bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel early detection test for breast cancer and its recurrence in blood through changes in immuno-biochemical signals (46.12)
Authors: Chandok Meena , Sakthivel Palanikumaran , Chaney Charles , Mccormack Michael , Wagner Kathryn , Mire Gary , Newby John , Parwani Anil , Renee Kathleen
Published: January 2023

We are developing an immune sensor for early detection of breast cancer and its recurrence in blood, based on identifying the earliest specific changes/alterations in the immuno-biochemical signals in different immune components that are distinctly different from those in a healthy immune system. The screening of different immuno-biochemical signals in their native and altered forms in different immune components/cells led to identification of a novel T cell population. Analysis of this T cell population and its immuno-biochemical signals on blood samples from the 4 groups - (i) healthy, (ii) recently diagnosed with invasive breast cancer stage 1{before the onset of treatment/surgery}, (iii) breast cancer treated individuals who had incidence of recurrence/relapse {BCR}, and (iv) breast cancer treated individuals who were disease free {BDF}- was done using multi-parameter flow cytometry. Sixteen molecules, assessed as the most likely candidates for sensing disruptions in the immune pathway, were used in developing a matrix. Care was taken to separate subjects that could confound the immuno-biochemical signals, potentially arising from other immune disorders. The method designed is different from conventional approaches and has the potential to be used as a cost-effective assessment to predict health status.
Defining the role of ICOS and CD28 costimulation in TH17 cell activation, differentiation and tumor immunity (46.29)
Authors: Nelson Michelle , Huff Logan , Rogers Carolyn , Kundimi Sreenath , Paulos Chrystal
Published: January 2023

TH17 cells play an essential role in regulating host defense and exacerbating various autoimmune diseases, but their role in tumor immunity remains incompletely elucidated. We found that CD28 abrogates TH17 expansion and function, while the inducible costimulator (ICOS) augments the generation of polyfunctional TH17 cells. Of clinical importance, ICOS-expanded TH17 cells were superior at killing human tumors compared to cells expanded with CD28. Our objective is to identify how ICOS regulates the differentiation and optimal expansion of TH17 cells that mediate tumor regression. We have polarized human CD4+ T cells towards TH17 with beads expressing anti-CD3 and CD28 and/or ICOS agonists. ICOS costimulation promotes the long-term generation of TH17 cells, determined by CD161 and CCR6 expression and IL-17A production. By sorting peripheral blood cells based on these two receptors, we identified a subset of cells that nearly all produce IL-17 when stimulated with an ICOS agonist. This innovative approach allows us to study the basic biology of IL-17-producing cells and affords us the ease of exclusively enriching them for adoptive immunotherapy for cancer. We are now uncovering new transcription factors uniquely regulated by ICOS that distinctly impact TH17 cells’ phenotype and functional fate. Understanding how ICOS signaling and optimal culture conditions promote TH17 cell function will be instrumental in advancing T cell-based therapies for cancer and infectious diseases.
Systemic 4-1BB activation induces a novel T-cell phenotype driven by high expression of Eomesodermin (46.30)
Authors: Curran Michael , Geiger Theresa , Montalvo Welby , Kim Myoungjoo , Reiner Steven , Al-Shamkhani Aymen , Sun Joseph , Allison James
Published: January 2023

Following treatment with 4-1BB agonist antibody, a novel population of KLRG1+ T-cells infiltrate murine melanoma. Compared to their KLRG1- counterparts, these T-cells express high levels of cytotoxicity associated genes in both the CD4 and CD8 lineages, and exhibit enhanced tumor-specific killing in vitro. The phenotype of these KLRG1+ cells is dependent on high expression of the T-box transcription factor Eomesodermin (Eomes). Interestingly, only activation of 4-1BB, not other TNFR family members generates this phenotype. The root of this difference appears to be that 4-1BB is expressed by antigen presenting cells (APC) which respond to its activation by producing cytokines which promote the development of these Eomes+KLRG1+ T-cells. By analyzing changes in APC cytokine production in vivo, and by using a series of gene knockout mice we have identified the factors necessary to generate this novel T-cell lineage. These T-cells represent a novel polarity we have termed ThEO (CD4) and TcEO (CD8) which resolve multiple questions associated with 4-1BB activation including how 4-1BB enhances tumor-specific cytotoxicity and how 4-1BB can promote tumor immunity while repressing autoimmunity. Understanding the nature of this novel lineage of highly tumoricidal T-cells in both tumor and pathogen-specific immunity may provide critical information for converting sub-optimal anti-tumor responses to therapeutically successful ones.
Enhancing cancer immunotherapy by combining Notch 1 and death receptor activation (46.38)
Authors: Shanker Anil , Malhotra Anshu , Dikov Mikhail , Biktasova Asel , Sayers Thomas , Carbone David
Published: January 2023

Notch signaling plays diverse roles in lymphocyte differentiation and tumor angiogenesis. Our recent findings implicate the role of Notch 1 pathway in promoting antitumor immune effector functions. We explored the effects of Notch 1 stimulation on T cell immunity and its potential pharmacological activation using Delta-like Notch 1 ligand DLL1 cluster in combination with the proteasome inhibitor bortezomib, which could stabilize Notch 1 activation. Bortezomib was found to selectively sensitize solid tumor cells to apoptosis by upregulating death receptors on tumor cells and amplifying caspase-8 activation in the death-inducing signaling complex. This resulted in reduced pulmonary nodules of injected tumor cells in mice. Additionally, in an in vivo tumor setting, no adverse effects of bortezomib were observed on the antigen-specific T cell proliferative or cytolytic functions. But no long-term survival benefits could be obtained, possibly due to the immunosuppressive effects of the tumor. In our more recent studies, we observed that DLL1-specific immune Notch activation could enhance antitumor T cell immunity by overcoming tumor-associated immunosuppression in D459 and Lewis lung carcinoma models. The results offer a novel combinatorial approach to abrogate the immunosuppressive circuitries operating in the tumor microenvironment with a potential to reduce tumor burden and strengthen tumor-specific immune responses.
Inducible B effector cells (iBECs) for cancer immunotherapy (46.4)
Authors: Ng Spencer , Penafuerte Claudia , Galipeau Jacques
Published: January 2023

