bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-22 Ameliorates Cerulein-Induced Pancreatitis in Mice by In-hibiting the Autophagic Pathway (44.15)
Authors: Park Ogyi , Feng Dechun , Radaeva Svetlana , Wang Hua , Yin Shi , Kong Xiaoni , Zheng Mingquan , Zakhari Sam , Kolls Jay , Gao Bin
Published: January 2023

Pancreatitis occurs when digestive enzymes are activated in the pancreas. Severe pancreatitis has a 10-30% mortality rate. No specific treatments for pancreatitis exist now. Here, we discovered that interleukin-22 (IL-22) may have therapeutic potential in treating acute and chronic pancreatitis. Wild-type and IL-22 knockout mice were equally susceptible to ceru-lein-induced acute and chronic pancreatitis, whereas liver-specific IL-22 transgenic mice were completely resistant to cerulein-induced elevation of serum digestive enzymes, pancreatic necrosis and apoptosis, and inflammatory cell infiltration. Treatment of wild-type mice with recombinant IL-22 or adenovirus IL-22 markedly attenuated the severity of cerulein-induced acute and chronic pancreatitis. Mechanistically, we show that the protective effect of IL-22 on pancreatitis was mediated via the induction of Bcl-2 and Bcl-XL, which bind to Beclin-1 and subsequently inhibit autophagosome formation to ameliorate pancreatitis. In conclusion, IL-22 ameliorates cerulein-induced pancreatitis by inhibiting the autophagic pathway. IL-22 could be a promising therapeutic drug to treat pancreatitis.
Interleukin-22 promotes liver progenitor cell proliferation in mice and patients with in viral hepatitis (44.16)
Authors: Feng Dechun , Kong Xiaoni , Weng Honglei , Park Ogyi , Wang Hua , Dooley Steve , Gershwin Eric , Gao Bin
Published: January 2023

The proliferation of liver progenitor cells (LPCs), which have bipotential capabilities to differentiate into hepatocytes and biliary epithelial cells, is often observed in the inflammatory region of chronic liver diseases; however, how inflammation promotes LPC proliferation remains obscure. We examined the role of IL-22 on LPC proliferation on human viral hepatitis patients and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet feeding mice model. We found that interleukin-22 (IL-22), a survival factor for hepatocytes, is highly elevated and correlates with the grade of inflammation and LPC proliferation in patients with viral hepatitis. In a murine model of LPC proliferation induced by feeding DDC diet, overexpression of IL-22 in the liver via the generation of liver-specific IL-22 transgenic (IL-22TG) mice or injection of IL-22 adenovirus markedly increases LPC proliferation. STAT3, the major downstream signal of IL-22, is highly activated in the LPCs from DDC-fed IL-22TG mice. An additional deletion of hepatocyte-specific STAT3 in IL-22TG mice diminishes LPC proliferation. In vitro IL-22 treatment activates STAT3 and increases cell proliferation of isolated EpCAM+CD45-LPCs from DDC-fed mice and BMOL cells (a mouse LPC line), both of which are detected expression of IL-22R1. Conclusion: inflammatory cell-derived IL-22 promotes LPC proliferation via an STAT3-dependent manner, and IL-22 is a key link between inflammation and LPC proliferation in viral hepatitis.
Interaction of vimentin and phosphorylated mitogen-activated protein kinases in FcϵRI and CC chemokine receptor 1 engaged mast cells (44.18)
Authors: Kuo Chuan-Hui , Toda Masako , Borman Satty , Richardson Ricardo , Inoko Akihito , Inagaki Masaki , Schneider Klaus , Ono Santa
Published: January 2023

Accumulating evidence points to cross-talk between FcϵRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein composing type III intermediate filament, participates in such cross-talk for CCL2/ monocyte chemotactic protein 1 production in mast cells. Co-stimulation via FcϵRI, with IgE/antigen, and CCR1, with recombinant CCL3/macrophage inflammatory protein-1α increased expression of phosphorylated, disassembled and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells), a model of mucosal type mast cells. Furthermore, the co-stimulation enhanced production of CCL2 and phosphorylation of mitogen-activated protein (MAP) kinases. Treating the cells with p38 MAP kinase inhibitor SB203580, but not with MAP kinase kinase (MEK) inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAP kinase, but not extracellular-signal-regulated kinase 1/2 (ERK1/2), plays a critical role in the chemokine production. Immunoprecipitation analysis showed association of vimentin with phosphorylated ERK1/2 and p38 MAP kinases in the co-simulated cells. Preventing formation of the soluble vimentin by aggregating vimentin filaments using beta,beta'-iminodipropionitrile reduced the association of vimentin with phospholylated MAP kinases, and CCL2 production in the cells. Taken together, soluble vimentin could need optimal CCL2 production in mast cells.
The Effect of Cytokine Biasing During CD8 T Cell Priming on Subsequent Memory Development (44.22)
Authors: Marshall Brett , Klonowski Kimberly
Published: January 2023

Following encounter with cognate antigen in the context of an APC, CD8 T cells undergo rapid expansion, contraction, and a select few will differentiate into memory T cells poised for rapid response upon subsequent antigen exposure. While several studies have described the phenotype of precursor memory T cells and the signals responsible for their survival and entry into the memory pool, it is becoming clear that the priming environments for each infection model are distinct and may influence memory development. Our studies address the physiological relevance of individual and combinations of common gamma chain cytokines, which may differentially be present during CD8 T cell priming and contribute to the subsequent development of specific subsets of memory T cells. Our studies use an in vitro model system in which CD8 T cells are primed with dendritic cells in the presence of IL-2, IL-7, and IL-15, as well as thymic stromal lymphopoietin (TSLP). These studies test the effects of various combinations of cytokine stimuli during T cell priming on the development of KLRG1-CD127+ memory precursors, as well as their further differentiation into CCR7+CD62Lhi central or CCR7-CD62Llo effector memory subsets following adoptive transfer into recipient animals. Together, our studies will help identify cytokine milieus best capable of driving development of specific subsets of CD8 memory, which may have implications for optimal immunity to distinct pathogens.
Effect of antimicrobial proteins/alarmins on DNA mediated activation of pDC and inflammation. (45.11)
Authors: Tewary Poonam , de la Rosa Gonzalo , Sharma Neeraj , Rodriguez Luis , Shirota Hidekatzu , Steinhagen Folkert , Klinman Dennis , Yang De , Oppenheim Joost
Published: January 2023

