bluuebunny/crossref_metadata_2025 huggingface dataset:
The Zinc binding site in the BH domain of Itk is required for interaction with Gα13 (50.10)
Authors: Huang Weishan , Morale Luis , Qi Qian , August Avery
Published: January 2023

The Tec family kinase, Itk has been shown to be critical in T cell activation and development downstream of the T cell receptor (TCR). Migratory responses to chemokines such as CCL11/Eotaxin and CXCL12/SDF-1α require Itk. However, the mechanism by which chemokine receptor signals connect to Itk is not clear. We speculate Gα13 may serve to couple GPCRs, Itk and downstream signals. Using a Bimolecular Fluorescence Complementation system, we show that Itk and Gα13 are in close physical proximity, less than 80Å. This interaction requires the membrane localization of Itk, as the membrane targeting deficient mutants (S15P, R29C), lost their association with Gα13. Moreover, we found that mutations in the Zn2+ binding Bruton’s tyrosine kinase homology (BH) motif (C132GC133G), disrupted the interaction, even though this Itk mutant was still able to get recruited to membrane associated clusters. However, this interaction is Itk kinase activity independent. We also show that both GTP bound and GDP bound forms of Gα13 can interact with Itk. Surprisingly, co-expression of Itk and Gα13 resulted in tyrosine phosphorylation of Gα13. Taken together, we propose that interactions between Gα13 and Itk may connect chemokine/chemokine receptor stimulation and downstream responses regulated by Itk. Itk may also modulate the function of Gα13 by tyrosine phosphorylation. These data provide evidence for better understanding of the mechanism by which chemokines regulate immune cell responses.
STAT5 is Critical to Maintain Effector CD8+, but not CD4+, T cell Responses (50.17)
Authors: Tripathi Pulak , Kurtulus Sema , Wojciechowski Sara , Sholl Allyson , Hoebe Kasper , Morris Suzanne , Finkelman Fred , Grimes H. , Hildeman David
Published: January 2023

During an immune response, most effector T cells die, while some are maintained and become memory T cells. Factors controlling the survival of effector CD4+ and CD8+ T cells remain unclear. Here, we assessed the role of IL-7, IL-15, and their common signal transducer, STAT5, in maintaining effector CD4+ and CD8+ T cell responses. Following viral infection, IL-15 was required to maintain the KLRG1hiCD127lo subpopulation of effector CD8+ T cells, while IL-7 and IL-15 acted together to maintain KLRG1loCD127hi CD8+ effector T cells. In contrast, effector CD4+ T cell numbers were not affected by the individual or combined loss of IL-15 and IL-7. Both IL-7 and IL-15 drove phosphorylation of STAT5 (pSTAT5) within effector CD4+ and CD8+ T cells. When STAT5 was deleted during the course of infection, both KLRG1hiCD127lo and KLRG1loCD127hi CD8+ T cells were lost, while effector CD4+ T cell populations were maintained. Further, STAT5 was required to maintain expression of Bcl-2 in effector CD8+, but not CD4+, T cells. Finally, IL-7 and IL-15 required STAT5 to induce Bcl-2 expression and to maintain effector CD8+ T cells. Together, these data demonstrate that IL-7 and IL-15 signaling converge on STAT5 to maintain effector CD8+ T cell responses.
TNFR1 functions as a survival receptor in TNFR2-/- CD8 T cells (50.19)
Authors: Twu Yuh-Ching , Teh Hung-Sia
Published: January 2023

TNF/TNFR superfamily members play central roles in host defense, inflammation, apoptosis, autoimmunity and organogenesis. Previous reports showed that TNFR2 regulates T cell activation by lowering the activation threshold and providing costimulatory signaling. Furthermore, activated TNFR2-/- CD8 cells are highly resistant to AICD. Here, we show that blocking of activated wild-type (wt) CD8 cells with anti-TNFR2 antibodies also renders these cells resistant to AICD. The resistance of activated TNFR2-/- CD8 cells to AICD correlates with the accumulation of TRAF2. Retroviral transfection studies showed that TRAF2 overexpression was partially effective in preventing AICD in activated wt CD8+ cells. Furthermore, neutralization of TNF-α inhibits the proliferation of anti-CD3-stimulated wt CD8 T cells and increases apoptotic death cells in activated TNFR2-/- cells. TNF-α blocking also reduced TRAF2 accumulation in activated TNFR2-/- CD8 cells, which correlates with their increase susceptibility to AICD. AICD-resistant TNFR2-/- CD8 cells expressed elevated levels of phosphorylated IκBα and neutralization of TNF-α blocked this increase. These results indicate that in activated TNFR2-/- CD8 cells, TNFR1 functions as a survival receptor by promoting TNF-α-mediated regulation of TRAF2 levels and the extent of phosphorylation of pro-survival signaling molecules such as IκBα. This work is supported by the Canadian Cancer Society.
CD8 T cells require intrinsic GITR signaling for their clonal expansion during influenza infection in vivo (50.21)
Authors: Snell Laura , McPherson Ann , Lin Gloria , Sakaguchi Shimon , Pandolfi Pier , Riccardi Carlo , Watts Tania
Published: January 2023