While cellular immunotherapy for malignances has traditionally focused on the development and manipulation of tumor targeted DCs, T and NK cells, B cells have remained vastly underutilized as a potential source for adoptive cell therapy. Here we demonstrate that B cells stimulated with FIST-2, a novel chimeric protein consisting of IL-2 fused to the ectodomain of the TGFβ receptor (type II), can adopt an effector phenotype with potent antitumor activity. Treatment with FIST-2 induces naïve splenic B cells to become B effector cells (iBECs), characterized by hyperphosphorylation of STAT3 and 5 downstream of the IL-2 receptor, upregulation of transcription factor, T-bet, and secretion of pro-inflammatory cytokines: IFNγ, TNFα and IL-6. iBECs retained their B cell identity by CD19 and PAX5 expression, but adopted an enhanced APC phenotype through upregulation of cell surface markers associated with antigen presentation and co-stimulation, including: MHC-II, CD80 and CD86. To determine whether iBECs conferred antitumor immunity, we utilized a mouse model of lymphoma expressing ovalbumin (EG.7-OVA). Syngeneic iBECs pulsed with OVA were able to activate OVA-specific OT-I and OT-II T cells in vitro, suggesting that they act as APCs. In vivo administration of OVA-pulsed iBECs protected immunocompetent C57BL/6 mice from EG.7-OVA tumor challenge, and promoted tumor regression in mice with pre-established tumors. These data support the concept of B cell-based adoptive immunotherapy.
Anti-cancer mechanism of the flavonoid icariin (ICA) and its derivative ICT on the tumor microenvironment (46.41)
Authors: Eksioglu Erika , Zhou Jun-Min , Chen Xianghong , Fortenbery Nicole , Latimer Helen , Rasche Sarah , Djeu Julie , Wei Sheng
Published: January 2023

Flavonoids are a group of polyphenolic compounds, commonly found on plants with many biological abilities. Those obtained from the natural derivatives of Herba Epimedii, specifically its active compound icariin (ICA) and a recently described derivative named 3, 5, 7-Trihydroxy-4'-methoxy-8-(3-hydroxy-3-methylbutyl)-flavone (ICT), have been shown to have strong immunomodulatory and anti-tumorigenic effects, including profound effects on myeloid-derived suppressive cells (MDSC), MRP8/14, TLR4 and STAT3 which correlated with a robust decrease in tumor burden in mice. These cells are a suppressive component of the tumor microenvironment that has been determined to play a pivotal role in the maintenance and progression of cancer. They contribute to the up-regulation of inflammatory mediators correlated with tumor progression as well as many other immune suppressive factors. While strides have been made in recognizing the role of these compounds on their therapeutic potential against cancer, their mechanisms of action are poorly understood. In particular we hypothesized that the effect on MDSC is linked to the inhibition of PDE5. We also hypothesized that the effect of these compounds on inflammatory pathways is mediated by the activation and perhaps upregulation of phosphatases which help modulate MDSC activation.
Effective age-specific tumor immunotherapy for B16 melanoma by targeting age-related immune dysfunction (46.42)
Authors: Hurez Vincent , Daniel Benjamin , Liu Aijie , Sun Lishi , Shin Tahiro , Zhang Bin , Curiel Tyler
Published: January 2023

Age is the biggest risk factor for cancer but most studies are in young hosts. We mitigated age-specific tumor-related immune dysfunction to develop immunotherapy for B16 melanoma effective in aged hosts. Young and aged CD4+CD25hi regulatory T cells (Tregs) had similar in vitro function and in vivo tumor-associated increases, but denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. Aged mice had more basal and tumor-related myeloid derived suppressor cells (MDSC). Depleting MDSC with anti-Gr-1 Ab improved tumor-specific immunity and clinical response better in aged versus young mice. Treg depletion significantly increased MDSC in aged but not young mice, suggesting age-related Treg control of MDSC. In vitro, Treg from aged tumor bearing mice suppressed GM-CSF-induced bone marrow MDSC differentiation. Treg depletion + anti-Gr-1 Ab was more efficacious than anti-Gr-1 alone in aged B16-bearing mice but was no better than Treg depletion alone in young mice. In MC-38 colon cancer, DT and anti-Gr-1 Ab effects were similar in young and aged hosts. Thus aged anti-tumor immune effector cells are competent to combat tumor when tumor-associated immune dysfunction is appropriately mitigated, and age-related immune dysfunction is tumor-dependent. Tailoring immunotherapy to counter age-related, tumor-associated immune dysfunctions remarkably improves cancer immunotherapy for aged patients with specific tumors.
Foxp1 Regulates Anti-tumor Effector Functions of CD8+ T cells in Ovarian Cancer (46.46)
Authors: Stephen Tom , Takata Hiroshi , Feng Xiaoming , Wang Haikun , Rutkowski Melanie , Allegrezza Michael , Hu Hui , Conejo-Garcia Jose
Published: January 2023

