bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR3 and TLR9 mRNA expression is impaired in macrophages from severely burned mice (44.31)
Authors: Kobayashi Makiko , Yoshida Tsuyoshi , Herndon David N , Suzuki Fujio
Published: January 2023

Classically activated Mϕ (CAMϕ) are not generated in mice with severe burn injuries even when they are stimulated with a mixture of poly I:C and CpG DNA (well-known inducers for CAMϕ). To investigate why the Mϕ conversion from burn mouse-derived Mϕ to CAMϕ was not induced after stimulation with a mixture of poly I:C and CpG DNA, the expression of receptors for poly I:C (TLR3) and CpG DNA (TLR9) in burn mouse-derived Mϕ were examined. BALB/c mice exposed to a flame burn injury (3rd degree, 25% TBSA burns) were used for these studies. Mϕ were prepared from the peritoneal cavities of mice 6, 24, 48, 120 and 168 hrs after burn injury using magnetic beads coated with anti-F4/80 mAb. Peritoneal Mϕ from unstimulated normal mice were utilized as a control. Then, Mϕ preparations obtained were analyzed for TLR3 and TLR9 mRNA expression using RT-PCR. In the results, TLR3 and TLR9 mRNAs were shown to be expressed by Mϕ from normal mice. However, TLR3 mRNA expression was not demonstrated in Mϕ from mice 6 to 168 hrs after burn injury. TLR9 mRNA expression was strongly impaired in Mϕ from mice 6 hrs after burn injury, and then it gradually recovered until 120 hrs after burn injury. These results indicate that burn mouse-derived Mϕ are shown to not be a carrier of TLR3 and TLR9. The unresponsiveness of burn mouse-derived Mϕ to the CAMϕ inducers may be related to the impaired expression of TLR3 and TLR9 that are receptors for poly I:C and CpG DNA, respectively.

This study was supported by SHC NA #8550.
Loss of TLR7 receptor expression in HCV viral infection as a potential mechanism to evade the innate immune system (45.13)
Authors: Chang Serena , Szabo Gyongyi
Published: January 2023

Hepatitis C Virus (HCV), a single stranded RNA virus that causes chronic inflammation in the liver, has evolved ways to interfere with the innate and adaptive immune system for survival. Recent studies show that Toll-like Receptor (TLR) 7 agonists decrease HCV expression in patients and infected cell lines, through a Type 1 IFN-mediated pathway. As it is unknown whether HCV interferes with TLR7 or its pathway, we investigated changes in TLR7 expression during HCV infection. Compared to 75% of the positively stained TLR7 expressing controls, human hepatoma cells infected with the replicating JFH-1 HCV clone, showed only 25% TLR7 expression by flow cytometry. Cells expressing full-length or sub genomic HCV replicons followed the same pattern as JFH-1 with 11% and 18% TLR7 expression compared to the wild-type expressing 56% and 42%. Micro array analysis of HCV infected livers showed a decrease in TLR7 mRNA expression suggesting in vivo correlation. TLR7-mediated signaling involves activation of IRF7 and IRF3 for type I IFN induction. IRF7 nuclear protein levels in JFH-1 cells were equivalent to control cells, as opposed to a drastic reduction in IRF3 levels and IP-10, an IFN-inducible gene, in JFH-1 cells vs. control cells. These results demonstrate an attenuation of TLR7 expression in Hepatitis C Virus infection that may represent a novel mechanism to prevent sufficient innate immune responses in HCV elimination.
Regulation of the CD8 T cell response to Influenza virus by Lymph Node Dendritic Cells (46.2)
Authors: Langlois Ryan Andrew , Legge Kevin L
Published: January 2023

Mice infected with high dose lethal inoculums of Influenza virus display increased morbidity and mortality when compared with sublethal inoculums. Previously, we have demonstrated that the increased disease observed in lethally infected mice is a result of a severe lymphopenia of influenza-specific CD8 T cells. This decrease in CD8 T cells following lethal inoculums is mediated through FasL+ lymph node dendritic cell (LNDC) driven apoptosis of activated Fas+ CD8 T cells. In contrast, during sublethal inoculums, FasL expression on LNDC is down regulated allowing for strong CD8 T cells responses and recovery from infection. Interestingly, our data suggests that only a fraction of LNDC down-regulate FasL expression during sublethal inoculums. Therefore, we herein determined which of the 6 subsets of LNDC mediate the apoptosis of influenza-specific CD8 T cells during lethal influenza virus inoculums. Our results demonstrate that while both plasmacytoid DC (pDC) and CD8α+DC down regulation FasL expression during sublethal inoculums, our initial studies show that CD8?+DC but not pDC are able to mediate apoptosis of influenza-specific CD8 T cells. Overall our results suggest that regulation of FasL expression on CD8α+DC may control the magnitude of the influenza-specific CD8 T cell response and ultimately the outcome of influenza virus infections.
Evasion of the innate response of circulating DC populations by foot-and-mouth disease virus (46.6)
Authors: Nfon Charles K. , Ferman Geoffery S. , Golde William T.
Published: January 2023

