bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-specific effector CD8 T-cells inhibit allergic responses via IFN-gamma secretion and dendritic cell interaction (103.4)
Authors: Tang Yafang , Chua Benson , Guan Shou Ping , Zhou Sophie , Ho Adrian , Wong Kenneth , Wong Kok Loon , Wong Fred , Kemeny David
Published: January 2023

Allergic inflammation of the airways causes changes in the lung wall that lead to inflammatory diseases such as asthma. Using a mouse model this response can be divided into an induction phase and an effector phase. Previously we have shown that transfer of CD8 T cells inhibits the induction of Th2 responses. In this study we have investigated the effect of CD8 T cells on the effector phase of the inflammatory lung response. In vitro activated OT-I CD8 T cells were transferred to ovalbumin (OVA)-alum immunized mice one day before challenge with OVA. Eosinophil infiltration and mucus secretion were inhibited by transfer of CD8 T cells. When OT-IxIFN-gamma-/-T cells were transferred, the inhibitory effect was abolished, suggesting an important role for IFN-gamma. In fact, without IFN-gamma, effector CD8 T cells were able to induce asthma-like inflammation which further proved the importance of IFN-gamma in the suppression of type II inflammation. We also investigated the effect of CD8 T cells on lung dendritic cell (DC) function. CD11c+CD11b+CD103- DCs from CD8 transferred mice increased largely in number and when co-cultured with CD4 T cells, they were able to induce more IFN-gamma production. These results suggest that in addition to regulating the induction of the allergic immune response, CD8 T cells can subsequently divert the local lung environment to one that favors Th1 immunity, through IFN-gamma and CD8-DC interaction.
Age-associated alterations of lipid metabolism and oxidative stress in the murine thymus are attenuated by in vivo growth hormone administration (104.13)
Authors: Mello-Coelho Valeria , Cutler Roy , Tammara Anita , Bunbury Allyson , Mattson Mark , Taub Dennis
Published: January 2023

The mechanisms by which GH facilitates the recovery of thymopoiesis during age-associated thymic atrophy are unknown. Herein, we analyzed the age-related lipid alterations in total thymus and thymocyte preparations derived from 04 to 18 month-old mice, and in thymi of GH-treated aged mice. Electrospray tandem mass spectrometry permitted us to detect increased amounts of tryglicerides (TG), free-cholesterol and C24:1 sphingomyelin in the total thymus of old mice as compared to those of young. Moreover, we verified a decrease in the sulfatide C24:1 and in unknown long-chain ceramides, and an increase in 4-HNE and 8-epi-prostaglandin F2α (PGF) in the thymi of older animals. Using immunohistochemistry, we verified an increase in the expression of TNFα and CD204 within the aged thymus sections. In analyzing thymocytes, we found a decrease in TG, free cholesterol and in sphingomyelin species with age. Interestingly, we detected a significant amount of ceramide C24:1 in thymocytes derived from old mice but not young. Following GH treatment, we observed a significant decrease in TG, free-cholesterol and PGF in the thymus of older animals. GH treatment significantly increased the amount of dihydroceramides and promoted a trend to decrease 4-HNE levels compared to thymi of age-matched denatured GH-treated control mice. These results suggest that increased oxidative stress associated with perturbed lipid metabolism within aged thymus can be attenuated after GH treatment.
Role for p53 in activation-induced cell death of T cell-independent B cell clones (104.16)
Authors: Mongini Patricia , Lee Hyunjoo , Nieto Jennifer , Chiorazzi Nicholas
Published: January 2023

B cell immunity involves clonal proliferation, differentiation of selected progeny into memory/effector cells, and activation-induced cell death (AICD) of others. Clonal expansion and contraction occur in human B2 cell cultures activated by surrogate C3dg-Ag and IL-4 ± BAFF (J Immunol 175:6143). The mechanism was here studied. Pro- and anti-apoptotic proteins were assessed by immunoblotting prior(d3-4) or during (d5-6) AICD and by immunostaining CFSE-labeled cells. Evidence of lesser Bim but elevated p53 and downstream proteins suggested p53-mediated apoptosis, perhaps triggered by expressed activation-induced cytosine deaminase (AID) (J Immunol 185:5300). This was reinforced by findings that AICD is reduced (but not ablated) by p53 siRNA or genetic deficiency in p53 or AID, in similarly activated mouse cells. A significant drop in Bcl-2 in dividing blasts preceded AICD and its importance was substantiated by greater clonal burst size in Bcl-2 TG-cultures. In germinal centers (GC), p53 is repressed by Bcl-6, resulting in infrequent p53 mutations in GC lymphomas (Phan et al., Nature 432:635). Our study suggests that B cell tumors with a higher incidence of p53 mutations may arise from inflamed peripheral tissues. In the latter, recirculating B cells will encounter C3dg-coated microbes or apoptotic cells and IL-4 and BAFF from innate immune system cells. The latter milieu would foster T cell-independent B cell growth and encourage positive selection of p53 mutants.
Mechanisms of thymic recovery and T cell reconstitution following sublethal ionizing radiation (104.21)
Authors: Këpuska Zana , Sempowski Gregory
Published: January 2023

