bluuebunny/crossref_metadata_2025 huggingface dataset:
PDZ-bm of influenza virus NS1 protein increases host susceptibility to super-infection via inhibition of IFNβ production
Authors: Rynda-Apple Agnieszka , Huber Victor C , Shepardson Kelly M. , Klonoski Joshua M. , Kyle Larson , Morton Rachelle
Published: January 2023

Type I interferon (IFN) signaling is a known contributor to increased host susceptibility to deadly bacterial super-infections (BSIs) following influenza A virus (IAV) pandemics. However, many IAV infections never develop secondary bacterial complications, suggesting that viral-derived factors can influence host susceptibility to BSIs. Utilizing reverse genetics we found that a natural variation in IAV nonstructural protein (NS1) could directly affect the duration of IFNβ signaling, which in turn affected susceptibility of mice to BSIs. Specifically, the presence of PDZ-binding motif (PDZ-bm) on the C terminus of PR8 IAV NS1 was responsible for suppression of IFNβ after day 3 post-PR8, which resulted in transition from reduced (day 3) to increased (day 7) BSI susceptibility determined by increased morbidity, mortality and lung bacterial burden. Introduction of stop codon in the position 220 of PR8 NS gene resulted in PDZ-bm deletion mutant (PR8-Trunc) that sustained production of IFNβ up to day 7 and protected WT mice from S. pnuemoniae BSI. We determined that IFNβ was required for this protective effect as unlike WT mice, IFNβ−/− mice were susceptible to BSI at day 7 post PR8-Trunc infection. Furthermore, treatment of WT mice at day 6 after WT PR8 infection with exogenous IFNβ reduced their susceptibility to BSI on day 7. Through utilization of both blocking antibodies and cell-specific type I IFN receptor −/− mice we found that reduced susceptibility to BSI required IFNβ signaling in both CD11c and Ly6G cells. Conversely, the increased susceptibility to BSIs at day 7 of PR8 infection resulted from IFNα signaling in Ly6G cells in the absence of IFNβ. Thus IAV-regulated duration of IFNβ signaling directly affects host susceptibility to BSIs.
Role of PD-1 in regulatory T cells during chronic virus infection
Authors: Ha Sang-Jun , Park Hyo Jin , Park Joon Seok , Jeong Yun Hee , Son Jinmin , Ban Young Ho , Chang Jun , Chung Doo Hyun
Published: January 2023

Regulatory T (Treg) cells act as terminators of T cell immunity during acute phase of viral infection; however, their role and suppressive mechanism in chronic viral infection are not completely understood. In this study, we compared the phenotype and function of Treg cells during acute or chronic infection with lymphocytic choriomeningitis virus. Chronic infection, unlike acute infection, led to a large expansion of Treg cells and their upregulation of programmed death-1 (PD-1). Treg cells from chronically infected mice (chronic Treg cells) displayed greater suppressive capacity for inhibiting both CD8+ and CD4+ T cell proliferation and subsequent cytokine production than those from naive or acutely infected mice. A contact between Treg and CD8+ T cells was necessary for the potent suppression of CD8+ T cell immune response. More importantly, the suppression required cell-specific expression and interaction of PD-1 on chronic Treg cells and PD-1 ligand on CD8+ T cells. Our study defines PD-1 upregulated on Treg cells and its interaction with PD-1 ligand on effector T cells as one cause for the potent T cell suppression and proposes the role of PD-1 on Treg cells, in addition to that on exhausted T cells, during chronic viral infection.
Tbet suppresses TH17 differentiation during viral infection
Authors: Hale J Scott , Mohammed Ata Ur Rasheed , Ahmed Rafi
Published: January 2023

Lineage-specifying transcription factors such as Tbet, GATA3, RORγt, and Bcl6 play critical roles in directing T helper lineage differentiation in response to various types of infections to orchestrate protective immune responses. Following acute LCMV infection, antigen-specific CD4 T cells differentiate to become either Tbet-expressing Th1 cells, or Bcl6+Tbet(low) T follicular helper cells. However, virus-specific CD4 T cells do not generate Th2 and Th17 responses. To determine whether Tbet plays a role in suppressing these alternative T helper differentiation programs in CD4 T cells during viral infection in a cell-intrinsic manner, we adoptively-transferred Tbet-deficient (Tbx21 knockout) SMARTA TCR transgenic CD4 T cells and then infected recipient mice with LCMV. Tbet deficient CD4 T cells generated 2 types of effector T helper cells: IL-17+CXCR5- Th17 cells and Bcl6+CXCR5+ Tfh cells. Tbet deficient Th17 effector cells expressed the transcription factor RORγt and the key cytokines IL-17 and IL-22. Co-transfer of wild-type and Tbet-deficient CD4 T cells revealed that the transcription factor Tbet represses the Th17 program in vivo in a cell-intrinsic manner. Together, these findings demonstrate the importance of the transcription factor Tbet in both promoting the useful Th1 response and suppressing the inappropriate Th17 response during viral infection.
Mast cells are present during the early stages of Chikungunya virus infection
Authors: Haese Nicole , Kreklywich Craig , Legasse Alfred , Heise Mark , Axthelm Michael , Messoudi Ilhelm , Streblow Daniel N
Published: January 2023