Plasmacytoid dendritic cells (pDCs) commonly known as IFN-α producing cells, detect microbial nucleic acids via endosomal Toll-like receptor (TLR)-7 and TLR-9. Antimicrobial peptides (AMPs) like LL37, derived from neutrophil granules and keratinocytes enhance the production of IFN-α by pDCs by forming complexes with self nucleic acids by converting them into a potent trigger of pDC activation (Lande K. et al. Nature 449-564, 2007). This report led us to study the effect of other antimicrobial proteins stored in neutrophils or produced by keratinocytes during inflammation. In this study we present opposing effects of two antimicrobial alarmins; Human Beta Defensin 3 (HBD3) and Lactoferrin (LF), on IFN alpha production by pDCs. HBD3, an epithelial cell product and a host defense molecule, acts as a potent activator of pDCs by promoting DNA induced IFN alpha production by pDC and subsequent activation of bystander myeloid cells. Hence HBD3 may be important contributor to ongoing inflammation and can exacerbate inflammation in autoimmune pathologies like psoriasis. On the other hand LF inhibited DNA induced IFN alpha production by preventing uptake of both self and CpG DNA indicating that it acts as a scavenger molecule for DNA motifs, potentially reducing the undesirable effects of high levels of IFN-α. We therefore propose that a delicate balance between different AMPs present at inflammatory sites determines the outcome of IFN-mediated pDC activation.
Visualization of the splenic iNKT cell effector program reveals cross-talk with dendritic cells at the marginal zone (45.13)
Authors: Leadbetter Elizabeth , King Irah , Tighe Michael , Veerapen Natacha , Besra Gurdyal , Mohrs Markus
Published: January 2023

Invariant Natural Killer T (iNKT) cells are an innate, CD1d-restricted T cell lineage that rapidly secretes cytokines such as interleukin (IL)-4 and interferon gamma upon activation. iNKT cells potentiate adaptive T cell immunity, in part, by activating or “licensing” dendritic cells (DCs). However, the choreography of DC licensing within secondary lymphoid organs remains unclear. Using a combination of gene reporter mice and mCD1d-specific tetramer staining in situ, we localize the endogenous splenic iNKT cell population and characterize their migration pattern and site of canonical cytokine production in response to cognate lipid antigen encounter. We show that iNKT cells are distributed throughout the spleen of naive mice, but upon activation with αGalCer or pathogen-derived lipids rapidly accumulate in the bridging channels and marginal zone where they secrete IL-4. Early iNKT cell cytokine production and migration depends on the presence of marginal zone DCs. iNKT activation acts reciprocally to upregulate CCR7 and relocalize marginal zone DCs to the splenic T cell zone, another means by which iNKTs may “license” DC-mediated adaptive immunity. Studies of iNKT migration and DC licensing during live bacterial infection are currently underway to further understand how iNKT cells exert their anti-microbial effects in vivo.
A fast and simple method for the isolation of untouched human gamma-delta T cells from PBMC (45.7)
Authors: Tabatabaei-Zavareh Nooshin , Le Fevre Tim , Thomas Terry , Fairhurst Maureen
Published: January 2023

T cells expressing the γδT cell receptor (γδTCR) comprise a minor subset of human circulating T cells (1-10%). γδT cells are distinct from αβT cells in that they exhibit limited combinatorial diversity of the TCR and recognize non-peptide antigens independent of HLA molecules. γδT cells exert innate effector functions including rapid release of cytokines and killing of target cells. Adaptive immune responses such as memory functions have also been attributed to γδT cells. Typically, elaborate purification protocols such as FACS-based cell sorting or expansion in culture are needed to obtain enough γδT cells for subsequent studies. Here, we describe a negative selection method to isolate untouched γδT cells from fresh or previously frozen peripheral blood mononuclear cells (PBMC). This method uses immunomagnetic, column-free cell separation technology (EasySepTM). Briefly, bispecific antibody complexes are used to cross-link non-γδT cells to dextran-coated magnetic particles. The unwanted cells are then removed using an EasySepTM magnet. Starting with 3±0.9% γδTCR+ T cells in PBMC, purities of 90±2% (n=7) are achieved. The isolated γδT cells produce IFN-γ when activated with non-peptide antigens. γδT cells have potential therapeutic applications in cancer and infectious diseases. This rapid method for the isolation of γδT cells will assist in the study of γδT cell biology and the development of γδT cell-based immunotherapies.
B16 melanoma inhibits cellular response to Interleukin-12 via multiple mechanisms including paracrine action of Wnt-inducible Signaling Protein-1 (46.10)
Authors: Klinke David , Kulkarni Yogesh , Chambers Emily , McGray A. J. Robert , Ware Jason , Bramson Jonathan
Published: January 2023