Proper thymic function is important for maintaining lifelong peripheral homeostasis of naïve and memory T cells. Age-induced thymic involution is chronic and does not recover. Acute thymus involution in settings of stress can spontaneously recover. The goal of this research was to define mechanisms of thymic damage and recovery following acute stress (irradiation). Young and old BALB/c mice were sublethaly irradiated (≤ 125 rad).Thymus function was monitored by thymus weight, cellularity, and TCR gene re-arrangement (mTREC). In young, irradiation significantly reduced thymocyte number and depleted mTREC levels by day 7, followed by full recovery (day 42). In old, radiation did not cause further decrease in thymocyte number or mTREC compared to that induced by age. To define mechanisms of thymic involution/recovery expression of thymostimulatory factors and indicators of healthy thymus were analyzed.While IL-7 and FoxN1 mRNA levels were not impacted in the young, KGF and AIRE mRNA expression levels were significantly reduced during irradiation-induced involution. These levels restored to baseline 21 days prior to full thymus functional recovery. Thus, suggesting a critical role of thymic-stromal derived KGF in promoting stroma/thymocyte crosstalk to mediate thymus recovery. Overall, these findings may lead to a better understanding of mechanisms involved in thymus recovery from damage and inform development of strategies to restore thymic function in the aged.
Impact of aging on CD4 T cell helper functions (104.6)
Authors: Lefebvre Julie , Maue Alexander , Petell Ashlee , Lanthier Paula , Tighe Michael , Eaton Sheri , Haynes Laura
Published: January 2023

Impairment of the immune system in older people leads to increased susceptibility to infection and reduced antibody production following immunization. We have previously shown that intrinsic defects in CD4 T cell function occur in aged mice and that this negatively impacts the antibody response. In addition, other age-related changes can impact CD4 T cell priming. Using an adoptive transfer model, we show that the trafficking of young TCR Tg CD4 T cells to the T cell zones of secondary lymphoid organs is impaired when they are transferred into aged hosts compared to young hosts. This correlates with delayed expansion and reduced activation as measured by CFSE dilution and CD69 expression, respectively. Using immunofluorescent staining of spleen sections, we demonstrate that the structure of the B cell follicles and the expression of CCL21 are disorganized in aged hosts compared to young hosts, likely causing this reduced trafficking in aged hosts. This also likely contributes to the reduced induction of Tfh cells in the lymph nodes of aged mice. Finally, we found that fewer aged cells produce cytokines and that the pattern of cytokine production is also different, aged mice having more cells producing IL-17 but less cells producing IL-4, IL-10 and IFNγ than young mice. Thus, not only is the CD4 T cell response reduced with age, it is also qualitatively different which could account for reduced induction of protection following immunization.
Sensitivity of CD4+ effector memory T cells to Fas-induced apoptosis is due to increased localization of Fas to lipid raft microdomains (104.9)
Authors: Cruz Anthony , Ramaswamy Madhu , Siegel Richard
Published: January 2023

Fas-FasL interactions play a critical role in TCR-induced apoptosis of activated CD4+ T cells. Fas deficiency prompts accumulation of CD44+ memory T cells and autoimmunity in mice, suggesting Fas plays a role in memory and/or autoreactive T cell apoptosis. We have recently shown that CD4+ effector memory cells were sensitive to Fas-induced cell death, and also a correlation between lipid raft-associated Fas and sensitivity to apoptosis. A critical cysteine residue near the transmembrane helix of Fas has been shown to be responsible for its association with lipid rafts. Whether the lipid raft association of Fas is regulated in different primary cell types is not known. We have compared the post-translational modification state and membrane localization of Fas receptor in purified naïve versus memory human CD4+ T cells. Memory T cells have increased lipid raft-associated Fas receptor versus naïve cells, which could explain the increased Fas sensitivity in the memory pool. Using a competitive inhibitor to palmitoylation, we found the sensitivity of effector memory T cells to Fas-induced apoptosis depended on Fas palmitoylation. Utilizing click chemistry to analyze the palmitoylation state of Fas showed increased receptor palmitoylation in cells sensitive to Fas-mediated apoptosis compared to resistant cells. These results suggest post-translational palmitoylation and lipid raft localization of Fas as a regulatory mechanism in Fas-induced apoptosis in memory T cells.
Functionally impaired memory CD8 T cells are generated after vesicular stomatitis virus infection (105.13)
Authors: Obar Joshua , Zickovich Julianne , Meyer Susan , Yagita Hideo , Noelle Randolph
Published: January 2023

Intracellular pathogens are capable of inducing vigorous CD8 T cell responses. However, it is unclear whether all memory CD8 T cell populations are capable of providing robust protective immunity against secondary infection. While both vesicular stomatitis virus (VSV) and Listeria monocytogenes infection result in the generation of large memory CD8 T cell populations that are capable of rapid secondary expansion, only memory cells induced by L. monocytogenes were protective against secondary infection. The lack of protective immunity after VSV priming correlated with expression of PD-1 on memory CD8 T cells. Importantly, mAb blockade of PD-1 at the time of re-challenge enhanced the ability of the VSV-induced memory CD8 T cell population to provide protective immunity. Moreover, overt activation of CD40, but not TLR9, signaling during priming enables the memory CD8 T cell population to provide strong protective immunity. CD40 signaling appears to alter the programming of the CD8 T cell response induced by VSV infection. Our data demonstrate that not all vaccine vectors are equal in their ability to generate highly protective memory CD8 T cell populations, even though they may generate memory population capable of undergoing robust secondary expansion. Thus, understanding how the CD40 signaling pathways regulate the generation of highly protective memory CD8 T cells will provide key insights into memory CD8 T cell biology and vaccine development.
CD8-EOMEShigh T cells define a subpopulation of CD8 memory cells that restore CD127high expression after cART treatment in HIV infected patients. (105.27)
Authors: Hasley Rebecca , Hong Changwan , Le Saout Cecile , Kim Grace , Friesen Travis , Nakamura Yoriko , Park Jung-Hyun , Sneller Michael , Roby Gregg , Rehm Catherine , Lane Clifford , Catalfamo Marta
Published: January 2023