Chikungunya virus (CHIKV) is a positive-sense RNA, arthropod-borne Alphavirus. In recent years the largest recorded outbreak of CHIKV occurred in the Islands of the Indian Ocean and India. CHIKV has continued to spread causing millions of cases across more than 40 countries. Acute symptoms of infection include the abrupt onset of fever, rash, fatigue, headache, backache, and arthralgia, with arthralgia lasting up to several years in some individuals. Ongoing studies analyzing cohorts of CHIKV infected humans have led to a well-developed understanding of the clinical manifestations of CHIKV disease. What is unknown is the role different components of the immune response play in controlling CHIKV dissemination and disease development. In our current study we utilized a non-human primate (NHP) model of CHIKV infection to characterize the early stage of CHIKV infection. Adult male and female rhesus macaques (RM) were infected subcutaneously with CHIKV-LR. Analysis of vRNA loads in joint tissues of infected RM revealed that the highest viral loads were detectable at 2 dpi with at least a log decrease in vRNA levels by 7 dpi. T cells, B cells, PMNs, macrophages, and mast cells were visible in the joints of CHIKV infected RMs at 2 and 7 dpi. To our knowledge this is the first time mast cells have been identified in the tissue of CHIKV infected NHPs. Levels of key proinflammatory cytokines/chemokines were also increased from 3–7 dpi. The gene expression profile in joint tissues reveled an increase in the expression of genes involved in recruitment, activation, and maintenance of mast cells. These findings suggest that infiltration of immune cells including the newly identified mast cells from 2 to 7 dpi assist in controlling CHIKV infection.
CD81: a novel lymphoma therapeutic target
Authors: Catalan Felipe Vences , Kuo Chiung-chi , Rajapaksa Ranjani , Duault Caroline , Levy Ronald , Levy Shoshana
Published: January 2023

Tetraspanin CD81 was initially described as a target of a monoclonal antibody (mAb) able to inhibit proliferation of lymphomas and activated lymphocytes, which suggests that CD81 plays a role in their survival. Indeed, studies in CD81 knockout mice have demonstrated its role in innate and adaptive immune responses, by regulating lymphocyte activation, proliferation and antibody production. Furthermore, CD81 deficiency leads to reduced anti-tumor immune suppression by MDSCs and Tregs, resulting in decreased tumor growth and metastasis. Moreover, treatment with anti-mouse CD81 mAbs reduces metastasis in tumor-bearing wild type mice.

Here we demonstrate that an anti-human CD81 mAb effectively reduces growth of lymphoma cell lines in vivo and improves survival. To further enhance the in vivo efficacy of our mouse IgG1 anti-CD81 mAb, we class switched the hybridoma to produce IgG2a and we also engineered a chimeric human IgG1 anti-CD81 mAb. Indeed, both isotypes when compared to Rituximab, show superior efficacy in eliminating tumor cells in vivo. And in vitro studies demonstrate superior direct killing, antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Finally, despite the expression of CD81 on normal lymphocytes which creates a challenge for antibody-based therapies, we demonstrate that B cell lymphoma cell lines were selectively sensitive to anti-CD81 therapy. Taken together, we proposed CD81 as a potential target for treating lymphoma.
Enhancement of Anti-Tumor Activities of Daratumumab Using ALT-803, an IL-15 Superagonist, in an Experimental Model and Against Primary Human Lymphoma Cells
Authors: Wong Hing C , Shrestha Niraj , Liu Bai , Alter Sarah , Jeng Emily K , Fehniger Todd A , Rhode Peter R
Published: January 2023