T cell based adoptive immunotherapy induces potent anti-tumor response in many cancers. Highly immunosuppressive tumor microenvironment, however, severely impairs effector functions and survival of transferred T cells. In order to gain the best possible immune elimination of tumor cells, specific molecular changes that impair T cell effector functions in the tumor microenvironment need to be identified and targeted. We recently reported that transcription factor Foxp1 critically regulates T cell effector functions as Foxp1-deficient CD8+T cells are better effectors. Here we demonstrate that immunosuppressive tumor microenvironment results in the upregulation of Foxp1 in tumor-reactive T cells, and impairs their effector functions. Consequently, mice bearing ovarian tumors survived significantly longer upon receiving adoptively transferred tumor-primed Foxp1-deficient T cells. In vitro studies further showed that Foxp1-deficient CD8+ T cells can proliferate better in the presence of immunosuppressive TGFb1. Analysis of adoptively transferred T cells showed that, compared to WT CD8+ T cells, Foxp1-deficient CD8+, but not CD4+, T cells survive better in the immunosuppressive tumor microenvironment. Tumor bearing mice receiving Foxp1-deficient effector T cells rejected secondary tumors indicating generation of anti-tumor T cell memory. Our findings indicate that targeting the transcription factor Foxp1 on effector T cells can dramatically improve anti-tumor immunotherapy.
Use of alloreactivity to generate in vivo anti-leukemic responses (46.48)
Authors: Fast Loren , Reagan John , Quesenberry Peter
Published: January 2023

A previous clinical trial demonstrated that infusion of haploidentical G-CSF mobilized WBC (1 - 2 x 10^8 cells/kg) into patients with refractory hematological malignancies (100 cGy irradiation) resulted in a high frequency of anti-leukemic responses including some complete responses despite a lack of detectable donor cell persistence beyond two weeks. Beyond a cytokine storm, no effector mechanisms were defined. It is possible that alloreactive responses were either capable of directly attacking the leukemic cells or reactivating the patient’s anti-leukemic effector cells that were already present via cross-reactivity. PBMNC were obtained from the blood of newly diagnosed leukemic patients, and separated into CD3- and CD3+ populations. The patient’s CD3+ cells were used as responder cells in a mixed lymphocyte culture and stimulated with mitomycin C treated patient’s CD3- cells or PBMNC from allogeneic normal donors chosen at random. On day 7, the effector cells were collected and tested for their ability to lyse 51Cr labeled patient CD3- cells. The results showed that the patient’s CD3+ cells stimulated with allogeneic PBMNC were able to generate anti-leukemic responses in a stimulator dependent fashion. Thus, this assay may be useful for predicting which donor would be best for patients receiving cellular immunotherapy and may also be useful for the defining the anti-leukemic effector cells that are present with this form of cellular immunotherapy.
Characterization and Manipulation of NZB miR15a/16-1 loci for CLL Therapy (46.50)
Authors: Kasar Siddha , Underbayev Chingiz , Yuan Yao , Raveche Elizabeth
Published: January 2023

Chronic Lymphocytic Leukemia (CLL) is a malignancy characterized by accumulation of mature CD5+ B-cells in peripheral lymphoid organs, bone marrow and blood and is incurable with conventional therapy. We have previously shown that similar to human B-CLL cells, NZB mice (de novo mouse model of CLL) have 50% less level of miR15a/16-1 that is associated with a mutation and deletion in the miR15a/16-1 region. The NZB loci lead to a processing defect inhibiting the conversion of pri-miR15a/16-1 to pre-miR15a/16-1. We hypothesized that exogenously increasing its expression would lead to increased apoptosis in tumor cells. Using a lentiviral delivery system, we were able to stably increase the level of these two microRNAs in LNC (NZB derived B-CLL cell line). The apoptosis observed in miR-GFP transduced LNC was twice as much as in LNC transduced with GFP lentivirus (control). In addition to the miR defect, B-CLL cells have a very high level of B-cell specific activator protein (BSAP) and this prevents their terminal differentiation. Recent reports have suggested that BSAP and miR15a/16-1 form an autoregulatory loop. Alternatively, we found that the miR defect can also be corrected by knocking down BSAP (using RNAi) leading to a cell cycle arrest. The second strategy is clinically relevant, since developing a BSAP inhibitor is more feasible than systemic miR delivery. Based on our findings we propose that targeting BSAP could be used as a novel therapeutic strategy for B-CLL.
SLAMF1/CD150 is a signaling receptor in a subset of chronic lymphocytic leukemia cells (46.9)
Authors: Bologna Cinzia , Buonincontri Roberta , Serra Sara , Vaisitti Tiziana , Audrito Valentina , Brusa Davide , Rossi Davide , Coscia Marta , Gaidano Gianluca , Terhorst Cox , Deaglio Silvia
Published: January 2023

Human SLAMF1 (CD150) is expressed on T, B, NK, and dendritic cells where it acts as a co-activator through self interactions. Gene expression profiling has identified SLAMF1 as part of the genetic signature characterizing chronic lymphocytic leukemia (CLL) patients with favorable prognosis. The analysis of SLAMF1 expression on the CD19+ fraction of 220 patients with CLL revealed highly variable levels. Statistical analyses indicate that patients characterized by a good prognosis express higher levels of SLAMF1 compared to the counterpart. Furthermore, patients with less than 6% SLAMF1+/CD19+ CLL cells had a significantly longer treatment free survival (median 6.4 in SLAMF1- vs 1.2 years in SLAMF1+ patients, P=.002). SLAMF1 expression was also inversely correlated with CD38, CD49d and ZAP-70, and positively associated with the presence of somatic mutations in the IgHV genes, all molecular hallmarks of good prognosis. Incubation of CLL cells with anti-SLAMF1 mAb induced a robust tyrosine phosphorylation pattern, mediated by the EAT-2 adaptor. Activated intracellular molecules included lyn, vav-1, p38 and jnk. Co-crosslinking of SLAMF1 with sIgM prolonged phosphorylation of p38 and jnk and increased the percentage of CLL cells undergoing apoptosis, as compared to either signal alone. In conclusion, SLAMF1 represents a novel marker for CLL patients with a favorable prognosis. Functional data suggest that it may function together with the BCR in regulating CLL apoptosis.
Caloric restriction responses can maintain immune responses during aging (47.14)
Authors: Ruby Carl , Jimenez Hugo , Goldufsky Josef , Chisholm Lana , Lukaesko Lisa , Weinberg Andrew , Kaufman Howard
Published: January 2023

