bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential pro-survival role for caspase-8 during T-cell activation and embryonic development through inhibition of RIPK3-dependent necrosis (159.9)
Authors: Dillon Christopher , Oberst Andrew , Weinlich Ricardo , Martinez Jennifer , McCormick Laura , Fitzgerald Pat , Pop Cristina , Hakem Razq , Salvesen Guy , Green Douglas
Published: January 2023

Caspase-8 is required for initiation of death receptor-mediated apoptosis, but counterintuitively is also required for normal T-cell proliferation and embryonic development. Recent work demonstrated that upon activation, caspase-8-deficient T cells fail to proliferate due to high rates of necroptosis, an alternative form of programmed cell death. To investigate these seemingly contradictory functions, we bred caspase-8 knockouts, which are embryonic lethal at E10.5, with necroptosis deficient RIPK3-/- animals. Unlike Casp8-/- animals, Casp8-/- RIPK3-/- double knockout (DKO) animals were generated at expected Mendelian frequencies and appeared grossly normal. DKO thymocytes responded normally to stimuli inducing intrinsic cell death, but resisted death induced by ligation of CD95. DKO animals, but not their normal littermates, were resistant to liver damage and death induced by injection of agonistic anti-Fas Ab Jo2. In contrast to casp8-/- T cells, DKO T cells proliferated normally in response to TCR ligation and displayed normal expansion and subsequent peripheral deletion in vivo when challenged with SEB. Interestingly, DKO animals display lymphoaccumulation of an aberrant B220+CD3+ population similar to that seen in gld or lpr mice. The survival of Casp8-/- RIPK3-/- mice and the normal proliferation of T cells from these animals point to an essential role for caspase-8 in limiting RIP3-dependent necrosis both during development and immune cell activation.
Chronic IFN-gamma expression alters the migration pattern of peripheral T cells (160.7)
Authors: Lin Fanching , Saleh Bahara , Hodge Deborah , Young Howard
Published: January 2023

We report here on preliminary data from an IFN-gamma(IFN-γ)ARE-deleted Balb/c mouse model developed in our laboratory.This mouse has a 162 nt deletion in the 3’untranslated region of the IFN-γ mRNA to eliminate an AU-rich sequence. The deletion has a positive impact on IFN-γ mRNA accumulation by dramatically increasing its half-life. In the absence of stimulatory factors,these mice express low level of IFN-γ constitutively. IFN-γ ARE-deletion results in a severe phenotype on Balb/c mice. The knockout (KO) and heterozygous (Het) mice are smaller in size than the littermate controls, have a rough coat and a low survival rate after weaning. Additionally, these mice have an enlarged thymus with an increased percentage of mature naïve T cells. The mature thymocytes from Het animals are able to leave the thymus and enter peripheral lymphoid tissues. However, the results from chimera experiments indicate that IFN-γ may impact the thymic environment, thus resulting in abnormal T cell development. In addition, we found the migration pattern of peripheral T cells from Het mice differs from that observed with cells from control animals, further suggesting an abnormality in the Het T cell physiology. Current experiments focus on analyzing changes in the thymic environment and T cell development in newborn mice. In addition, we are analyzing chemokine receptor expression to determine if changes in these receptors may be a mitigating factor in the T cell migration patterns.
The elucidation of the oral/respiratory axis in inducing protective immunity against Francisella tularensis (161.22)
Authors: Signarovitz Aimee , Yu Jieh-Juen , Ray Heather , Guentzel M. Neal , Williams Ifor , Yagita Hideo , Berton Michael , Klose Karl , Arulanandam Bernard
Published: January 2023

Using a Francisella tularensis subspecies novicida live attenuated vaccine strain (U112ΔiglB), we have shown that oral vaccination of C57BL/6 mice induces significant protective immunity against pulmonary challenge with the highly human virulent F. tularensis strain SCHU S4. This protection is primarily mediated by antigen-specific CD4+ T cells and IFN-γ production and is dependent on mucosal IgA expression. We are examining the mechanism(s) by which the oral/respiratory axis induces optimal protective immunity against F. tularensis. Intestinal M-cells play an important role in oral vaccination by trafficking antigens from the lumen to the lamina propria and in presentation of antigens to underlying APCs for the subsequent induction of mucosal immunity. We have shown that mCherry-labeled U112ΔiglB co-localizes to intestinal mouse M-cells by 90 minutes following vaccination. Localization to M-cells was further verified with mouse ileal loops. Translocation of U112ΔiglB has also been demonstrated through M-cell-like-cells in an in vitro transwell co-culture system similar to M-cell trophic enteric bacteria. Current studies are examining the functional importance of intestinal M-cells in induction of mucosal immunity for the rational development of an effective oral vaccine against pulmonary tularemia. These studies will provide information for the general design of oral vaccines using live attenuated bacterial strains.
Immediate induction of enhanced CD8 suppressive function following glatiramer acetate therapy in multiple sclerosis (164.20)
Authors: Ayers Chris , Mendoza Jason , Greenberg Benjamin , Frohman Elliot , Karandikar Nitin
Published: January 2023