After acute viral infection a small population of effector CD8 T cells upregulate CD127 expression and develop into long lived resting memory T cells. During chronic infections, such as HIV, there is an accumulation of CD8 T cells with effector/memory-like phenotype. These observations led to the hypothesis that there is a developmental impairment in the long lived resting memory T cells within that setting. The transcription factors RUNX3 and Eomesodermin (EOMES) are important in the development of long lived memory CD8 T cells. We studied the dynamics of CD127 expression, RUNX3 and EOMES in patients with HIV infection and different levels of viremia. Consistent with other reports, CD127 expression was decreased in the memory CD8 T cell subset. The CD8 memory CD127low T cells showed increased expression of RUNX3 and EOMES mRNA, as well as effector molecules perforin and granzyme B. In patients with suppressed viremia, we could identify two populations of CD8 T cells expressing EOMEShigh and EOMESint. The EOMESint cells showed a short-lived effector phenotype (perforin+, granzyme B+, CD127low) and the EOMEShigh cells showed a memory-like phenotype (perforin-, CD127high). These results suggest that in human CD8 T cells, EOMES is necessary for the maintenance of long lived memory CD8 T cells and its expression was influenced by HIV-RNA levels and the associated inflammatory environment (R=0.4, p=0.01). These findings provide new insights into the pathogenesis of HIV infection.
Memory CD8+ T cells deficient in IFN-γ protect from a lethal viral disease (105.3)
Authors: Remakus Sanda , Rubio Daniel , Sigal Luis
Published: January 2023

Ectromelia virus (ECTV), the causative agent of mousepox, is a lethal mouse pathogen of the Orthopoxvirus genus, which also includes variola virus. C57Bl/6 (B6) mice are resistant to ECTV infection while mice such as DBA2/J are susceptible. B6.D2-D6 mice, a B6 congenic strain with the NK complex from DBA2/J are sensitive to ECTV and can be protected by adoptive transfer of memory CD8+ T cells (MTCD8+) from convalescent mice. To ascertain the role of IFNγ in MTCD8+ mediated protection, we transferred MTCD8+ from IFNγ-/- mice (Mγ-/-) into B6.D2-D6 mice and challenged them with ECTV. All naïve CD8+ (NB6) recipients died and all recipients of memory B6 CD8+ (MB6) or Mγ-/- recipients survived. Seven dpi, MB6 and Mγ-/- recipients had significantly less liver damage and virus titers in the spleen and liver than NB6 recipients. This demonstrates that MTCD8+ do not have to produce IFNγ to protect from a viral disease. Of interest, we also found that donor virus-specific cells promoted an endogenous virus-specific TCD8+ response in the spleens and livers of recipients. Next we determined if IFNγ was necessary when MTCD8+ cells were present by transferring MB6 or Mγ-/- CD8+ into IFNγ-/- mice. All NB6 and Mγ-/- recipients succumbed to mousepox while all recipients of MB6 recipients survived. Thus, IFNγ is required for resistance to mousepox but can be produced in sufficient quantities for protection by MTCD8+ or other non-memory cells, probably NK cells or primary T cell effectors.
Comprehensive evaluation of the CD8 responses to HSV-1 in humans (105.41)
Authors: Jing Lichen , Haas Juergen , Dann Greg , Dong Lichun , Liang Kerry , Wald Ann , Koelle David
Published: January 2023

CD8-only vaccines can be protective on some mouse models. HSV-1-specific CD8s localize to sites of lesion and healed skin, and to chronically infected ganglion. We enriched polyclonal HSV-1-specific CD8s from human blood using a novel cross-presentation/activation marker-based protocol. After expansion, these cell populations were quite oligoclonal as assessed by TCR beta chain CDR3 deep sequencing. To query fine specificity, artificial APC were created by transfecting Cos-7 cells with each HLA A or B heavy chain from the subject. Each HSV-1 ORF was cloned into an expression vector and separately co-transfected with each HLA allele. Activation of CD8+ cells in the polyclonal T-cell population to specific ORFs was detected by interferon-gamma ELISA. The complexity of the response ranged from 7 to 23 reactive ORF/HLA combinations per subject. Most such epitopes are HSV-1 type-specific, but some are identical within the related pathogen HSV-2. One type-common epitope was studied in HLA A*0201-transgenic mice, in which DNA vaccination with the HSV-1 full-length ORF lead to very strong epitope-specific responses. Overall, we observed a remarkably similar complexity for the CD8 response to HSV-1 by using either the deep TCR sequencing or antigenic specificity method. Novel candidate immunodominant HSV-1 CD8 antigens were identified, and no apparent skewing to virion input or immediate early proteins due to TAP inhibition was detected.
The tug-of-war between dendritic cells and human chronic viruses (105.47)
Authors: Jain Pooja
Published: January 2023

