bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycan-induced antiviral immune response inhibits HIV replication (154.19)
Authors: Ring Jennifer , Harn Don
Published: January 2023

Several viruses have developed strategies to evade recognition by the host immune response, thereby establishing chronic infection. In these cases, an immunotherapeutic drug that triggers an antiviral immune response could produce a more favorable outcome. We evaluated the immunomodulatory human milk glycan Lacto-N-fucopentaose III (LNFPIII) for its ability to trigger an antiviral immune response and inhibit HIV replication. Human PBMCs were treated with LNFPIII-HSA, a glycoprotein consisting of on average, 8-10 molecules of LNFPIII conjugated per molecule of HSA via an APD linker. This treatment resulted in the upregulation of several antiviral cytokines including TNF-α, IFN-γ, and major HIV-suppressive chemokines MIP-1α, MIP-1β, and RANTES. Upon infection, we observed reduced HIV replication in treated PBMCs, likely due to the multitude of antiviral cytokines present. When monocytes were removed from culture, or CD14 was neutralized, the antiviral effects of LNFPIII-HSA were negated. These results indicate that LNFPIII-HSA triggers an antiviral immune response capable of controlling HIV replication by activating the antiviral activity of CD14+ monocytes.
Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels (154.32)
Authors: Lee Andrew , Hornell Tara , Wang Nan , Harding James , Deshpande Chetan , Hertel Laura , Lacaille Vashti , Pashine Achal , Macaubas Claudia , Mocarski Edward , Mellins Elizabeth
Published: January 2023

Human cytomegalovirus (HCMV) productively infects CD34+ progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. CIITA mRNA levels were significantly lower in HCMV-infected matLC than in mock-infected cells. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV were decreased by HCMV, a result not seen after incubation of matLC with LPS. Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. The stability of CIITA mRNA was not diminished by HCMV, arguing that diminished transcription is the mechanism for CIITA mRNA reduction. HCMV-infected matLC expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. Thus, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, resulting in reduced MHC II biosynthesis as a candidate immunoevasive mechanism.
Abrogation of human CD59 function regains neutralizing and non-neutralizing antibody functions in triggering complement-mediated virolysis of HIV-1 (154.7)
Authors: Yu Qigui , Hu Ningjie , Amet Tohti , Byrd Daniel
Published: January 2023

Patients infected with HIV-1 are well known to mount a vigorous and sustained antibody (Ab) response to the virus, yet fail to control virus proliferation or protect them from developing AIDS. Recent studies have suggested that resistance of HIV-1-infected cells and virions to Ab-dependent complement-mediated lysis (ADCML) is dependent on regulators of complement activation (RCA), particularly CD59 that controls the formation of the membrane attack complex (MAC) at the terminal stage of the complement activation cascades via all three activation pathways. Abrogation of CD59 function renders HIV-1 virions and infected cells sensitive to ADCML. In the current study, we used the well-characterized anti-HIV-1 nAbs (2G12, 2F5, 4E10 and b12) and non-nAbs (5F3, A32 and F240) to investigate whether the enhancement of ADCML by inhibition of CD59 is mediated by nAbs, non-nAbs or both. We found that these Abs, regardless of their neutralizing activity, significantly enhanced ADCML of many HIV-1 lab strains and 6 primary isolates examined in the presence of CD59 blockers. Direct addition of CD59 blockers into plasma from 16 HIV-1-infected individuals triggered ADCML of endogenous plasma virions, and the effect correlated to Ab titers rather than Ab neutralizing activity, indicating that patient plasma Abs are able to recognize endogenous virions, and abrogation of CD59 may represent a novel approach to regain the function of both nAbs and non-nAbs naturally present in patient’s blood.
Identification and characterization of CD8+ T helper cells based on CD40L expression (155.1)
Authors: Frentsch Marco , Stark Regina , Listopad Joanna , Meier Sarah , Schulz Axel , Durlanik Sibel , Blankenstein Thomas , Thiel Andreas
Published: January 2023

CD8+ T cells are primarily regarded as cytotoxic cells. We have identified a substantial subset of CD40L expressing non-cytotoxic T-cells among primary human CD8+ T cells. CD40L+ CD8+ T cells exert diverse characteristic Th-cell functions such as activation of B cells, induction of DC maturation and secretion of cytokines such as IL-2, IFNγ, TNFα or IL-4. Up to 25% of total human central and effector memory CD8+ T cells express CD40L including cells specific for pathogens such as influenza, CMV and EBV, as well as yellow fever virus specific cells after primary vaccination. At next we assessed the functional relevance of CD40L expression on CD8+ T cells during a anti-tumor response. We challenged Rag1-/- mice with SV40 Tag expressing tumor cells and injected in parallel wt or CD40L-/- CD8+ T cells. Only application of CD40L competent wt CD8+ T cells prevented the establishment of tumors, whereas injection of CD40L-/- CD8+ T cells resulted in non-controlled tumor progression similar to non-treated tumors, although tumor-specific T cells were primed in these mice. Our results disclose an essential functional relevance of CD40L expressed by CD8+ T cells. Especially, in situations of reduced CD4+ T-cell help and MHC-II antigen presentation and/or limited danger signals CD40L+ CD8+ T cells may exert essential helper responsibilities for immunity and thus are potent candidate T cells to execute or support effective anti-tumor or anti-pathogen immune therapies.
Immune adjuvant and antitumor activity of baccatin III, a synthetic precursor of taxol (155.15)
Authors: Kim Ki-Hyang , Park Chan-Su , Park Dong-Eun , Im Sun-A , Lee Chong-Kil
Published: January 2023