The evolution among viruses of multiple mechanisms to evade the innate immune response, particularly the actions of interferons, has been described for FMDV infected cells. However, mechanisms involving the response of cells of the innate immune system, especially uninfected cells, are not well understood. Here we report the analysis of IFN producing dendritic cell subsets of PBMC. When pigs were infected with two serotypes of FMDV, viremia developed and serum IFNa peaked at 2–3 days after inoculation. IFNa in serum is no longer detectable 4 to 5 days post infection. The IFNa response to CpG of plasmacytoid dendtritic cells (pDC) was significantly diminished. In addition, monocyte derived dendritic cells (MoDC) of pigs produce IFNa in response to poly I:C but not CpG. During FMDV infection, the MoDC response to poly I:C stimulation is blocked with similar kinetics to viral inhibition of pDC. Analysis shows no infection of PBMC taken from these animals. We propose that this virus has evolved a mechanism to shut down the IFNa response of DC in the periphery. The virus induces a rapid IFN response from dendritic cells which is then blocked by anti-inflammatory signals from viral infected cells. This skews the balance of the host/pathogen interaction in favor of lateral viral spread during acute infection. We are currently evaluating potential mechanisms by which the virus blocks cytokine responses in non-infected cells.
Binding of the green tea catechin, epigallocatechin gallate, to the CD4 receptor on human immune cells resulting in inhibition of HIV-1-gp120 binding and HIV-1 infectivity (47.18)
Authors: Nance Christina L , Siwak Edward , Westerfield Susan , Willis Van , Shearer William T
Published: January 2023

Binding of HIV-1 envelope glycoprotein, gp120, to the CD4 T cell receptor results in HV-1 infection. We previously presented evidence of high affinity binding of the green tea catechin, epigallocatechin gallate (EGCG) to the CD4 molecule. We now present evidence in parallel studies that EGCG inhibits the binding of gp120 on human CD4+ T cells and PBMC and prevents the HIV-1 infectivity of macrophages and PBMC. Binding studies utilized spectroscopy and flow cytometry. HIV-1 infectivity was assessed by HIV-1 p24 EIA. Studies indicated that EGCG binds to CD4 at the same binding pocket as gp120. EGCG markedly inhibited the binding of HIV-1-gp120 to CD4+T cells in a dose-dependent manner at physiologically relevant levels (32% at 0.2uM p<0.05, 42% at 2uM, 47% at 20uM p<0.01). EGCG significantly inhibited the HIV-1infectivity of human primary macrophages in a dose-dependent manner. There was 100% inhibition of p24 production by EGCG at 25–100uM (p<0.0001), 95% at 12uM, and 79% at 6uM (p<0.001). The response by human PBMC was similar. The control catechin did not alter gp120 binding nor inhibit HIV-1 infectivity. We conclude that EGCG is able to significantly reduce the attachment of HIV-1-gp120 to CD4, along with a decrease in the infectivity of HIV-1 to target cells. The competitive binding properties of EGCG for the CD4 binding sites by gp120 may translate to an HIV-1 preventative strategy.

Supported by NIH grant R21AT003084.
Lethally-irradiated Yersinia pestis immunization protects against the plague: a novel vaccine candidate (47.23)
Authors: Lambert Nathaniel Dane , Nilles Matthew L. , Bradley David Sherman
Published: January 2023

We provide evidence here that immunization with lethally γ-irradiated Yersinia pestis provides an effective and novel vaccine against the plague. Traditionally, neither chemical nor heat killing of bacteria has been successful in inducing an appropriate immune response to provide protection against further infection. It has been postulated, however, that inactivation via γ-irradiation would allow for retention of both adjuvant and antigenic components necessary for an effective vaccine. We hypothesize that, unlike many vaccines that induce predominately an antibody response, γ-irradiated whole bacteria will result in both a protective humoral and cellular immune response. Recent evidence that γ-irradiated Listeria monocytogenes protected mice from future exposure to L. monocytogenes supports this hypothesis.

Y. pestis, the cause of the plague, is endemic in much of the world. It creates a considerable health risk and is now also considered a putative agent for bioterrorism, although current vaccine candidates have potential pitfalls. Immunization with lethally γ-irradiated Y. pestis (600 kRads from a 137Ce source) provides significant protection against the plague in the absence of any additional stimulatory adjuvant in both inbred B10.T(6R) mice and out-bred Swiss Webster mice, LD50’s of greater than 106 CFUs in both strains, compared to 103 CFUs and 20 CFUs in sham immunized B10.T(6R) and Swiss Webster mice respectively. These data suggest both a new and effective vaccine for Y. pestis, as well as potential vaccines that have remained elusive for other bacteria.
CD8+ T cells primed by a non-replicating adeno-associated virus vector undergo apoptosis at the site of antigen expression (47.4)
Authors: Best Victoria , Bowen David , Walker Christopher
Published: January 2023

Adeno-associated virus (AAV) vectors facilitate prolonged expression of proteins in tissues of rodents and primates despite induction of cellular immune responses. To address the paradox of how these antigens persist in the face of a CD8+ T cell response, we delivered an AAV vector expressing the enhanced green fluorescent protein (eGFP) to the quadriceps muscle of Balb/c mice. Expression of eGFP in the muscle was stable for months even though high frequencies of CD8+ T cells targeting an immunodominant epitope were detected as early as two weeks after vector administration. MHC class I tetramer staining revealed effector CD8+ T cells in multiple tissues for at least 9 months. Long-lived eGFP-specific CD8+ T cells in spleen and liver maintained an effector phenotype, suggesting a block in the development of memory populations. Importantly, antigen-specific CD8+ T cells were enriched in the muscle, the site of antigen expression, when compared to spleen and liver. Moreover antigen-specific CD8+ T cells in the muscle, but not spleen or liver, bound annexin V, suggesting that localized apoptosis might also prolong expression of AAV-encoded eGFP. The possibility that programmed death of CD8+ T cells at the site of antigen expression may contribute to antigen persistence has important implications for both gene therapy and vaccine applications.
The TLR3 agonist poly (I:C) acted directly on mouse CD8 T cells and augmented their antigen-specific responses upon adoptive transfer into naïve recipient mice (48.31)
Authors: Salem Mohamed Labib , Diaz-Montero C. Marcela , Chen Yian , EL-Naggar Sabry A. , Kadima Andre N. , Gillanders William E. , Cole David J.
Published: January 2023