Several members of the tumor necrosis factor receptor family regulate T cell expansion by influencing T cell activation and survival. Among these, glucocorticoid-induced TNF receptor family-related gene (GITR) is of interest, as it is expressed on both effector and regulatory T cells. While much work has focused on the role of GITR on regulatory T cells, little is known about its role on effector T cells in an infectious disease context. We sought, therefore, to examine the intrinsic role of GITR on CD8 T cells during a viral infection. To do this, we employed an adoptive transfer model where WT and GITR-/- TCR transgenic OT-I CD8 T cells were forced to compete with each other in response to infection with a recombinant influenza virus carrying the ovalbumin epitope recognized by OT-I, Influenza A/X31-OVA. In both the primary and secondary response to virus profound defects in GITR-/- OT-I T cell numbers were detected in all organs examined. Further investigation revealed that the decreased number of GITR-/- OT-I T cells was not due to a homing or proliferative defect, but rather that GITR was important for TRAF2 and TRAF5 mediated T cell survival signaling. Moreover, we also used a similar adoptive transfer model to show that GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR antibody. These results highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection.
The Effector phase of the CD8+ T cell response is not programmed during initiation (50.25)
Authors: Duttagupta Priyanka , Dolfi Douglas , Boesteanu Alina , Mueller Yvonne , Katsikis Peter
Published: January 2023

It is believed that CD8+ T cell responses are programmed at initiation in the lymph nodes by activated antigen presenting cells to undergo further proliferation in the absence of antigen and costimulation signals. We examined the effector CD8+ T cell response in the lungs of influenza virus infected mice during the late phase of infection, in the absence of costimulation, antigen and dendritic cells (DC). Blocking CD28-CD80/CD86 signaling with antibodies or depleting DCs after day six of the infection results in a 6-fold reduction in the absolute number of lung NP366-374- specific CD8+ T cells at the peak of the infection on day 10. Adoptive transfer of effector OVA257-264- specific CD8+ T cells into WSN-OVA infected wild type mice, CD80/CD86 knockout mice and DC depleted CD11c-DTR mice showed a 7-fold reduction in the number of effector CD8+ T cells in the lungs. Importantly effector CD8+ T cells need antigen to expand. Transferred effector OVA257-264 specific CD8+ T cells from lungs of WSN-OVA infected mice fail to expand in mice infected with influenza virus that does not carry cognate antigen. Moreover the absence of costimulation and dendritic cells leads to increased apoptosis of effector CD8+ T cells. In conclusion our results show that antigen-specific CD8+ T cells at the effector site need the continuous presence of dendritic cells, antigen and costimulation for expansion and survival during the late effector phase of the immune response
Genome-wide transcription profiling identifies the mechanisms of LFA-1 contributing to CD8+ T Cell Activation and immune signal network (50.26)
Authors: Li Dan , Li Hua , Liang Shoudan , Molldrem Jeffery , Ma Qing
Published: January 2023

Our previous data demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signal in CD8+ T cell activation. However, the detailed molecular pathways that regulate these processes and global impact on immune functions are poorly defined. The purpose of this genomic study is to explore and understand the LFA-1 and TCR signaling network with CD8+ T cell transcription profiling. CD8+ T cells from C57BL/6 mice and LFA-1-KO mice before and after stimulation were applied to the Mouse Whole Genome Oligo Microarray. Statistic analysis identified a list of LFA-1-associated genes differentially expressed in activated LFA-1-KO CD8+ T cells. Pathway analysis indicated that most of these genes are enriched in IL-10 signaling/ p38 MAPK signaling/ cell cycle regulation/ notch signaling/ NK signaling/ chemokine signaling. Furthermore, biological function analysis implicated these genes enriched in the functions of antigen presentation/ cell-mediated immune response/ inflammatory response/ cellular development/ cell-to-cell signaling and interaction/ immune cell trafficking/ cellular growth and proliferation/ inflammatory disease/ lymphoid tissue structure and development. Our results indicated that LFA-1 plays an important role in the immune signal network and has a global impact on immune system, and provided a complete genome-wide database to further characterize the molecular mechanisms of LFA-1 during T cell activation.
Signaling via PD-1 controls the expression of cyclins and the activation of cdk2 in human primary CD4+ T cells (50.30)
Authors: Brown Julia , Boussiotis Vassiliki
Published: January 2023

The Programmed Death 1 (PD-1) pathway plays a critical role in the inhibition of T cell activation and the maintenance of T cell tolerance. Expressed by activated T cells, PD-1 downregulates T cell effector function upon engagement with its ligands PD-L1 and PD-L2. Although vital for the inhibition of self-directed immune responses in several models of autoimmunity, expression of PD-L1 and PD-L2 by malignant tissues can prevent effective tumor-specific immunity. Furthermore, the ligation of PD-1 results in T cell exhaustion in several chronic viral infections. PD-1 mediated signaling via SHP-2 decreases Akt activation via inhibition of PI3K, but little is known about mechanisms of cell cycle regulation. Using anti-CD3 with or without anti-PD-1 conjugated to magnetic beads plus soluble anti-CD28, we have examined the expression of cell cycle mediators upon stimulation of human primary CD4+ T cells. Analysis of cell lysates by western blot revealed that signaling via PD-1 inhibited the upregulation of Skp2 and prevented the degradation of p27kip1 and the expression of cyclin E and cyclin A. Furthermore, despite abundant levels of cdk2, ligation of PD-1 prevented cdk2 phosphorylation on Thr 160 thereby inhibiting the G1/S phase transition and progression through S phase. We conclude that inhibition of T cell activation via PD-1 signaling has direct consequences for the expression and regulation of integral proteins involved in the cell cycle.
Effects of bystander inflammatory cytokines on CD8 T cell response to future antigenic encounter. (50.33)
Authors: Thomas Sunil , Kolumam Ganesh , Thompson Lucas , Kaja Murali-Krishna
Published: January 2023