Baccatin III (BA-III), a precursor for the synthesis of taxol, is widely considered to be an inactive derivative of taxol. Here we show that baccatin III exerts immune adjuvant activity. When added to cultures of immature dendritic cells (DCs) generated from bone marrow cells with GM-CSF, baccatin III increased the expression of MHC II molecules and the allostimulatory activity of the DC. The immune adjuvant activity of baccatin III was examined in mice injected with a model antigen, OVA, emulsified with Freud’s incomplete adjuvant. Inclusion of baccatin III significantly increased the production of OVA-specific IgG, but not IgM. The in vivo combination effects of baccatin III and gemcitabine, an antitumor agent, were examined in mice bearing mammary carcinoma 4T1 cells. Combination of baccatin III with gemcitabin efficiently inhibited the tumor growth and the accumulation of myeloid derived suppressor cells (MDSC, CD11b+Gr-1+ cells), while increasing cellularity of CD4 T cells and CD8 T cells in the spleen, compared to baccaatin III only- or gemcitabine only-treated groups. These results demonstrate that baccatin III exerts immune adjuvant activity, and further show that baccatin III in combination with gemcitabine, a nucleoside analog used as chemotherapy, may be useful in the treatment of tumors that induce the accumulation of MDSCs.
Computer analysis and identification of antigenic determinants from five glycoproteins of HSV2 (155.17)
Authors: Yuexi Li , Xingsheng Wang , Jianmin Liao , Dengke Yin , Suqin Li , Zhengmao Wang , Lin Li , Guangyan Xie
Published: January 2023

To screen the antigenic determinants from glycoprotein B (gB2), C (gC2), E (gE2), G (gG2) and I (gI2) of herpes simplex virus II (HSV2) for designing novel vaacine, the aminao acid sequences of the five proteins were analysed with several software algorithms, DNAstar, Biosun, and Antheprot. The common antigenic determinants screened by all the software algorithms were synthesized chenically and identified by animal immune experiments. Sixteen synthesized antigenic determinants selected from the five proteins were linked to carrier protein BSA respectively to enhance their immunogenicity, and uesed as immunogens to immunize mice. The antibodies in the sera of the immunized mice were detected with the coreesponding immunogens and the parent glycoproteins by EIA and western blotting. The virus-neutralizing activity of the antisera were tested with vero cell in vitro. The antisera reacted strongly with the parent glycoproteins, and also the peptides reacted strongly with antibodies against the parent glycoproteins. The antisera against five antigenic determinants from gB2 ,gC2, gE2, gG2, and gI2 can neutralize HSV2 infectivity in vitro respectively, which has not been reported up to now. These results suggested that the antigenic determinants from the five glycoproteins screened by software algorithms and validated by experiments are promising for vaccine design.
Delivery of tumor-targeted immunotoxin by tumor-specific T cells (155.19)
Authors: Swanson Anna , Wu Danli , Kuiatse Isere , Kim Hong-Sung , Heslop Helen , Brenner Malcolm , Yotnda Patricia
Published: January 2023

Immunotherapy of cancer using monoclonal antibodies and adoptive transfer of lymphocytes is showing considerable promise. Based on experience with other cancer therapeutics immunotherapeutic modalities, targeting discrete molecular components of the tumor, will prove superior to a single effector system. “Multitargeting” approaches will not only prevent tumor immune escape, they will also allow safer and potent anticancer therapies. We have engineered antigen specific T cells so that they can target tumor cells directly, and then release a potent toxin linked to a monoclonal antibody molecule thereby targeting an independent tumor antigen to the tumor reactive T cell. These engineered T cells were tested in vitro and in vivo in a xenograft model of B-lymphoma. We found that tumor antigen specific T cells retain their anti-tumor activity and tumor homing abilities even when transduced with a gene encoding a chimeric monoclonal antibody-toxin. The release of the targeted immunotoxin molecule occurs only after the T cell has engaged its tumor target. The combination of these two distinct effector systems (T cells and immunotoxin) targeting two distinct tumor antigens have superior anti-tumor activity than a single system targeting a single antigen, with no greater toxicity. By combining two highly specific and partially effective strategies for tumor destruction, we achieved selective and more effective tumor killing.
Bivalent HPV16/18 L1 VLP vaccine induces cross-neutralizing antibodies that may mediate cross-protection (155.27)
Authors: Pinto Ligia , Kemp Troy , Hildesheim Allan , Safaeian Mahboobeh , Pan Yuanji , Porras Carolina , Schiller John , Lowy Douglas , Rolando Herrero
Published: January 2023

Human papillomavirus (HPV) L1 VLP-based vaccines are protective against HPV vaccine-related types; however, correlates of protection have not been defined. We observed that vaccination with the HPV-16/18 L1 VLP vaccine induced cross-neutralizing antibodies for HPV types for which evidence of vaccine efficacy has been demonstrated (HPV31/45) but not for other types for which no cross-protection was observed (HPV52/58). In addition, HPV31/45 cross-neutralizing titers showed a significant increase with number of doses (HPV31, p<0.001; HPV45, p<0.001) and correlated with HPV16/18 neutralizing titers, respectively. Cross-neutralizing antibody titers were much lower than those observed for the homologous vaccines types but persisted over 36 months of follow-up. Increases in antibody avidity occurred with increased doses of vaccine but levels of avidity did not correlate well with antibody titers. Our findings support the hypothesis that cross-neutralizing antibodies are effectors of the cross-protection observed for the HPV16/18 vaccine. Further studies are needed to better understand the potential role of avidity in protection against infection.
Development of three murine models of Staphylococcus aureus infection to aid in the characterization of a broadly protective vaccine and elucidation of immune responses to different routes of infection (155.29)
Authors: Prabhakara Ranjani , Mocca Christopher , Brady Rebecca , Plaut Roger , Burns Drusilla , Stibitz Scott , Merkel Tod
Published: January 2023