Age-related defects accumulate in both the innate and adaptive arms of immunity to generate a state of immune deficiency. Our laboratory has previously shown a decrease in immune responses in aged mice compared to younger mice in the context of OX40 costimulation. This loss of immunity during aging underscores the need for simple approaches to boost immunity in older individuals. We hypothesize that immune responses in the context of OX40 activation or blockade of CTLA-4 in older mice can be restored and/or maintained through caloric restriction (CR) or dietary supplementation with a CR mimetic. Two CR dietary regimens were used to determine if immunity in old immune-compromised mice could be restored and if immunity could be maintained during aging. In addition mice were fed diets that were formulated with resveratrol (100 ppm), a CR mimetic. Tumor immune responses and Ag-specific T cell responses in these mice were then assessed and compared to young mice and the appropriate dietary controls. Our results demonstrate that caloric restriction cannot restore immune function in old mice. However, CR or resveratrol dietary supplementation initiated at a young age can maintain both OX40-mediated anti-tumor immunity and T cell responses as these mice age. Thus, the induction of a caloric restriction response (via traditional CR or by a CR mimetic) appears to maintain immunological fitness and may increase the effectiveness of immune-based cancer therapies in older subjects.
Loss of nuclear Pro-IL-16 imparts resistance to apoptosis in CTCL T cells (47.5)
Authors: Tuzova Marina , Memari Patrick , Mosaheb Munir , Cruikshank William
Published: January 2023

Cutaneous T-cell Lymphoma (CTCL) is a rare type of non-Hodgkin’s lymphomas characterized by increased T cell proliferation and decreased apoptotic responses. We have reported on the loss of nuclear Pro-IL-16 in CTCL T cells due to a mutation restricting nuclear translocation, which results in dysregulation of Skp2 and p27kip1, and promotes hyper-proliferative state. We have now investigated the relationship between the loss of nuclear Pro-IL-16 and apoptosis. RT-PCR analysis of a panel of apoptosis-related genes was conducted using T cells obtained from IL-16 knock out and wild type control mice. The IL-16 null cells demonstrated a significant reduction in pro-apoptotic genes such as Bax, Bcl10, Bcl2110, and caspases 7, 8 and 9, with an increase in expression of anti-apoptotic genes such as Bag1, Tsc22d3 and Pim2. Annexin V staining of these cells indicated a 43% reduction in the apoptotic response to CD3/CD28 stimulation in the knock-out cells. To confirm the anti-apoptotic effect of nuclear Pro-IL-16, human CTCL-derived T cell line (HUT 78) was transfected to express normal full-length IL-16. Based on Annexin V staining, the apoptotic response increased by 35% in cells, expressing normal IL-16. These studies indicate that loss of nuclear Pro-IL-16 in abnormal T cells correlates not only with increased cell proliferation, but with a decrease in apoptosis as well.
Naringenin modulates intracellular signaling pathways and cell surface receptors in Chlamydia trachomatis-infected mouse J774 macrophages to control concomitantly elicited inflammation (48.3)
Authors: Yilma Abebayehu , Singh Shree , Dennis Vida
Published: January 2023

Sexually transmitted Chlamydia trachomatis infection is of public health concern because of its potential devastating reproduction consequences. C. trachomatis invades the mucosal surface of the female genital tract after it is recognized by cell surface receptors. Increasing evidence now suggests that inflammatory mediators, which are produced by infected cells, contribute to the chronic inflammatory conditions. Controlling excess inflammatory mediators during the early stage of an infection may be a viable alternative to combat the disease. Previously we reported that naringenin, a natural flavonone, reduced IL-6 and TNF levels by up to 70% in C. trachomatis-infected mouse J774 macrophages. Here we hypothesize that naringenin reduces inflammatory mediators by inhibiting intracellular signaling pathways and cell surface receptors. Naringenin inhibited the phosphrylation of p38 MAP kinase and NFκB as induced by C. trachomatis-infected J774 macrophages. Flow cytometric analyses further revealed the ability of naringenin to reduce the cell surface receptor expression of TLR2, TLR4 and CD86 but not CD80 on infected macrophages. These findings suggest the potential of naringenin not only to control inflammatory mediators produced by innate immune cells but also those produced by C. trachomatis-activated T cells during the adaptive immune response. Overall our data imply the positive role played by naringenin in controlling an early C. trachomatis-induced inflammation.
Contact time dependent Effect of human mesenchymal stem cells on IFN-r and IL-17 secreted by CD4+ T effector memory cells (48.4)
Authors: Kim Mee Kum , Jeong Hyun Jeong , Lee Young Eun , Ryu Jin Sook , Lee Won Woo , Wee Won Ryang
Published: January 2023