Daratumumab has recently been approved by FDA for treatment of patients with relapsed or refractory multiple myeloma. Daratumumab has been shown to reduce the tumor burden by targeting CD38 expressing tumor cells. In this study, we examined the in vitro as well as in vivo ability of ALT-803, a novel IL-15 superagonist-complex, to enhance the tumor targeting antibody dependent cellular cytotoxicity (ADCC) of daratumumab in combination therapy. Using freshly isolated PBMCs, we demonstrated that ALT-803 significantly enhanced the ADCC activity of Daratumumab against Daudi, human B lymphoma, cells. We also observed that purified human NK cells, which were activated by ALT-803, enhanced daratumumab-mediated ADCC activity against primary human B-lymphoma cells. We also assessed the in vivo effects of ALT-803 on daratumumab-mediated antitumor activity against Daudi cells in the SCID xenograft model. We observed that both ALT-803 and daratumumab alone were capable of reducing Daudi tumor burden in tumor-bearing mice. However, the combination of ALT-803 with daratumumab increased antitumor activity which significantly reduced Daudi cells in the bone marrow. These observations indicate that the addition of ALT-803 to daratumumab treatment enhances the antitumor efficacy of Daratumumab compared to daratumumab alone. In addition, we have evidence that ALT-803 also augment the Antibody Dependent Cytotoxicity of Phagocytosis of Daratumumab against tumor cells. In a current clinical study, our correlative studies also show that ALT-803 augment the ADCC activity of Daratumumab against Daudi cells. This also demonstrates that ALT-803 possesses synergistic antitumor activities with tumor-specific antibodies in vivo.
A novel, dual-specific antibody conjugate targeting CD134 and CD137 costimulates T cells and elicits antitumor immunity
Authors: Ryan Joseph , Mittal Payal , Svedova Julia , Menoret Antoine , Wasser Jeffrey S. , Adler Adam J. , Vella Anthony T.
Published: January 2023

Checkpoint inhibiting antibodies targeting CTLA-4 and PD-1, along with costimulatory agonists, exhibit impressive anticancer efficacy and evidence of durable tumor regression in patients. Despite this success however, many patients fail to respond, likely due to multiple immunosuppressive tactics employed by cancer cells. Combination therapies designed to counteract these immunosuppressive features show promise, but strategies relying on multiple agents suffer from logistical and regulatory challenges, as well as increased risks of adverse events. We developed a novel immunotherapeutic agent by fusing two TNFR family costimulatory agonists (anti-CD134/CD137) into a single biologic encompassing the therapeutic benefits of both agents while avoiding many of the drawbacks associated with traditional combination therapies. Our preliminary data show that this dual-specific, tetravalent antibody conjugate induces potent and differential in vitro potentiation of cytokine secretion from CD3-stimulated splenocytes, compared to that observed with single agonists alone or in combination. We hypothesize that this a consequence of unique signaling and hybrid costimulatory events. Secondly, this conjugate, referred to as OrthomAb, potently delayed tumor growth and reduced tumor burden in the aggressive B16 melanoma model. In vivo antitumor efficacy exceeded that of either individual unconjugated agonist. Importantly, this work establishes a framework for fusion of immunomodulatory biologics into a single drug with the benefit of combination immunotherapy to treat cancer.
CC-122 is more potent but transcriptionally mirrors Lenalidomide in Chronic Lymphocytic Leukemia
Authors: Do Priscilla , Cheney Carolyn , Yu Lianbo , Mo Xiaokui , Awan Farrukh , Muthusamy Natarajan , Byrd John C , Blachly James S
Published: January 2023

Lenalidomide (Len) is an immune modulatory drug for the treatment of hematological malignancies which contrasts with a large repertoire of immunosuppressive treatments. Len is FDA approved for Multiple Myeloma (MM), yet its development in Chronic Lymphocytic Leukemia (CLL) has been hampered by a potentially fatal, dose-limiting toxicity known as tumor flare. Tumor flare consists of enlarged, painful lymph nodes (LN) and cytokine release and is managed by lower dosage/prophylactic measures. Len and next-generation reagent CC-122, target cereblon, leading to degradation of transcription factors IKZF1/IKZF3. We compared CC-122 to Len using primary CLL B and T cells and CLL-derived cell line, OSU-CLL. We present a key difference between CC-122 and Len treated activated CLL T cells.