Multiple Sclerosis (MS) is an immune-mediated, demyelinating disorder of the CNS. Glatiramer acetate (GA/Copaxone) is an FDA-approved treatment for relapsing-remitting MS that induces functional changes in antigen-presenting cells (APC) and CD4 and CD8 T-cells. We have previously shown that treatment-naïve MS patients have deficient CD8 T-cell responses to GA, which are restored after GA therapy. This is associated with enhanced cytotoxic/suppressive function in these cells. In this study, we assessed the immediate immunologic changes induced by GA therapy in treatment-naïve MS patients by multiparametric monitoring of T-cell and APC subsets prior to and at 4, 12, 24, 72 hrs of treatment. In untreated healthy control subjects quantitative and functional readouts showed stable dynamics over time. MS patients showed greater proportions of memory/effector CD4 and CD8 populations at baseline, which did not vary. Similarly, cytokine production by APC subsets was also stable. Interestingly, as early as 4 to 12 hrs after the initial GA injection, we observed significantly enhanced suppressive ability in CD8 T-cells. This rapid functional change consistently occurred in GA, as well as an IFN-b-treated control cohort. These studies show that the induction of T-cell suppressive function is an important early event during therapy. Future studies will delineate whether these changes are predictive of sustained longterm immunologic and clinical responsiveness.
Macromolecular prodrug conjugate P-Dex attenuates nephritis in lupus-prone (NZBxNZW)F1 mice (164.5)
Authors: Nelson Richard , Yuan Fang , Wang Dong , Gould Karen
Published: January 2023

Corticosteroids are used in the treatment of nephritis, a severe manifestation of systemic lupus erythematosus. Untreated, nephritis results in chronic glomerular inflammation, loss of renal function, albuminuria, and ultimately leads to renal failure and death. Unfortunately, current therapeutic strategies are problematic, as the prolonged systemic exposure of corticosteroids yields severe side effects, including hypertension, osteoporosis, and immunosuppression. To remedy this, we developed a polymeric macromolecular prodrug (P-Dex) that targets inflamed glomeruli and other sites of inflammation by sequestering the corticosteroid dexamethasone via an acid-labile bond. In this study, we evaluated the efficacy of P-Dex in treating nephritis in lupus-prone (NZBxNZW)F1 mice. After cessation of treatment, the P-Dex treatment group displayed a lower incidence of albuminuria and greater histological preservation of glomerular structure than control groups. Further, P-Dex mitigated the side effects associated with the use of corticosteroids. Compared to control groups, mean arterial pressure was significantly lower in the P-Dex group, approaching normal levels. Finally, in contrast to mice treated with free Dex, mice treated with P-Dex displayed no significant loss of bone mineral density. Together, these data suggest that P-Dex may be a more effective therapy in treating nephritis, while simultaneously minimizing side effects associated with prolonged exposure to corticosteroids.
Inhibition of differentiation, amplification and function of human Th17 cells by intravenous immunoglobulin (164.7)
Authors: Maddur Mohan , Janakiraman Vani , Hegde Pushpa , Lacroix-Desmazes Sebastien , Kaveri Srini , Bayry Jagadeesh
Published: January 2023

Th17 cells play a critical role in the pathogenesis of several autoimmune and inflammatory diseases. Intravenous immunoglobulin (IVIg), a therapeutic preparation of polyclonal IgG, increasingly used in the treatment of diverse autoimmune and inflammatory diseases, may target Th17 cells to exert therapeutic effects. Therefore, we examined whether IVIg interferes with the development and function of human Th17 cells. To address this, Th17 cells were differentiated from naïve human CD4+ T cells in the presence of TGFβ and IL-21. Th17 cells were amplified by stimulating memory CD4+ T cells in the presence of IL-1β and IL-6. The effect of IVIg was examined on differentiation and amplification of Th17 cells, expression of Th17 lineage-specific transcription factor RORC, secretion of Th17 effector cytokines and phosphorylation of STAT3, a transcription factor that plays an important role in Th17 development and function. IVIg inhibits the differentiation and amplification of human Th17 cells, as well as the production of their effector cytokines IL-17A, IL-17F, IL-21 and CCL20. The inhibitory effects of IVIg on Th17 cells are F(ab')2-dependent and involve interference with the expression of RORC and activation of STAT3. Also, IVIg significantly enhanced Foxp3+ regulatory T cells (Treg) among the memory CD4+ T cells. These results reveal a novel mechanism of action of IVIg in achieving therapeutic effect in autoimmune and inflammatory diseases.
Targeting of pre-existing and induced breast cancer stem cells with HER2-specific immunotherapeutics and NK cells (165.18)
Authors: Wischhusen Jörg , Diessner Joachim , Becker Kathrin , Hönig Arnd
Published: January 2023

Trastuzumab (Herceptin®) has substantially improved the overall survival of patients with mammary carcinoma. While recruitment of NK cells and antibody-dependent cell-mediated cytotoxicity (ADCC) contribute to this therapeutic effect, immunoselection of cultured breast cancer cells with trastuzumab and NK cells resulted in selective survival of individual sphere-forming cells. Moreover, epithelial to mesenchymal transition was induced which transformed previously differentiated cancer cells into cells with a CD44highCD24low “cancer stem cell-like” phenotype. Immunoselected breast cancer cells thus showed increased clonogenicity and differentiation potential in vitro and enhanced tumorigenicity in vivo. Their insufficient immunotherapeutic targeting might be due to low HER2 expression and the virtual absence of ligands for the activating NK cell receptors NKG2D and DNAM 1. Susceptibility towards NK cells and trastuzumab, however, was restored upon differentiation. Since relapse after antibody therapy could be due to such trastuzumab- and NK cell-refractory tumor initiating cells, we explored if treatment of these cells with the novel HER2-targeting drugs T-DM1 or the HER2 dimerisation inhibitor pertuzumab could improve NK cell mediated killing or counteract induction of the stem cell-like phenotype. This translational approach might help to identify the most promising drug candidate for the targeted second and third line treatment of HER2-positive metastasized breast cancer.
IFNγ mediates the antitumor effects of radiation therapy (165.34)
Authors: Gerber Scott , Cron Kyle , Lord Edith
Published: January 2023