Staphylococcus aureus is a common etiological agent of nosocomial and community acquired infections. Pan-antibiotic resistant strains of S. aureus have emerged at an alarming rate, making vaccination an important defense against S. aureus. S. aureus causes a variety of infections that differ in route of infection and disease progression, but previous experimental vaccines have focused on only one type of infection. Hence, multiple animal models are an important tool in studying broadly protective S. aureus vaccines. We sought to develop several murine models that mimic both planktonic and biofilm-mediated S. aureus disease. We focused on three infections that are of the greatest concern: biofilm-mediated implant infection, skin and soft tissue infection (SSTI), and sepsis. We have reproducibly generated a local, chronic, biofilm-mediated implant infection in 100% of C57BL/6 mice, and have determined the minimum dose that results in a chronic infection rate of 100%. We have also made progress towards determining the LD50, maximum non-lethal dose, and minimum lethal dose of S. aureus in a BALB/c sepsis model, as well as reproducibly produced SSTI in BALB/c mice. We plan to use these models to elucidate the memory responses associated with each infection type, and to characterize putative vaccines. If protective immune responses are elicited after vaccination in each of these models, those antigens will be combined into a multivalent vaccine to be tested in all three models.
Yet another phenotypic strategy for defining human IL-17 + CD4 T cells (155.33)
Authors: Li Yongqing , Yee Cassian
Published: January 2023

IL-17-secreting T cells represent a distinct lineage of CD4+ T effector cells (Th17) essential in the pathogenesis of numerous inflammatory disorders, autoimmune diseases and tumor immunity. A means to define the population of human Th17 cells has been confounded by the heterogeneity and plasticity of helper and regulatory T cell subsets. Although combinations of chemokine and surface receptors have been associated with the Th17 phenotype, these markers are not unique to Th17 T cells. In this study, we adapt a murine-based strategy to enrich for a population of human IL-17-producting CD4 T cells from PBMC. Isolation of IL-17+ and IL-17-negative T cell clones provided uniform lineage-specific populations that enabled us to identify differentially expressed genes. Consistent with previously published data, enhanced expression of IL-23R, RORC and CCR4 were well-represented in IL-17+ CD4 T cell clones confirming their lineage specificity. A closer analysis of these clones reveals a functional phenotype that excludes Rhodamine and differentially expresses CCR6 and CD161. We extrapolate these findings to the unselected PBMC population and using a combination of surface and functional markers, demonstrate that a uniquely defined, non-destructive strategy can be used to enrich for IL-17+, Treg-resistant T cells; furthermore, antigen-specific IL-17+ effectors can be expanded using this approach to achieve numbers of cells sufficient for adoptive cellular therapy of cancer.
A modified form of a tumor-associated phosphorylated peptide derived from BCAR3 generates CD8 T cells that are highly crossreactive to the natural phosphopeptide and capable of recognizing tumor cells (156.13)
Authors: Obeng Rebecca , Zarling Angela , Engelhard Victor
Published: January 2023

Using metal ion affinity chromatography and mass spectrometry, we identified a phosphopeptide (BCAR3pT130) derived from BCAR3 among numerous phosphopeptides presented by HLA-A2 on human melanoma cells. BCAR3 activates the PI3K/Akt pathway and mediates migration and estrogen resistance in breast cancer cells, processes that are associated with malignancy. In evaluating the immunotherapeutic use of BCAR3pT130, we found that BCAR3pT130 had a moderate HLA-A2 binding affinity but was nonimmunogenic in HLA-A2 transgenic mice. Substitution of the P9 anchor residue Leu with Val had no impact on HLA-A2 binding affinity but the modified BCAR3pT130 proved to be significantly immunogenic. This suggests that the modification altered the conformation of the phosphopeptide and that the immune response against the natural phosphopeptide is restricted by tolerance. CD8 T cells generated using the modified BCAR3pT130 produced IFNγ, TNFα, and were cytotoxic based on CD107a expression. The T cells were equally reactive with the natural and modified phosphopeptides. This suggests that the natural BCAR3pT130 phosphopeptide is a weak agonist and that the sequence modification creates a stronger agonist. These T cells also recognized endogenously processed BCAR3pT130 on HLA-A2 human melanoma and breast cancer cells. Thus, the association of BCAR3 with malignant processes makes BCAR3pT130 an appealing target for cancer immunotherapy that can be evaluated using the modified form of the phosphoepitope.
IGKV3 proteins as candidate “off-the-shelf” vaccines for kappa-light chain-restricted B cell non-Hodgkin’s lymphomas (156.15)
Authors: Dolcetti Riccardo , Martorelli Debora , Guidoboni Massimo , De Re Valli , Muraro Elena , Turrini Riccardo , Merlo Anna , Pasini Elisa , Caggiari Laura , Romagnoli Luca , Spina Michele , Mortarini Roberta , Gasparotto Daniela , Mazzucato Mario , Carbone Antonino , Rosato Antonio , Anichini Andrea
Published: January 2023