Interleukin-12 (IL12) enhances anti-tumor immunity when delivered locally within the tumor microenvironment. However, immunoregulatory elements within the tumor microenvironment are thought to dampen the effect of IL12 as an adjuvant. The objective of this study was to identify biochemical mechanisms of tumor-immune cell cross-talk that locally suppress the action of IL12. A high content in vitro assay was developed to quantify tumor-mediated suppression of the action of IL12 using a T helper type 1 cell line. Following an induction period, the melanoma cell model, B16F0, inhibited the cellular response to IL12 in vitro. This paracrine effect was not explained by induction of apoptosis or creation of a cytokine sink, despite both mechanisms at work within the co-culture assay. Secreted protein, acidic and rich in cysteine (SPARC); exosomes; and Wnt-inducible signaling protein-1 (WISP-1) were identified using a proteomics workflow and confirmed to be enriched in B16F0-conditioned media. Neutralization of WISP-1 recovered and recombinant WISP-1 dose-dependently inhibited the activity of Signal Transducer of Activated Tyrosine 4 (STAT4), a key transcription factor within the IL12 pathway, within the in vitro co-culture assay. Moreover, WISP-1 was expressed in vivo following intradermal challenge with B16F10 cells and exhibited an inverse dependence on tumor size.
Identification of HER-2/neu peptide p373-382 as a naturally processed immunodominant HLA-A2 binding epitope (46.11)
Authors: Henle Andrea , Erskine Courtney , Benson Linda , Knutson Keith
Published: January 2023

HER2 is a prime target for cancer therapeutics due to its high overexpression in tumors. Advanced clinical trials, in breast and prostate cancer, investigating the use of the HLA-A2-binding HER2 peptide p369-377 (E75) as a vaccine are ongoing. In the present study, we investigated the proteasomal processing of p369-377 and other epitopes from HER2. We did not detect processing of p369-377 by either the proteasome or immunoproteasome in vitro, suggesting that tumor cells do not naturally process this epitope from HER2 for presentation on HLA-A2. However, other HER2 peptides were found to be consistently processed by the proteasome and immunoproteasome. One newly identified processed peptide, HER2 p373-382, was found to bind HLA-A2 stronger than p369-377, despite algorithms that predicted p369-377 as a stronger binder. Cytotoxic T lymphocytes (CTL) specific for p373-382 recognized target cells pulsed with either p373-382 or p369-377. CTL specific for p373-382 also lysed HER2+HLA-A2+ human breast cancer cells more efficiently than p369-377-specific CTL. These results suggest that cancer cells are naturally processing and presenting p373-382. Furthermore, our results suggest that T cells generated against p369-377 in previous studies may have been cross-reacting with p373-382. Thus, the newly identified p373-382 peptide may be a more appropriate candidate for vaccines with this new evidence that it is processed and presented by HLA-A2 for recognition by HER2-specific CTL.
A CD28 based bi-directional molecular bridge that support plasma cell survival in the bone marrow. (46.16)
Authors: Nair Jayakumar , Murray Megan , Koorella Chandana , Rozanski Cheryl , Carlson Louise , Shaughnessy John , Bergsagel P. , Boise Lawrence , Lee Kelvin
Published: January 2023

Dendritic cells (DC) are essential for the long term survival of plasma cells (PC) in the bone marrow and for the maintenance of persistent antibody titers. But the molecular basis for these interactions is not very clear. Both normal and malignant PC (like myeloma) express the T-cell co-stimulatory molecule CD28. Our data show that CD28 expressed on PC play a major role in CD80/CD86 (B7) mediated DC induction of growth factors such as IL-6 and immunosuppressive enzyme indoleamine 2,3 dioxygenase (IDO) as well as in antibody or DC mediated protection of PC in vitro. While IL-6 protected PC in vitro, IDO induced the formation of regulatory T cells in culture. Use of myeloma drug lenalidomide to inhibit DC-B7 expression or anti-CD28(Fab)2 fragments to block CD28-B7 interactions reduced DC ability to protect MM cells or produce IL-6/IDO. Moreover, just like in T-cell models, CD28 activation in malignant PC triggers similar molecular events such as tyrosine phosphorylation and association of p85. We believe that downstream of CD28, while PI3K/Akt governs survival, the Vav1/SLP76 pathway regulate PC proliferation and these form the molecular basis for a bi-directional molecular bridge between the PC and bone marrow microenvironment. Targeting this bridge could form the basis for future therapeutic strategies. Our data suggests a role for CD28-B7 in plasma cell survival by modulating the bone marrow microenvironment.
Protease activated interleukin-2 cytokine fusion proteins for tumor immunotherapy (46.17)
Authors: Skrombolas Denise , Puskas John , Sedlacek Abigail , Narrow Wade , Mastrangelo Michael , Bowers William , Lord Edith , Frelinger John
Published: January 2023

Cytokines are being used for tumor immunotherapy, but when delivered systemically, negative side effects limit their utility. To mitigate these side effects but maintain their anti-tumor effects, we have constructed a panel of fusion proteins (FPs) with IL-2 joined to a specific inhibitor, separated by specific protease cleavage sequences. The rationale is that at the tumor site, where the concentration of a protease is high, the FP will be cleaved, allowing the IL-2 to dissociate from the inhibitor, increasing the biological activity of the cytokine and enhancing anti-tumor effectors. We have expressed these FPs and showed that cleavage in vitro results in increased biological activity of IL-2. In the Colon 38 peritoneal tumor model, delivery of a FP results in increased frequency of T cells (p< 0.0001) and NK cells (p< 0.01) compared to vehicle control treatment, and a corresponding decrease in tumor growth. To test for systemic side effects and evaluate the effects of FP delivery, we used an adeno-associated virus (AAV) delivery system to achieve sustained, high systemic levels of the FPs in mice. AAV-expressed FPs persist at high levels (~500 pg/ml) for at least 9 weeks and do not appear immunogenic. Importantly, unlike injection of free IL-2, expression of the FPs did not generate an inflammatory cytokine response as determined by a 22-analyte Luminex assay. Thus, protease activated cytokine fusion proteins may be a valuable means to enhance tumor immunotherapy.
Development of protease activated cytokines that can be used to alter cytokine tumor microenvironments (46.20)
Authors: Frelinger John , Skrombolas Denise , Puskas John , Sullivan Mark
Published: January 2023