Radiation (IR) therapy has long been an effective cancer treatment acting primarily through the induction of tumor cell damage at a molecular level. However, a new concept is emerging suggesting that the immune system plays a pivotal role in determining the effectiveness of IR therapy. We have previously demonstrated that IR therapy promotes antitumor immunity through the release of tumor antigens and induction of a pro-inflammatory tumor microenvironment. Our work here has identified IFNγ as an essential cytokine which mediates the efficacy of IR therapy. Local IR (15Gy) to mice bearing colon38, a colon adenocarcinoma, resulted in decreased tumor burden in wild type animals. Interestingly, IR therapy had no effect on tumor burden in IFNγKO mice. We further determined that intratumoral (i.t.) levels of IFNγ spiked two days following IR which directly correlated with a decrease in tumor burden that was not a result of direct cytotoxic effects of IFNγ on tumor cells. CD8+ T cells and NK cells were the predominant producers of IFNγ, however both radiosensitive populations were depleted by IR; a paradox not yet completely understood. IR treated tumors could be further divided into responders and non-responders. Responders were shown to possess higher IFNγ levels, and i.t. treatment with IFNγ further enhanced the effectiveness of IR therapy. These data suggest that IFNγ may play a pivotal role in mediating the antitumor effects of IR therapy.
Early Treg suppression by a Listeriolysin-O-expressing E. coli vaccine in heterologous prime-boost vaccination against cancer (165.49)
Authors: Dai Ming-Shen , You Ren-In , Hsieh Yu-Feng , Lo Kai-Yu , Sytwu Huey-Kang , Vassaux Georges , Chao Tsu-Yi
Published: January 2023

[Background] Earlier studies have shown that higher CD8+ T-cell response and better tumor protection can be achieved by heterologous prime-boost vaccination in mice. We previously demonstrated that a Listeriolysin-O (LLO)-expressing E. coli vaccine can enhanced CD8-cytotoxic T cell (CTL) responses by mitigating regulatory T cells (Treg)-mediated suppression. We herein employed this Treg-inhibiting vaccine into the prime/boost immunization strategy. [Methods] Bacteria E. coli-LLO expressing Ovalbumin (OVA) and plasmid pcDNA—encoding OVA were used as specific antigen immunization. C57B6 mice and syngenic melanoma cell line B16-OVA were used in tumor challenge model. CD25 monoclonal antibody (PC-61) was used for Treg depletion. [Results] Higher OVA-specific CD8+ T-cell responses and superior tumor inhibition were seen in the bacterial-prime/plasmid-boost setting than homologous or reversed sequence. This tumor protection effect from heterologous prime/boost remained significant in the therapeutic model. When examining the Treg effect during the prime/boost immunization, we found that only early Treg-suppression/depletion could lead to better antigen-specific CTL and tumor response. [Conclusion] Our studies offer the first evidence that a Listeriolysin-O E. coli vaccine can induce superior anti-tumor effect in conjunction with DNA in a heterologous prime-boost protocol and proposed that early Treg inhibition is crucial to a successful immunization against cancer.
Severe herpesvirus-induced pulmonary pathology post-syngeneic bone marrow transplant is related to effects of transforming growth factor-beta (169.39)
Authors: Coomes Stephanie , Farmen Sara , Wilke Carol , Laouar Yasmina , Moore Bethany
Published: January 2023

Pulmonary infections and pneumonitis are frequent complications of hematopoietic stem cell transplant. We intranasally infected syngeneic bone marrow transplant (BMT) mice at 5 weeks post-BMT and non-transplanted control mice with the murine gammaherpesvirus, γHV-68. At d7 post-infection, BMT mice have increased levels of lytic virus compared to control. By d21 post-infection, the virus is latent and there is no difference in viral load between BMT and control mice. However, BMT mice develop virus-induced pneumonitis characterized by fibrosis, peripheral inflammation, and hyaline membranes. Alveolar macrophages from BMT mice are foamy and express increased levels of both alternative and classical activation markers. BMT mice have reduced oxygen saturation and an infiltration of neutrophils and T cells in the alveolar space, as well as increased levels of hydrogen peroxide, nitrite, and TGFβ. Using mice expressing the dominant negative TGFβRII transgene (DN-TGFβRII) as BMT donors allowed us to determine the effects of TGFβ in the development of pneumonitis on multiple cell types. T cell DN-TGFβRII BMT mice had a drastic reduction in pulmonary pathology, while CD11c DN-TGFβRII BMT mice had only a modest improvement. These data suggest that development of herpesvirus-induced pneumonitis post-BMT can be significantly reduced by blocking TGFβ signaling, especially in T cells.
Induction of PP2A Bβ, a novel regulator of IL-2 deprivation-induced T cell apoptosis, is deficient in patients with systemic lupus erythematosus. (44.17)
Authors: Crispin Jose , Apostolidis Sokratis , Finnell Melissa , Tsokos George
Published: January 2023