The human immune system is under constant challenge from many viruses, some of which the body is successfully able to clear. Other viruses have evolved to escape the host immune responses and thus persist, leading to the development of chronic diseases. Dendritic cells (DCs) are professional antigen presenting cells that play a major role in both innate and adaptive immunity against different pathogens. For the past few years our efforts have been focused on exploring the participation of DCs in the pathogenesis of HTLV-1, HIV-1 and HCV. We observed previously that depletion of DCs in CD11c-DTR transgenic mice enhanced the susceptibility to cell-free HTLV-1 infection. We further performed the host-pathogen interaction studies utilizing Flt3 ligand derived murine bone marrow DCs (FL-DCs). First, the kinetics of viral entry, proviral integration, and expression of the viral protein Tax was established and then effects of cell-free HTLV-1 was examined on these cells. Phenotypically, FL-DCs demonstrated activation and produced an array of proinflammatory cytokines as well as IFN-α. Virus-matured FL-DCs also stimulated proliferation of autologous CD3+ T cells and IFN-γ production. Gene expression studies revealed upregulation of interferon-stimulated genes, most cytokines, and transcription factors but a distinct downregulation of many chemokines. Overall, these results highlight the critical interaction of DCs with a human chronic virus important for the early immune responses.
A novel, protective Toll-like receptor independent innate immune response to HSV-1 in the cornea (105.51)
Authors: Conrady Christopher , Zheng Min , Wuest Todd , Mandal Nawajes , Tran Julie , Liu Chuanju , Carr Daniel
Published: January 2023

Toll-like receptors (TLRs) are innate sentinels required in clearance of bacterial and fungal infections of the cornea, but their role in viral immunity is currently ambiguous. To evaluate the role of TLRs in ocular immunity to viral infection, TLR adaptor protein deficient mice (MyD88-/- and Trif-/-) were infected with HSV-1. MyD88-/- and Trif-/- mice had similar viral loads in the cornea as those of wild type (WT) mice but substantially lower levels than type 1 interferon receptor A1 deficient (CD118-/-) mice 48-120 hours post infection (pi). A chimeric mouse model utilizing WT and CD118-/- mice was then employed and identified both a resident and bone marrow (BM)-derived immune component in viral containment 48 hours pi. The BM contribution was linked to infiltrating F4/80+ GR1+ monocytes that were severely diminished in CD118-/- mice compared to all other groups, corresponding to a loss of CCL2 production. CCL2 deficient mice were found to possess significantly more virus, consistent with a loss of F8/80+ GR1+ infiltrating monocytes in the cornea compared to WT controls. To identify the key sentinel in viral recognition of the cornea, the DNA sensor, p204, was first identified in mouse corneal epithelium by confocal microscopy and Western blot. In vivo siRNA knockdown of the sensor resulted in significant elevation of virus. Taken together, these results describe a novel TLR-independent innate immune sensor critical in the initial control of acute HSV-1 infection.
Nanolipoprotein particles as vaccine platforms for co-localization of adjuvants and subunit antigens (106.10)
Authors: Blanchette Craig , Hoeprich Paul , Fischer Nicholas
Published: January 2023

Subunit vaccines provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, these antigens require immunostimulatory adjuvants to generate the appropriate adaptive immune response. We hypothesize that the efficacy of subunit antigens will be enhanced if co-localized with adjuvant during delivery in a single delivery platform. Thus, our goal is to develop a robust vaccine delivery platform that will allow for the co-delivery of both subunit antigens and adjuvants using nanolipoprotein particles (NLPs). NLPs are discoidal HDL analogs comprised of lipid bilayers stabilized by scaffold proteins. These biocompatible, nanoscale particles can be assembled to incorporate nickel-chelating lipids to form nickel-chelating NLPs (NiNLPs). Thus, any His-tagged antigens can be conjugated to the NiNLPs. The amphipathic adjuvants monophosphoryl Lipid A (MPLA) and cholesterol-modified CpGs (Chol-CpGs) were anchored to the lipid bilayer of the NiNLPs. His-tagged influenza hemagglutinin 5 was conjugated to adjuvant:NiNLPs and injected into mice. Antibody titers increased 32 and 8 fold (MPLA and CpG, respectively) compared to co-administered formulations. Also, the immune response was skewed towards a Th1 response, with up to 16-fold increases in IgG2a:IgG1 ratios. Our findings demonstrate that co-localized delivery of adjuvant with antigens provides significantly greater immune stimulation in mice compared to co-administered formulations.
Dendritic cell function and T cell responses to influenza viruses in patients with X-linked agammaglobulinemia (106.12)
Authors: Liu Yinping , Yuet Wu , Tu Wenwei , Lau Yu-Lung
Published: January 2023