CC-122 0.1–10uM or 1uM Len are not directly cytotoxic to primary CLL B cells (avg. fold change 0.94–1.49 normalized to vehicle, N=8). CC-122 was more potent than Len by degradation of IKZF1/IKZF3, and immune activation measured by CD86 induction (7.2% more CD86+ induction, p=0.0026, N=12). RNA-seq analysis of the OSU-CLL cell line showed that at physiologically relevant doses, CC-122 (0.1uM) and Len (1uM) have no significant differences. Microarray analysis of 0.1uM CC-122 v. 1uM Len on αCD3 activated CLL T cells showed only two significantly different genes: CXCL13 and HLADQ-A1, p<0.0001. CXCL13, a B cell chemoattractant in CLL, was ≥ 3 fold increased with 1uM Len v. 0.1–1uM CC-122 (N=3). Overexpression of the CXCL13 receptor, CXCR5, in CLL but not MM may contribute to migration of CLL cells to the LN and potentially tumor flare. Retaining the anti-tumor activity of Len while mitigating tumor flare is of clinical interest and critical to the development of CC-122.
CD19/CD3 bispecific antibodies induce potent response against chronic lymphocytic leukemia cells ex vivo
Authors: Robinson Hannah Ruth , Qi Junpeng , Baskar Sivasubramanian , Rader Christoph , Wiestner Adrian
Published: January 2023

While treatment of chronic lymphocytic leukemia (CLL) has been advanced by introduction of targeted therapies such as tyrosine kinase inhibitors, there remains a need for adjunct therapies capable of inducing response in the setting of relapse on or resistance to first-line agents. Blinatumomab is a bispecific antibody (bsAb) against CD19/CD3, designed to target endogenous T cells against B cells via the formation of cytolytic synapses. It is approved for treatment of relapsed/refractory B-cell acute lymphoblastic leukemia, and has shown promise for use in non-Hodgkin’s lymphoma. Designed in the relatively small 54.1 kDa BiTE format, blinatumomab has a half-life of 2.1 hours and requires continuous intravenous infusion for clinical efficacy. In contrast, our novel anti-CD19/CD3 bsAb, designed in the 100 kDa single chain Fv-Fc format (CD19/CD3-scFv-Fc), has an approximately 100-fold longer half-life and could therefore be suitable for weekly dosing. We found that both blinatumomab and CD19/CD3-scFv-Fc induce potent killing of CLL cells ex vivo compared to cells treated with control HER2/CD3-scFv-Fc bsAb or medium alone. This response was associated with robust expansion of autologous CD4+ and CD8+ T cells, as shown by absolute cell counts and CFSE-detected population doubling. Notably, while HER2/CD3-scFv-Fc induced T cell expansion, no CLL directed cytotoxicity was observed. Our results identify CD19/CD3-scFv-Fc as a possible novel immune-based treatment for CLL.
Systemic Stimulatory Cancer Immunotherapy Induces Corticosterone-mediated Thymic Involution
Authors: Minnar Christine Mall , Sckisel Gail D. , Beerthuijzen Johanna M.T. , Aguilar Ethan G. , Mirsoian Annie , Crittenden Marka , Blazar Bruce R. , Monjazeb Arta M. , Murphy William J.
Published: January 2023

Cancer immunotherapies (IT) are quickly becoming frontline therapies for a variety of cancers and are often immunostimulatory in nature. We have previously demonstrated that systemic administration of strong immunostimulatory regimens in mice resulted in memory T cell activation but also concurrent immune paralysis of naïve T cell responses. The thymus is critical for generation of naïve T cells. While it is known that thymic involution occurs with age along with a decrease in the naïve T cell pool, there have been few studies investigating the effects of IT on the thymus. Here we demonstrate that immunostimulatory regimens (αCD40/IL-2 or systemic LPS) lead to marked, but transient, thymic involution in young mice. As soon as 48 hours after therapy initiation, the thymic cellularity decreased by 60%. Importantly, we show that this thymic hypocellularity correlated with significant increases in systemic corticosterone immediately following the initiation of therapy. Prior adrenalectomy prevented thymic involution emphasizing the role of glucocorticoids post IT. Interestingly, stress induced thymic involution was followed by a ‘rebound effect’ with significant increase in thymic cellularity compared to control mice occurring two weeks after IT cessation. Importantly, similar effects on corticosteroid levels in cancer patients receiving high dose IL2 therapy was observed and this was correlated with later decreases in T cell receptor excision circles (TRECs) as indicators of recent thymic emigrants. These results suggest that thymic involution following strong immunostimulatory regimens acts as a negative feedback loop for developing T cells which may be more pronounced in aged recipients where such a pool is already limited.
Synergistic Anti-tumor Activity of Local SS1P with Systemic CTLA-4 Blockade Causes Complete Regression of Both Injected and Un-injected Tumors in Mice
Authors: Leshem Jasmin , O’Brien James , Reiter Yoram , Pastan Ira
Published: January 2023

SS1P consists of an anti-mesothelin Fv attached to Pseudomonas exotoxin A and kills tumor cells by inhibiting protein synthesis. Because of its unique mechanism of cell killing, SS1P does not inhibit the immune system like other anti-cancer drugs. Here we determined if SS1P can cooperate with anti-CTLA-4 to induce anti-tumor immunity and cause complete tumor regressions.