The activity and specificity of the ubiquitously expressed phosphatase PP2A is determined by the type of regulatory (B) subunit that couples to the catalytic/scaffold core of the enzyme. We determined that the Bβ subunit (PPP2R2B) -considered brain specific- is expressed in T cells during late phases of activation (48 to 72 h). To determine its function, we cloned and expressed it in non-stimulated human T cells where it triggered apoptosis. To identify which conditions promote the expression of PP2A Bβ, we induced apoptosis in T cells through Fas ligation, restimulation, or IL-2 deprivation. Transcription of PP2A Bβ was induced exclusively by IL-2 deprivation. Since T cell apoptosis is known to be altered in T cells from patients with systemic lupus erythematosus (SLE), we analyzed the regulation of PP2A Bβ in this autoimmune disease. We found that levels of PP2A Bβ did not increase upon IL-2 deprivation in 2/3 of the patients. Remarkably, this defect was accompanied by resistance to apoptosis. Moreover, kinetics of cell death were normal in cells that upregulated PP2A Bβ. We have identified a novel role for the phosphatase PP2A, particularly the holoenzyme formed by PP2A Bβ. Bβ appears to trigger apoptosis of T cells in the absence of IL-2 and probably contributes to the termination of a no-more-needed immune response. We propose that in SLE patients, defective apoptosis contributed by faulty upregulation of PP2A Bβ contributes to longer survival of autoreactive T cells.
Innate memory phenotype CD8+ T cells exhibit prompt and potent antigen specific response (46.2)
Authors: Huang Weishan , August Avery
Published: January 2023

Innate memory phenotype CD8 T cells (IMP CD8+CD44hiCD122int) develop in thymus and carry abundant preformed mRNA for cytokines such as interferon-γ (IFN-γ). These cells rapidly produce IFN-γ when stimulated with PMA/Ionomycin or IL-12/IL-18. Whether they can also contribute to antigen specific responses is unclear, as it is difficult to distinguish them from “true” memory cells. We developed a novel method to derive antigen specific IMP CD8, using murine bone marrow chimeras to separate naïve and IMP antigen specific CD8 T cells recognizing the model antigen ovalbumin (OVA). WT or OTI bone marrow transplanted into irradiated MHCI-/- mice gave rise to predominantly IMP CD8 T cells. By contrast, MHCI-/- or OTIMHCI-/- bone marrow transplanted into irradiated WT mice gave rise to predominantly naïve CD8 T cells. IMP CD8 T cells thus derived significantly up-regulated IFN-γ 6 days post OVA restimulation in vitro, following primary exposure to transgenic Listeria-OVA, compared to analogously derived naïve CD8 T cells alone or WT controls. More importantly, OTI IMP CD8 T cells exhibited significantly enhanced early response to OVA or OVA257-264 peptide without primary stimulation. Our findings indicate that the dynamics of the antigen specific response of IMP CD8 T cells is enhanced compared to antigen specific naïve CD8 T cells. Furthermore, this work provides a promising murine model for future investigation of IMP CD8 T cell memory development and antigen response.
Verification of immunophenotyping markers for naïve & memory mouse T cells (46.22)
Authors: Hingorani Ravi , Ernst David
Published: January 2023

The immune system responds with greater intensity upon antigen re-exposure. This forms the basis of immunological memory. Mouse memory T cells form a heterogeneous group of long-lived cells that express high CD44 levels. Naïve and central memory T cells express CD62L and recirculate through secondary lymphoid tissues while effector memory cells do not express CD62L and do not recirculate through the lymphoid tissues. Effector memory T cells express homing receptors enabling their entry into nonlymphoid tissues and are prevalent during certain infections. Clear understanding of mechanisms that mediate immunologic memory is essential for effective vaccine development. Tremendous effort has been devoted to characterizing markers on the surface of T cells to help identify cells responding to vaccines. To date no single marker that segregates T cell subsets involved in immunological memory has been described. Based on the literature we developed a 4-color fluorescent antibody panel that identifies naïve, memory and effector mouse T-cells. The panel consists of antibodies towards CD4, CD3, CD44 and CD62L. This panel enables identification of core subsets and provides flexibility to incorporate additional probes/antibodies labeled for the FL1 channel. Optimal set up procedures and gating strategies for different flow cytometers will be presented. The panel was used to differentiate naïve and memory T cells from old and young mice, in different tissues and different mouse strains.
Intrinsic interferon signature in myeloid dendritic cells from lupus-prone mice precedes disease onset (47.1)
Authors: Gallucci Stefania , Varghese Linda , Bennett Heather , Jog Neelakshi , Shivers Debra , Ning Yue , Behrens Edward , Caricchio Roberto , Sriram Uma
Published: January 2023

Systemic Lupus Erythematosus (SLE) patients over-express Type I Interferon (IFN) responsive genes but the cellular source of this IFN Signature is unclear. We found that ex vivo dendritic cells (DCs) from Sle1,2,3 lupus-prone mice express the IFN Signature. To test if the IFN Signature was intrinsic to DCs, we grew myeloid DCs in vitro from bone marrow in GM-CSF medium (BMDCs), and found that IFN responsive genes, analyzed by real-time RT-PCR, are constitutively over-expressed in Sle1,2,3 BMDCs as compared to non-autoimmune C57BL/6 BMDCs: expression of IFN-b, signaling molecules IRF-7, STAT1 and STAT2, and IFN responsive genes ISG-15, IL-15, Oas-3 and Mx1, was significantly higher in lupus BMDCs than in C57BL/6 BMDCs in absence of any stimulation. The IFN Signature was similar in BMDCs from mice negative for autoAbs and from mice with high autoAb titers, suggesting that the IFN Signature precedes disease onset and is not affected by the autoimmune process. Sle1,2,3 BMDCs were hyper-sensitive to nucleic acids CpGs and R848, confirming a role for TLR7 and TLR9 in lupus pathogenesis. However, treatment with oligonucleotide sequences inhibitory for TLR7/TLR9 did not suppress the constitutive over-expression of IFN responsive genes, indicating that DC IFN Signature is independent from chronic exposure to TLR7/9 ligands. We suggest that myeloid DCs are a source of IFN Signature in Sle1,2,3 mice and have an intrinsic dysregulation in the IFN signaling/response.
CDR3 analysis of the TCRVβ repertoire in individual pancreatic lymph nodes of nonobese diabetic mice: evidence for public clonotypes throughout diabetes development. (47.12)
Authors: Marrero Idania , Vong Allen , Dai Yang , Davies Joanna
Published: January 2023