We have reported that in vivo administration of the toll-like receptor 3 ligand (TLRL) poly (I:C) induced rapid stimulation of the innate mediators, including NK cells, Mϕ, dendritic cells, and inflammatory cytokines. These effects strikingly augmented post vaccination CD8 T cells responses. It is not clear, however, whether poly (I:C) acts directly on CD8 T cells. Therefore, we analyzed the expression of TLRs on both CD4 and CD8 T cells and tested whether CD8 T cells respond directly to poly (I:C). Real-time PCR analysis revealed that TLRs (TLR1-TLR13) are expressed in the purified CD4 and CD8 T cells, and their in vitro treatment with 50 μg/ml poly 10 nM PMA, or with OVA peptide-pulsed splenocytes modulated the expression of the tested TLRs. Utilizing OT-1 TCR adoptive transfer transgenic mouse model, in vitro treatment of purified naïve OT-1 cells with 50 μg/ml poly (I:C) for 18 hours in the absence of antigen stimulation triggered their activation as evidenced by upregulation of the early activation markers CD69 and CD25, and downregulation of CD62L expression. Importantly, when these cells were adoptively transferred into naïve recipient followed by peptide vaccination, they showed superior expansion and activation to their naïve counterparts. These results clearly reveal a direct effect of poly (I:C) on CD8 T cells and supports the notion of direct involvement of TLRs in adaptive immune responses.
Origin and Dynamics of Tumor-Associated Foxp3+ Regulatory T Cells (49.4)
Authors: Duramad Omar , Perng Olivia , Zal M. Anna , Liu Yong-Jun , Zal Tomasz , Qin F. Xiao-Feng
Published: January 2023

Regulatory T cells (Tregs) expressing the forkhead transcription factor Foxp3 play a central role in the dominant control of immunological tolerance. Compelling evidence obtained from both animal and clinical studies have now linked the expansion and accumulation of Foxp3+ Tregs at the tumor site to the failure of immune-mediated tumor rejection. However, little is known regarding tumor-associated Foxp3+ Tregs within tumor tissues. Using both subcutaneous (s.c.) tumor and lung metastasis models of MCA-205 fibrosarcoma, we have found that tumor-associated Foxp3+ Tregs are highly activated and undergo vigorous cellular proliferation. Tumor-associated Foxp3+ Tregs were also found to be more potent functionally by in-vitro suppression assays and express higher levels of membrane-bound TGF-b1. To visualize endogenous Foxp3+ Tregs, we have generated Foxp3-GFP transgenic reporter mice by inserting GFP into a bacterial artificial chromosome (BAC) encompassing the Foxp3 locus. Using multi-photon microscopy, we observed a rapid recruitment and accumulation of Foxp3+ Tregs to the nascent lung tumor metastasis as early as 3 days after tumor inoculation. Intravital imaging of established s.c. tumors revealed a distinctive behavior of Foxp3+Tregs: the suppressor cells form a well-defined periperipheral layer encasing the tumor mass, moving rapidly within the superficial plain but not perpendicularly in and out of the tumor. Together, these data highlight that tumor-associated Foxp3+ Tregs are highly dynamic in function, behavior, and origin, perhaps contributing to overall poor immunogenicty of tumors.
Tumor induced senescence of T-lymphocytes (TIST): a novel mechanism of immune evasion (49.7)
Authors: Montes Carolina Lucia , Orhue Vbenosa , Chapoval Andrei , Strome Scott , Gastman Brian
Published: January 2023

Tumor immune evasion exhibits passive and active components. Passive components include tumors becoming inconspicuous and resistant to immune-mediated killing. The active components consist of tumors induced cellular dysfunction, like apoptosis of immune effectors cells. We hypothesized that tumor infiltrating lymphocytes that encounter cancer at a low tumor:T cell ratio, for a short time, undergo senescence instead of apoptosis. Senescence is a form of cellular dysfunction that leads to the demise of the cell. We demonstrated that a high percentage of human T cells from healthy donors incubated with different tumor cell lines for 6 hours at a 1:1 Tumor/T cell ratio and then cultured for 7 days in media only, undergo senescence as judged by the incremental loss of CD27 and CD28 expression and telomere shortening. Cell to cell contact does not seem necessary in the induction of this process. We also showed increases in the senescence associated molecules (p53, p21 and p16) in TIST. In addition, TIST occurs equally in CD4+ and CD8+ cells and both cells types suppress the proliferation of CD3− stimulated or allogenic effector cells but do not express CD25 nor Foxp3. Increased levels of INF gamma, IL10 and IL6 suggest a role for these cytokines in TIST mediated immunosuppression. This novel mechanism of immune evasion has impact in both diagnostic and treatment aspects of cancer.

Supported by Otorhinolaryngology Department.
A murine model for studying the immunobiology of basal cell carcinoma (50.12)
Authors: Mavers Melissa , Shah Maulik
Published: January 2023

The incidence of basal cell carcinoma (BCC), the most frequent human tumor, is rising. BCC incidence and aggressiveness are greatly increased in immunocompromised patients, indicating a role for the immune response in their prevention, formation, and growth. Human biopsies have exhibited a peritumoral infiltrate of lymphocytes with low numbers within the tumor itself. The Ptc1 mouse, a phenocopy of nevoid BCC syndrome, in which patients may acquire thousands of BCCs, develops tumors ~12 months after radiation exposure. To further study the immunobiology of BCC, a more practical tumor model was needed. We evaluated the use of ASZ001, a murine BCC cell line isolated and cultured from a Ptc mouse. Injection of 2.5x106 cells s.c. into Ptc1 mice elicited rapid tumor development with an average tumor volume of 58mm3 on day 2. Microscopic analysis showed an aggressive phenotype and a paucity of intratumoral T cells, similar to BCCs in the Ptc1 mouse and human biopsies. Thus, injection of ASZ001 cells into Ptc1 mice is a rapid tumor model that will be used to study the immune response to BCC.