Inflammatory cytokines produced during infection act on a wide variety of cells-these include infected as well as uninfected professional antigen presenting cells, T and B cells. Very few of these cells bear receptors specific to the pathogen derived peptides and a majority are bystander cells. Some of these bystander- activated lymphocytes may potentially encounter cognate antigen at later phases. Here we asked what would be the effect of bystander activation on CD8 T cells if they encountered cognate antigen later. We tested this by subjecting ovalbumin-specific TCR transgenic CD8 T cells (OT-1) to Lymphocytic choriomeningitis virus (LCMV) induced bystander activation in vivo and then assessing their response to ovalbumin. Our results show that prior bystander activation drastically diminishes their ability to expand following cognate antigenic exposure. Further experiments show that Type I interferons, a set of inflammatory cytokines produced predominantly during the early infection are mainly responsible for this inhibitory effect on bystander cells. Paradoxically the action of Type I interferons on T cells around the time of antigenic exposure has a markedly positive effect on clonal expansion. These observations suggest that the behavior of T cells is drastically different depending upon whether they receive inflammatory signals prior to or during TCR encounter, and warrants further studies on effects of inflammation in relation to the time of TCR engagement.
A novel role for Egr-3 in promoting IL-17 producing T cells (50.37)
Authors: Parkinson Rose , Powell Jonathan
Published: January 2023

Our lab has previously shown that anergic CD4+ T cells upregulate the transcription factor Egr-3 and conversely, that overexpression of Egr-3 inhibits a Th1 response and induces anergy. Using a transgenic mouse model of T cell-specific Egr-3 overexpression (Egr-3 Tg), we attempted to induce anergy in Th17 cells and instead made the unexpected finding that Egr-3 Tg T cells produce increased amounts of IL-17 when cultured in non-skewing conditions. Egr-3 Tg Th17 cells coexpress very little IL-10, even when cultured with TGF-b and IL-6. Further examination of these mice revealed that in addition to CD4+ IL-17 producing cells, there was a marked increase double negative IL-17 producing T cells. This prompted us to examine TCR gene usage. Indeed, the IL-17 producing double negative cells, and to a lesser degree CD8+ cells, all employed the γδ TCR. Interestingly, when the Egr-3 Tg mice were bred to CD4+ TCR transgenic mice, the double negative IL-17 producing cells expressed the αβ TCR. These findings suggest that Egr-3 is playing a role in differentiation toward IL-17 producing cells rather than primarily directing γδ T cell differentiation. Overall our findings demonstrate that under different circumstances Egr-3 can act either as a promoter of T cell anergy or a promoter of Th17 differentiation.
The cell-specific induction of CXCL9 by interferon (IFN)-γ in CNS microglia is determined by the myeloid transcription factor PU.1 (51.6)
Authors: Campbell Iain , Carter Sally , Gysbers Vanessa
Published: January 2023

The IFN-γ-inducible chemokines CXCL9 and CXCL10 bind to CXCR3 and are implicated in the pathogenesis of T-cell-mediated immunity in the CNS. However, the cellular localization of these chemokines differs with CXCL9 produced by macrophage/microglia (M/M) while CXCL10 is produced by both M/M and astrocytes. Here we determined the mechanism for the M/M-restricted expression of the Cxcl9 gene induced by IFN-γ. Of a number of transcription factors involved with IFN-γ-mediated gene regulation, PU.1 was present constitutively in murine M/M but not in astrocytes. Both in the brain and in culture, PU.1 was localized exclusively to the nucleus of M/M cells. ChIP analysis revealed that STAT-1, PU.1 and IRF8 were bound to the CXCL9 promoter in IFN-γ-treated M/M, whereas in astrocytes STAT-1 alone bound to the CXCL9 promoter. STAT-1 but not IRF8 was critical for IFN-γ-induced expression of both the Cxcl9 and Cxcl10 genes in M/M and in M/M and astrocytes, respectively. The siRNA-mediated knockdown of PU.1 in M/M markedly impaired IFN-γ induced CXCL9 but not STAT1 or IRF8. D1A astrocyte cells showed partial reprogramming to a myeloid phenotype after transduction with PU.1 with expression of CD11b and acquisition of IFN-γ-induced CXCL9. Thus, while IFN-γ is a pivotal mediator of both Cxcl9 and Cxcl10 gene expression, this cytokine differentially induces the expression of CXCL9 in myeloid cells through a mechanism that is determined by PU.1. Support: NIH NS044905.
2nd generation anti-HIV designer T cells cure HIV-infected cultures and preferentially survive versus 1st generation designer T cells (52.19)
Authors: Selliah Nithianandan , Skowron Gail , Bais Anthony , Gomes Erica , Lu Li , Yang Wen , Junghans Richard
Published: January 2023

Immunotherapies that have the potential to eradicate reservoirs of HIV infected cells may be useful to suppress or cure HIV/AIDS. T cells expressing chimeric immune receptors (CIR) through gene modification (designer T cells) can be redirected to kill HIV-infected cells. Previously, an anti-HIV CIR, CD4ζ (1st generation), was created to kill HIV+ cells. However, the adoptive transfer of autologous CD4ζ T cells failed to control the viral infection in clinical trials. We created a 2nd generation CIR for enhanced potency by providing co-stimulatory signal (CD4CD28ζ). Our data show that both 1st and 2nd generation CIRs expressed well on retrovirally transduced human T cells and kill HIV+ targets with high efficiency, whereas CD28 in the CIR confers a distinct advantage in the expansion potential and cytokine IL2 secretion. Interestingly, our preliminary data show that all 1st generation designer T cells died in 7 days when co-cultured with HIV+ cells, whereas 2nd generation designer T cells survived with cure of the infected cultures. This stunning result shows dramatic benefit in the new 2nd generation CIR versus the 1st generation that was used in human clinical trials. We are presently exploring whether this cell death in the 1st generation designer T cells is due to heightened sensitivity to AICD and/or due to HIV infection. Either way, these data show a striking benefit to our new 2nd generation designer T cells as a promising therapy for HIV/AIDS.
Highly Immunogenic JVRS-100 Adjuvanted Universal Influenza A Vaccine (52.4)
Authors: Callejo Bernadette , Monath Tom , Lay Marla , Chang Stella , Fairman Jeff
Published: January 2023