The aim of this study was to test the hypothesis that the natural T cell response during chronic spontaneous Type 1 Diabetes (T1D) is part of an ordered sequence of events that results in the expansion of a predetermined TCR repertoire. We analyzed the TCRVβ repertoire ex-vivo in the pancreatic lymph nodes (PLN) of 4 and 10-week-old pre-diabetic female NOD mice, as well as in NOD mice at the onset of the disease, using CDR3 length spectratyping. The TCRVβ repertoire was compared with the repertoire found in diabetes-resistant (NOR) mice and in NOD mice after cyclophosphamide (CYP) treatment. We show that public clonotypes are common in NOD mice at all stages tested and that the responding public T cell repertoire is dynamic and changes during disease development giving a different signature for prediabetic and diabetic mice. Also, we show that public clonotypes are not restricted to disease susceptible mice. In addition, acute diabetes induced in pre-diabetic mice with CYP is associated with a shift in the responding public T cell repertoire to resemble that of mice with spontaneous T1D. Expansions in T cells expressing TCRVβ5.2, and Vβ12 families are strongly associated with diabetes onset, whether disease is acute or chronic. In conclusion, we show that the progression of T1D is associated with the generation of public T cell clonotype signatures that are specific to early prediabetic, late prediabetic and diabetes onset.
Expansion of peritoneal B1a cells in NZM2410 lupus-prone mice is regulated by the cell cycle regulator gene Cdkn2c (47.3)
Authors: Potula Hari Hara , Xu Zhiwei , Morel Laurence
Published: January 2023

Here, we identified the cyclin-dependent kinase inhibitor Cdkn2c (p18) gene in the Sle2c1 NZM2410-derived lupus susceptibility locus by the combination of genetic mapping and candidate gene analysis, that is responsible for an expansion of the B1a cell compartment. A novel -74 T/C SNP in the p18 promoter is associated with a significantly reduced p18 expression in the splenic B cells and peritoneal cavity (Pc) B1a cells from Sle2c1-carrying mice, as evidenced by micro array, RT-PCR and immunoblot analysis. Compared to B6 controls, this reduced p18 expression leads to a defective G1 cell cycle arrest in splenic B cells that impairs plasma cell function, and to an increased proliferation of Pc B1a cells which results to an age-dependent expansion of the B1a cell compartment. To determine the function of the -74 T/C SNP in p18 expression, we have cloned and characterized the proximal promoter region of the mouse p18 gene in reporter vector. Functional analysis of the 5' flanking region by sequential deletions revealed crucial elements between -300 and +1, confirming the in silico prediction, that two conserved YY-1 sites located in the Sle2c1 allele negatively regulates the p18 expression in SLE prone mouse. EMSAs and ChIP analysis demonstrated that YY-1 interacts in vitro and in vivo with the p18 promoter. These results provide evidence that p18 plays a critical role in B1a cell self-renewal, and its impaired expression leads to high autoreactive accumulation of B1a cells.
CD4+ T cells are critical to the therapeutic efficacy of TAA-based cancer vaccines and control of recurrences (48.11)
Authors: Sharma Rajesh , Srivastava Abhishek , Yolcu Esma , Shirwan Haval
Published: January 2023

The role of CD4+ T cells remains ambiguous in cancer immunotherapy. We herein addressed this issue using a novel form of soluble 4-1BBL (SA-4-1BBL) costimulatory molecule as an effective adjuvant component of HPV E7 and survivin as TAA-based vaccines in E7 expressing TC-1 cervical and survivin over expressing 3LL lung cancer preclinical models, respectively. Vaccination of C57BL/6 mice on day 6 having established tumors with SA-4-1BBL admixed with E7 or survivin TAAs resulted in the eradication of TC-1 and 3LL tumors in 70% and 80% of mice, respectively, along with generation of tumor specific long-term memory that prevented tumor recurrence on second challenge. The in vivo depletion of CD4+ T cells one day before tumor challenge significantly compromised vaccine efficacy in both TC-1 (25%) and 3LL (12.5%) models. In marked contrast, the depletion of CD4+ T cells one day prior to vaccination did not significantly alter the therapeutic efficacy of the vaccines. However, these mice had compromised immunological memory since a second challenge of 3LL on day 60 resulted in tumor development in the majority (85.7%) of mice. Taken together, this study demonstrates an indispensable role of CD4+ T cells in priming immune responses against cancer as well as establishment of long-term memory.
Protective role of T-bet in anti-IL17A treatment of lung cancer (48.13)
Authors: Reppert Sarah , Boross Ildiko , Koch Sonja , Koslowski Michael , Türeci Özlem , Lehr Hans-Anton , Finotto Susetta
Published: January 2023