This work was supported by the Saint Louis University Cancer Center and Department of Pediatrics.
T and B Cell Immune Responses to Tumor Antigen MUC1 are Induced by Targeting Antigen to B Lymphocytes (50.20)
Authors: Ding Chuanlin , Wang Li , Al-Gwahi Hayma , Marroquin Jose , Hansen Richard , yan jun
Published: January 2023

To test the hypothesis that MUC1 specific T cell responses may be induced by targeting MUC1 to B cells in the presence of CpG ODN adjuvant, we first conjugated a model Ag ovalbumin (OVA) with anti-CD19 mAb as a means to target Ag to B cells. OVA TCR transgenic CD8 T cells (OT-I) and CD4 T cells (OT-II) were used to exam if targeting OVA to B cells through CD19 leads to Ag presentation in the context of MHC class I and II for T cell activation. The results indicated that OVA conjugates could significantly induce CD4 and CD8 T cells proliferation and activation. These expanded T cells were not deleted or anergized, and were characterized by proliferation (OT-II T cells) and IFN-γ release (OT-I T cells) in response to OVA peptide. CpG ODN could significant enhance the survival of CD8+ T cells and IFN-γ production. To further extend these observations to tumor Ag, MUC1 transgenic (Tg) mice that are tolerized to MUC1 Ag, resembling cancer patients, were immunized with anti-CD19 conjugated MUC1 with or without CpG ODN. The data demonstrated that MUC1 conjugates in combination with CpG ODN elicit high-titer anti-MUC1 Abs. In addition, MUC1-specific cytotoxicity and vigorous IFN-γ secretion was generated. In contrast, Tg mice immunized with MUC1 peptide, even in the presence of CpG ODN, did not elicit Ab production and T cell responses. These results indicated that Ag targeted to B cells via CD19 in combination with TLR agonist could reverse immunological tolerance to MUC1 Ag and generate MUC1-specific B cell and T cell responses.
In vivo synergy of TLR ligands through activation of distinct signaling pathways in dendritic cells (52.1)
Authors: Zhu Qing , Belyakov Igor M , Klinman Dennis M , Berzofsky Jay A
Published: January 2023

Toll-like receptors (TLRs) play a critical role for the host to generate cellular immunity against invading microorganisms. Each TLR is specific for a certain range of microbial components and more than one is likely to be involved in any natural infection. We found that certain TLRs can act synergistically in response to microbial stimuli in specific, non-random, combinations. Vaccination of mice with antigenic peptide together with pairs of ligands for TLR2 and 3 or 3 and 9 effectively primed T cells in vivo, while each individual ligand or paired TLR2 and 9 ligands at the same doses were ineffective. Dendritic cells (DCs) appeared to play a central role in response to the synergistic combinations of TLR ligands by amplifying production of inflammatory cytokines and chemokines, such as IL-12, TNFα and RANTES, through MyD88-dependent signaling pathway. Costimulatory molecules like CD86 were upregulated by TLR3 ligand alone but not further increased when either TLR2 or 9 ligand was added. Also, no synergy was observed between TLR2 and 9 ligands in DC activation. We conclude that triggering of TLR3, which signals only through TRIF, enhanced the potency of the MyD88-dependent pathway used by TLR2 and 9, leading to a synergistic effect of DCs on T-cell priming. Our results suggest a strategic use of TLRs in host defense mechanisms, and may also have important implications in the design of vaccine adjuvants.
Expression of Perforin-2 is regulated via transcriptional and translational mechanisms (52.18)
Authors: SHIRATSUCHI MOTOAKI , Ramos Jahir , Deyev Vadim V , Podack Eckhard R
Published: January 2023

At the last AAI meeting we identified a macrophage-expressed protein as a pore-forming protein and called it Perforin-2 (P2). We also reported that P2 translation from the cap-structure is impaired. We further assessed the regulatory mechanisms of P2 translation.

The P2 gene has a long 5′-UTR which contains a short intron near the translation start site. Various lengths of 5′-UTR sequences were cloned into the discistronic luciferase vector, transfected into 293 or RAW cells, and luciferase activity was measured. We also performed promoter assay by using luciferase constructs containing various lengths of P2 promoter. Finally, bone marrow cells from mice were differentiated into mature dendritic cells with GM-CSF and LPS, and mRNA levels were quantified by real-time RT-PCR.

Bicistronic reporter assays showed that P2 protein expression is under translational control by an internal ribosome entry site (IRES) in the 5′-UTR. Part of the IRES is included in an intron, indicating that control of splicing affects translation.

Promoter assays demonstrated much stronger promoter activity in RAW cells than in 293 cells. The P2 gene has an ectopic promoter in the 5′-UTR. P2 transcription is upregulated dramatically during differentiation of bone marrow precursors into mature dendritic cells.

Since P2 is a pore-forming protein, the expression of P2 protein thus is tightly regulated by transcriptional and post-transcriptional mechanisms to avoid self-killing.
LPS-mediated microRNA expression in mouse macrophage (52.19)
Authors: Mi Qing-Sheng , Seo Kook-Heon , Zhou Li
Published: January 2023

MicroRNAs (miRNAs) are a recently discovered class of evolutionarily conserved small noncoding RNAs that lead to post-transcriptional repression, which is involved in the regulation of diverse biological processes including cell development and proliferation. Macrophage activation is an important event in the innate immune responses. To examine the potential involvement of miRNAs in regulation of the innate immune response, we analyzed the expression of over 270 mouse miRNAs, using miRNA array, after exposure of mouse macrophage cell line RAW264.7 to LPS. We found that more than 25 miRNAs were differentially regulated in LPS-treated RAW264.7 cells compared to untreated cells, including the up-regulation of miR-155, miR-132, miR-22, miR-342 and miR-146 genes in LPS-treated cells and the down-regulation of miR-696, miR-805, miR-706, miR-710 and miR-214 genes. These founding suggests that the miRNAds are probably involved in the regulation of mouse innate immune responses. (JDRF, 5-2006-918)
Retinoids accelerate B lineage lymphoid differentiation (83.12)
Authors: Chen Xinrong , Kincade Paul W.
Published: January 2023