Mesenchymal stem cells(MSCs) has known to have antiinflammatory properties in various inflammatory disease, however the effect of MSCs on the inflammation is reported to be variable depending on the disease entity, contact time, application time and administration site for the delivery. The study was aimed to analyze the contact time-dependent effect of human mesenchymal stem cells(hMSCs) on IL-17 and INF-r secreted by CD4+ T effector memory cells. Human peripheral blood mononuclear cells(PBMCs) was obtained from the healthy voluntary donors with informed consents(n=5). The study was performed in accordance with the guidelines of the Declaration of Helsinki. CD4+ T cells were purified through MACS separator with the human CD4+ T Cell Isolation Kit II (Miltenyi Biotec). CD4+ T cells were stimulated with anti-CD3/CD28 and cultured in medium supplemented with IL-1β and IL-23. The bone marrow derived hMSCs got in contact with CD4 T cells on 0 day, 3 days, and 5 days after stimulation at a T cell-to-MSC ratio of 10:1. We found that the earlier hMSCs were contacted to CD4+ CD45RO+ T cells, the more effect was suppressive on both IFN-γ and IL-17A secreted by CD4+ effector memory T cells. In fact, later contact of hMSCs to activated T cells dropped most suppressive effect of hMSCs down on effector memory CD4 T cells. It suggested that the earlier time point contact might be the better for hMSCs to down-regulate the functions of effector memory CD4 T cells after activation.
Staphylococcus aureus protein A mediates innate immune responses during nasal colonization in humans (49.25)
Authors: Cole Alexander , Muthukrishnan Gowrishankar , Eade Colleen , Wood Matthew , Lamers Ryan , Ellis Austin , Cole Amy
Published: January 2023

Mucosa of the anterior human nares can harbor Staphylococcus aureus (SA), an etiologic agent that causes severe infections. Nasal carriage of SA is a complex host-pathogen interaction involving bacterial and host mediators that influence innate host defenses. We have revealed that the exoproteome of SA nasal carrier strains contains unique determinants, which enable SA to colonize the nares. In the current study, we assess how one of these bacterial mediators, Staphylococcal protein A (Spa), affects host innate immune response to SA nasal carriage. Functional disruption of spa was performed in an SA nasal carrier strain using Targetron insertion of a group II intron. Subsequent in vitro growth kinetics analysis and ex vivo culture on human nasal epithelial cells (HNECs) revealed no differences between wild type and spa-disrupted (Δspa) SA strains. However, the Δspa mutant induced lower expression of pro-inflammatory cytokines including IL6, IL8 and TNFα from HNECs compared to the levels elicited by the wild type strain. Additionally, IL17, a crucial regulator of mucosal immunity, was induced at significantly lower levels in HNECs inoculated with Δspa SA compared to those inoculated with the wild type SA strain. Furthermore, expression of the chemokines MIP1β and MCP1, as well as HL60 neutrophil migration, were significantly reduced in conditioned media from HNECs inoculated with Δspa mutants. Spa is likely involved in mediating the mucosal response to SA colonizing the nose.
Building a toolkit: An undergraduate teaching laboratory sequence in immunology (50.1)
Authors: Chatterjea Devavani , Linder Grace
Published: January 2023

We have designed a laboratory curriculum in immunology where students build and use a toolkit of basic techniques. In the first 10 weeks, students learn how to isolate human and mouse immune cells, label cells with antibodies, perform magnetic enrichment, flow cytometry analysis, T cell proliferation, and ELISA assays. We emphasize keen observation, excellent record keeping, and protocol development. We ensure that each team has at least two opportunities to perform each new technique. Students survey the published literature and formulate a novel question about effects of a dietary supplement of choice on in vitro T cell stimulation. Teams develop experimental designs that are critiqued in a group workshop. Independent experiments are carried out, data collected and analyzed by teams, and a detailed, reflective lab notebook entry compiled by each student. Teams present their findings in a culminating mini-symposium. Our iterative emphasis on skill building, critical record-keeping, application of newly learned skills, and teamwork closely simulates the learning that happens in authentic research laboratories. Students encounter and comprehend these basic procedures in classroom discussions, research seminars, and journal articles. According to students surveyed, this plays a critical role in the productive integration of classroom and laboratory learning, and gives students a strong sense of engagement with the professional world of immunology research.
Anti-MS4a4B treatment abrogates MS4a4B-mediated protection in T cells and ameliorates experimental autoimmune encephalomyelitis (51.13)
Authors: Xu Hui , Yan Yaping , Li Zichen , Zhang Guang-Xian , Williams Mark , Carey Gregory , Zhang Jianke , Rostami Abdolmohamad
Published: January 2023

MS4a4B is a membrane protein that highly expressed in T cells. We have demonstrated previously that MS4a4B has a regulatory role in proliferation and apoptosis of T cells. In this study, we further analyzed the mechanisms underlying MS4a4B-mediated anti-apoptotic effects in T cells and tested the feasibility of using anti-MS4a4B antibody as a therapeutic approach for experimental autoimmune encephalomyelitis (EAE). Our data showed that knockdown of MS4a4B expression by MS4a4B-specific siRNA markedly enhanced levels of anti-CD3 stimulation-induced cleavage of caspase 3, caspase 8 and caspase 9 in pre-activated primary T cells. Interestingly, binding of anti-MS4a4B antibodies to T cells induced activated T cells to undergo apoptosis and administration of anti-MS4a4B antibody in vivo caused T cell depletion in the peripheral blood. To test the therapeutic effects of anti-MS4a4B antibody in suppression of T cell-mediated autoimmune responses, we injected anti-MS4a4B antibodies into EAE mice. The results show that anti-MS4a4B treatment ameliorated the severity of EAE, accompanied by decreased Th1 and Th17 cell responses and reduced levels of pro-inflammatory cytokines in the central nervous system (CNS), suggesting that MS4a4B may serve as a target of antibody-based therapy for T cell-mediated diseases. Supported by American Heart Association grant AHA-0830072N (to H.X)
Durable pharmacological responses from the peptide drug ShK-186, a specific Kv1.3 channel inhibitor that suppresses T cell mediators of autoimmune disease (51.5)
Authors: Munoz Ernesto , Tarcha Eric , Londono Luz , Norton Kayla , Banks Amy , Chi Victor , Beeton Christine , Chandy K. , Iadonato Shawn
Published: January 2023