IL17A is a cytokine mainly produced by Th17 cells. The function of the Th17 cell lineage in lung cancer remains to be elucidated. In our study we found increased expression of the Th17 transcription factors RORα4 and RORγt and the cytokine IL-17A in the lungs of patients with lung adenocarcinoma compared to healthy control tissue. In lung cancer tissues IL-17A inversely correlated with the Th1 specific factor T-bet. Targeting of IL-17A in a murine model of lung adenocarcinoma by intranasal application of anti IL17A antibodies resulted in reduction of lung tumor load and prolonged survival of wild-type mice. Studies in T-bet (-/-) mice revealed up-regulation of lung IL-23/IL-23-R and IL-17A/IL-17A-R in T cells infiltrating the tumor. Local anti-IL-17A antibodies treatment improved (50%) survival in treated T-bet (-/-) mice, reduced the number of IL-17R expressing lung CD4+ T cells, induced the number of natural killer cells and CD8+ T cells producing IFNγ compared to wild type littermates-bearing tumor and treated in the same way. These data might indicate that local anti IL-17A antibody therapy could be successful for the treatment of lung tumor also in the absence of T-bet.
Activation of endogenous CD4 T cells stimulates favorable immunologic changes in the tumor milieu and enhances the antitumor effect of lentivector immunization (48.15)
Authors: He Yukai , Hong Yuan , Peng Yibing , Xiao Haiyan , Mi Michael , Munn David , Wang Gui-Qiang
Published: January 2023

CD4 T cells are known to help the induction, migration, and effector functions of CD8 antitumor responses. Here, we reported an additional layer of CD4 helper function, i.e. activation of endogenous CD4 T cells could modulate the tumor milieu. We engineered a lentivector to express a nominal fusion Ag composed of hepatitis B surface protein (HBsAg) and IgG2a Fc domain (HBS-Fc-lv) that induced potent CD8 as well as robust CD4 response. Remarkably, immunization with this vector caused regression of established tumors. Immunological analysis revealed that functional CD8 and CD4 T effector (Teff) was markedly increased while the Treg ratio was substantially decreased in the regressing tumors. HBS-Fc-lv immunization also shifted the suppressive tumor lesion microenvironment to one that is immune stimulating. Mechanistically, the decrease of tumor Treg ratio and the improvement of antitumor effect of HBS-Fc-lv immunization were dependent on CD4 activation and IFNg production. CD4 Teff cells were preferentially expanded in the vaccine draining lymph nodes and selectively recruited into tumor lesions as the chemokines for Teff recruitment were significantly increased in the tumor lesions after HBS-Fc-lv immunization. These data indicate that immunization strategies capable of activating both CD8 and CD4 T cells can improve the tumor milieu and generate an enhanced antitumor effect.
Transient mTOR blockade augments vaccination induced CD8 memory responses for tumor immunity (48.21)
Authors: Shrikant Protul , Li Qingsheng , Rao Rajesh , Vazzano Joseph , Gibbs John , Odunsi Kunle
Published: January 2023

Vaccination aims to generate functional CD8+ T cell responses for tumor immunity. However, the CD8+ T cell functions that empower tumor efficacy have not been clearly established. In this study, we demonstrate that a vaccination strategy that generates memory rather then effector CD8+ T cells shows enhanced tumor efficacy. A short-course of rapamycin treatment transiently blocks viral vaccination induced mTOR in CD8+ T cells for reduced early clonal expansion; due to dampened proliferation, and transitions T-bet induced type 1 effector cells to memory precursors. Strikingly, longer-course of rapamycin treatment abrogated the enhanced viral vaccination induced CD8+ memory generation; persistence and antigen-recall, generated by transient mTOR blockade. Although, the phenotype of day 40 antigen-specific CD8+ memory responses produced by vaccination alone or vaccination plus rapamycin appeared to be identical, they were quantitatively and qualitatively distinct and demonstrated greater tumor efficacy. These findings identify the use of rapamycin in vaccination strategies to enhance CD8+ T cell memory and demonstrate the augmented tumor efficacy of memory rather than effector CD8+ T cells.
Role of MDA5 in triggering of potent antiviral responses in the absence of type I IFN positive feedback (49.1)
Authors: Kim Won-keun , Tapia Karla , Barria Maria , Moran Thomas , Lopez Carolina
Published: January 2023

Viral recognition by specialized cellular sensors is critical for the innate immune response to virus infection. Detection of viral components by these sensors triggers the production of type I interferon (IFN), the earliest hallmark of viral infection, which plays an essential role in inducing an antiviral state in both infected and non-infected cells. Highly pathogenic respiratory viruses replicate in the lungs to high titers before a detectable anti-viral response begins, indicating that the viruses have devised effective mechanisms to avoid the innate anti-viral response. We have shown that dendritic cells can be potently activated in response to Sendai virus that contains a high dose of defective viral genomes (SeV HD) even in the absence of type I IFN signaling. The induction of anti-viral cytokines is driven by a quick and potent activation of IRF3 and NF-κB independently of IRF7. This response depends on the presence of MAVS that is essential for the signaling of the viral RNA sensors RIG-I and MDA5. Interestingly, our study indicates that infection with SeV HD triggers MDA5, but not RIG-I, in a type I IFN independent manner. This supports the hypothesis that the type I IFN-independent up-regulation of MDA5 is involved in the recognition of viral RNA and the subsequent induction of antiviral genes. In conclusion, this study suggests a previously unappreciated type I IFN independent function of MDA5 in the innate immune response to respiratory viruses.
Parasite induced IFN-γ treats severe H5N1 influenza infection (49.11)
Authors: O'Brien Kevin , Knoll Laura , Schultz-Cherry Stacey
Published: January 2023