Retinoids are known to have potent effects on hematopoietic cells, and all trans retinoic acid (ATRA) is widely used to treat patients with acute promyelocytic leukemia. Retinoids may normally maintain stem cell integrity, and our main objective was to learn if B lineage lymphoid progenitors express functional receptors. Total CD19+ cells increased substantially in the bone marrow, while early lymphoid progenitors (ELP) and pro-lymphocytes (Pro-L) were reduced in mice treated with time-release ATRA pellets. Culture experiments with highly enriched progenitors demonstrated they were direct drug targets. Overall cell yields at short culture intervals were reduced, but CFSE labeling indicated this did not result from reduced proliferation. ELP are very primitive progenitors and 10 days is normally required for them to generate CD19+ cells in culture. Remarkably, lymphoid cells and IL-7 responding progenitors emerged in just 7 days when ATRA was present. Treatment with a panel of agonists suggested that RAR rather than RXR mediates these responses. ATRA had similar effects on human B cell differentiation. That is, it inhibited the expansion of human CD34+CD38− progenitor cells and accelerated the differentiation of human CD19+ cells. There may be previously unsuspected side effects of ATRA therapy, and retinoids might normally contribute to the lymphopoietic environment in bone marrow.
Structural Transitions that Regulate the Apoptotic Activity of BAX are Dependent on its C-terminal Alpha-9 Helix (84.10)
Authors: Khaled Annette R. , Tschammer Nuska , Zhang Ge , Selby Thomas A.
Published: January 2023

Apoptosis is essential for lymphocyte development and the maintenance of cellular homeostasis. Previously, we showed that the apoptotic protein, BAX, is a critical mediator of IL-7-withdrawal induced death; yet, how this deadly protein is activated is poorly understood. Under apoptotic stress, BAX translocates to mitochondria, causing cell death. To explain this transitional behavior, we tested the hypothesis that the C-terminal α9 helix of BAX is essential for mitochondrial membrane binding. Using a multi-disciplinary approach, we demonstrated that the lysine at position 189, near the end of the α9 helix, is required for membrane association. Hence, the α9 helix could function as a membrane anchor. However, computational docking analysis revealed a new function for the α9 helix. By binding in reverse orientation to the internal hydrophobic grooves of adjacent BAX molecules, this helix enables dimerization and the release of additional membrane-spanning domains. This reverse orientation of binding places the lysine at position 189 in binding proximity to conserved acidic residues in the BH3 domain of BAX, enabling the interaction between the α9 helix and the pocket. We have, therefore, identified a novel and indispensable role for the C-terminal α9 helix of BAX, demonstrating that, by binding in trans to a hydrophobic groove, this domain unleashes the protein’s lethal activity.
IL-4 PROTECTS THE CH31 B-CELL LINE FROM ANTI-IgM-INDUCED GROWTH ARREST AND APOPTOSIS: CONTRIBUTION OF THE PI-3′ KINASE/AKT PATHWAY (84.11)
Authors: Carey Gregory B. , Semenova Elena , Qi Xiulan , Keegan Achsah D.
Published: January 2023

Interleukin-4 (IL-4) promotes lymphocyte survival and protects primary lymphomas from apoptosis. Previous studies reported differential requirements for the STAT6 and IRS2/PI-3′ kinase signaling pathways in mediating the IL-4-induced protection from Fas-mediated apoptosis. In this study, we characterized IL-4-activated signals that suppress anti-IgM-mediated apoptosis and growth arrest of CH31, a model B-cell lymphoma line. In these cells, anti-IgM treatment led to mitochondrial dysfunction and the loss of activation-specific phosphorylation of Akt and of CDK2. These were followed by caspase activation, loss of BAD phosphorylation, growth-arrest and then apoptosis. Strikingly, IL-4 treatment prevented or reversed these changes, dramatically rescuing the cells from Go/G1-arrest and driving them back into S- and G2/M- phases of the cell cycle. Furthermore, IL-4 suppressed the activation of caspases 9 and 3 and, in contrast to previous reports, induced the phosphorylation (deactivation) of BAD. IL-4 treatment also induced expression of BclxL, a STAT6-dependent gene. Finally, pharmacologic inhibitors of PI-3′ kinase, of Akt and of JAK/STAT signaling and dominant inhibitory forms of PI-3′ kinase and Akt, abrogated IL-4 anti-apoptotic function. These results suggest that the IL-4 receptor activates several signaling pathways that act to suppress the apoptotic program activated by anti-IgM.
B Cell Receptor Crosstalk: The IL-4-induced Alternate Pathway for BCR Signaling Operates in Parallel with the Classical Pathway, Is Sensitive to Rottlerin, and Depends on Lyn (86.14)
Authors: Guo Benchang , Blair Derek , Chiles Thomas , Lowell Clifford , Rothstein Thomas
Published: January 2023

B cell exposure to IL-4 alters subsequent B cell receptor signaling such that ERK phosphorylation becomes signalosome-independent; however, the nature of this new, alternate signaling pathway and its relationship to the classical, signalosome-dependent signaling pathway, are not known. Here we report that the alternate and classical pathways for BCR signaling are differentially affected by rottlerin, and by Go6976 or LY294002, respectively. Further, in B cells lacking PKCâ, the classical pathway for BCR signaling is blocked, whereas the alternate pathway is little affected. Conversely, in B cells lacking Lyn, the alternate pathway for BCR signaling is blocked, whereas the classical pathway is little affected. The rottlerin-sensitive element is not PKCä, inasmuch as the alternate pathway as not blocked in PKCä-deficient B cells. These results indicate that the rottlerin-sensitive, Lyn-dependent alternate pathway, and the classical pathway, for BCR signaling operate in parallel when BCR engagement follows IL-4 exposure.
B-Cell-Receptor Signal-Transduction in Antigen Internalization and Presentation (86.15)
Authors: Malhotra Shikha , Cogeshall K Mark
Published: January 2023