A vaccine based on a conserved external ectodomain of M2 (M2e) offers the potential for inducing broad-immunity against divergent influenza A strains including pandemic viruses. Cohorts of mice were vaccinated 3x (IM) with M2e in the form of a multiple antigenic peptide with 4 copies of M2e (MAP4), with or without cationic lipid DNA complex adjuvant (JVRS-100). Lethal challenge using different strains of virus (H1N1, H3N2) were done to establish efficacy based on survival. The addition of JVRS-100 to M2e-MAP4 resulted in a significant decrease in morbidity and mortality following lethal challenge with H1N1 (survival 100% vs. 30% without adjuvant) or H3N2 (80% vs. 20%). Adjuvanted vaccine resulted in higher levels of IgG (p=0.0159), IgG1 (p<0.02) and IgG2a (p<0.005) vs. M2e-MAP4 alone. A dose titration of M2e-MAP4 inoculated with a constant amount of JVRS-100 resulted in 100% survival after challenge even at a vaccine dose of 25ng. Addition of M2e-MAP4/JVRS-100 to trivalent inactivated influenza vaccine (TIV) confers full protection with a single vaccination prior to lethal challenge. Competitive binding ELISA confirmed that the sera from vaccinated mice contained M2e conformational epitopes. Vaccination with MAP2 and MAP4 indicated the protective epitopes were dependent on dimeric and not tetrameric configuration of the MAP construct. The experiments demonstrate the potency of MAP configured M2e peptide with the JVRS-100 adjuvant in the development of universal flu vaccine.
Reduced IL-4 production by murine T cells after B cell depletion (83.10)
Authors: Li Yongmei , Dunn Robert , Cohen Philip
Published: January 2023

Anti-mouse CD20 antibody, clone 18B12, depletes B cell effectively in vivo. Previously, we observed that IgG2a-18B12, but not the IgG1 isotype, resulted in partial reduction of CD4+ memory T cells from mice that exhibit Sjogren’ syndrome-like autoimmune disease. To determine the effect of B cell depletion on CD4 T cell function in normal mice, B6 mice were given three weekly i.p. injection of 250 ug each IgG2a-18B12, IgG1-18B12 or control mIgG2a antibodies (n=4 to 5 per group). 14 days after the last injection, both follicular and marginal zone B cells were depleted >99% in IgG2a-18B12 treated group. MZ B cells were reduced by 66% in mice treated with IgG1-18B12. CD4+ T cells were stimulated for 63 hours by plate-bound anti-mouse CD3 and soluble anti-CD28. CD4+ T cells isolated from IgG2a-18B12 treated mice produced levels of IL-2 and IFN-gamma similar to cells from control IgG2a treated mice, but showed 75% reduction in IL-4 production (p=0.014). CD4+ T cells from IgG1-18B12 treated mice showed similar level of IL-4 production as compared to control IgG2a treated group. These findings suggest that in vivo B cell depletion can result in alteration of T cell cytokine production and this effect seems to depend on the isotype of B-cell targeting antibody.
The Role of Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) in Experimental Autoimmune Encephalomyelitis (EAE) (83.20)
Authors: Chang Rhoda , Miller Stephen , Longnecker Richard
Published: January 2023

Multiple Sclerosis (MS) is an inflammatory, autoimmune disease of the central nervous system. The cause of MS is still unknown but epidemiological and immunological studies have implicated Epstein-Barr Virus (EBV), which infects B cells, as a possible etiological agent. Of particular interest is EBV Latent Membrane Protein 2A (LMP2A). Previous work in our lab using transgenic mice in which B cells express receptors for Hen Egg Lysozyme (BCRHEL) and LMP2A has demonstrated that LMP2A enhances the expansion and differentiation of B cells upon immunization with HEL, revealing a potential contribution of LMP2A in autoimmunity. As B cells may be a key player in MS, we wanted to examine the role of LMP2A in the animal model Experimental Autoimmune Encephalomyelitis (EAE). Recombinant MOG protein was immunized into transgenic mice in which B cells express LMP2A. LMP2A mice show increased severity and incidence of disease, and faster onset of disease. LMP2A also affects lymphocyte recruitment into the CNS and enhances activation of lymphocytes in immunized mice. Further in vitro analysis will address the role of LMP2A in lymphocyte activation and antigen presentation.
Ag Specific T Effector (TE), Ag specific Foxp3+ regulatory T cells (Treg), and immune complex (IC) interact in a special testicular microenvironment and promote disease progression in a new spontaneous autoimmune orchitis (EAO) model. (83.25)
Authors: Tung Kenneth , Paul Alberta , Qiao Hui , Grafer Connie , Li Jonathan , Pramoonjago Patcharin , Sparks Josh , Bui Elise , Sun Wei
Published: January 2023