Outbreaks of influenza A viruses are a continued burden to society in terms of both human health and economic costs. Given the continuous evolution of the virus away from our limited antiviral therapies, new treatments are desperately needed. An alternative approach is through the identification of immune enhancement treatments. We show that mice chronically infected with the immune modulating protistian parasite Toxoplasma gondii survive lethal influenza infection. Because a non-infectious fraction of parasite extract elicits an immunologic response similar to live parasites, it was tested as a post-infection treatment for severe influenza virus. Extract administration two days post- lethal influenza infection enhanced survival, lowered viral titers, and reduced clinical disease. Examination of treated, influenza virus infected mice lacking lymphocytes showed that while the adaptive immune response is not absolutely required for enhanced survival, it is necessary for extract-mediated viral clearance. Mechanistic analysis of the extract induced-innate immune responses showed that interferon gamma (IFNγ) production is both necessary and sufficient for survival. These studies demonstrate that T. gondii may have beneficial properties for its warm-blooded host and a non-infectious component from one microbe can be used to enhance the immune response to a second microbe.
Augmentation of immunity to influenza A virus by manipulating a host counter inflammatory pathway (49.5)
Authors: Sharma Shalini , Sundararajan Aarthi , Rouse Barry
Published: January 2023

Reactions to pathogens are usually tuned to effect immunity and limit tissue damage.With chronic infections, where immunity is unsuccessful, tissue damage would be more severe were it not for the operation of numerous host counter inflammatory events. One such event is the inhibitory effects of some galectins binding to their receptors on effector T cells. A potential downside of counter inflammatory events is that they may constrain the effectiveness of immunity to acute infections. We present evidence for this situation in mice acutely infected with Influenza A virus (IAV). We show that virus specific CD8+T cells in both bronchoalveolar lavage (BAL) and spleen up regulate Tim-3 following IAV infection. In an ex-vivo apoptosis assay, addition of Galectin-9 (Gal-9) to the splenocytes (d10pi) caused apoptosis of both Tim-3+and DbNPtet+ CD8+T cells. Furthermore, Galectin-9 knockout (G9KO) mice mounted more robust acute phase virus-specific CD8+T cell response and cleared virus more rapidly than WT mice. G9KO mice also generated higher virus specific serum IgM, IgG and a more rapid IgA response than WT. When IAV immune mice were challenged with a heterologous IAV, G9KO mice mounted a 4 fold stronger IAV specific CD8+T cell response than WT mice. Tim-3 fusion protein infusion into infected WT mice enhanced CD8+T cell responses to IAV. Our results could indicate that manipulating galectin signals may represent a convenient approach to improve immune responses to some vaccines.
Differential role of UVB-induced photolesions in skin and immune response. (50.10)
Authors: Bombardieri Cíntia , Quayle Carolina , Rebel Heggert , Rijksen Yvonne , de Gruijl Frank , Hoeijmakers Jan , Menck Carlos Frederico Martins , van der Horst Gilbertus
Published: January 2023

UV-induced DNA damage is an essential step in the process by which UVB can alter host resistance to infections diseases. UV-light induces two types of photolesion on DNA that are repaired by photolyases (absent in placental mammals) or Nucleotide Excision Repair. Photolyases are capable of repair these lesions using a photon of light as source of energy. Although the deleterious effects of UV are widely known, the specific role of each photolesion in the induction of changes in the immune response remains unknown. It has been shown in vivo that CPDs are responsible for the majority of the effects after UVB skin irradiation of DNA repair proficient mice, such as erythema, apoptosis, hyperplasia, tumorigenesis and immunosuppression. However the role of 6-4PP is still unknown. We show that the removal of CPD lesions prevents hyperplasia and induces dark skin pigmentation. When 6-4PPs were removed, UVB promoted hyperplasia and tanning. Also, unlike 6-4PP removal, CPD removal is capable of preventing the development of p53 epidermal patches, which have a direct correlation with carcinoma development. Furthermore, the CPD removal is sufficient to suppress the induction of Treg (CD4+CD25+Foxp3) in draining lymph nodes and spleen indicating a minor state of immunosuppression. This study can provide new information as how immune responses are regulated depending of the lesion that is repaired and improve our understanding the immune system role in skin cancer induction.
TLR2 stimulation drives human CD4+CD25+CD127negFOXP3+ Tregs into a Th17-like phenotype with reduced suppressive function. (50.4)
Authors: Gran Bruno , Sanvito Lara , Darlington Peter , Constantinescu Cris , Bar-Or Amit , Nyirenda Mukanthu
Published: January 2023

Naturally occurring CD4+CD25hiFOXP3+ Tregs suppress the activity of pathogenic T cells and prevent development of autoimmunity. TLRs are involved in modulating Treg functions both directly and indirectly. Specifically, TLR2 stimulation can reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 production has been associated with reduced Treg function. We examined the effect of different TLR2 ligands on the suppressive functions of human CD4+CD25hiCD127negFOXP3+ Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of Tregs on CD4+CD25- responder T cell (Tresp) proliferation while at the same time enhancing the expression of IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralisation of either IL-17 or IL-6 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that TLR2 stimulation in combination with TCR activation drives naïve human T helper precursors to Th17 differentiation. We conclude that TLR2 stimulation can induce human Tregs to a Th17 phenotype skewing and identify this plasticity as a new mechanism of regulation of Treg function by TLRs, which could enhance microbial clearance by releasing T effector functions.
TLR agonist, R848, induces further differentiation of myeloid-derived suppressor cells (50.5)
Authors: Im Sun-A , Lee Moon-Kyu , Kim Ki-Hyang , Hwang Hyun-Jee , Kim Ae-Ran , Lee Chong-kil
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) accumulate in cancer patient and tumor-bearing mice and potently suppress T-cell activation. MDSCs represent a group of immature myeloid cells expressing CD11b and Gr-1. Here we show that TLR agonist R848, which binds to TLR7/8, induces further differentiation of MDSCs into mature myeloid cells. MDSCs were isolated from the spleens of mice bearing mammary carcinoma 4T1 cells, and then cultured in the presence of R848. Phenotypic analysis of the MDSCs showed that they differentiate into cells expressing higher levels of F4/80, CD11c and class II MHC molecules. Consequently, the MDSCs cultured in the presence of R848 induced anti-CD3/anti-CD28-induced T cell proliferation strongly. The differentiation-inducing activity of R848, however, was abrogated in the presence of high concentration of GM-CSF. The differentiation-inducing activity of R848 on immature myeloid cells was also confirmed using immature bone marrow-derived dendritic cells and peritoneal macrophages. These results show that R848 could be useful in the induction of further differentiation of MDSCs into mature myeloid cells, and further suggest that neutralization of GM-CSF activity might be necessary in developing therapeutics inducing further differentiation of MDSCs.
NKG2D signaling on CD8+ T cells represses T-bet and rescues CD4-unhelped CD8+ T cell memory recall but not effector responses (52.12)
Authors: Guevara-Patino Jose-Alejandro , Zloza Andrew , Lyons Gretchen , Kohlhapp Frederick , A. O'Sullivan Jeremy , Lacek Andrew , Bellavance Emily , Zafirova Biljana , Polić Bojan
Published: January 2023