Humoral immune response is initiated by triggering B cell receptor (BCR). Antigen-induced clustering of the BCR results in the assembly of a multimolecular signaling complex. We are investigating the link between antigen receptor-triggered signal transduction and receptor internalization in mouse B- lymphocytes. BCR internalization requires formation of clathrin-coated pits and actin polymerization. The dynamin GTPase provides energy for clathrin-mediated endocytosis of various receptors. We found that transfection of B cells with a GTPase defective Dynamin mutant prevents BCR internalization. Additionally, dynamin co-localized with the BCR during BCR internalization. Vav is a guanine nucleotide exchange factor for Rho GTPases (regulators of actin polymerization). We found Vav is similarly co-localized with the BCR, and transfection with inhibitory Vav mutants block activation of Rac and prevent BCR internalization. We propose a model in which Vav regulates the activity of Rac during BCR internalization. Once Rac is activated, actin nucleation proteins cause F-actin assembly, which is required for both BCR internalization and BCR movement to endosomal compartments where the antigen is processed and finally presented to a T cell. Studies are in progress to see if dynamin regulates Vav recruitment to the BCR, or if they are independently regulated molecules, but both are required for internalization.
Expression of bone specific alkaline phosphatase on human B cells (86.18)
Authors: Hossain Ashfaque , Jung Larry K
Published: January 2023

A complex relationship exists among the molecules involved in the development of the immune system and of the bony skeleton. Molecules involved in one system can exert effects on the other system. Alkaline phosphatase (AP) is produced at high levels by osteoblasts and it plays an important role in bone remodeling. AP activity is also found on human B cells although the exact nature is not clearly understood. The purpose of this study is to determine the nature of expression of AP in human lymphocytes. Using a monoclonal antibody against bone specific alkaline phosphatase (BAP), its expression was detected in approximately 50% of circulating B cells but not T cells. These cells were distinct from the circulating osteoblasts. Lymphocyte stimulation by TPA with or without ionomycin, did not change BAP expression. Using RT-PCR, BAP specific transcripts were detected in the B cells expressing surface BAP but not in those not expressing BAP. The expression of BAP on B cells was similar in children as in adults. Its expression on transformed B cell lines was variable and EBV-transformed B cells did not express BAP. BAP was predominantly expressed in the CD27negative subpopulation. As CD27 is thought to be a marker for memory lymphocytes, it might be inferred that BAP is down-regulated in the memory B cells. Collectively, these data suggest that BAP may play an important role in the differentiation and maturation of human B cells.
Negative Regulation of CD40-mediated B Cell Responses by E3 Ubiquitin Ligase Cbl-b (87.21)
Authors: Zhang Jian , Qiao Guilin , Lei Minxiang , Li Zhenping
Published: January 2023

CD40 is a TNF receptor (TNFR) superfamily member which is essential for B cell function. Cbl-b, an adapter protein and ubiquitin ligase, has been shown to regulate activation of T and B cells through their antigen receptors. In this study, we report that CD40-induced proliferation is significantly augmented in mice lacking Cbl-b. Furthermore, Cbl-b−/− mice display enhanced thymus–dependent (TD) Ab responses and germinal center (GC) formation, whereas introduction of CD40 deficiency abolishes the hyper-Ab responses and heightened GC formation in response to TD Ag challenge. Mechanistically, Cbl-b selectively down-modulates CD40-induced activation of NF-κB and JNK. Cbl-b associates with TNFR-associated factor 2 (TRAF-2) upon CD40 ligation, and inhibits the recruitment of TRAF-2 to the CD40. Together, our data suggest that Cbl-b attenuates CD40-mediated NF-κB and JNK activation, thereby suppressing B cell responses.
Role of IFN-γ and IL-12 in Silencing the IL-4-Producing Potential in Th1 Cells (87.29)
Authors: Zhuang Yonghua , Huang Zan , Nishida Jun , Masuda Jennifer , Gapin Laurent , Brown Melissa , Zhang Lin , Huang Hua
Published: January 2023

To develop into committed Th1 cells, naive CD4+ T cells not only need to acquire the capacity to produce IFN-γ, but also need to silence the IL-4-producing potential. The role of IFN-γ is thought to primarily upregulate the IL-12β2 chain expression and thus enhances the IL-12 responsiveness. The direct effect of IFN-γ in Th1 cell development remains less clear. Furthermore, it is not clear whether both the IFN-γ and the IL-12 pathway are critical in silencing the IL-4-producing potential in Th1 cells. In this study, we investigated the role of the IFN-γ and the IL-12 pathways in silencing the IL-4-producing potential in Th1 cells. We found that IFN-γ was essential in silencing the IL-4-producing potential in Th1 cells, while IL-12 only partially suppressed the IL-4-producing potential. IL-12 completely depended on STAT4, while IFN-γ completely depended on STAT1 to suppress the IL-4-producing potential. Interestingly, T-bet is only partially required for IFN-γ to silence the IL-4-producing potential, whereas it is absolutely required for IL-12 to suppress the IL-4-producing potential. Our study clarifies the role of IFNγ-STAT1-T-bet and IL-12-STAT4-T-bet pathway in silencing the IL-4-protential during Th1 cell development, indicating that an additional factor, whose induction is STAT1 signaling, may be critical in Th1 cell commitment.

