activation, and both SOCS-1 and SOCS-3 mRNA expression in naïve CD4+ T cells, in a time-dependent manner. These results indicate that IL-27 differentially induces SOCS-1 and SOCS-3 expression in primary astrocytes and naïve CD4+ T cells by activation of STAT-1α and STAT-3 in a cell-type specific manner. We propose that SOCS-1 and SOCS-3 may participate in IL-27-mediated suppression of TH17 cell production and autoimmune brain inflammation.
Disruption of Rxra Gene in Thymocytes and T Lymphocytes Modestly Alters Lymphocyte Frequencies, Proliferation, Survival and Th1/Th2 Balance (87.12)
Authors: Stephensen Charles Bolt , Borowsky Alexander D. , Lloyd Kevin C. Kent
Published: January 2023

Retinoid X receptor (RXR) agonists, including the vitamin A metabolite 9-cis retinoic acid, decrease T lymphocyte apoptosis and promote Th2 development ex vivo. To examine the in vivo role of RXR-alpha in T lymphocytes we disrupted the Rxra gene using Lck-targeted, cre-loxP-mediated excision of Rxra exon 4. Successful disruption was seen in thymocytes and T lymphocytes. Mice were healthy and the thymus, spleen and lymph nodes appeared normal. However, knockout mice had a lower percentage of double-positive (CD4+CD8+) and a higher percentage of double-negative thymocytes than wild type mice. The percentages of splenic T and B lymphocytes were lower in knockout mice. Ex vivo proliferation was decreased and apoptosis increased in T lymphocytes from knockout mice. Memory CD4+ T lymphocytes from knockout mice produced more IFN-γ and IL-2 and less IL-5 and IL-10 than memory cells from wild type mice, indicating a Th1 bias in vivo. However, Rxra disruption did not similarly bias ex vivo differentiation of naïve CD4+ T lymphocytes, nor did Rxra disruption alter the serum IgG1/IgG2a response to immunization. In summary, disruption of Rxra altered T and B lymphocyte percentages, produced a modest Th1 bias in vivo, and altered T lymphocyte proliferation and apoptosis ex vivo.

This work was supported by NIH grant # R01 AI 50863, USDA CRIS Project # 5306-51530 and USDA cooperative agreement # 585306-0-233.
The Tec kinase Itk regulates conventional versus non-conventional CD8 T cell development (87.14)
Authors: Horai Reiko , Handon Robin , Broussard Christine , Mueller Kristen , Venegas Ana M. , Fowlkes B.J. , Schwartzberg Pamela L.
Published: January 2023

Mutations affecting the Tec kinases Itk and Rlk impair both positive and negative selection and alter CD4 and CD8 T cell development. We previously demonstrated that mature CD8 SP thymocytes and peripheral CD8 T cells in Itk−/− and Rlk−/−Itk−/− mice do not resemble conventional CD8 T cells. Instead, these cells express memory markers and can be selected on MHC class I expressed on hematopoietic cells, suggesting these cells resemble non-conventional MHC class Ib-selected “innate-type” cells. Indeed, Itk-deficiency increases positive selection on MHC class Ib in the Kb−/−Db−/− background. We further find that either

improving TCR signaling with stronger signals orblocking CD28 costimulation, partially corrects memory phenotypes of Itk−/− CD8 T cells, arguing that different strength or quality of signals may lead to development of this distinct CD8 T cell population.

Our results suggest that Tec kinases differentially regulate development of conventional versus non-conventional lymphocytes. The requirement for signals from hematopoietic cells for selection of these “innate-type” CD8 T cells in Itk−/− mice will be discussed.
CD28:CD80 interactions mediate anitgen independent ring junction formation and signalling in T cells (87.15)
Authors: Thomas V. K , Dustin Michael L.
Published: January 2023

The two-signal model of T cell activation has been focused on what costimulatory signals are necessary to facilitate and enhance T cell response to a specific antigen. However, it is also intriguing to study the ability for costimulatory interactions to induce signaling in the absence of specific antigen recognition. We examine the signaling of CD4+ T cells engaging high densities of CD80 molecules in the presence of ICAM-1 in an antigen-independent T cell response.