We are developing an immunotherapy approach in which cytokines can be preferentially activated by proteases at the site of growing tumors, thus altering the tumor microenvironment. This approach employs a novel fusion protein (FP) in which a cytokine is joined to a specific binding moiety, such as a single-chain antibody (scFv), separated by a protease cleavage site. The rationale is that before cleavage, the cytokine is largely inactive, but that after cleavage by a protease preferentially expressed at the tumor site, the cytokine becomes biologically available as the inhibitory scFv dissociates from the cytokine. To test this approach, we have isolated scFvs using phage display that bind to human IL-2, including one that can be inhibited in its binding to IL-2 by the neutralizing anti-human IL-2 antibody MQ1-17H12. This scFv has been used to construct and express a FP containing a protease cleavage site. Immunoblot analyses revealed that the FP can be cleaved in vitro and that cleavage results in increased functional activity of the IL-2. To investigate how the increase in functional activity of the IL-2 depends upon the binding characteristics of the scFv, we have created mutations that alter its affinity for IL-2, which are being evaluated for how they affect the characteristics of the FP. Since scFvs that bind to essentially any molecule can be readily isolated, this should be a general approach for increasing the biologic activity of immuomodulators at tumor sites.
The immune system is pivotal in mediating the anti-tumor effects of radiation therapy (46.3)
Authors: Gerber Scott , Sedlacek Abigail , Lord Edith
Published: January 2023

A new paradigm has emerged demonstrating that the immune system mediates many of the anti-tumor effects of radiation therapy (RT). RT continues to be a cornerstone in the treatment for many cancers, unfortunately not all individuals respond effectively to RT resulting clinically into two groups consisting of non-responders (progressive disease) and partial/complete responders (tumor control/cure). We have designed a unique model that mirrors this clinically relevant phenomenon in which mice bearing colon38, a colon adenocarcinoma, were treated locally with 15Gy RT resulting in both non-responders and responders. Interestingly, our data uncovered a window of interest four days following RT in which critical events occurred that ultimately determined the effectiveness of therapy. Intratumoral IFNγ was increased in responsive tumors and licensed CD8 T cells to exhibit potent lytic activity against tumor cells; a response that was greatly diminished in tumors refractory to RT. We explored whether enhancing IFNγ and CD8 T cells via immunotherapy would increase the responder to non-responder ratio. Indeed, multi-modality therapy comprised of IL-12 and RT in mice bearing colon38 tumors resulted in complete remission of cancer in 100% of cases compared to a cure rate of only 40% with RT alone. Our data suggests that the efficacy of RT may depend on the immune response generated shortly after treatment and targeting this component may greatly enhance the effectiveness of RT.
A novel anti-B7-H4 human recombinant antibody overcomes TAM-mediated T-cell inhibition (46.43)
Authors: Dangaj Denarda , Zhao Aizhi , Lanitis Evripidis , Joshi Shree , Sandaltzopoulos Raphael , Powell Jr Daniel J , Scholler Nathalie
Published: January 2023

Tumor polarization of macrophages represents an essential immune escape mechanism thus targeting tumor-associated macrophages (TAMs) could boost anti-tumor immune response. To model tumor-induced macrophage polarization, we developed an in vitro system of transwell co-culture with human macrophages, T cells and ovarian cancer cells (OVCAR3) where in vitro-matured macrophages and CD3/CD28 subactivated T cells were separated from tumor cells; in vitro-generated TAMs overexpressed the immunosuppressive molecule B7-H4 and inhibited T cell functions. We then hypothesized that targeting B7-H4 could block T cell inhibition mediated by B7-H4+ TAMs. We isolated anti-human B7-H4 scFvs from a novel yeast-display scFv library derived from B cells of ovarian cancer patients’ ascites. T cell proliferation and activation were restored in presence of anti-B7-H4 scFv #56 during transwell co-culture, suggesting that scFv #56 could block B7-H4 inhibitory signals mediated by TAMs. To further address whether scFv #56 activity was TCR-dependent, we set up experiments with antigen-specific T cells (MART1) and wild type or B7-H4-transduced artificial APCs (T2) loaded with MART1 peptide. T cell cytokine response was downregulated by B7-H4 expression on T2 and partially restored in presence of scFv#56. Our results indicate that competing with B7-H4 inhibitory signals on TAMs can promote T cell activity. We propose that in vivo use of anti-B7-H4 scFv could increase anti-tumor T-cell immunity.
Overcoming obstacles to naïve T lymphocyte trafficking in tumor-draining lymph nodes (46.47)
Authors: Muhitch Jason , Ito Fumito , Fisher Daniel , Diehl Michael , Appenheimer Michelle , Evans Sharon
Published: January 2023