BALB/c mice expressing spermatid-specific transgenic ovalbumin (OVA), when crossed to DO11.10 mice, develop spontaneous EAO that begins at 8 wks, which progresses from preclinical stage (8-16 wks) to clinical stage with infertility (>17 wks). Progression time can be reduced to 3 wks by day 3-thymectomy. In preclinical EAO, DC, TE, Treg and OVA-IC are co-localized in, and confined to, discrete non-invasive cell clusters outside the blood-testis barrier (BTB). In clinical EAO, while remaining confined to the clusters, cells become invasive, penetrate the BTB, and deplete germ cells. Why does EAO progress despite persistent expansion and co-localization of activated Ag specific Treg and TE? To address this conundrum, we have accrued the following findings: 1) Changes in the intrinsic TE function: IFNγ-dominance in preclinical EAO versus IL17-dominance in clinical EAO; and adoptively, preclinical T cells transfer preclinical EAO while clinical T cells transfer clinical EAO; 2) only the TE from clinical EAO resist suppression by Treg, 3) In vivo conversion of Treg to Th1 or Th17 TE occurs in testes with clinical EAO; and 4) Infusion of OVA autoAb rapidly results in new IC deposition in the cell clusters, and promotes EAO progression. We conclude that intra-testicular events, within testis cell clusters, critically modify intrinsic properties of both TE and Treg, with the net effect strongly favoring EAO progression. This may depend on activation by co-localized IC.
Cytokine priming enables triggering of autoreactive CD8 T lymphocytes by low affinity TCR ligands (83.7)
Authors: Ilangumaran Subburaj , Dubois Stephanie , Chen Xi Lin , Leblanc Chantal , Ohashi Pamela , Ramanathan Sheela
Published: January 2023

The cellular and molecular events preceding the initial activation of autoreactive CD8 T cells in several autoimmune diseases remain unclear. Recent reports have implicated IL-6, IL-21, IL-15 and IL-7 in the triggering of autoreactive CD8 T cells in mouse models of autoimmune diseases. We have shown that IL-21 and IL-6 synergize with IL-7 or IL-15 to induce antigen-independent proliferation of naive CD8 T cells, with a concomitant increase in antigen responsiveness. We postulated that the cytokine-induced augmentation of TCR sensitivity (referred herein as ‘cytokine priming’) might enable stimulation of potentially autoreactive CD8 T cells by low affinity self antigens. Here, we show that cytokine priming enables naive TCR transgenic CD8 T cells to respond to low affinity TCR ligands, resulting in proliferation and acquisition of effector functions. We have used a transgenic mouse model of autoimmune type 1 diabetes to show that cytokine-primed autoreactive CD8 T cells acquire the capacity to cause disease following stimulation with weak TCR agonists, and that the diabetogenic potential of these cells is dependent on the continuous availability of IL-15. These findings demonstrate a novel mechanism by which cytokines contribute to the triggering of autoreactive CD8 T cells and have important implications for autoimmune diseases associated with infections and chronic inflammation.
Intrinsic defects in B cell response to influenza vaccination in elderly humans (84.11)
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Landin Ana Marie , Frasca Daniela
Published: January 2023

We have previously shown that elderly humans have fewer percentages of CD19+ total B cells, switch memory B cells, and increased percentages of naïve B cells. Activated CD19+ B cells have less E47, AID and Igγ1 circle transcripts (CTs) with age. AID is critical for class switch recombination (CSR) and somatic hypermutation, both necessary for optimal function of immunoglobulin (Ig). More recently we have initiated a series of experiments to measure the antibody response to influenza vaccination by hemagglutination inhibition assay (HI) and associated this with the isolated/intrinsic B cell response to these antigens (containing adjuvants) in vitro. Our results show that the specific in vitro AID response of B cells to vaccination and the in vivo serum HI response to vaccination are both decreased with age. We are currently pursuing these studies with more subjects and with both seasonal as well as H1N1 vaccine response. These results indicate that these biomarkers (decreased E47, AID, CSR, IgG) could more accurately track optimal immune responses and activity, and would be better predictors of vaccine and therapeutic agent effectiveness in humans.
Treg and rapamycin effects on humoral immunity in young and old mice (84.12)
Authors: Benavides Adriana , Fernandez Elizabeth , Sharp Z. , Strong Randy , Kraig Ellen
Published: January 2023

Recently, the mTOR inhibitor rapamycin was shown to extend lifespan in mice even when administered at 20 months of age (Harrison et al. Nature 2009). Although studies have shown that it enhances vaccine efficacy and immune memory, others demonstrated an increase in Tregs and a depression of T cell proliferation. Thus, to assess the effect of rapamycin on humoral immunity, young and old mice were fed a rapamycin or control diet and then immunized 3 times with the protein antigen Omp34 from Aggregatibacter actinomycetemcomitans (Aa), a periodontal pathogen. As expected, rapamycin impaired antibody responses to Omp34 in young mice. In older mice, the humoral response was low and not enhanced by the drug. Serum cytokines were also measured and treatment with rapamycin had no effects. In order to address the role of Tregs, mice in each group were given 3 injections of αCD25 (functionally inactivates Tregs) or an isotype control antibody prior to each immunization. However, this treatment did not reverse the effects seen in antibody titers or cytokine levels, suggesting that Tregs alone do not account for the effects seen. After the last immunization, mice were challenged with viable Aa and protection was assessed. Rapamycin had no effect on protection, but surprisingly, antibody production to Aa in older mice was higher in those fed rapamycin. In younger mice, drug treatment had no effect. In conclusion, rapamycin’s effects on immunity should be assessed in old and young mice.
Kinetic and function of c-Rel in naïve B cells (84.4)
Authors: Damdinsuren Bazarragchaa , Grumont Raelene , Zhang Yongqing , Wood William , Becker Kevin , Gerondakis Steve , Sen Ranjan
Published: January 2023