Immunodeficiency patients have high risks for influenza infection. X-linked agammaglobulinemia (XLA) is a primary immunodeficiency characterized by the deficiency of humoral immunity. Hence, cellular immune response becomes more important for these patients. However, little is known about the non-humoral immune responses to influenza virus in XLA patients. Here, we examined the APC function and T-cell immune responses to influenza viruses in XLA patients. Monocyte-derived immature dendritic cells (imMDDCs) from XLA patients had normal maturation upon inactivated influenza virus stimulation in terms of surface molecule expressions and cytokine productions. The T-cell stimulatory capacity of influenza virus-treated imMDDCs determined by mixed lymphocyte reactions from XLA patients was also similar as that from normal controls. There were no obvious side effects in XLA patients after administration of trivalent inactivated influenza vaccine (TIV). TIV significantly induced influenza virus-specific CD4 and CD8 T-cell responses in XLA patients. The frequencies of influenza virus-specific memory/effector CD4 and CD8 T cells in XLA patients are comparable to that in normal controls during 6 months of observation after vaccination. Our results indicated that XLA patients have normal APC function and T cell responses to influenza virus. Influenza vaccination may provide some benefits to XLA patients for influenza infection, at least reducing the severity of illness.
Control of MRSA sepsis by CCL1 antisense ODN in severely burned mice (106.23)
Authors: Asai Akira , Kogiso Mari , Herndon David , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Severely burned mice are very susceptible to MRSA infection. M1Mϕ, a major effector cell in host antibacterial innate immunity, were not generated in mice 1 to 3 weeks after severe burn injury, because these mice carried M2bMϕ predominantly. Previously, CCL1 was shown to be needed to maintain M2bMϕ properties, and the elimination of M2bMϕ was demonstrated in burned mice treated with CCL1 antisense ODN. Therefore, in this study, we tried to control MRSA infection by CCL1 antisense ODN treatment in mice 1 to 3 weeks after burn injury. Burned mice (25% TBSA, 3rd degree flame burn) were treated s.c. with 10 μg/mouse of CCL1 antisense ODN twice a day for 2 days beginning 13 days after burn injury. Fourteen days after burn injury, these mice were infected i.v. with 107 CFU/mouse of MRSA. The severities of MRSA infection were evaluated by (i) the growth of the pathogen in organs and blood and (ii) their mortality rates, as compared with controls (burned mice treated with scrambled ODN). In the results, all burned mice treated with CCL1 antisense ODN survived after MRSA infection, while 83% of burned mice treated with scrambled ODN died. After infection, MRSA grew in the kidneys and blood of burned mice treated with scrambled ODN, while greatly diminished growth of the pathogen was shown in these organs of burned mice treated with CCL1 antisense ODN. These results indicate that MRSA sepsis is effectively controlled in burned mice subjected to CCL1 gene therapy.
Anti-lipopolysaccharide IgM antibody exhibits vibriocidal activity against Vibrio cholerae in the clinical sera (106.4)
Authors: Yang Jae Seung , Kang Seok-Seong , Kim Kyoung Whun , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Serum vibriocidal antibody assay has been used to evaluate the immunogenicity of cholera vaccines formulated with killed whole-cell of Vibrio cholerae. However, serum antibodies representing the vibriocidal activity are not fully characterized. In this study, we examined which isotype is associated with the vibriocidal activity using human convalescent sera with high virbiocidal activity and 20 clinical serum samples obtained from human subjects who had been vaccinated with a killed, whole-cell cholera vaccine. Total IgG, IgA or IgM antibody levels were not correlated with the vibriocidal activity in the 20 human clinical sera. When IgG, IgA and IgM antibodies specific to each of the heat-killed V. cholerae, lipopolysaccharide (LPS), or outer membrane protein U were measured in the 20 clinical serum samples, anti-LPS IgM antibody showed the highest correlation with the vibriocidal activity (Pearson correlation coefficient r = 0.818). Depletion of IgM antibodies in the convalescent sera produced a significant (P<0.05) decrease in the vibriocidal activity (16-fold decrease), whereas little change was observed with depletion of IgG and/or IgA. Furthermore, size-exclusion column chromatography followed by vibriocidal assay and analysis of antibody isotypes demonstrated the co-elution of vibriocidal activity and IgM antibody. Collectively, these results suggest that LPS-specific IgM could be a potential serologic marker for the vibriocidal antibody assay.
Anti-CD3 mAb therapy promotes tolerance by selectively depleting pathogenic cells while preserving regulatory T cells (107.12)
Authors: Penaranda Cristina , Tang Qizhi , Bluestone Jeffrey
Published: January 2023

Monoclonal anti-CD3 antibodies have been used clinically for two decades to reverse steroid-resistant acute graft rejection. In autoimmune diabetes, short course treatment with FcR non-binding anti-CD3 mAb in mice with recent onset of diabetes induces long-term disease remission. Induction of tolerogenic regulatory T cells (Tregs) has been implicated to be one of the mechanisms of action by FcR non-binding (FNB) anti-CD3 mAb in these settings. In this study, we examined the effect of FNB anti-CD3 mAb treatment on the homeostasis of CD4+CD25+Foxp3+ Treg cells in vivo. Anti-CD3 mAb treatment induced a transient systemic rise in the percentage but not absolute number of Tregs due to selective deletion of CD4+CD25- naïve and effector T cells. T cell depletion induced by FNB anti-CD3 mAb was independent of pro-apoptotic proteins Fas or Bim and was caspase 3 independent. Tregs were not preferentially expanded and we found no evidence of convertion of conventional T cells into Tregs suggesting that Tregs are resistant to anti-CD3-induced cell death. Interestingly, we found increased Helios expression on Tregs further showing that adaptive Tregs are not induced by anti-CD3 treatment. Taken together, our results suggest that FNB anti-CD3 mAb therapy selectively induce death of potentially pathogenic CD25- T cells and stabilized, although did not induce, Tregs, thus promoting tolerance by restoring the balance between pathogenic T cells and regulatory T cells.
Structural requirements for the inhibition of calcium mobilization and mast cell activation by the pyrazole derivative BTP2 (107.16)
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 and their contribution to disease. We find that the pyrazole compound BTP2 inhibited FcϵRI-triggered sustained Ca2+ influx in RBL-2H3 cells and murine BMMCs 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. In vitro, BTP2 inhibited FcϵRI-triggered degranulation and cytokine secretion, correlating with inhibition of NFAT nuclear translocation. Consistent with this effect, BTP2 inhibited antigen-induced histamine release in vivo. 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 BMMCs, 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.
The effect of B-cell depletion in Theiler's viral model of multiple sclerosis (107.17)
Authors: Li Libin , Matsumoto Makiko , Pachner Andrew
Published: January 2023