An increasing set of B-cell non-Hodgkin’s lymphomas (B-NHLs) show a biased usage of IGKV3-20 and IGKV3-15 immunoglobulin genes, but it is not known whether the corresponding proteins are immunogenic in the majority of patients. We herein demonstrate that IGK+ lymphoma patients show spontaneous T cell responses to these proteins and that IGKV3-specific cytotoxic T lymphocytes (CTLs) can be easily induced ex vivo. IGKV3-20-specific CTLs cross-react against different IGKV3 proteins, an effect mediated by the presence of 21 shared, sometimes promiscuous, T cell epitopes, presented by the most common HLA class I allele products, thus assuring a broad HLA coverage of IGKV3-based vaccines. Many natural variants of these epitopes are carried by IGK light chains expressed by a broad spectrum of B-NHLs and we show that IGKV3-20-specific CTLs cross-react also against several of these variant epitopes. Both humoral and CTL specific responses were induced by KLH-conjugated IGKV3-20 protein in HLA-A2-transgenic mice and co-injection of IGKV3-20-specific CTLs with IGKV3-20+ lymphoma cells into SCID mice totally prevented tumor growth, thus confirming the ability of these effectors to mediate efficient cytotoxic responses also in vivo. These results provide the rationale to exploit IGKV3 proteins as “off-the-shelf” vaccines for a large fraction of lymphoma patients.
Bi-specific TCR anti-CD3 fusion for the immunotherapy of malignant melanoma (156.16)
Authors: Jakobsen Bent , Ashfield Rebecca , Bossi Giovanna , Adams Katy , Harper Jane , Buisson Sandrine , Paston Samantha , Baker Debbie , Williams Daniel , McGrath Yvonne , Hassan Namir
Published: January 2023

The human immune system can theoretically identify malignant cells by ‘scanning’ peptide antigens presented on the cell surface by Class I HLA molecules, and indeed many cancer patients generate CD8 cyto-toxic T cell responses to tumour-specific antigens. However, the majority of patients fail to clear tumours, since T cell avidity for self-antigens tends to be weak, and cancer cells employ escape mechanisms for avoiding destruction by T cells. We have engineered novel, bi-functional protein therapeutics termed ImmTACs which re-direct the immune system to target and destroy tumour cells with a high degree of potency and specificity. IMCgp100 comprises a soluble, high affinity T cell Receptor specific for the HLA-A2 presented melanoma-associated epitope gp100 280-288, fused to an anti-CD3 scFv domain. The anti-CD3 moiety activates a polyclonal T cell response when targeted to melanoma cells presenting as few as twenty gp100 epitopes, predominantly through activation of effector memory CD8 lymphocytes, leading to killing of the tumour cells. Extensive in vitro testing of IMCgp100 with a panel of tumour cells and normal human tissue cells demonstrates pico-Molar potency against cancer cells and specificity for HLA-A2 gp100 positive cells. The agent is able to inhibit tumour growth in mouse xenograft models. A phase I dose-escalation trial in late stage melanoma patients was initiated in October 2010 and interim results will be presented at this meeting.
MHC II-restricted peptides derived from invariant chain negative breast cancer cell-based vaccines activate healthy donor T-cells (156.17)
Authors: Chornoguz Olesya , Grmai Lydia , Gapeev Alexei , O’Neill Michael , Ostrand-Rosenberg Suzanne
Published: January 2023

We have generated cell-based MHC II vaccines to activate patients’ CD4+ T-cell to facilitate tumor immunity. This was based on the hypothesis that invariant chain (Ii) negative, MHC II and CD80 positive tumor cells present novel MHC II peptides and circumvent patients’ tolerance to their cancer. To confirm that absence of Ii facilitates presentation of novel immunogenic tumor peptides we sequenced MHC II peptides from MCF10/DR7/CD80, MCF10/CIITA/CD80/Ii siRNA (Ii-) and MCF10/DR7/CD80/Ii, MCF10/CIITA/CD80 (Ii+) vaccines using LC-MS/MS. 116 Peptides were identified for MCF10/DR7/CD80, 228 peptides for MCF10/DR7/CD80/Ii. 52 Peptides were in both cell lines. 108 Peptides were identified for MCF10/CIITA/CD80, 28 peptides for MCF10/CIITA/CD80/Ii siRNA, 6 peptides in both cell lines. This confirms that Ii+ and Ii- MHC II vaccines present distinct peptide repertoires and agrees with in vitro studies showing that human MHC II Ii- and Ii+ vaccines activate distinct T-cell subsets. 7 peptides with the highest MHC II binding affinity were tested for their ability to activate tumor-specific CD4+ T-cells. 5 of the peptides activated CD4+ T-cells to the same extent as breast cancer peptide Her2 p776, (measured by IFNγ production). This is the first study to compare the human MHC II peptide repertoire in the absence or presence of Ii and to identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.
Requirements for common γ chain family cytokines for adoptively transferred γδ T cell function and homeostasis in vivo (156.19)
Authors: Kakimi Kazuhiro , Izumi Takamichi , Kondo Makoto , Fujieda Nao , Kondo Atsushi , Morishita Takeharu , Maekawa Ryuji , Matsushita Hirokazu
Published: January 2023