The Potassium Kv1.3 channel on effector memory T cells is a target to treat autoimmune diseases. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the development of methods to establish a pharmacokinetic (PK) - pharmacodynamic (PD) relationship for ShK-186, and test the hypothesis that the drug has a sustained PD effect in autoimmune disease models. The PK of ShK-186 was evaluated in rats and cynomolgus monkeys using HPLC-MS/MS and ELISA methods to measure the free fraction in plasma. These results were compared with SPECT/CT data collected with an 111In-DOTA-conjugate of ShK to assess whole blood PK parameters as well as the drug’s absorption, distribution and excretion. Our data suggest ShK-186 is absorbed slowly from the injection site resulting in blood concentrations above the Kv1.3 channel-blocking Kd for up to 7 days in monkey. PD studies on human peripheral blood mononuclear cells show that transient ShK-186 treatment results in a sustained suppression of cytokine responses and may contribute to prolonged drug effects. Moreover, in rat delayed-type hypersensitivity and chronic relapsing experimental autoimmune encephalomyelitis, a single dose of drug every 3 - 5 days is as effective as daily administration. These data suggest the PD effects of ShK-186 are governed primarily by slow distribution from peripheral tissues and long residence time on the Kv1.3 channel.
Evaluation of a HLA-DR2 Inhibitor for the Treatment of MS Using Human Cellular Immunologic Models (51.6)
Authors: Ji Niannian , Olson Gary , Self Christopher , Forsthuber Thomas
Published: January 2023

Multiple sclerosis (MS) is the most common human neurological disease affecting approximately 400,000 Americans with no cure available. It is believed to be driven by autoimmune CD4+ T cell responses directed against myelin antigens in the CNS. CD4+ T cell activation and response require antigen presentation by MHC (HLA in human) class II molecules. The HLA class II allele HLA-DRB1*1501 (HLA-DR2) is highly associated with MS, therefore we tested the immunological properties and activity of PV-267, a small molecule DR2 inhibitor in vitro in human cellular immunologic models related to MS using flow cytometry analysis, human cytokine ELISPOT and Bio-Plex assays. In vitro studies of human PBMCs from MS patient or healthy control subjects and testing a myelin basic protein specific T cell line showed that: 1) DR2 inhibitors can inhibit myelin antigen-specific human T cell responses mediated via HLA-DR2; 2) inhibit the production of pro-inflammatory cytokines; 3) treatment with the inhibitors does not have undesired non-specific immune-activating effect on human PBMCs expressing either HLA-DR2 or other HLA-DR molecules. In sum, our preclinical study has established the immunologic efficacy and assessed the safety of DR2 inhibitors in vitro. The results provide a strong rationale for the application of MHC inhibitors for the treatment of MS and potentially other T cell-mediated autoimmune diseases based on the immunological efficacy and good safety profile.
Disparate adjuvant properties among three formulations of “alum” (52.11)
Authors: Cain Derek , Sanders Sergio , Cunningham Michael , Kelsoe Garnett
Published: January 2023

Aluminum salts, commonly referred to as “alum,” are the most common adjuvants in human vaccines and serve as a benchmark in the development of novel adjuvants. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to induce inflammatory signals that, in turn, promote antibody production. It has been noted that some formulations of alum used in animal studies, notably Imject alum®, are not the chemical equivalents of the aluminum used in human vaccines, raising concerns where Imject serves as the comparator for novel adjuvants. Here, we compared the capacity of Imject alum, Alhydrogel®, and a traditional alum-antigen (Alum-Ag) precipitate to induce humoral responses in mice to the hapten-carrier NP-CGG. Primary humoral responses elicited by Alhydrogel and the Alum-Antigen precipitate were significantly greater than those induced by Imject alum. The three alum formulations elicited comparable responses in terms of acute eosinophil and monocyte infiltration at the immunization site, but neutrophil recruitment by Imject alum was signficantly reduced compared to Alum-Ag precipitate or Alhydrogel. These findings reveal substantial differences in the adjuvant properties and inflammatory effects of distinct alum preparations, an important consideration in the analysis of new adjuvants and mechanistic studies of alum’s adjuvanticity.
Inhibition of mTOR promotes dendritic cell activation and survival and enhances therapeutic autologous vaccination in mice (52.4)
Authors: Amiel Eyal , Everts Bart , Freitas Tori , King Irah , Curtis Jonathan , Pearce Erika , Pearce Edward
Published: January 2023