Introduction: B cell depletion (BCD) has been utilized to treat relapsing-remitting multiple sclerosis (MS), but it is also not clear whether BCD ameliorates disability progression. We used an anti-mouse CD20 mAb (5D2), and tested its effects in Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TIDD), a viral CNS infection considered the best experimental animal model of disability progression in MS. Methods: SJL mice were injected intraperitoneally with 5D2 15 days before intracerebral infection of TMEV BeAn strain. 5D2 bioactivity was assessed by flow cytometry assay of CD19+ cells in peripheral blood mononuclear cells (PBMC). Mouse disability was measured by Rotarod, and viral load was measured by real-time reverse transcriptase PCR (RT-PCR) for TMEV RNA. Antibody levels were determined in mouse sera and CSF by ELISA, and in CNS tissues by real-time RT-PCR for IgG RNA. Results: Bioactivity of 5D2 was confirmed by nearly absent CD19+ cells in the peripheral blood from animals with persistent BCD. Antibody responses in both CNS and peripheral system were suppressed during 5D2 treatment. Relatively higher viral loads were detected in 5D2-treated mice than in PBS animals, and the viral levels correlated negatively to IgG production in the brain. 5D2 caused marked worsening of the early encephalitis, possibly from deletion of a B cell phenotype which downregulates inflammation.
Combined antibody therapy for type 1 diabetes (107.2)
Authors: Hu Changyun , Ding Heyuan , Wong F. Susan , Wen Li
Published: January 2023

Studies have shown that oral tolerance is effective in treating different models of autoimmune diseases including type 1 diabetes (T1D). Both T and B cells play important roles in the development of T1D. Antibodies targeting either T or B cells have shown to be effective in treating T1D in both patients and NOD mice. However, individual antibodies are only effective in a proportion of those treated. Furthermore, the beneficial effects have not been long lasting. To test whether the combination of anti-CD20 and oral anti-CD3 treatment can improve the efficacy in prevention and reversal of T1D and prolong the beneficial effects, we treated a group of human CD20 transgenic NOD mice with anti-hCD20 monoclonal antibody and oral anti-CD3. The results suggested that anti-CD20 and anti-CD3 have synergistic effects in the diabetes prevention and reversal. Mechanistic studies have demonstrated that the combination treatment induced FoxP3+ Tregs and enhanced the regulatory function of Treg. A subset of IL-10+ CD4 T cells was also induced in peyer’s patches of the mice treated with combined therapy. Our preliminary study may provide a novel therapeutic approach to treating T1D.
Development of antigen-loaded, immunosuppressive microparticles to modify dendritic cell behavior for the prevention of type 1 diabetes in NOD mice. (107.9)
Authors: Keselowsky Benjamin , Lewis Jamal , Carstens Matthew , Xia Chang Qing , Clare-Salzler Michael
Published: January 2023

Antigen-specific immunomodulation remains an important goal in the treatment of type 1 diabetes (T1D). We hypothesize that in vivo targeting of biodegradable microparticles (MPs) to DCs delivering immunomodulatory agents can promote a tolerogenic DC phenotype and induce Tregs in vivo, in order to ameliorate type 1 diabetes in NOD mice. To test this hypothesis, poly(lactide-co-glycolide) MPs were loaded with antigen - insulin B:9-23, and immunomodulatory factors - vitamin D3 (D3), TGF-B1 and GM-CSF. These MP formulations were injected subcutaneously into 4 week old female NOD mice followed by a booster at 5 weeks of age. Formulations (n = 10) consisted of a.) blank MPs, b.) MPs loaded with insulin peptide only, c.) MPs loaded with GM-CSF + insulin peptide, d.) MPs loaded with D3+TGF-B1+insulin peptide, e.) MPs loaded with GM-CSF+D3+TGF-B1 + insulin peptide. Blood glucose was measured once a week until 32 weeks of age (currently at 22 weeks). Kaplan-Meier analysis at this point reveals an overall p-value of 0.094 for survival proportions among groups, with the highest proportion of non-diabetic mice attributed to the formulation of MPs loaded with GM-CSF+D3+TGF-B1+insulin peptide (60% non-diabetic), compared to the blank MPs with the lowest proportion (20% non-diabetic), suggesting delayed onset of T1D. In vitro mechanistic assessments demonstrate MP formulations are capable of promoting tolerogenic DC phenotype and inducing syngeneic CD4+CD25+foxp3+ regulatory Tcells.
Immunity to secondary bacterial pneumonia caused by Streptococcus pneumoniae following Influenza A infection (108.10)
Authors: Bernui Mariana , Liboon Jennie , Shen Hao
Published: January 2023