We have conducted clinical trials of adoptive γδ T cell therapy for the treatment of esophageal, colorectal, pancreatic and non-small-cell lung cancers, and cholangiocarcinoma. Patients received ≥1x109 γδ T cells every 2 weeks. These cells had been expanded ex vivo for 14 days by culture with zoledronate (5 μM) and IL-2 (1000 IU/ml). Infused γδ T cells gradually accumulated in patients’ peripheral blood and accounted for 10% of PBMC as late as 3 months after the final injection, even without IL-2 administration in vivo. These cells maintained the ability to release cytotoxic granules (detected by CD107 assay) and produce IFN-γ. To determine the factors contributing to γδ T cell survival in vivo, we investigated their expression of receptors for common γ chain (γc) family cytokines at the time of infusion. IL-2Rβ and γc, but not IL-7Rα, IL-15Rα or IL-21R, were present on these cells, and although the IL-2Rα was upregulated at the initiation of culture, it was only very weakly expressed at the time of transfer. When γδ T cells were maintained for longer periods in culture, IL-15 but not low dose IL-2 and IL-7 supported their survival. These results suggest that tissue-specific γc-dependent signals in response to IL-15 might affect the survival and function of infused γδ T cells, and hence materially influence the success of immunotherapy.
Activating and tolerizing signaling “fingerprint” in T cells responding to DC/tumor MUC1 vaccine (156.23)
Authors: Reichenbach Dawn , Finn Olivera
Published: January 2023

Early biomarkers of efficacy of vaccines are lacking. We hypothesize that intracellular signaling in T cells following administration of the vaccine yields a “fingerprint” predictive of an effective or a tolerogenic immune response and could be used as a biomarker. Our lab has explored immune responses to human tumor MUC1 peptide vaccine in wildtype (WT) versus human MUC1 transgenic mice (MUC1.Tg) and found that they were significantly lower in MUC1Tg. To “fingerprint” signaling differences between the effective immune response in WT versus the lower (tolerogenic) response in MUC1.Tg mice, we vaccinated both with DC loaded with MUC1 peptide, harvested spleens (n=3) at 1,6,12, and 24 hours post vaccination and analyzed phosphorylation downstream of TCR and costimulatory molecules, as well as activation of cell survival, proliferation, and apoptosis signaling cascades. We saw early increases in pSTAT1, pSTAT4, and p38MAPK with a trend towards higher phosphorylation in WT mice that did not reach statistical significance. There was also a trend towards higher phosphorylation in WT mice in pLCK and pAKT downstream of the TCR and costimulatory molecules. Interestingly, at certain timepoints we observed increases in signaling in MUC1.Tg mice, which may serve as compensatory mechanisms that we will further investigate. We are challenging these two vaccinated groups of mice with MUC1+ tumor in an attempt to correlate early T cell activation with later protection from tumor growth.
Increased lethal cytokine-mediated toxicity following immunotherapy in aged versus young mice (156.24)
Authors: Bouchlaka Myriam , Alderson Kory , Sckisel Gail , Chen Mingyi , Murphy William
Published: January 2023

We and others have previously demonstrated that agonistic αCD40 and IL2 immunotherapy (IT) treatment in young mice results in synergistic anti-tumor effects in multiple tumor models. However, efficacy of this and other ITs can be limited by their induction of systemic toxicity. The majority of mouse tumor studies use younger mice (8-12 weeks) which do not accurately represent the age of a typical cancer patient (>50 yrs or 12 months in a mouse). Therefore, we sought to determine the potential toxicity of IT in aged versus young mice. Within 2-3 days after IT treatment, marked toxicity was observed in aged (17-22 months) and middle aged mice (9-12 months) resulting in 100% mortality as opposed to no mortality in young mice (2-4 months). Aged mice had significantly higher serum levels of TNFα, IFNγ, IL6 and IL17a after IT compared to young treated mice, with a proportionate increase with age. Histological evaluation of aged treated mice revealed disseminated lymphocytic infiltrates to the gut, lung and liver; multiple organ damage with hepatic piecemeal necrosis; extensive GI mucosal damage and peribronchiolitis in the lung compared to young treated mice. Finally, IT-treated aged IFNγ-receptor and TNFα-KO mice showed significant decreases in mortality compared to treated WT-aged mice. This data suggests that IT with αCD40/IL2 induces a cytokine storm culminating in multiple organ damage and rapid lethality that correlates with increased serum cytokines and worsens with age.
Cytokine-based cancer immunotherapies induce differential effects on CD4+ and CD8+ T cell phenotype and function (156.9)
Authors: Sckisel Gail , Hsiao Hui-Hua , Bouchlaka Myriam , Wilkins Danice , Tietze Julia , Blazar Bruce , Murphy William
Published: January 2023

We have recently observed that cytokine-based immunotherapy (IT) regimens induced an Ag-nonspecific expansion of the memory T cell pool which was responsible for anti-tumor efficacy. We therefore sought to determine the effects of systemic IT on subsequent Ag-specific responses. During IT, there was a significant expansion of the memory CD8 T cell population with the cells in active cell cycle. Splenocytes from treated mice proliferated less in response to TCR engagement, yet they proliferated to a greater extent when stimulated with IL2 alone. Flow cytometric analysis of stimulated splenocytes revealed that CD8 T cells from IT treated mice appropriately upregulated activation markers (ie, CD25, CD44, NKG2D) and expanded normally. Conversely, CD4 T cells from the same mice exhibited markedly less proliferation, activation marker upregulation, and IFNγ production in response to the same stimuli. In a vaccine model where mice were immunized against allogeneic splenocytes while undergoing IT, CD8 T cells were capable of responding to antigen earlier and more robustly upon secondary in vitro restimulation than immunized, untreated mice. Together, these data show that immune stimulatory therapies allow CD8 T cells to function as Ag-specific or nonspecific effectors while functionally exhausting CD4 T cells. We also show that neither CD4 exhaustion after IT, nor the nonspecific expansion of memory CD8 T cells impairs the ability of CD8 T cells to generate new Ag-specific responses.
Activation of the inflammsome inhibits the development of cell-mediated immunity (157.10)
Authors: Sauer John-Demian , Pereyre Sabine , Portnoy Daniel
Published: January 2023