This work is funded by an NIH grant (RO1 AI48568).
Monomeric Agonist pMHC Ligand Triggers TCR Signaling Independent of Endogenous Ligand (87.31)
Authors: Ma Zhengyu , Sharp Kim A , Janmey Paul A , Finkel Terri H
Published: January 2023

At the contact interface between T-cell and antigen presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to T-cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. A critical yet controversial issue is whether monomeric agonist pMHC is sufficient to trigger TCR, or if simultaneous engagement of TCRs by two pMHCs is required. Here, we show that CD4+ T-cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid planar lipid bilayers. T-cells responded to bilayer-anchored agonist MCC-IEk in a sub-linear fashion, further indicating the sufficiency of monovalent ligand. Moreover, on bilayers, fixed plastic surfaces, or live APCs, endogenous pMHC did not enhance TCR triggering by agonist pMHC. Therefore, agonist pMHCs trigger TCR as individual monomers, independent of endogenous pMHC, supporting a model of TCR triggering by conformational change or kinetic segregation, and not via pMHC pseudodimers.
Wound repair mediated by γδ T cells requires mTOR (87.43)
Authors: Jameson Julie Marie , Mills Robyn Elaine
Published: January 2023

γδ TCR-expressing lymphocytes in the skin play roles in biological processes such as wound repair, tissue homeostasis, and tumor rejection.

γδ T cells regulate their epithelial neighbors by producing growth factors for keratinocyte proliferation, epithelial homeostasis, and inflammatory cell migration. Rapamycin is a triene macrolide, which inhibits mTOR signaling by binding FKBP12, thus inhibiting αβ T lymphocyte function. Rapamycin is approved by the U.S. Food and Drug Administration for the prophylaxis of acute kidney rejection, however patients that receive rapamycin report an increased incidence of wound healing complications. We hypothesize that rapamycin not only targets allograft-specific αβ T lymphocytes, but also suppresses skin γδ T cells. mTOR plays key roles in growth factor and cytokine signal reception, therefore rapamycin administration may mimic nutrient insufficiency. Our preliminary studies indicate that rapamycin arrests skin γδ T cell proliferation in G1 phase and causes them to undergo autophagy. We have established skin organ cultures to administer rapamycin to wounded murine skin. Our results demonstrate that when rapamycin is present, there is a defect in wound closure over time. We propose that rapamycin blocks skin γδ T cells from receiving survival signals, which results in the inhibition of skin γδ T cell proliferation and function negatively impacting wound repair.

Support from Leukemia and Lymphoma Society and NIH DK073098.
Adoptively transferred tumor-specific T cells expanded ex vivo using HSV amplicons encoding 4-1BBL persist in the host and demonstrate anti-tumor activity in vivo. (88.11)
Authors: Yi Kyung Hee , Nechushtan Hovav , Zhang Yu , Bowers William J , Tolba Khaled A , Podack Eckhard R , Federoff Howard J , Rosenblatt Joseph D
Published: January 2023

4-1BB is a T cell costimulatory receptor which promotes T cell expansion and survival. We studied the anti-tumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using herpes simplex virus (HSV) amplicons encoding either 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7). Lewis Lung Carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7, or control HSV and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1). Naïve or ex vivo-stimulated OT-1 were then transferred into LLC/OVA-bearing mice. Mice given 4-1BBL-stimulated OT-1 had significantly decreased tumor volumes compared to untreated mice (P<0.0001) or mice given naïve OT-1 (P=0.0003). Transfer of B7-stimulated OT-1 did not control tumor growth. Higher percentages of OT-1 were seen in the blood, draining lymph nodes, and tumor for the 4-1BBL-stimulated OT-1 group compared to other groups at 8 days post transfer. BrdU incorporation of 4-1BBL-stimulated OT-1 was markedly increased within the tumor bed. Splenocytes from the 4-1BBL-stimulated OT-1 group showed increased cytolytic activity against LLC/OVA and higher numbers of OT-1 cells, which expressed CD44 and the memory marker Ly-6C, compared to controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand efficiently in vivo resulting in tumor eradication and the generation of tumor-specific memory T cells.
Costimulation Through ICAM-1 Induces Differentiation of Human Naïve CD4+ T Cells to Regulatory T Cells (88.32)
Authors: Cool Kelli M , Dotson Abby L. , Otto Amber R. , Kohlmeier Jacob E. , Benedict Stephen H
Published: January 2023

Inducible regulatory T cells can be generated from precursor cells in the periphery. However, the specific precursors and the mechanism of Treg differentiation are not well characterized. We investigated the participation of Intercellular Adhesion Molecule-1 (ICAM-1) in the process of Treg induction beginning with human naïve CD4+ T cells, defined as CD45RA+CD45RO-CD11aloCD27+CCR7+. Costimulation of naïve CD4+ T cells through ICAM-1 (but not CD28) resulted in the appearance of a sizeable population of Foxp3+CD25+CD4+ Treg cells. This stimulation treatment also generated substantial quantities of the immunosuppressive cytokine IL-10. The Treg cells that differentiated following costimulation through ICAM-1 strongly inhibited responder cell proliferation in co-culture experiments. The ability of naïve CD4+ T cells to undergo this differentiation process was examined using cells from both young individuals and older individuals. These results indicate that ICAM-1 resident on the surface of naïve CD4+ T cells can mediate the differentiation of Tregs from naïve cells and suggest that ICAM-1 may be a signaling molecule to target for Treg therapies.
HVEM-BTLA interactions regulate early CD8+ T cell response against Listeria monocytogenes (88.33)
Authors: Sun Yonglian , Ruddy Matthew , Miller Mendy , Wang Yang , Lee Youjin , Kaye Jonathan , Murphy Kenneth , Pfeffer Klaus , Fu Yang-Xin
Published: January 2023