A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and implanted in two different locations. SS1P was injected directly into one tumor and anti-CTLA-4 given IP.

In mice treated with anti-CTLA-4 and SS1P, tumor regressions occurred in 11 out of 12 SS1P injected tumors (91%) and in 7 out of 12 un-injected tumors (53%). No tumor regressions occured with drugs given separately. Pathological evaluation showed a large sterile central abscess in injected tumors surrounded by a collar of inflammation containing a mixture of lymphocytes and other mononuclear cells. Analysis of responding tumors showed a time dependent increase in CD8+ cells located mainly in the inflammation collar. RNA analysis of responding tumors showed that 385/768 immune related gene transcripts were elevated by twofold or more including CD8a, PDL2 and IL1 alpha (13, 21 and 27-fold).

Altogether, combining local SS1P with anti-CTLA-4 promotes massive inflammation that cures multi-centered cancer disease providing a strong rational to explore this combination therapy in patients.
TCR-like antibody duocarmycin conjugates promote cytotoxicity of tumor cells expressing low peptide/HLA targets
Authors: Herrera Christian E , Lowe Devin B , Bivens Camille K , Mobley Alexis S , McCormick Amanda , Wichner Timea , Sabnani Manoj K , Wood Laurence M , Weidanz Jon A
Published: January 2023

Immunotherapeutic strategies such as unmodified antibodies have significantly advanced treatment options for individuals with cancer. Yet, to further improve the efficacy of this approach, engineered platforms such as antibody drug conjugates (ADCs) are under development. Current FDA-approved ADC designs incorporate tubulin inhibitors that abate cancer growth and mediate clinical protection for patients. However, successful ADC treatment typically requires high tumor antigen (TA) surface expression to bind/internalize ADC material, destabilize cytoskeletal networks, and suppress tumorigenesis. Unfortunately, few known TAs are localized on cancer cells at elevated densities, potentially minimizing the therapeutic index and widespread application of current ADC regimens. Our group sought to overcome this ADC limitation of TA expression by crosslinking the DNA alkylating compound duocarmycin with T cell receptor (TCR)-like antibodies, which specifically bind TA peptide/human leukocyte antigen (HLA) complexes analogous to a TCR. In select assays, a panel of TCR-like ADCs were capable of distinguishing human breast and colon cancer lines and promoting potent cell cytotoxicity even under common scenarios of low TA peptide/HLA densities. Additionally, TCR-like antibody TA peptide/HLA binding appeared to play an important role in ADC-mediated cytotoxicity, supporting the notion that spatial TA ligand interactions influence ADC function. Overall, our data highlight the proof-of-principle that TCR-like ADCs induce cancer cell death under physiologically-relevant TA expression profiles and support their translational development, particularly alongside agents that disrupt DNA replication.
Engineering of Antibody Conjugated Nanogel Platform for Targeted Drug Delivery to CD4+ T lymphocytes
Authors: Canakci Mine O , Thayumanavan Sankaran , Osborne Barbara A
Published: January 2023

In an ideal systemic therapy, therapeutic agents travel through the bloodstream, reaching cells all over the body and preferentially killing abnormal cells. However, hydrophilic or lipophilic properties of a potential drug affects its ability to reach cells from the bloodstream. Thus, only a small amount of the drug reaches the diseased tissue. Moreover, selective killing approaches to minimize side effects usually fail and many normal cells and tissues are exposed to the drug upon treatment. Development of antibody drug conjugates (ADC) revolutionized the targeted approach for drug delivery. ADCs are composed of cytotoxic drugs covalently linked to monoclonal antibodies which selectively bind to tumor-markers and, by doing so, decrease the side effect of the drug on healthy tissue. Currently there are two FDA approved ADCs in the market. The number of ADCs in clinical trials continuously increases however some fail in efficacy studies. Our approach is to use antibody-nanogel conjugates in order to increase the drug load by avoiding modifications on the therapeutic agent. Nanogels are chemically cross-linked, water-soluble polymer nanoparticles and can encapsulate or trap the lipophilic drug molecules. Selective uptake of these nanogels can be achieved by functionalization of the surface of the nanogel with monoclonal antibodies that can recognize the surface markers of the abnormal cells in which the drug needs to be delivered. Overall aim of this project it to engineer anti-CD4 conjugated nanogels to enhance the antibody mediated targeted drug delivery approach by combining our knowledge in both immunology and polymer chemistry for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) in mouse models.
Understanding how immune checkpoint modulators alter pre-existing rheumatologic disorders in cancer patients
Authors: Nelson Michelle , Shirai Keisuke , Gupta Ridhi , Gilkeson Gary , Paulos Chrystal M
Published: January 2023