The inflammasome is a multiprotein complex that leads to the activation of Caspase-1, resulting in secretion of IL-1β and IL-18 as well as the induction of a programmed cell death termed pyroptosis. To understand the role of the inflammasome in the development of CD8+ T-cell mediated adaptive immune responses, we utilized Listeria monocytogenes as a model system. Wild type L. monocytogenes infection resulted in negligible activation of the inflammsome, therefore we engineered a strain of L. monocytogenes to robustly activate the inflammasome. A strain of L. monocytogenes (LpFlaA-L. monocytogenes) engineered to ectopically express Legionella pneumophila flagellin, a well described agonist of the Nlrc4 inflammsome, resulted in strong activation of the inflammasome in vitro. Activation of the inflammasome resulted in attenuation of L. monocytogenes virulence during acute infection. In addition, mice immunized with LpFlaA-L. monocytogenes failed to provide protection against a subsequent lethal challenge. Protection from challenge was rescued in Caspase-1 mice immunized with LpFlaA-L. monocytogenes. In addition to lack of protection, there was a decrease in the development of both antigen specific CD4+- and CD8+-IFNγ producing T-cells following immunization with LpFlaA-L. monocytogenes compared to wild type immunization. Taken together our data suggests that activation of the inflammasome is detrimental to the development of cell-mediated adaptive immune responses.
RIG-LIKE receptor signaling modulates the innate immune response and astrocyte activation after injury to the spinal cord (157.6)
Authors: de Rivero Vaccari Juan Pablo , Minkiewicz Julia , Wang Xiaoliang , German Ramon , Marcillo Alex , Dietrich W. , Keane Robert
Published: January 2023

Injury to the central nervous system (CNS) induces a glial response, in which astrocytes become activated and produce inflammatory mediators. However, the molecular basis for glial activation and the signaling pathways regulating glial/innate immune responses remains poorly understood. Here, we examine the activation of retinoic acid inducible gene-like (RIG) receptors (RLRs) and the involvement of RLR signaling in the regulation of type I IFNs following spinal cord injury (SCI). Immunohistochemical analysis followed by confocal microscopy procedures reveals that astrocytes express two intracellular RLRs, RIG-I and melanoma-associated gene 5 (MDA5). Immunoblotting of spinal cord protein lysates after SCI and stretch injury of culture astrocytes indicates that injury to the CNS activates RLR signaling as determined by phosphorylation of IRF3 leading to production of type I IFNs. Stimulation of RIG-I and MDA5 with synthetic RNA (poly(I:C)) results in RLR signaling activation, phosphorylation of IRF3, and increased expression of glial fibrillary acidic protein and the intermediate filament protein vimentin, two hallmarks of reactive astrocytes. To the best of our knowledge this is the first time that the RLR signaling pathway is described as being involved in regulating astrocyte activation and modulation of the innate immune response after SCI.
The Protein Kinase C alpha regulates RIG-I mediated IRF3 activation through HDAC6 and beta-catenin (158.18)
Authors: Zhu Jianzhong , Sarkar Saumendra
Published: January 2023

RIG-I like receptors (RLR) are key cytoplasmic receptors to recognize viral RNA, and induce type I interferons via transcription factors such as IRF3 and NF-κB. Here we describe a novel signaling pathway involving PKCα, HDAC6 and β-catenin, which is essential for IRF3-mediated gene induction after virus infection. Stable knockdown of PKCα in various human cell lines inhibited Sendai virus-mediated IFN induction and enhanced virus replication. Sendai virus infection activated PKCα, which enhanced HDAC6 activity in a signal-dependent manner. HDAC6 physically interacted with PKCα and virus infection increased the deacetylation activity of HDAC6, which was absent in PKCα knockdown cells. HDAC6-mediated deacetylation of β-catenin enhanced its nuclear translocation and promoter binding, which was also attenuated in PKCα knockdown cells. We show that in the absence of PKCα/HDAC6-mediated β-catenin nuclear translocation, IRF3, although activated, does not bind to its promoter and is unable to support transcription.
Lung natural helper cells are an important source of innate TH2 cytokines (158.2)
Authors: Halim Timotheus , Sun Ann , Takei Fumio
Published: January 2023

Recently, novel innate TH2 cytokine producing lymphocytes termed nuocytes or natural helper cells have been found in the gut-associated mucosa tissues. Similar TH2 cytokine producing innate cells have also been detected in other organs, including the lung, but their functional significance is not well understood. We have identified TH2 cytokine producing innate lymphocytes, termed lung natural helper (NH) cells in B6 mouse lungs and elucidated their role in lung eosinophilia and asthma. Lung NH cells are Lineage-negative but express of Sca-1, c-Kit, CD127 and CD25. Lung NH cell gene expression microarrays indicate that they likely constitute a novel lymphoid lineage. When purified and stimulated in vitro, they produce large amounts of IL3, 4, 5, 13, and IL17A. They also rapidly produce IL5 and IL13 in lung explant cultures stimulated with IL25, an alarmin released by stressed lung epithelial cells. Lung NH cells are present in Rag-/- mice that lack T and B cells but absent in Rag-/-cγ-/- mice. Intranasal administration of protease allergen papain rapidly induces eosinophil infiltration and mucus hypersecretion, which are hallmark asthma symptoms, in the lung of B6 and Rag-/- mice but not in Rag-/-cγ-/- mice. Papain treatment of lung explants also induces IL13 production by lung NH cells. Thus, lung NH cells are unique lymphocytes of the innate immune system and an important source of T cell-independent TH2 cytokines. They likely play an important role in initiating asthma.
Dynamic changes of gene expression in concordance with histone modifications in CD8 T cells after activation (159.2)
Authors: Subedi Kalpana , Araki Yasuto , De Supriyo , Wood William , Sharov Alexei , Zang Chongzhi , Schones Dustin , Lhotsky Brad , Dudekula Dawood , Becker Kevin , Ko Minoru , Peng Weiqun , Zhao Keji , Weng Nan-ping
Published: January 2023