Negative regulation of T cell responses at the early phase of an immune response is largely unknown. Here, we show that the newly discovered co-inhibitory interaction between B and T lymphocyte attenuator (BTLA) receptor and herpesvirus entry mediator (HVEM) ligand is essential and sufficient to negatively regulate the early CD8+ T cell response against Listeria monocytogenes infection. BTLA expression is transiently up-regulated on bacteria-specific CD8+ T cells shortly after infection. Both HVEM−/− and BTLA−/−, but not LIGHT−/− mice, demonstrate resistance to Listeria infection. Accordingly, blocking BTLA pathway by antagonist anti-BTLA monoclonal antibody promotes antigen-specific CD8+ T cell expansion and IFN-γ production as well as bacteria clearance in vivo. In contrast, engagement of BTLA inhibitory signal with HVEM-Ig inhibited antigen-specific CD8+ T cell responses as well as bacteria clearance in a CD8+ T cell dependent manner, indicating the critical role of CD8+ T cell in early bacteria clearance and that cross-linking BTLA is sufficient to suppress CD8+ T cell response at an early stage. These studies reveal that HVEM-BTLA co-inhibitory interactions play a critical regulatory role in the early stages of the CD8+ T cell response against bacteria infection.
IL-15 administration to human CD4+ T cells leads to expansion of CD4+CD25− cells but to a decrease of CD4+CD25highFoxp3+T cells via apoptosis (88.44)
Authors: Strauss Laura , Bergmann Christoph , Whiteside Theresa L.
Published: January 2023

Objective: Adoptive transfer of CD4+CD25highFoxp3+ regulatory T cells (nTreg) for immune therapy will require the development of strategies to expand these cells on a clinical scale. We have shown that rapamycin promotes the survival of nTreg. IL-15 protects human T cells from apoptosis. However, effects of IL-15 on nTreg remain unknown.

Methods: CD4+CD25+ and CD4+CD25− T cells were sorted from PBMC of normal controls using AutoMACS. All separated T cells were expanded in the presence of anti-CD3/CD28 Abs, 1000 IU/mL IL-2 or 20 IU/mL IL-15 for 21 d. Rapamycin (1 nM) was added to half of the cultures. After 21 d sensitivity to apoptosis (AnnexV+) was determined. Regulatory function was tested by co-culture analysis with CFSE-labeled responders.

Results: TCR-stimulated CD4+CD25− T cells were sensitive to rapamycin-induced apoptosis, whereas Treg were resistant (90±7.5% vs. 12±10.5%). IL-15 + activated CD4+CD25− T cells were resistant to apoptosis relative to activated CD4+CD25− T cells alone (2±1.5 %). In contrast, Treg + IL-15 and rapamycin became sensitive to apoptosis (98±2.5% vs. 2.8±1.7%). Rapamycin enhanced the suppressor function of Treg (p ≤ 0.001). CD4+CD25− T cells were not suppressive, but expanded with IL-15 + rapamycin did suppress T cell proliferation (60±15%).

Conclusion: Our results indicate that proliferation and survival of Treg and T responders are mediated by different molecular pathways.
ROLE OF TLR4 TYROSINE PHOSPHORYLATION IN SIGNAL TRANSDUCTION AND ENDOTOXIN TOLERANCE (89.2)
Authors: Medvedev Andrei , Piao Wenji , Rhee Sang Hoon , Chen Haiyan , Basu Subhendu , Wahl Larry M , Fenton Matthew J , Vogel Stefanie N
Published: January 2023

This study examined if tyrosine phosphorylation of TLR4 is required for signaling. Introduction of the P712H mutation into the TIR domain of constitutively-active mouse ΔTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Y674A and Y680A mutations within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK, IκB-α degradation, and activation of NF-κB and RANTES reporters. Full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed LPS-inducible cell activation that correlated with altered MyD88-TLR4 interactions, increased association with a short IRAK-1 isoform, and decreased levels of activated IRAK-1 in complex with TLR4. Pretreatment of HEK293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-κB activation. Induction of endotoxin tolerance markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. These data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin tolerance, and suggest involvement of Lyn kinase in these processes.

This study was supported by NIH grants AI-059524 (AEM), AI-057490 and AI-44936 (SNV).
HMGB1 secretion is regulated by protein kinase C (89.26)
Authors: Oh Young Joo , Youn Ju Ho , Shin Jeon Soo
Published: January 2023

High mobility group box-1 protein (HMGB1) is secreted from activated monocytes/macrophages and known as a key late mediator of sepsis, so the regulation of secretion is important to control the sepsis. Phosphorylation of HMGB1 is one of major secretion pathways in our previous report. To investigate the phosphorylation-related pathway of HMGB1, LPS-induced HMGB1 secretion was observed with various inhibitors in RAW264.7 cells. Wortmannin and LY294002 inhibited HMGB1 secretion, suggesting the involvement of phosphoinositide 3-kinase (PI3K). Akt inhibitor IV and rapamycin failed to inhibit HMGB1 secretion, suggesting no relevance of Akt-mTOR axis pathway. In contrast, protein kinase C (PKC) inhibitor Gö6983 could inhibit HMGB1 secretion. And we confirmed that HMGB1 was directly phosphorylated by PKC but not by casein kinase II or cdc2 in in vitro kinase assay using autoradiography and □-scintillation counting. When RAW264.6 cells were pre-treated with JNK inhibitor, LPS-induced HMGB1 secretion was decreased but not by PD 98059 and SB 203580, p42/44 and p38 pathway inhibitors respectively. These results suggesting that PKC is effector kinase for HMGB1 phosphorylation for secretion and PI3K may act in concert to control of HMGB1 secretion via a pathway independent manner of p42/44 and p38 MAPK. Which specific isoform of PKC is involved in HMGB1 phosphorylation is now under investigation.