Effective tumor immune responses rely on trafficking of rare tumor-specific T cells to tumor-draining lymph nodes (TdLN) where they undergo activation and expansion, and subsequently to tumor tissues to gain access to neoplastic targets. Here, we evaluated the trafficking of naïve CD8 T cells to TdLN. Lymphocyte entry into TdLN was diminished by ~50% in mice bearing B16 melanoma, suggesting a localized mechanism of immune escape. Intravital microscopy revealed that while normal proportions of lymphocytes initiated tethering and rolling in high endothelial venules (HEV) of TdLN (34%), the fraction of lymphocytes that transitioned from rolling to firm arrest was reduced compared to unchallenged lymph nodes (8 and 20%, respectively). Diminished trafficking in TdLN was attributed to suboptimal presentation of the CCL21 chemokine in HEV. We further determined that inflammatory cues triggered by systemic thermal therapy (STT, 39.5 ± 0.5°C, 6 h) could restore normal T cell entry in TdLN, paralleling our previous findings in non-tumor-challenged mice. Improved homing was due to a reduction in rolling velocity and an increase in proportion of T cells undergoing firm arrest. STT was further shown to enhance the display of trafficking molecules required for firm arrest, i.e., CCL21 and ICAM-1 in HEV. These findings suggest that therapeutic targeting of HEVs for improved entry of naïve T cells in TdLN may be a novel strategy to overcome immune evasion.
CD28-mediated pro-survival signaling in multiple myeloma (46.6)
Authors: Murray Megan , Nair Jayakumar , Lee Kelvin
Published: January 2023

Multiple myeloma (MM) is a plasma cell neoplasm which requires molecular bridges to the bone marrow microenvironment, one of which may be CD28. It has been observed that CD28 is expressed on MM cells and is correlated with progressive disease and worse outcomes. We have previously shown that activation of CD28 protects MM cells from chemotherapy. We have also observed that co-culture of MM cells with bone marrow stromal cells such as dendritic cells (which express the CD28 ligands CD80 and CD86) protect MM cells from chemotherapy or serum starvation. This protection is entirely abrogated by either a CD28 blocking antibody (CD28.6) or by CTLA4-Ig, which binds to the ligands CD80 and CD86. Since CD28 in T cells signals via a PI3K-Akt axis, we interrogated whether or not this pathway is crucial for CD28-mediated MM cell survival. We observed that blockade of PI3K signaling using chemical inhibitors abrogates CD28-mediated survival. Since the pro-apoptotic molecule Bim is both critically important in MM biology and downstream of PI3K-Akt, we next examined whether or not it is regulated by CD28. When CD28 is activated, Bim mRNA and protein levels are downregulated, and when CD28 signaling is blocked, Bim protein is upregulated. Moreover, when Bim is knocked down with siRNA, MM cells are no longer sensitive to CD28 blockade. Taken together, these data show that CD28-mediated survival is regulated (at least in part) by downregulating Bim and thus preventing apoptosis.
DC enzymes as early biomarkers of an effective vaccine (46.7)
Authors: Marvel Douglas , Finn Olivera
Published: January 2023

Previous work by our lab and others has shown that human MUC1 transgenic mice (MUC1.tg) are hyporesponsive to vaccination with some forms of MUC1 and fail to mount a spontaneous immune response against MUC1 positive tumors. An interesting correlate of hyporesponsiveness to MUC1 vaccination is that as early as 24 hours post vaccination, splenic dendritic cells (DC) in wild type (WT) mice upregulate expression of a group of proteolytic enzymes previously characterized as functioning primarily in pancreatic catabolism (e.g. Trypsin-1, Trypsin-4, Carboxypeptidase B1). This upregulation does not occur in the MUC1.tg animals. Using an in vitro co-culture system and in vivo antibody depletion, we show that regulation of enzyme expression in DC is dependent on their interaction with CD4 T cells such that CD4 effector T cells (Teff) up-regulate these enzymes, whereas the presence of activated CD4 regulatory T cells (Treg) actively suppress this activity in an IL-10 dependent manner. TLR agonists do not upregulate enzyme expression, suggesting differences are due to alternative DC activation pathways following their interaction with T cells. We propose that one or more of these enzymes, which appear to be co-regulated, could serve as early markers of, and most likely also as contributors to, DC activation by helper T cells. In contrast, lack of their expression marks insufficient activation or active suppression by Tregs.
Effects of aging on the human adaptive immune system revealed by high-throughput DNA sequencing of T cell receptors (47.16)
Authors: Robins Harlan , Desmarais Cindy
Published: January 2023

As we age, our adaptive immune system undergoes changes that affect vaccine efficacy as well as susceptibility to disease and infection. Identifying the factors that determine immune repertoire diversity and studying the impact of changes in diversity through the life span is therefore an important public health concern. The objective of our study was to characterize alterations that occur in the T cell receptor repertoire as we age. To address these questions we utilized a high-throughput sequencing (HTS) approach developed by Adaptive Biotechnologies. This technology (immunoSEQ) amplifies rearranged T-cell receptor CDR3 chains and uses HTS to capture millions of CDR3 sequences. CD4+ Memory, CD4+ Naïve, CD8+ Memory and CD8+ Naïve T cell populations from peripheral blood were isolated from a total of 15 healthy volunteers, ranging in age from 25 to 80. The CDR3 region of each TCRB chain was then sequenced, generating a comprehensive profile of the T cell receptor repertoire for each cell population. We observed a reduction in the relative contribution of CD8+ naïve T cells with advancing age. This effect was exacerbated by CMV infection. These results provide a basis to further characterize the effects of immunosenescence on the adaptive immune system.
The forkhead transcription factor, Foxo1, coordinates an adaptive, pro-survival response to calorie restriction in T cells (47.3)
Authors: Gubbels Bupp Melanie , Edwards R.
Published: January 2023