Memory T cells are characterized by their ability to exhibit a rapid response to the recall antigen. Our previous study shows that histone methylation provides a chromatin basis for the rapid transcriptional response of memory CD8 T cells. To further understand how the histone methylation patterns are established and maintained in memory T cells, we conducted a parallel analysis of gene expression changes with histone methylation changes in naïve and memory (central and effector memory cells) CD8 T cells after in vitro stimulation. Naïve and memory CD8 T cells were isolated from normal adult and stimulated in vitro with antibodies against CD3 and CD28. Gene expression changes (microarray) and histone methylation (H3K4me3 and H3K27me3) (ChIP-Seq) were analyzed. We observed substantial changes in gene expression (~25% increase and ~25% decrease of total expressed genes) after 72 hours of stimulation. Similar trends of histone methylation (H3K4me3 and H3K27me3) changes in corresponding genes in CD8 T cells were observed, suggesting a dynamic change of histone methylation in CD8 T cells after activation. Furthermore, we found that the open chromatin of some poised genes in memory CD8 T cells were established in activated naïve CD8 T cells. Together, these results indicate that histone methylation is dynamic after activation in CD8 T cells and suggest that activation-induced change of chromatin in naïve T cells is part of differentiation process to establish memory T cells.
Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration (159.6)
Authors: Kurachi Makoto , Takamura Shiki , Abe Jun , Ueha Satoshi , Matsushima Kouji
Published: January 2023

Strength of inflammatory stimuli during the early expansion phase plays a crucial role in effector and memory cell fate-decision of CD8+ T cells. But it is not known how early lymphocyte distribution after infection impacts this process. Here we demonstrate that chemokine receptor CXCR3 is involved in CD8 T-cell commitment to effector rather than memory fates by regulating T-cell recruitment to an antigen/inflammation site. After systemic viral (VV-OVA) and bacterial (LM-OVA) infection, contraction of CXCR3KO antigen-specific CD8 T cells is significantly attenuated, resulting in massive accumulation of fully functional memory CD8 T cells. Unlike WT cells, CXCR3KO antigen-specific CD8 T cells fail to cluster early after infection at the marginal zone in the spleen, where inflammatory cytokines such as IL-12 and IFNα are abundant, thus receiving relatively weak inflammatory stimuli. Consequently, CXCR3KO CD8 T cells exhibit shortened expression of CD25, and preferentially differentiate into memory precursor effector cells as opposed to WT CD8 T cells. This series of events has important implications for development of vaccination strategies to generate increased numbers of antigen-specific memory CD8 T cells via inhibition of CXCR3-mediated T-cell migration to inflamed microenvironments.
Miz-1 is required to coordinate the pre-TCR-dependent transitional step in DN3/DN4 thymocytes. (160.1)
Authors: Saba Ingrid , Kosan Christian , Möröy Tarik
Published: January 2023

Miz-1 (Myc-interacting zinc finger-1) is a BTB/POZ domain transcription factor that can activate or repress gene transcription. To explore the function of Miz-1, we have generated mice that express a non-functional protein lacking the POZ domain, Miz-1ΔPOZ. These mice show a very small thymus owing to a dramatic reduction of early T cell progenitors and DN4 pre-T cells. DN1-3 cells have higher apoptotic rate indicating that Miz-1 is coordinating survival signals. Miz-1ΔPOZ mice also have a DN3/DN4 transitional block and DN3 cells showed a dramtic up-regulation of the cell cycle regulator p21Waf1. Surprisingly, neither the introduction of a Bcl-2 transgene nor the deletion of p21Waf1 rescued the DN3/DN4 block in Miz-1ΔPOZ mice. The block at the pre-TCR-dependent selection stage rather suggested that Miz-1 might regulate pre-TCR signaling. Although the pre-TCR signaling does not seem to be altered by the expression of a non-functional Miz-1, the intracellular expression of the TCRβ is reduced. Introduction of a rearranged TCR transgene rescued the DN3/DN4 cell numbers and their function in Miz-1ΔPOZ mice. Our data suggest that Miz-1 is required for the transport or the stability of the pre-TCR in order to support the proliferative burst of DN3 cells. Comparative gene expression analysis will determine which are the direct effector genes of Miz-1 and contribute to a better understanding of the critical factors involved in this precise step of T cell development.
The role of germinal centers in T cell receptor revision (160.10)
Authors: Higdon Lauren , Fink Pamela
Published: January 2023