IAV is highly contagious and responsible for both seasonal and pandemic flu. However, mortality associated with IAV is mainly due to secondary bacterial infections. The bacterial and host factors that mediate lethality, as well as the protective immune mechanisms against co-infections, are largely unknown. By sequentially infecting mice with IAV and Sp (IAV/Sp), we show that IAV-infected mice are highly susceptible to secondary Sp infections (<10 CFU). Mice lacking IL-12, Type 1 IFN receptors, or both were just as susceptible to an IAV/Sp infection, suggesting these inflammatory cytokines are not involved in lethality. IAV-specific CD8 T cells from IAV/Sp co-infected mice had increased PD-1 expression, a marker associated with dysfunctional/exhausted CD8 T cells. When we immunized mice with different strains of IAV or Sp and challenged them with IAV/Sp co-infection, IAV-immunized mice were fully protected against an IAV/Sp infection. Immunization with one IAV strain provided cross-protection against a co-infection challenge with a different serotype strain of IAV. Immunization with Sp prolonged survival of co-infected mice, and was IL-12 dependent. We conclude that 1) Early inflammatory cytokines are not responsible for death caused by IAV/Sp co-infections, 2) Secondary Sp infections do not affect the magnitude but the quality of IAV-specific CD8 T cell responses, and 3) cross-protective immunity against IAV/Sp co-infections can be induced by immunization with IAV or Sp.
RhoH is a key functional determinant of the “stop and go” nature of CD4+ T cells during activation (109.17)
Authors: Baker Christina , Comrie William , Fedorchuk Christine , Hyun Young-Min , Meier-Schellersheim Martin , Kim Minsoo
Published: January 2023

CD4+ T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by APCs, and then cells quickly recirculate through the bloodstream to another lymph node. Therefore regulation of a CD4+ T cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients (“go” signals) as well as to receive appropriate TCR activation signals upon cognate antigen recognition (“stop” signals). The mechanism(s) by which a CD4+ T cell regulates these “go” and “stop” signals are not well defined. We find that over-expression of the hematopoietic specific RhoH protein in the presence of chemokine signals results in decreased Rap1-GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T cell chemotaxis, while in the presence of TCR signals there is enhanced and sustained Rap1-GTP and LFA-1 activation as well as prolonged T:APC conjugates. RhoH-/- CD4+ T cells appear to circulate through the blood and lymphoid tissue more quickly corroborating our in vitro migration data. RT-PCR analyses of activated CD4+ T cells and live images of T cell migration and immunological synapse formation have revealed that functions of RhoH take place primarily at the levels of transcription and intracellular distribution. Thus, we hypothesize that RhoH expression may provide a single molecular determinant that allows a CD4+ T cell to switch between sensing chemokine mediated “go” signals and TCR dependent “stop” signals.
PIM kinase activity is required for B cell function in vitro and in vivo (109.5)
Authors: Jackson Leila , Pheneger Jed , Wright A. , Robinson John , Allen Shelley , Hicken Eric , Carter Laura
Published: January 2023

The PIM family of serine/threonine kinases has roles in proliferation, differentiation, and cell survival. In lymphocytes, their activity is regulated by inducible expression following cytokine or antigen receptor signaling. Using small molecule inhibitors, we have examined the role of PIM1/3 kinases in B cells in vitro and in vivo and in the MRL/LPR model of lupus. PIM kinases were expressed 1-3 hours after B cell activation with anti-IgM or anti-CD40 but not BAFF. Inhibition of PIM-1/3 kinase activity decreased anti-IgM- and anti-CD40-induced B cell proliferation without inducing apoptosis. AR-254 also prevented IL-4-, and IFNy-induced B cell survival, but had no affect on BAFF-induced B cell survival consistent with the inability of BAFF to upregulate PIM expression. AR-770 administration in the HEL immunization model resulted in a 90% reduction of HEL-specific IgG2a and IgG3, but had no effect on IgG1 suggesting a role for PIM kinases in IFNy-mediated class switching. In the MRL/MpJ-Fas lupus model, mice treated with AR-770 had >90% reduction in proteinuria AUC, normalized anti-dsDNA antibody titers and ~80% decrease in kidney glomerulo- and interstitial nephritis as well as vessel and protein cast formation. Under specific conditions, Taken together, the data suggests that inhibition of PIM kinases may be a useful strategy for patients with B cell-mediated diseases such as lupus.
Role of C-reactive protein in regulatory monocyte induction and activation in trauma patients (110.18)
Authors: Mold Carolyn , West Sonlee , Du Clos Terry
Published: January 2023

C-reactive protein (CRP) is an acute phase reactant. Its plasma concentration increases from <5 to >100 µg/ml within 48 h of injury due to increased synthesis by hepatocytes responding to IL-6. Highly elevated CRP levels are seen in trauma, and maintenance of these levels is associated with a poor prognosis, but the role of CRP in trauma is poorly understood. We measured a panel of monocyte markers and cytokines in 27 trauma patients for 3 days following admission and analyzed their correlation with CRP levels. We identified a subpopulation of monocytes expressing high levels of CD14 and CD16 that were not found in healthy volunteers. These cells also expressed CD163 and HLA-DR. The percentage of monocytes found in this subpopulation was highly correlated to plasma CRP levels (p=0.0001) and to intracellular heme oxygenase (HO-1) (p=0.0009). CRP levels also correlated with monocyte HO-1, and plasma anti-inflammatory cytokines, IL-10, IL-1RA, and TGF-β. We hypothesized that CRP activates monocytes to a regulatory phenotype as part of the compensatory anti-inflammatory response in trauma. Incubation of isolated monocytes with trauma plasma, but not CRP-depleted trauma plasma, increased CD16 and HO-1. Differentiation of monocytes with M-CSF also induced CD16 and HO-1, and M-CSF-differentiated monocytes responded to CRP by secretion of IL-10 and IL-1RA. These results suggest a role for CRP in the generation and activation of regulatory monocytes during the acute phase response.
Transgenic expression of MDA5 enhances interferon responses, CD8 activation, and viral clearance (110.19)
Authors: Crampton Steve , Deane Jonathan , Otubusin Owokunile , Hasty Karen , Bolland Silvia
Published: January 2023