In addition to longevity enhancement, calorie restriction has been reported to slow the appearance of aging-related T cell abnormalities. However, the molecular mechanism underlying this phenomenon in T cells is ill-defined. We report that short-term calorie restriction in wild type mice inhibited the spontaneous apoptosis of naïve CD8+ T cells by approximately three fold, as compared to cells isolated from mice fed ad libitum. An ortholog of the forkhead transcription factor, Foxo1, is required for the longevity benefits of calorie restriction in C. elegans. Therefore, mice with a conditional, T cell-specific deficiency of Foxo1 (Foxo1 cKO) were also subjected to calorie restriction. Indeed, the pathway through which calorie restriction results in reduced naïve CD8+ T cell apoptosis appears to involve Foxo1, as calorie restriction in the absence of Foxo1 did not result in reduced naïve CD8+ T cell apoptosis. Instead, naïve CD8+ T cell apoptosis was similarly high for cells isolated from ad libitum wild type, ad libitum Foxo1 cKO, and calorie restricted Foxo1 cKO mice. Thus, Foxo1 coordinates an adaptive, pro-survival transcriptional response to calorie restriction. To our knowledge, Foxo1 is the first transcription factor identified to contribute to this process in mice.
IL-15 inhibits caspase-3-mediated T cell apoptosis (47.9)
Authors: Thai Phan , Fortner Karen , Budd Ralph
Published: January 2023

Active caspase-3 is an effector cysteine protease crucial for apoptosis. T lymphocyte survival impinges on the ability of the cell to keep the levels of active caspase-3 in check. Previous studies have shown that despite IL-2 and IL-15 sharing γc and IL-2Rβ (CD122) receptor subunits and being capable of stimulating proliferation and activation of T cells, these cytokines differentially regulate the survival of T cells. Although IL-2 plays a crucial role in activation-induced cell death (AICD) of peripheral T cells whereas IL-15 extends survival of T cells by inhibiting AICD, the mechanism behind this difference in susceptibility to AICD is not well understood. In this study, we report that IL-2-cultured primary murine T cells manifest considerably higher levels of active caspase-3 compared to IL-15-cultured T cells. This was independent of initiator caspases, such as caspase-8 and -9. Furthermore, there was a very robust increase in caspase activity as well as cell death following α-CD3 restimulation of IL-2-cultured T cells, whereas the IL-15-cultured T cells manifested almost no increase in caspase activity or cell death. Taken together these data support the hypothesis that IL-15 can inhibit T cell apoptosis and therefore prolong T cell survival by maintaining low levels of active caspase-3.
Monocytic lineage myeloid-derived suppressor cells are the principal suppressor population (48.11)
Authors: Haverkamp Jessica , Smith Amber , Qualls Joseph , Balouzian Liza , Bronte Vincenzo , Opferman Joseph , Murray Peter
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells and are identified in mice as Gr-1+ (Ly6G/C) CD11b+ cells with the ability to inhibit T cell proliferation. However, the key functional immunosuppressive MDSC population remains controversial. Functional dissection of MDSCs is limited by population heterogeneity because Ly6C is expressed on mature and immature monocytic and granulocytic cells, each of which could potentially mediate immunosuppression. Here we describe genetic and chemical strategies allowing us to ablate the entire population of granulocytic or monocytic MDSCs specifically. Our data define the monocytic-macrophage MDSC population as the predominant suppressor population. Furthermore, our data have implications for the engineering of MDSCs as active suppressors for autoimmune diseases and targeted therapies aimed at selective ablation of monocytic MDSC in the treatment of cancer.
Tolerogenic DC differentiation and development of immunosuppressive DC precursors (48.13)
Authors: VanGundy Zachary , Taylor Cassandra , White Angela , Baker Julie , Muth Dillon , Papenfuss Tracey
Published: January 2023

Tolerogenic dendritic cells (tDCs) are important myeloid cells which potently regulate innate and adaptive immune responses. We have previously found that the steroid hormone estriol (E3) generates tDCs in vivo which protect mice from developing autoimmune disease. To better understand tDC generation, we developed an in vitro tDC differentiation model. Myeloid progenitors were differentiated with GM-CSF in the presence of steroid hormones E3 and retinoic acid (RA) and both differentiated tDCs and their precursors (pre-tDCs) were phenotypically and functionally evaluated. We found that both E3 and RA tDCs had increased expression of stimulatory and inhibitory costimulatory markers and suppressed proliferation of responder immune cells in a dose-dependent manner, similar to in vivo tDCs. Interestingly, we also found that pre-tDC had enhanced immunosuppressive abilities over differentiated tDCs without marked changes in phenotypic cell composition. Although CD11b+Gr1+ populations were unchanged, increases in FoxP3+ expression were seen in responder splenocyte populations when co-cultured with pre-tDCs. These results suggest that pre-tDCs may be acting as myeloid derived suppressor cells (MDSCs) and are a potently immunosuppressive immature myeloid population that gives rise to tDCs. A better understanding of how tDCs are generated has potential therapeutic relevance for the treatment of numerous inflammatory diseases.
Role of C-type lectin receptor CLEC12A in human basophil activation (48.6)
Authors: Divekar Anagha , Conklin John , Yang Xifeng , Ransom John
Published: January 2023

CLEC12A is a member of the C-type lectin receptor family with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif. Murine CLEC12A is expressed on variety of cell types including dendritic cells, monocytes/macrophages, B cells, neutrophils and basophils but not NK cells. In humans, CLEC12A is reported to be expressed on dendritic cells (DC, both myeloid and plasmacytoid), monocytes, B cells (normal and leukemic) and NK cells, but not on T cells or basophils. Performing flow cytometric analysis of human peripheral blood leukocytes using an anti-human CLEC12A antibody (clone 50C1), we report CLEC12A expression on a subset of CD3, CD19 and CD56 negative cells. Further analysis revealed these cells to be CCR3+ basophils. Antibody mediated ligation of CLEC12A on DCs results in cytokine production and cellular maturation and can inhibit NK cell mediated cytotoxicity. In response to IgE and IL-3, basophils can induce Th2 differentiation and amphiregulin production, respectively. We hypothesize that ligation with CLEC12A will alter IL-3 and/or IgE mediated basophil activation, which will be studied in our ongoing work.
IL-27 and B7-H1 mediate liver plasmacytoid DC ability to suppress delayed-type hypersensitivity responses (48.9)
Authors: Matta Benjamin , Raimondi Giorgio , Rosborough Brian , Thomson Angus
Published: January 2023