Temporal control of NF-κB activation has been proposed to play an important role in response of cells to NF-κB -inducing stimuli. Our earlier studies found two phases of NF-κB activation in primary B lymphocytes stimulated via the B cell receptor. Phase I NF-κB is induced via the canonical pathway and leads to transient nuclear expression of RelA and c-Rel. Phase II NF-κB activation is mediated by de novo c-Rel gene transcription and translation. We have followed up these observations to determine the function and mechanism of c-Rel during each phase. Using a combination of biochemical, gene expression studies and gene reconstitution studies we propose that c-Rel provides unique survival functions during both phases of NF-κB activation, and supports cell cycle progression in response to CD40 stimulation in phase II. Our studies help to de-convolute functions of c-Rel that are unique from those that are redundant with RelA in B lymphocytes.
A cyclooxygenase-2 (COX-2) pathway promotes expression of activation-induced cytosine deaminase (AID) within replicating human B cells (84.7)
Authors: Mongini Patricia , Lee Hyunjoo , Trott Joshua , Chiorazzi Nicholas
Published: January 2023

Within an inflammatory milieu, B cells specific for autoantigens or microorganisms can develop tertiary lymphoid foci exhibiting AID, downstream class switching, somatic mutation, and occasionally malignant transformation. This study has examined whether COX-2, recently found elevated within cycling human B lymphoblasts, can contribute to the above. We report that after stimulation with surrogate C3dg-bound Ag, IL-4 and BAFF, CFSE-labeled IgM+ human B cells develop clones expressing division-dependent AID, accompanied by elevated levels of mPGES-1 (an inducible prostaglandin E2 (PGE2) synthase), and the PGE2 receptor, EP2. The contemporaneous expression of these proteins was functionally relevant. Thus, COX-2 inhibitors diminished both AID and class switching to IgG. Furthermore, exogenous PGE2, or selective EP2 agonist, increased AID protein. While PGE2 supplementation consistently augmented daughter cell viability and absolute yield of IgG+ progeny, somewhat surprisingly, within cultures showing higher baseline IgG switching, added PGE2 diminished the proportion of IgG+ viable progeny. Available data suggest that while PGE2-augmented AID promotes IgG switching, this process may also lead to greater activation-induced death. Taken together, these novel findings show that during a T cell-independent burst of proliferation, B cells can upregulate both AID and several COX-2 pathway proteins that constitute a positive amplification route for AID expression.
Actions of novel inflammation-resolving lipid mediators on human B cells (84.9)
Authors: Ramon Sesquile , Serhan Charles , Phipps Richard
Published: January 2023

The resolution of inflammation is an active and dynamic process. Newly identified lipid mediators have been recognized as key players during the process of inflammation resolution. These lipid-derived molecules constitute three classes of compounds (lipoxins, resolvins and protectins), all derived from essential fatty acids. New data demonstrates that these lipid mediators regulate aspects of the immune response, including inhibition of neutrophil infiltration, reduction of T cell cytokine production and stimulation of macrophage phagocytic activity. However, their effects on B lymphocytes are unknown. We show for the first time that the novel lipid mediator lipoxin B4 and 17-HDHA increase the ability of normal human B cells to produce IgM and IgG when activated with CpG plus anti-IgM. The two lipid derived molecules along with lipoxin A4 also enhance B cell differentiation, measured by an increased frequency of CD38+ cells. In addition, resolvin D1 and AT-resolvin also increase antibody production in CpG-stimulated B cells. None of the inflammation resolution lipid mediators affect proliferation and are non-toxic to the cells. Increase of plasma cell differentiation and antibody production coincides with the known involvement of pro-resolving mediators during the late stages of inflammation and pathogen clearance.
Repertoire selection and effector functions of neonatal γδ T cells (85.12)
Authors: Cairo Cristiana , Sagnia Bertrand , Cappelli Giulia , Leke Rose , Leke Robert , Colizzi Vittorio , Pauza C.
Published: January 2023

The neonatal T cell repertoire includes a low frequency of Vγ2Vδ2 T cells. This population is poorly reactive to phosphoantigens or aminobisphosphonates, compared to the adult repertoire in blood. The neonatal Vδ2 cell population also shows a reduced proportion of Vγ2-Jγ1.2 rearrangements; this TCR chain combines with the Vδ2 to endow phosphoantigen responsiveness. We are interested in how the neonatal repertoire is selected to produce the dominant Vγ2-Jγ1.2 rearrangement seen in adults. IL-15 did not improve responses to stimulation, but improved survival. Both IL-2 and IL-15 up-regulated NK receptors and perforin production, although IL-2 preferentially down-regulated CD27 expression, that may be linked to the development of cytotoxicity. Curiously, aminobisphosphonate stimulation in a single round selected a Vγ2 repertoire similar to adults. Thus, neonatal γδ T cells found in cord blood are an attractive model for understanding the genesis of cytotoxic T cells lineages.
Decline of influenza-specific T cell repertoire in healthy geriatric donors (85.2)
Authors: Lee Jessica , Oelke Mathias , Ramachandra Lakshmi , Canaday David , Schneck Jonathan
Published: January 2023

While the goal of influenza vaccination is to develop antibodies to protect against a primary infection, clearance of the infection is primarily mediated through CD8+ T cells. The study of the CD8+ T cell response to influenza epitopes is crucial in understanding the disease and associated morbidity and mortality. The age-associated decline of the immune system is believed to be responsible for the increased risk of influenza infection in the geriatric population. Since the CTL response is important for the clearance of influenza infection, we compared the CTL response to immunodominant and subdominant influenza epitopes in HLA-A2+ control adult donors, aged 21-42, to the response in healthy geriatric donors, aged 65 and older. This was done using an artificial Antigen Presenting Cell (aAPC) based stimulation assay which revealed responses that could not be detected by ELISpot. Control donors showed a broad, multi-specific response to the subdominant epitopes, and each donor’s subdominant influenza-specific response was unique. When comparing the control donors’ CTL response to the geriatric donors’, the immunodominant M1 responses were preserved, however, the geriatric donors lacked the broad multi-specific response to influenza-specific subdominant epitopes seen in the control donors. These data indicate that aging leads to a decrease in the subdominant influenza-specific CTL responses which may contribute to the increased morbidity and mortality in older individuals.
Regulation of mast cell signaling by SHP-1 as a phosphatase and an adaptor (86.1)
Authors: Mizuno Kazuya , Nakata Kazuko , Yakura Hidetaka
Published: January 2023