Dendritic cells (DCs) are potent inducers of T cell immunity and autologous DC vaccination holds promise for the treatment of cancers and chronic infectious diseases. In practice, however, therapeutic vaccines of this type have had mixed success. Here we show that brief exposure to inhibitors of mechanistic Target Of Rapamycin (mTOR) in DCs during the period that they are responding to TLR agonists makes them particularly potent activators of naïve CD8+ T cells, and able to induce enhanced immune responses capable of controlling melanoma in a therapeutic autologous vaccination model in the mouse. The improved performance of DCs in which mTOR has been inhibited is correlated with an extended lifespan following activation and prolonged, increased expression of costimulatory molecules. In this work, we define molecular and metabolic processes that determine DC lifespan and identify that how mTOR inhibition alters these pathways to extend the post-activation lifespan of DCs. These findings define mTOR as a molecular target for augmenting DC survival and activation and document a novel pharmacologic approach for enhancing the efficacy of therapeutic autologous DC vaccination.
Candidate adjuvant restores DC-NK cross-talk and improves immunization outcomes in nicotine-exposed hosts (53.14)
Authors: Nouri-Shirazi Mahyar , Nourishirazi Erika , Lang Kevin , Guinet Elisabeth
Published: January 2023

Vaccines are aimed at all target populations including smokers, who constitute one billion people worldwide. There have been several studies published on the link between reduced vaccine effectiveness and cigarette smoke. However, there have been no studies targeted at developing vaccine formulas to increase the effectiveness of vaccines in this population. Recently, we reported that exposure to nicotine significantly diminishes the development of antigen-specific effector memory Th1 cells and antibody production, leading to poor host response to an otherwise protective and therapeutic vaccine. We also reported that defects in the immunobiology of the DC system in vivo underlie this poor outcome (Accepted in The Journal of Immunology). In this study, we further demonstrate that among TLR agonists tested, a candidate adjuvant overcame the impact of nicotine and restored DC-NK cross-talk in vitro. Furthermore, the addition of this adjuvant to vaccine formulation significantly improved vaccination outcome in nicotine-exposed mice as evidenced by clonal expansion and differentiation of adoptively transferred OVA-specific T cells into IFNg-secreting effector Th1 cells in the spleen and lymph nodes of both control and nicotine-exposed mice. In summary, our research has revealed the physiological changes triggered by nicotine on host immunity and opened the possibility of developing a vaccine formulation that works optimally in all target populations.
Immunostimulatory properties of dynamic oligonucleotide micelles (53.9)
Authors: Liu Haipeng , Irvine Darrell
Published: January 2023

Synthetic immunostimulatory oligonucleotides such as double-stranded RNA or unmethylated cytosine-guanosine motifs (CpG-ODNs) mimic molecular signatures of pathogens and trigger an immunostimulatory cascade; as a result, these synthetic ODNs have been extensively studied as therapeutic agents for cancer and as vaccine adjuvants. Using a self-assembly approach, we designed lipo-CpG-ODN molecules composed of CpG oligos conjugated with diacyl lipid tails. These dynamic amphiphiles form compact micelles ~20 nm in diam. in water but can also disassemble and insert their acyl tails into the plasma membrane when incubated with cells. These two key elements of the CpG micelle-ultra-small physical size and cell membrane insertion capability- combine to greatly enhance the adjuvant properties of CpG or other co-associated TLR agonists. Injection of CpG micelles s.c. resulted in a 5-15-fold enhancement of draining lymph node (dLN) retention compared with free CpG or stabilized CpG-micelles incapable of inserting into membranes. Immunization with soluble antigen mixed with CpG micelles induced 10-fold increased frequencies of antigen-specific T-cells in blood and elicited higher antibody production than equivalent doses of soluble CpG, and also elicited stronger responses than CpG-micelles lacking membrane insertion potential. Thus, dynamic micellar TLR agonists may be of interest as novel vaccine adjuvants.
In vivo induced deletion of the mitochondrial protein stomatin-like protein-2 is associated with development of an inflammatory motor deficit disorder (54.10)
Authors: Mitsopoulos Panagiotis , Christie Darah , Welch Ian , Madrenas Joaquin
Published: January 2023

Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein identified in proteome analysis of glycolipid-enriched membrane microdomains of mammalian cells, including lymphocytes. Our preliminary data suggest SLP-2 recruits prohibitins to cardiolipin-enriched domains in the mitochondrial inner membrane to form specialized membrane microdomains, required for optimal cellular respiration. We hypothesized that deletion of the SLP-2 gene will cause defective mitochondrial function that may result in organ-selective disease. We found that a conventional SLP-2-deficient mouse was embryonic lethal at the pre-implantation stage. Thus we generated viable tamoxifen-inducible systemic SLP-2 knockout mice. Administration of tamoxifen (3mg/40g; 10 days; i.p.) resulted in a motor phenotype characterized by poorer SHIRPA scores, weight loss and death (68% at day 28, p<0.05). This phenotype correlated with SLP-2 gene deletion and decreased protein levels in select tissues. Histological analysis revealed multifocal myofibrillar degeneration of the diaphragm with a mononuclear inflammatory infiltrate. In addition, apoptosis throughout the exocrine pancreas and pancreatic atrophy due to loss of normal acinar tissue were observed. In conclusion, in vivo SLP-2 deletion induces a mitochondrial disease-like phenotype, suggesting this protein is required for optimal metabolic function. SLP-2-deficient mice may be useful to assess the function of SLP-2 in vivo and to study mitochondrial diseases.
Targeting CD44 in SEB-induced acute respiratory distress syndrome (54.13)
Authors: McKallip Robert , Bridges Christy , Law Gabriela , Sun Jingping
Published: January 2023