IL-27 and B7-H1 are regulatory molecules that modulate immune responses. Plasmacytoid (p) dendritic cells (DC) can promote tolerance. We investigated the contribution of IL-27 and B7-H1 to the ability of murine pDC to regulate T cell responses and to suppress delayed-type hypersensitivity (DTH) reactions to ovalbumin (OVA). PDCA-1+ pDC were purified from livers and spleens of Flt3L-mobilized, C57BL/6J wild type (WT), Ebi3 knockout (KO), or B7-H1 KO mice. pDC phenotype was evaluated by flow cytometry. IL-27p28 was quantified by ELISA and flow cytometry. Ebi3 expression was evaluated by Western blot. T cell allostimulatory capacity of pDC was determined in CFSE MLR. For DTH, mice were immunized with OVA-pulsed bone marrow-derived conventional DC with or without pDC from mice fed OVA. Mice were challenged with heat-aggregated OVA and footpad swelling and T cell responses quantified. Liver pDC express high levels of IL-27p28 and Ebi3 (the components of IL-27) but low IL-12p35 (that forms IL-35 with Ebi3). They express high levels of B7-H1 compared to spleen pDC. Ebi3 KO and B7-H1 KO pDC show enhanced T cell allostimulatory capacity compared to WT pDC. Liver but not spleen pDC suppress DTH responses to OVA, a phenomenon dependent on IL-27 and B7-H1. In conclusion, liver pDC exhibit high expression of IL-27 and B7-H1 that mediate their regulatory ability in vitro and in vivo. Liver pDC, IL-27, and B7-H1 may be important therapeutic targets to promote tolerance or enhance immunity.
The role of a cx3cr1+/egfp mucosal resident dendritic cell network in mucosal homeostasis (49.10)
Authors: Velazquez Peter , Moriel Gabriela , Xu Cassie
Published: January 2023

Cxcr1 is expressed in all CD11c+ mucosal dendritic cells (DC). Mucosal DCs are perivascular and are interconnected to form a network. However, the importance of the network in maintaining mucosal homeostasis is poorly understood. We first aimed to gain insight on the structure of the DC network. Using multi-photon intravital microscopy, we demonstrate that the cx3cr1+/egfp DC network interconnects DCs between villi and throughout small and large bowel. The network overlaps a radiation resistant non-hematopoietic cell network. We next aimed to examine the role of DCs in vascular permeability. cx3cr1+/egfp DCs were selectively depleted by targeting depletion with CD11c-diptheria toxin receptor (DTR) transgenic intercross. Preliminary data suggests that DCs are not necessary to maintain vascular integrity, as measured by permeability of 70kDa dextran fluor. Critical for maintaining adaptive immunity is spatial temporal regulation of T cell migration as T cells survey antigen present cells (APC). Since DCs are a critical APC, T cells patrolling was examined following depletion of the mucosal DC network. Adoptive transfer studies demonstrate that CD4 and CD8 T cell migration remains intact despite selective depletion of DCs. In summary, the mucosal DC network represents a higher order of organization of DCs than previously appreciated. Resident DCs may play a role in maintaining vascular permeability but surprisingly do not play a role in regulating mucosal T cell migration.
Th17 responses driven via PPARγ blockade lead to faster recovery from enteroaggregative Escherichia coli infection (49.13)
Authors: Washington Casandra , Bassaganya-Riera Josep , Viladomiu Monica , Pedragosa-Marin Mireia , Guerrant Richard , Roche James , Hontecillas Raquel
Published: January 2023

Enteroaggregative Escherichia coli (EAEC) is a recognized cause of enteric disease in diverse clinical settings. Mucosal immunity towards EAEC infections is poorly understood. To better characterize immunoregulatory mechanisms underlying EAEC infections we constructed a computational model mimicking host responses to EAEC at the gut mucosa. Preliminary model calibration efforts demonstrated remarkable fitting to experimental data. Nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) has been targeted to modulate mucosal immune responses to EAEC. Wild type and conditional knockout mice lacking PPARγ in T cells were fed protein-deficient diets at weaning and challenged with 5x109cfu EAEC strain JM221. Quantitative RT-PCR data reveal that administration of GW9662, a potent PPARγ antagonist, for 7 days post infection or the deletion of PPARγ in T cells results in upregulation of pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β when compared to non-treated infected animals. Histological analysis of colons divulges lower leukocyte infiltration and decreased mucosal thickness 14 days following EAEC infection after pharmacological blockade of PPARγ or deletion of PPARγ in T cells. These findings were accompanied by faster clearance of colonic EAEC in tissue-specific PPARγ null mice. In conclusion, we present a fully integrated approach to study host responses to EAEC that provides new insights on the pathogenesis and treatment of enteric infections.
Allergic Eosinophils Alter Immune Responses to Influenza A Virus. (49.24)
Authors: Samarasinghe Amali , Woolard Stacie , McCullers Jonathan
Published: January 2023