To clarify how SHP-1 regulates mast cell signaling, we generated BMMC from motheaten (me), viable motheaten (mev), and control (WT) mice and examined signaling events and final outputs following FcϵRI and IL-3 stimulation. FcϵRI-induced tyrosine phosphorylation of SLP-76 and LAT, MAPK activation, and cytokine production were increased in me-/mev-BMMC. In addition, phosphorylation of SHP-1 was increased in mev. Since the activity of Lyn and Syk was comparable in all BMMC, these molecules are substrates of SHP-1. me-BMMC showed impaired PLCγ phosphorylation, calcium mobilization, and degranulation, which could not be observed in mev-BMMC. This result suggests that the expression of SHP-1 is sufficient for the positive regulation of these pathways. The absence of SHP-1 expression disrupted the association between SLP-76 and PLCγ. Next, we examined IL-3-dependent cell proliferation and cell death. When BMMC were stimulated with IL-3, ERK activation was increased and sustained in me-/mev. In accordance with increased ERK activation, apoptosis was decreased in me-/mev-BMMC. In contrast to the predicted role of ERK as a pro-survival molecule, IL-3-induced cell proliferation was much lower in me-/mev-BMMC than WT-BMMC. Addition of PD98059 resulted in the suppression of decreased apoptosis and cell proliferation in me-/mev-BMMC. These results suggest that SHP-1 positively regulates IL-3-dependent mast cell proliferation and apoptosis by inhibiting ERK activity.
Anti-IgE monoclonal antibodies that bind to IgE bound by CD23 but not to IgE bound by IgE Fc receptors on basophils (86.10)
Authors: Shiung Yu-Yu , Chiang Chen-Yi , Chen Jiun-Bo , Wu Pheidias , Hung Alfur , Chang Tse Wen
Published: January 2023

IgE is a central mediator responsible for immediate-type hypersensitivity reactions. The anti-IgE monoclonal antibody (mAb), omalizumab, has been shown in numerous clinical trials to be efficacious in the treatment of severe allergic asthma and other allergic diseases. Omalizumab was designed to bind to free IgE in blood and membrane-bound IgE (mIgE) on B cells, but not to IgE bound by the high-affinity IgE Fc receptors (FcϵRI) on basophils and mast cells and by the low-affinity IgE Fc receptors (CD23) on B cells and many other cell types. Additionally, omalizumab can bind to IgE and prevent it from binding to both FcϵRI and CD23. Studies by other groups have also shown that CD23 is involved in the synthesis of IgE and that interfering CD23 function, such as by cross-linking CD23 on B cell surface, can cause the down-regulation of IgE production. We have developed anti-IgE mAbs that mimic omalizumab in various aspects except their ability to bind to IgE on CD23. These newly developed mAbs bind to linear epitopes on IgE. They do not induce the degranulation of IgE-pulsed rat basophilic leukemic cells (RBL SX-38), which had been transfected with the genes of human FcϵRI. The ability of the mAbs to cross-link IgE-occupied CD23 on the cell surface of B cell lines is being tested. The results suggest that these anti-IgE mAbs may render a set of pharmacological mechanisms, which are somewhat different from that of omalizumab, for controlling IgE in patients with allergic diseases.
Adenosine receptor A3AR is responsible for augmenting FcϵRI-induced degranulation of human lung mast cells by extracellular adenosine. (86.17)
Authors: Gomez Gregorio , Schwartz Lawrence
Published: January 2023

Inhaled adenosine induces a bronchoconstrictive response in asthmatics that is attributed to enhanced activation of mast cells in the airways. In this study, we demonstrate that extracellular adenosine augments FcϵRI-mediated degranulation of human mast cells from lung via the adenosine A3 receptor (A3AR). We found that low concentrations (10-7 - 10-6 M) of adenosine and its stable analog NECA enhanced degranulation of mast cells from lung, but not skin. Quantitative real time PCR analysis revealed that lung and skin mast cells expressed equal amounts of A2aAR and A2bAR (A1AR was not detectable), but that lung mast cells expressed a 5-fold greater amount of A3AR mRNA transcripts compared with skin mast cells. This suggested that A3AR was responsible for augmenting degranulation of lung mast cells. Indeed, targeted stimulation with the A3AR-specific agonist HEMADO significantly enhanced mediator release from lung mast cells following FcϵRI crosslinking with an anti-FcϵRIα MAb, but had little effect on skin mast cells. In addition, the enhancing effect of adenosine on lung mast cells could be completely inhibited by inactivating Gi-coupled A3ARs by ADP ribosylation with pertussis toxin. These data provide direct evidence that extracellular adenosine enhances IgE-induced degranulation of human lung mast cells through an A3AR-mediated mechanism; thus supporting a role for A3AR in the bronchoconstrictive response to inhaled adenosine in asthmatics.
Tyrosines in the carboxy-terminal region regulate Syk function (86.18)
Authors: Castro Rodrigo , Zhang Juan , Jamur Maria , Oliver Constance , Siraganian Reuben
Published: January 2023