The aim of cancer immunotherapy is to reinvigorate an exhausted immune system. Checkpoint modulators PD1/PDL1 and CTLA4 antagonists “release the breaks” on ineffective T cells and subsequently bolster tumor-targeted T cells. These checkpoint modulators are increasingly available for numerous cancers and therefore the number of patients on immunotherapy are ever increasing. In contrast, patients with autoimmune diseases are prescribed immune therapies that dampen the immune system. Of note, immune suppressive therapies increase the incidence of various cancers. The interesting question that has not been fully addressed- what is happening immunologically in patients who have pre-existing autoimmune diseases and are treated with checkpoint modulators for cancer? We have been evaluating the immune correlates in patients with pre-existing rheumatoid arthritis (RA; often being treated with TNF-α blocker) who later receive checkpoint therapy when diagnosed with cancer. Following checkpoint therapy, we saw an increase in central memory T cells, a decrease in PD1 and CTLA4 expression, and a decrease in Th1 and Th17 cells in the peripheral blood. Additionally, we have been working on a dual disease animal model- collagen-induced arthritis (CIA) and lung cancer models. This will has allowed us to study the direct immunological consequences of giving treatments that either dampen or boost the immune system on both cancer and CIA disease states. Outcomes from this work will continue to expand our understanding of the immune system in patients with dual diseases and thus be better equipped to evaluate immunological correlates for exacerbated autoimmune disease and to productively treat patients with both autoimmune and cancer diagnoses.
Autocrine IL-2 programs survival of exhausted CD8 T cells during chronic infection
Authors: Yuzefpolskiy Yevgeniy , Barman Soumik , Smith Kendall A , Sarkar Surojit , Kalia Vandana
Published: January 2023

Loss of T cell function is a hallmark of chronic infections (such as HIV, HCV) and cancers. Chronic antigenic stimulation leads to a progressive, hierarchical loss of function in T cells – properties such as autocrine IL-2 production and ability to lyse infected target cells are lost first, followed by the loss of TNF-α and then IFN-γ production. The sum total of these events, known as exhaustion, is followed by the eventual physical deletion of antigen-specific CTLs. Our current clinical checkpoint blockade therapies are heavily hinged upon the very presence of the exhausted T cells that can be re-invigorated to regain function. Hence, it is critical to define the survival signals that prevent the deletion of exhausted antigen-specific T cells.

While IL-2 is historically known to be a key survival cytokine for T cells, autocrine expression of IL-2 is extinguished early during T cell exhaustion. Thus, it has been long believed that IL-2 is dispensable for the maintenance of exhausted CTLs during chronic infections. Surprisingly, deletion of il2 specifically in antigen-specific CD8 T cells (CD8IL2−/−) led to a precipitous decline in the numbers of these exhausted CD8 T cells compared to their wild-type counterparts (CD8IL2+/+) within the same environment. This paradoxical need for autocrine IL-2 in the long-term sustenance of an exhausted CD8 T cell pool is ascribed to its instructional role in programming key aspects of CD8 T cell survival and metabolism early during priming. These findings are of high consequence to checkpoint blockade immunotherapeutic strategies, where success is heavily dependent on the presence of an exhausted T cell population that can be re-functionalized to fight off the chronic infection or tumor.
A spontaneous mutation in NCR1 locus reveals an inhibitory role for IFNγ response by NCR1 via inhibiting dok-1 phosphorylation
Authors: Jang Youngsoon , Gerbec Zachary , Choi Bongkum , Subramaniam Malarkannan , Moore Bethany , Laouar Yasmina
Published: January 2023