WHO estimates that 235 million people globally suffer from asthma, which was among the important risk factors for hospitalization during the 2009 influenza pandemic. Using a novel co-morbidity model of acute allergic asthma and influenza, we demonstrate that mice with acute asthma are protected from influenza-induced morbidity. Asthmatic mice did not lose weight or exhibit typical, severe, clinical signs of infection. In spite of having a greater infiltration of cells into the airways, these mice had suppressed Th1- and Th2-type cytokines compared to flu-only controls. Resistin-like molecules were highly expressed during early timepoints that correlated with eosinophil influx indicating that responding eosinophils may be mediating protection against influenza. Eosinophils were harvested from allergic airways and adoptively transferred to matched mice infected with pandemic influenza virus. Eosinophil transfer into flu-infected non-allergic mice markedly altered cellular influx into the airways. Significantly more CD4+ Th2 lymphocytes, macrophages, neutrophils, and eosinophils were present compared to control mice. Viral titers were reduced 5 days after eosinophil transfer, and the host immune response was altered. We conclude that allergy-induced eosinophils modulate immune responses to influenza virus. The role of eosinophils in protecting the acutely allergen-sensitized host from viral mediated immunopathology merits further investigation.
The intranasal delivery of non-specific virus-like particles elicits heightened and accelerated immune responses to influenza virus (49.27)
Authors: Richert Laura , Rynda-Apple Agnieszka , Wiley James , Douglas Trevor , Harmsen Allen
Published: January 2023

We have previously shown that the intranasal delivery of a small heat-shock protein cage nanoparticle, a virus-like particle (VLP), elicits the formation of inducible bronchus-associated lymphoid tissue (iBALT), which provides protection against many diverse high-dose pathogen challenges. We demonstrate here that the presence of non-specific, VLP-induced iBALT facilitated accelerated influenza virus clearance and enhanced immunity through accelerated airway (CD103+) and parenchymal (CD11b+) dendritic cell antigen processing and trafficking. These responses were dependent upon the presence of CD11c+ cells, which had been directly exposed to VLPs, and migrated into the lung in a CCR2, VCAM-1, and ICAM-1-dependent manner. The initial innate response to influenza challenge was then followed by an accelerated expansion of influenza-specific CD4+ T cells. The combination of these events resulted in accelerated viral clearance and significantly ameliorated host-derived collateral damage. Furthermore, we show that the presence of iBALT, which is not specific to influenza virus, was efficient at clearing high-dose virus, independently of the TBLN’s help. Thus, we herein demonstrate the mechanism by which the presence of iBALT, derived from non-specific, non-pathogenic VLPs, and lacking any memory cells to agents of subsequent challenge, elicited alternative cellular trafficking and expansion patterns, and thus cleared virus at an accelerated rate, with less harmful consequence.
Commensal flagellated A4 bacteria promote intestinal Th1/Th17 cell development but inhibit Th2 responses (49.3)
Authors: Liu Houpu , Yao Suxia , Cong Yingzi , Elson Charles
Published: January 2023

Host and microbiota interact interdependently to maintain intestinal immune homeostasis. As Th1, Th17, and Tregs are present in lamina propria (LP) in the steady condition, mucosal Th2 cells are hardly detected. Some specific commensal organisms have defined effects on mucosal immune functions, e.g., SFB promotes intestinal Th17 whereas B. fragilis and certain Clostridium spp induce Tregs. However, it is still unclear if there are specific commensal organisms downregulating Th2 responses to microbiota. A4 bacteria, a commensal isolated from mouse cecum, produce CBir1 flagellin, a dominant antigen in Crohn’s disease patients and in experimental colitis. In CBir1 TCR transgenic mice, Th1, Th17 and Treg but no Th2 cells are present in intestinal LP. Upon transferred into TCRβxδ-/- mice, CBir1 Tg T cells differentiated into IFN-γ+, IL-17+, FoxP3+ cells but not IL-4+ cells in LP. When stimulated with CBir1 flagellin in vitro, CBir1 Tg CD4+ T cells were able to differentiate into Th1, Th17 and Tregs but resisted Th2 differentiation. Pretreatment of APC with A4 bacterial lysates or CBir1 flagellin, but not with control E. coli, promoted IFN-γ but inhibited IL-4 production of OTII T cells stimulated with OVA and of wild-type T cells stimulated with anti-CD3 under neutral or Th2 polarizing conditions. Collectively, our data reveal that A4 bacteria inhibit Th2 and favor Th1/Th17 responses in intestinal mucosa via production of flagellin, probably through stimulating IFN-γ production.
CD4+ T cells regulate skin resident memory CD8+ T cells (TRM) (49.8)
Authors: Jiang Xiaodong , Clark Rachael , Liu Luzheng , Seneschal Julien , Purwar Rahul , Tian Tian , Fuhlbrigge Robert , Kupper Thomas
Published: January 2023

The requirement of CD4+ T cell help for CD8+ T cell responses has been extensively explored in secondary lymphoid organs using systemic infection models. Whether such help also exists in peripheral tissues like the skin is unknown. We recently reported that skin resident memory CD8+ T cells (TRM) are non-recirculating and are superior to central memory CD8+ T cells (TCM) in protecting against re-infection. Interestingly, we also found that the acute CD8+ T cell migration into infected skin does not require CD4+ T cell help. Further study showed that the acute protection response of CD8+ T cells in the skin was equivalent in wild type and CD4-deficient mice. We next asked the role of CD4+ T cells in CD8+ TRM protective responses in the skin. To our surprise, the absence of CD4+ T cells did not impair the survival of CD8+ TRM in the skin. However, the recall response of CD8+ TRM in previously infected skin was significantly compromised in CD4-/- mice. The viral load assay further showed that the recall response of CD8+ TRM was significantly compromised when memory CD4+ T cells in the skin were depleted, suggesting that signals from memory CD4+ T cells in the skin are delivered to CD8+ TRM locally. Taken together, these data imply that while CD4+ T cells are not required both for acute CD8+ T cell response and maintenance CD8+ TRM in the skin, they do play an important role in regulating recall responses of CD8+ TRM.