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). In the current study we investigated the role of CD44 in ALI/ARDS. Intranasal exposure of CD44 wild type (CD44 WT) mice to SEB led to a significant increase in the expression of CD44 on lung mononuclear cells. CD44 knockout (CD44 KO) mice developed significantly reduced SEB-induced ALI/ARDS, compared to similarly treated CD44 WT mice. Deletion of CD44 did not have a significant effect on lymphocyte activation, apoptosis or inflammatory cytokine production. However, mononuclear cell migration to the lungs of SEB-exposed CD44 KO mice was significantly reduced when compared to mononuclear infiltration in the lungs of SEB-exposed CD44 WT mice. Mechanistically, deletion of CD44 led to reduced ability of SEB-exposed spleen cells to bind to lung epithelial cells. Finally, treatment of SEB-exposed mice with anti-CD44 mAbs led to a significant reduction in vascular permeability, prevented inflammatory cells infiltration in the lungs and reduced the ability of SEB-exposed splenocytes to adhere to epithelial cells. Together, these results demonstrate an important role of CD44 in SEB-induced lung injury and suggest the possibility of targeting CD44 for the treatment of SEB-induced ALI/ARDS.
Glycogen synthase kinase-3 facilitates IFN-γ-mediated phorbol ester-induced skin inflammation (54.25)
Authors: Hsieh Chia-Yuan , Chen Chia-Ling , Tsai Cheng-Chieh , Huang Wei-Ching , Tseng Po-Chun , Lin Yee-Shin , Chen Shun-Hua , Wong Tak-Wah , Lin Chiou-Feng
Published: January 2023

Activation of glycogen synthase kinase (GSK)-3 facilitates IFN-γ signaling, and IFN-γ generation is positively correlated with disease severity in inflammatory skin diseases, such as psoriasis and atopic dermatitis. In this study, we investigated a pathogenic role for GSK-3 in phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA)-induced IFN-γ-mediated skin inflammation. TPA induced acute skin inflammation in the ears of C57BL/6 mice, including edema, the infiltration of granulocytes but not T cells, and the deregulation of adhesion molecule CD54 expression, in an IFN-γ receptor (IFNGR) 1-regulated manner. TPA/IFN-γ induced GSK-3 activation and caused the activation and expression of signal transducers and activators of transcription 1 in a GSK-3-regulated manner. Inhibiting GSK-3 through either pharmacological inhibition or a genetic approach using lentiviral-based short hairpin RNA reduced acute TPA-induced skin inflammation, but not T cell infiltration. Notably, inhibiting GSK-3 decreased the TPA-induced IFN-γ production as well as the nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, in CD3+ T cells. In chronic TPA-induced skin inflammation, inhibiting GSK-3 attenuated epidermis hyperproliferation and dermis angiogenesis. These results demonstrate the dual and indispensable role of GSK-3 in TPA-induced skin inflammation by facilitating IFN-γ signaling.
Contribution of an aging environment towards a proinflammatory tumor-like milieu (54.4)
Authors: Mirza Noweeda , Hoelzinger Dominique , Myers Cheryl , Pollock Kevin , Dominguez Ana , Lustgarten Joseph , Cohen Peter , Gendler Sandra
Published: January 2023

Chronic inflammation associated with aging is termed inflamm-aging. It manifests as a rise in many proinflammatory mediators and contributes to a decline in immune function. Unregulated inflammasome activation initiates an inappropriate inflammatory response. It is particularly imperative to understand inflammasome dysregulation in the elderly (>65 yrs), since it is a likely basis of immune dysfunction. Analyses of freshly isolated memory CD4+ and CD8+ splenic T cells demonstrated that even without further activation, inflammasome genes were already significantly up-regulated in old mice compared to young, including factors critical to myeloid-derived suppressor cell (MDSC) expansion including S100a8, S100a9, IL-1β, IL-13 and IL-6. A corresponding 2-3 fold increase in CD11b+Gr1+cells was observed in the spleens of old naïve mice compared to young. Importantly, cells with the CD11b+Gr1+ phenotype from old mice, unlike young, were functionally MDSCs and suppressed T cell proliferation. Therefore, the immunosuppressive environment of the tumor-bearing host also appears to be operative in aging, even in the absence of tumor. We hypothesize that constituitive activation of inflammasome complex results in a chronic inflammatory state, altering immune responses in the elderly similarly to the tumor-bearing state. Preemptive reversal of inflammasome-induced MDSCs may enhance T cell immunosurveillance in the elderly, a population with a heightened susceptibility to malignancy.
Phospholipase A2 plays a key role in PMN transepithelial migration during S. pneumoniae infection (55.18)
Authors: Bhowmick Rudra , Leong John , McCormick Beth
Published: January 2023

Streptococcus pneumoniae lung infection is characterized by a massive alveolar infiltration of neutrophils (PMNs). Using an in vitro model of lung epithelium and human PMNs, we have shown that S. pneumoniae-induced PMN transepithelial migration is mediated by hepoxilin A3 (HXA3), an arachidonic acid (AA)-derived eicosanoid. Generation of AA is thought to be the rate-limiting step in generation of eicosanoids. Therefore, we studied the role of phospholipase A2 (PLA2), which generates AA from membrane phospholipids, on S. pneumoniae-elicited PMN transpeithelial migration. We found that pneumococcal infection of in vitro lung epithelium resulted in release of AA. This event was partially inhibited by pan-PLA2 inhibitors ONO-RS-082 and ACA, and also by diacylglycerol (DAG) lipase inhibitor RHC 80267. However, while the pan-PLA2 inhibitors significantly reduced S. pneumoniae-induced PMN transmigration, RHC 80267 had no effect on this event. We found that, cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) are important for generation of free AA and inhibition of these enzymes lead to dramatic reduction in PMN transepithelial migration. On the other hand, secretory PLA2 (sPLA2) did not seem to pay a key role in this event. We observed that S. pneumoniae infection caused an increase in the phosphorylation of cPLA2, suggesting a mechanism whereby S. pneumoniae activates PLA2 to generate free AA that may ultimately be converted to the PMN chemoattractant HXA3.