Immunosurveillance by Natural killer (NK) cells provides an efficient effector mechanism to eliminate tumor and virally infected cells. A well-defined characteristic of NK cells is their ability to lyse target cells deficient in MHC class I expression or target cells that express non-MHC ligands as a result of infection or transformation. This observation has been the basis for identification of a number of inhibitory and activatory NK cell receptors. Although the collective effort during the last decade shed light on the function of many NK cell receptors, the role of NCR1 in NK cell biology remains highly controversial. We have recently identified a spontaneous mutation of NCR1 in our C57BL/6 mouse colony. In this mutant, NCR1 expression was absent on conventional NK cells of all lymphoid organs as well as on innate lymphocytes (ILC3) of the gut lamina propria. Of note, NCR1 expression was absent on the surface as well as in the intracellular compartment of NK cells. Likewise, lack of NCR1 expression was confirmed in mixed BM chimera indicating a cell intrinsic phenotype. Only genetic backcrossing could correct the lack of NCR1 in these mice. ORF sequencing of NCR1 gene in mutant mice revealed a point mutation (C14R) in the region of signal peptide of NCR1 protein. However, while development and homeostasis of NK cells was not affected by the lack of NCR1, infection of NCR1C14R mutant mice with murine cytomegalovirus resulted in increased resistance, an advantage that correlated with NK cell hyperresponsive production of IFN-γ, but not cytotoxic activity. Moreover, NCR1 limits NK cell IFNγ response via regulation of Dok-1 phosphorylation and p38 activation. Collectively, our data reveal the role for NCR1 in IFNγ production of NK cell.
ADAMTS5 and its substrate versican play a critical role in influenza virus immunity
Authors: Stambas John , Ye Siying , Izzard Leonard , Dlugolenski Daniel Aaron , Tripp Ralph A , McCulloch Daniel , McMahon Meagan
Published: January 2023

Members of the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) metalloproteinase family remodel the extracellular matrix (ECM). In this current study we demonstrate that Adamts5−/− mice infected with X31 influenza virus (104pfu/mouse) show enhanced weight loss, have higher virus titres and perturbed T cell immunity when compared to WT counterparts. These responses correlate with the accumulation of a key ADAMTS5 substrate, versican, in the MLN of Adamts5−/− mice and underpin disruption of T cell migration as evidenced through the use of both in vitro and in vivo models. Our research emphasizes the importance of ADAMTS5 activity in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce overall morbidity and mortality.
Performance of the HIV diagnosis screening algorithm using two immunoassays and a molecular test
Authors: Balada-Llasat Joan-Miquel , Dougherty Jenna , Goldenbaum Brian , Wang Shu-Hua
Published: January 2023

Advances in HIV diagnostic testing have improved detection of HIV infections. HIV can be detected earlier so patients benefit from treatment and negative results reassure uninfected patients. Because first line immunoassays lack specificity, positive results need to be confirmed. The aim of this study was to evaluate the algorithm used at The Ohio State Wexner Medical Center for the diagnosis of HIV infection. The algorithm begins with a combination immunoassay that detects HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen [ADVIA Centaur® HIV Ag/Ab Combo (CHIV), Siemens]. All positive specimens are confirmed with an immunoassay that differentiates HIV-1 from HIV-2 antibodies [Geenius HIV-1/HIV-2 Confirmatory assay (GHIV), Bio-Rad]. Specimens that are positive by CHIV and negative or indeterminate by GHIV proceed to HIV-1 nucleic acid testing for resolution. A total of 14,994 patients were evaluated using CHIV assay, of these 99.7% were negative (n= 14,950) and 0.3% were positive (n=44). The 44 positive specimens were tested by GHIV, only 4 confirmed positive and 40 were negative. Of the 40 that tested negative, only 32 had enough serum left to be tested by RNA HIV molecular test and all tested negative. Of the 8 that there was not enough serum left, 6 patients had a follow up viral load and they were all negative. In summary, of the 44 CHIV positive, only 4 were confirmed by GHIV, and 38 confirmed negative by the molecular tests, questioning the utility of the additional molecular step.
T follicular helper cell-derived IL-10 sustains humoral immunity during chronic viral infection
Authors: Zander Ryan A , Xin Gang , Schauder David , Cui Weiguo
Published: January 2023