CD4+ T cells in Vβ5 transgenic mice become tolerant to an endogenous superantigen through either deletion or T cell receptor (TCR) revision. In the revision process, T cells downregulate surface Vβ5 expression and undergo RAG-mediated rearrangement and expression of endogenous TCRs. Revision occurs in germinal centers (GCs), suggesting that revising T cells may be follicular helper T cells (Tfh). We are studying whether revising T cells have a Tfh phenotype and whether GC interactions are required for revision. Preliminary data indicate that revising T cells have an RNA phenotype closely resembling that of Tfh and distinct from that of post-revision T cells. Studies on the intracellular and surface phenotype of revising and post-revision T cells are ongoing. The importance of GCs to TCR revision is being analyzed using SAP and CXCR5 null mice. SAP is required for prolonged interactions of B and T cells in the GC, and CXCR5 is required for cellular migration into the follicle. Analyses of mixed chimeras generated using CXCR5 null and wildtype bone marrow donors are ongoing. CXCR5 and SAP null mice have reduced numbers of post-revision T cells, but no reduction in revision intermediates. These results suggest that migration into the follicle and prolonged B-T cell interactions are both required for completion, but not initiation, of TCR revision.
Essential role of IL-12 signaling for NK cell expansion and memory formation during viral infection (160.15)
Authors: Sun Joseph , Bezman Natalie , Beilke Joshua , Lanier Lewis
Published: January 2023

Although natural killer (NK) cells are generally classified as innate immune cells, several recent studies demonstrated that NK cells become long-lived memory cells and contribute to secondary immune responses like T and B cells. However, the precise signals that promote generation of long-lived NK cells are unknown. Using cytokine receptor deficient mice, we show that the pro-inflammatory cytokine IL-12 is indispensible for virus-specific NK cell expansion and generation of memory cells during MCMV infection. In contrast to wildtype NK cells which proliferated robustly and resided in lymphoid and non-lymphoid tissues for months following MCMV infection, IL-12 receptor-deficient NK cells failed to expand and were difficult to detect soon after infection. In the absence of IL-12 signaling, NK cells were also unable to mediate protection following MCMV challenge. We have evidence that a STAT4-dependent, IFN-gamma-independent mechanism contributes toward the generation of memory NK cells during viral infection. Understanding the full contribution of inflammatory cytokine signaling in the NK cell response to viral infection will be of interest for development of vaccines and therapeutics.
Investigating the involvement of Foxo1 in T cell repertoire diversity (160.8)
Authors: Gubbels Bupp Melanie , Horseman Erica
Published: January 2023

Advanced age is associated with increased susceptibility to and mortality from infectious diseases, especially influenza and pneumonia; and vaccination responses in the elderly are diminished. Declining resistance to infection in aging individuals is correlated with a progressive decline in the size and repertoire of the T cell pool. A better understanding of the mechanisms underlying age-related deterioration of T cell immunity could inform therapies that enhance general immune function and vaccination effectiveness in this vulnerable population. The forkhead transcription factor, Foxo1, has recently been identified as a major regulator of naïve T cell homeostasis. Moreover, a Foxo1 homolog, DAF-16, promotes longevity in C. elegans. Therefore, these studies documented the repertoire diversity of splenic CD4+ and CD8+ T cells in mice selectively deficient for Foxo1 in the T cell compartment (Foxo1 cKO mice). Our studies indicate that T cell-specific Foxo1 deficiency impacts TCR diversity in both the CD4+ and CD8+ splenic T cell populations of young mice. These observations suggest the intriguing possibility that in mice, Foxo1 may participate in aging-related changes in T cell diversity.
B cell traffic through skin increases during inflammation (161.13)
Authors: Geherin Skye , Fintushel Sarah , Lee Michael , Alt Carsten , Young Alan , Hay John , Debes Gudrun
Published: January 2023

B cells infiltrate skin in many chronic inflammatory diseases caused by autoimmunity or infection. Despite their potential to contribute to disease processes, skin-associated B cells remain poorly characterized. Here, we find in an ovine granuloma model of cutaneous inflammation, B cells increase during the chronic phase of inflammation in both relative and absolute number in the skin and skin-draining afferent lymph. Surprisingly, these B cells are a heterogeneous population that is phenotypically distinct from the B cell population in lymph nodes with more ‘innate-like’ B cells co-expressing high levels IgM and CD11b. Furthermore, skin B cells have increased MHCII and CD80/86 expression compared to lymph node B cells, suggesting that they are well-suited for T cell activation at the site of inflammation. Additionally, unlike co-isolated T cells, skin B cells are not responsive to ligands of chemokine receptors typically associated with skin homing, implying that they utilize alternative chemokine receptors to migrate to the skin. In summary, our data show that a population of B cells recirculates through uninflamed and inflamed skin, fulfilling a so far unappreciated role in skin immunity.
Antigen-dependent surveillance of the lungs by pathogen-specific CTL prolongs protective cellular immunity after influenza infection. (161.18)
Authors: Lee Young Tae , Wu Tao , Bouchard Keith , Cauley Linda
Published: January 2023

Influenza viruses are segmented negative-strand RNA viruses which do not integrate into the genome of their host and are incapable of latent infection. Although infectious virus is cleared from the lungs within ~7-10dpi we have shown that trace quantities of processed peptide antigen remain in the draining lymph nodes of the respiratory tract for ~2 months and correspond with local changes in the migration patterns of pathogen-specific CTL in the respiratory tract. Prolonged antigen presentation is a common characteristic of infection with multiple strains of influenza virus, but antigen clearance can occur with much faster kinetics after infection with some isolates. Comparisons between these different isolates show that the processed peptide antigens reinforce expression of two homing receptors (CD69 and CD103) in the lungs which help activated virus-specific CD8 T cells colonize the mucosal tissues during a mild inflammatory response. Furthermore, virus-specific CTL which lacked CD103 expression were less efficient in clearing infectious virus from the lungs, than wild type cells, after heterosubtypic challenge. These data indicate that prolonged antigen presentation is required to reinforce local CTL responses in the lungs and thus explains why protective cellular immunity is short-lived.