In the absence of CD4+ T cell help, primed CD8+ T cells mount defective responses. However, the description of priming cues capable of rescuing these defective functions is lacking. Here, we show that concurrent delivery of DNA plasmids encoding antigen and NKG2D ligand, Rae-1ϵ, to antigen presenting cells rescues CD4-unhelped CD8+ T cell memory recall but not effector phase responses. NKG2D-mediated rescue of CD4-unhelped CD8+ T cell memory recall responses is characterized by reversal of elevated T-bet expression, recovery of granzyme B, IL-2, and IFNγ production and recovery of CTL function. NKG2D-mediated rescue requires NKG2D expression on CD8+ T cells and is dependent on IL-15. These findings have the potential to address limitations of current CD8+ T cell regiments used in the absence of CD4 help against intracellular pathogens and cancer, by generating effective CD8+ T cell memory recall responses.
Extracellular matrix from porcine small intestinal submucosa as immune adjuvants (52.21)
Authors: Aachoui Youssef , Ghosh Swapan
Published: January 2023

Porcine small intestinal submucosa (SIS) of Cook Biotech is widely used in tissue remodeling. It also proved safe and effective in enhancing efficacy of a prostate cancer vaccine. In this study, we compared SIS-adjuvanticity with alum’s in mobilizing innate immunity via inflammasomes, and in boosting antibody response to soluble proteins, ovalbumin, and a hapten-protein conjugate, phthalate-KLH. This evaluation carried out in BALB/c, C57Bl/6 and lupus-prone NZB/WF1 mice was done by both Intraperitoneal and subcutaneous routes of immunization. Methods involved determination of antibody titer, isotyping, microarray profiling of cytokines and chemokines and qPCR of inflammasomes-related genes. Results clearly highlighted the difference between SIS and alum. SIS provoked neither pro-inflammatory cytokines (IL-6, IL-1β, and TNFα) nor NLRP3 inflammasomes as alum did, but it did up-regulate IL4 and CD30-ligand. SIS also activated chemotactic factors LIX and KC (neutrophil chemotactic factors), MCP 1 (monocyte chemotactic factors), MCSF and MIP 1-α (macrophage chemotactic factor) and Lympotactin, albeit a little less than Alum. SIS, however, was as effective as alum in engendering lasting and specific antibody responses, primarily of IgG1 type, but proved inconsequential in induction of anti-DNA response or renal pathology. In conclusion, although both alum and SIS induced Th2 type immune response, SIS was much less inflammatory than alum.
Display of conserved Human Immunodeficiency Virus Type 1 gp41 membrane-proximal external region epitopes on bovine papilloma virus-like particles (53.19)
Authors: Zhai Yougang , Zhong Zhenyu , Zariffard Reza , Spear Gregory , Qiao Liang
Published: January 2023

Human immunodeficiency virus type 1 (HIV-1) is mainly transmitted via mucosa; an effective vaccine must induce appropriate mucosal immunity to contain virus transmission. To induce 2F5/4E10-like broadly neutralizing antibody at mucosa sites, the HIV-1 epitopes ELLELDKWASLWN and NWFDITNWLWYIK, recognized by broadly neutralizing antibodies 2F5 and 4E10, respectively, and a short membrane-proximal external region (MPER) (LELDKWASLWNWFDITNW) of HIV gp41 (containing both epitopes) were displayed on three surface loops of bovine papillomavirus (BPV) 1 L1 protein. BPV L1 can form virus-like particles which are mucosa-tropic. Chimeric virus-like particles (CVLPs) were assembled when the fusion proteins were expressed in sf9 cells. CVLPs had sizes similar to those of BPV particles and could be recognized by HIV broadly neutralizing antibodies 2F5 and 4E10. By competition ELISA, CVLP showed strong abilities for competitively binding to 2F5 in the presence of competitors, the peptide ELLELDKWASLWN or to 4E10 in the presence of peptide NWFDITNWLWYIK. Epitope-specific serum IgG and intestinal secretory IgA responses were elicited after oral immunization of mice with the CVLPs. The antibodies specifically recognized HIV-1 gp160 envelope glycoprotein expressed on the cell surface. These findings provide valuable information for an HIV vaccine immunogen design to induce 2F5/4E10-like broadly neutralizing antibody via presenting HIV-1 gp41MPER epitope on BPV VLPs.