MDA5 is a pattern recognition receptor responsible for detecting double stranded viral RNA in the cytoplasm. Ligation of MDA5 leads to the induction of inflammatory cytokines and type one interferons (IFN-I) that help coordinate the adaptive and innate immune response against the virus. Here, we describe the phenotype of mice overexpressing MDA5 under its endogenous promoter. MDA5 transgenic (MDA5 Tg) mice have normal numbers of B and T cells, yet expanded numbers of CD11b+ cells expressing Ly6C. Splenocytes isolated from MDA5 Tg display an interferon gene expression signature. MDA5 Tg CD8+ T cells have increased surface expression of CD44, CD69, Ly6C and Ly6A/E, indicative of an activated phenotype. In vitro, CD8+ T cells from MDA5 tg hyperrespond to stimulation. Surprisingly, MDA5 tg mice fail to mount a CD8+ T cell-driven antigen-specific immune response against Vesicular stomatitis virus (VSV). MDA5 tg mice also lacked VSV neutralizing antibodies 16 days after infection with the virus. Although MDA5 tg mice failed to mount a full immune response against the virus, they were resistant to a lethal dose of VSV. This result suggests that increased viral resistance of MDA5 Tg is independent of antibody-neutralization and CD8+ T cell-mediated clearance, and might be due to high expression levels of interferon inducible inhibitors of virus replication. MDA5 tg mice could serve as a model for diseases involving type one interferon and CD8+ T cells, such as Type One Diabetes.
The role of autophagy and amphisome formation in virus recognition and virus-stimulated IFN-α production by human plasmacytoid dendritic cells (111.30)
Authors: Deng Jing , Dai Jihong , Fitzgerald-Bocarsly Patricia
Published: January 2023

pDC secrete large quantities of IFN-α in response to viral nucleic acid stimulation via TLR7/9. However, little is known about virus trafficking in pDC after entry. To investigate the subcellular trafficking of viral particles, we examined intracellular events after viral stimulation. The formation of double-membrane autophagosomes was observed by EM within 2hr stimulation of pDC with HSV, Flu, HIV-1 or CpGA. Class I/III PI3K inhibitors 3-methyladenine, wortmannin and LY294002 significantly suppressed virus-induced IFN-α production at an early stage. However, using Amnis imaging flow cytometry, we found that uptake of HSV-GFP was not inhibited, suggesting virus-induced autophagy rather than uptake was specifically blocked by PI3K inhibitors. To further investigate events related to virus-induced autophagy, we studied the colocalization of HSV-GFP particles, early endosomal marker CD71 and autophagagosomal marker LC3B. CD71 co-localized with LC3B after pDC stimulation with virus or CpGA, indicating amphisomes, which are autophagic vacuoles formed from fusion of autophagosomes and endosomes, were induced. With chloroquine, an inhibitor of endosomal fusion and acidification, there was accumulation of the internalized HSV-GFP particles and amphisomes. Collectively, our studies demonstrate that amphisomes are induced by virus or TLR9 ligand stimulation in pDC. Further studies will be conducted to characterize the role of these amphisomes in virus-induced IFN-α production.
Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria (111.33)
Authors: Miao Edward , Leaf Irina , Treuting Piper , Mao Dat , Sarkar Anasuya , Wewers Mark , Aderem Alan
Published: January 2023

Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of IL-1β and IL-18. While wild type Salmonella typhimurium infection is lethal to mice, a strain that persistently expresses flagellin was cleared by the cytosolic flagellin detection pathway via NLRC4 activation of caspase-1; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1 induced pyroptotic cell death released bacteria from macrophages, exposing them to uptake and killing by reactive oxygen species in neutrophils. Similarly, caspase-1 cleared Legionella and Burkholderia by cytokine independent mechanisms. Our results show, for the first time, that caspase-1 can clear intracellular bacteria in vivo independent of IL-1β and IL-18, and establish pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.
RGS2 is a negative regulator in STAT3-mediated Nox-1 expression (111.5)
Authors: Baek Suk Hwan , Lee Hyung-Kyoung , Kim Jin-Sik , Park Dae-Weon , Chin Byung-Rho , Bae Yoe-Sik
Published: January 2023

RGS2 protein is well known as a negative regulator of GPCR signaling. In our previous research, we understood that RGS2 mRNA expression is decreased by TLR4, and thereby foam cell formation is induced. The present study proved how the decreased RGS2 regulates specific gene, Nox-1, expression. While TLR2 stimulation increased Nox-1 expression and decreased RGS2 expression in wild type mice macrophages, such phenomena did not occur with TLR2 KO mice macrophages. Nox-1 expression increase by TLR2 was intensified by RGS2 siRNA, and was inhibited by RGS2 overexpression. TLR2 stimulation caused phosphorylation of tyrosine 705 and serine 727 residues, and STAT3 siRNA inhibited Nox-1 expression by TLR2. RGS2 directly associated with STAT3 acting as a negative regulator by inhibiting Nox-1 expression by TLR2. PKC-eta and PLD2 also participated in Nox-1 regulation by controlling RGS2 expression. Overall, these results suggest that TLR2 stimulation decreases RGS2 expression mediating PKC-eta and PLD2, and thereby will increase Nox-1 expression by releasing the transcription ability of STAT3 restrained by RGS2.