Examination of freshly isolated naive mouse T cells interacting with CD80 and ICAM-1 reveals a novel role for CD28:CD80 interactions in antigen independent ring junction formation in which CD80:CD28 interactions were located in the central region. The unengaged T-cell receptor is also localized to this central region. These cells exhibit increased cytoplasmic Ca++ compared to cells interacting with ICAM-1 alone. With as many cells fluxing Calcium as seen in antigen dependent stimulation. Mouse T cells interacting with ICAM-1 and CD80 also show an increased level of phosphorylated tyrosine residues. Although there is signaling in an antigen independent T cell response, it is not sufficient for inducing IL-2 secretion. The higher expression levels of CD80 achieved in the bilayer distinguish these results from earlier studies (Bromley et al, 2001; Markiewicz et al 2005) showing no interaction of mouse CD28 and CD80 in the absence of antigen.

Thus, CD28, as well as NKG2D (Somersalo et al, 2004; Markiewicz, 2005), can stimulation formation of antigen independent ring junctions. These experiments also demonstrate that CD4+ T cells can be induced to form antigen independent adhesion rings under conditions of high CD80 density. The function of these structures is under investigation. NIH AI44931
MUC1 Mucin Expressed no Activated T Cells has a Regulatory Function (87.17)
Authors: Su Ee W , Kane Lawrence P , Finn Olivera J
Published: January 2023

MUC1 (CD227) is a large (>400kDa), heavily glycosylated, type-I transmembrane protein present most notably on the apical surface of ductal epithelia. In epithelial adenocarcinomas MUC1 is overexpressed as a hypoglycosylated protein in a non-polarized fashion. Because of these attributes, MUC1 has been studied foremostly as a tumor-associated antigen. The role of MUC1 in other cell types remained largely unexplored until MUC1 was found to be expressed by activated human T cells from healthy donors. Preliminary studies we have performed on the role of MUC1 in T cells point towards MUC1 as a negative regulator of effector T cell function. Crosslinking of MUC1 limits the proliferative capacity of primary human T cells activated with anti-CD3, as well as the ability of these cells to make IL-2 and upregulate IL-2Rα. Studies using the Jurkat T cell line have yielded intriguing results. When MUC1 is present and crosslinked, Jurkat T cells activated with anti-CD3 do not secrete IL-2. Paradoxically, knockdown of MUC1 in Jurkat T cells with siRNA also renders them unable to produce IL-2 when similarly activated. Because failure to secrete IL-2 and proliferate following TCR ligation are characteristics of T cell hyporesponsiveness, elucidating the exact role of MUC1 in T cell activation in the context of other co-inhibitory or co-activating receptors is important. To do so, we have been employing luciferase reporter assays to determine the effect of MUC1 expression and crosslinking on well-characterized signaling pathways activated during the engagement of TCR/CD3 and CD28. This will add to our current, limited, knowledge on MUC1 signaling in T cells.
Generation and characterization of GIT1 and GIT2 deficient mice (87.18)
Authors: Phee Hyewon , Weiss Arthur
Published: January 2023

The G-protein-coupled receptor (GPCR)-kinase-interacting proteins 1 and 2 (GIT1 and GIT2) are multidomain proteins involved in diverse cellular processes. In fibroblasts, GITs are thought to regulate focal adhesion (FA) turnover, cell spreading, and motility through interaction with various proteins, including Pak-interacting Rac/Cdc42 guanine exchange factor, PIX. In T lymphocytes, GITs interact with alpha and beta PIX and PAK (p21-activated kinase) through their interaction with PIX. Previously, we showed that the GIT/PIX/PAK complex is recruited to the contact site between T cell and antigen presenting cell and GIT and/or PIX seems to play a crucial role in PAK recruitment and T cell activation. However, the physiological roles and activation mechanism of GITs in TCR-activated signal transduction pathways and T cell immune responses have not been addressed thoroughly. GIT1 and GIT2 both are expressed in human and mouse T cells, although the major form expressed in hematopoietic lineage is GIT2.

To determine the role of GIT1 and GIT2 in immune cell activation in vivo, we generated GIT1 and GIT2 deficient mice. Both deficient mouse lines were backcrossed to C57BL/6 and Balb/C backgrounds in order to perform functional immunological studies. In the meantime, we performed biochemical studies that do not require pure background.