The Syk tyrosine kinase family has an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine phosphorylated ITAMs in the subunits of FcϵRI results in a conformational change, with an increase in the enzymatic activity of Syk. This conformational change exposes the COOH terminal region of Syk which has three conserved Tyr residues (Y623, Y624, Y625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe were expressed in Syk-deficient mast cells. There was decreased FcϵRI induced degranulation, together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. There was a parallel decrease in NFAT and NFκB activation with the mutated Syk. In non-stimulated cells, there was increased tyrosine phosphorylation of the mutated Syk, however, this still increased after FcϵRI activation. In vitro mutated Syk had decreased ITAM-binding and reduced kinase activity. In testing Syk mutated singly at each one of the tyrosines, Y623F had minimal effect while Y624F but specially Y625F had the most reduction in these assays. Also, tyrosines Y624 and Y625 controlled Syk capacity for autophosphorylation. Therefore these tyrosines in the tail region of Syk regulate its kinase activity and their phosphorylation is important for Syk function in mast cell FcϵRI-mediated signaling.
Potent inhibition of calcium mobilization and mast cell activation by the pyrazole derivative BTP2 (86.9)
Authors: Law Mankit , Mottram Laurie , Morales Jose , Peterson Blake , August Avery
Published: January 2023

Cytosolic Ca2+ changes in mast cells are central for driving their activation. Thus, pharmacological tools that inhibit this process represent unique strategies for inhibiting mast cell function. We find that the pyrazole compound BTP2 inhibited FcϵRI-triggered sustained Ca2+ influx in a mast cell-like cell line RBL-2H3 with little effect on the tyrosine phosphorylation of cellular proteins. It also did not affect FcϵRI-induced phosphorylation of mitogen-activated protein kinases Erk1/2, JNK, and p38 nor downstream c-fos expression. This suggests that BTP2 inhibits Ca2+ mobilization without affecting the FcϵRI signal transduction cascade. BTP2 inhibited degranulation and β-hexosaminidase release in RBL-2H3 cells. Consistent with this effect, BTP2 inhibited antigen-induced histamine release in vivo. BTP2 also inhibited cytokine secretion of IL-2, IL-3, IL-4, IL-13, and TNF-α by BMMCs. Analysis of structure-function relationships between the BTP2 parent compound and its derivatives implicate a role for the trifluoromethyl group at both carbon positions 3 and 5 of pyrazole in the inhibition of degranulation of RBL-2H3 cells, with a more dominant effect evoked by the trifluoromethyl group at C3. In conclusion, our studies indicate that pyrazole compounds can provide the molecular framework for the design of specific inhibitors of Ca2+ entry mechanisms and further BTP2 as a medicinal candidate for the treatment of allergic reactions and other mast cell-mediated diseases.
EGCG Inhibits Inflammation Independently of AMPK (87.10)
Authors: Reilly Christopher , Regna Nicole , Peairs Abigail
Published: January 2023

Epigallocatechin-3-gallate (EGCG), a bioactive component of green tea, has been reported to exert anti-inflammatory effects in immune cells. EGCG has been shown to activate the metabolic regulator, AMP-activated protein kinase (AMPK). Mesangial cells from MRL/lpr lupus-like mice are hyper-responsive to immune stimulation and overproduce nitric oxide (NO) and other inflammatory mediators when stimulated. In our current studies, we sought to determine if EGCG would inhibit inflammation in AMPK deficient mesangial cells. Cultured mesangial cells from MRL/lpr mice were treated with siRNA for mouse Prkaa2-Design A, specific for AMP-activated protein kinase-alpha (AMPK-α) or siRNA negative control for mouse. After 48 hours, the cells were serum-starved for 2 hours, treated with 50 μM epigallocatechin-gallate (EGCG) for one hour, and stimulated with lipopolysaccharide (LPS)/interferon (IFN)-γ for 30 minutes. The addition of siRNA inhibited AMPK expression in mesangial cells. LPS/IFN-γ induced inflammatory mediator production (iNOS expression, supernatant NO, and interleukin-6) as expected. Interestingly, EGCG blocked inflammatory mediator production in the AMPK deficient cells suggesting EGCG inhibits inflammatory pathways independently of AMPK to decrease inflammatory mediatory production. Taken together, these studies show that EGCG attenuated inflammation in MRL/lpr mouse mesangial cells independent of AMPK.
Bio-distribution and in vivo antioxidant properties of cerium oxide nanoparticles (87.26)
Authors: Hirst Suzanne , Karakoti Ajay , Tyler Rom , Sriranganathan Nammalwar , Seal Sudipta , Reilly Christopher
Published: January 2023

Cerium oxide nanoparticles have oxygen defects in their lattice structure that enable them to scavenge oxygen radicals. Recent reports have shown nanoceria to act a regenerative free radical scavenger in a physiological environment. The goal of our study was to perform a comprehensive analysis of the biological distribution and in vivo antioxidant capabilities of nanoceria. We examined three peroral (PO), intravenous (IV), and intraperitoneal (IP) administration routes along with deposition, clearance, histology and WBC counts in nanoceria administered mice. We also examined if nanoceria would decrease ROS production in mice treated with carbon tetrachloride (CCl4). As determined by inductively coupled plasma-mass spectrometry (ICP-MS), nanoceria organ deposition for IV and IP mice was greatest in the spleen followed by the liver, lungs, and kidneys. Elimination for all administration routes was through feces. Nanoceria distribution was confirmed with IVIS in vivo imaging, and deposition was confirmed with transmission electron microscopy (TEM). Nanoceria administration did not induce histopathology, however it did raise WBC counts. Nanoceria administration to mice with induced liver toxicity reduced liver 8-isoprostane, urine 8-OHdG, and plasma MDA levels markers of oxidative stress. Our studies show that nanoceria remains deposited in tissues and decreases ROS, suggesting that cerium oxide nanoparticles may be a useful antioxidant treatment for oxidative stress.