Although it is well-appreciated that Tfh cells play a critical role in regulating antiviral humoral immunity, the detailed cellular and molecular pathways that govern their differentiation and function during chronic viral infection remains incompletely understood. In this study, we generated IL-10 and IL-21 double-reporter mice to study the dynamic expression of the immunoregulatory cytokines IL-10 and IL-21, which are known to differentially regulate T cell-mediated control over viral replication. Infection of these double-reporter mice with either an acute or chronic strain of LCMV, revealed a unique subset of IL-10+IL-21+co-producing Tfh cells that arise only during chronic viral infection. Notably, compared to IL-21-single producing Tfh cells, IL-10+IL-21+co-producing Tfh cells exhibited an enhanced germinal center (GC) Tfh-like profile, as exemplified by their increased per cell expression of PD-1, CXCR5, and Bcl-6. Additional analyses identified that IL-10-producing Tfh cells have an increased capacity to form stable Tfh-B cell conjugates compared to their IL-10− Tfh counterparts, suggesting that IL-10+ Tfh cells may specialize in providing help signals to B cells during chronic infection. Importantly, depletion of IL-10+IL-21+ co-producing CD4 T cells or deletion of Il10 specifically from Tfh cells resulted in impaired GC B cell responses, LCMV-specific antibody production and viral control. Mechanistically, we show that B cell intrinsic IL-10 signaling is indispensable for GC B cell differentiation and function. Collectively, our findings delineate a heterogeneous population of Tfh cells and elucidate a critical role for Tfh-derived IL-10 in promoting humoral immunity during persistent viral infection.
CD8 T cells initiate blood-brain barrier disruption during experimental cerebral malaria through non-cytotoxic alterations to endothelial cells
Authors: Huggins Matthew A , Jin Fang , Johnson Aaron J
Published: January 2023

Cerebral malaria is a serious health complication that can arise during Plasmodium infections. Symptoms of human cerebral malaria (HCM) include coma, permanent neurological deficits, and potentially death. While the mechanism is not well understood, brain swelling and edema have been shown to correlate with a poor disease outcome for afflicted individuals. This work attempts to identify the cellular changes occurring during breakdown of the blood-brain barrier (BBB) by utilizing the murine model of experimental cerebral malaria (ECM). Previous work determined that the tight junctions claudin-5 and occludin, proteins essential for regulating BBB integrity, were disorganized on cerebral endothelial cells during ECM. Here, we focus on the constituents of the BBB to characterize the changes occuring within endothelial cells during disease progression. Importantly, endothelial cell changes were not strictly induced by parasitic infection itself, but also required the presence of functional CD8 T cells. Using perforin deficient mice to impair CD8 T cell effector function, we demonstrate that brain endothelial cells retain the characteristics of uninfected animals in contrast to wild-type infected mice. Interestingly, the CD8 T cells appear to be acting independently of direct killing as there is not an increase in activation of the apoptotic pathway nor is there an increase in dead or dying endothelial cells. From these data we propose that non-cytotoxic CD8 T cell functions are required for ECM induction.
Activation of histone acetyltransferase p300 by MAPK pathway is required for optimal production of PGE2 in primary human monocytes activated with muramyl dipeptide adjuvant (MDP).
Authors: Liu Fengjie , Romantseva Tatiana , Golding Hana , Zaitseva Marina
Published: January 2023

Prostaglandin E2 (PGE2) is a proximal mediator of fever produced by human monocytes and macrophages activated by microbial pathogens and, in some cases, by adjuvants that contain analogues of microbial products. Here we studied the signaling pathway of PGE2 production in primary human monocytes activated with MDP, the minimal structure unit responsible for the adjuvant activity of peptidoglycans.

MDP alone did not induce COX-2 mRNA expression and PGE2 protein production in monocytes. However, COX-2 mRNA and PGE2 were dramatically up-regulated when MDP was combined with T cell conditioned medium (TCM) prepared from CD3 bead-purified human T cells. MDP-induced nuclear NF-kB was not further increased by TCM. However, TCM induced increase in cytosolic Ca2+ and in phosphorylated ERK1/2 in monocytes, suggesting a role for MAPK signaling. In agreement with this, inhibition of Ca2+, of Ca2+-dependent PKC, and of MAPK kinase enzymes MEK1/2 suppressed PGE2 production in MDP/TCM-treated monocytes. Chromatin immunoprecipitation analysis showed that TCM alone or combined with MDP facilitated association of histone acetyltransferase p300 to the promoter regions of COX-2, IL-1b, and of IL-6 genes. Inhibition of p300 by C646 suppressed the recruitment of p300 and the acetylation of H3 and H4 histones at the COX-2 promoter, prevented COX-2 gene transcription, and PGE2 production. Taken together these results demonstrate that MDP-induced COX-2 gene transcription and PGE2 protein production in primary monocytes requires a second signal delivered by a TCM through a Ca2+-dependent activation of PKC/MEK1/2 signaling pathway followed by phosphorylation of ERK1/2 and p300-facilitated acetylation of histones H3 and H4 at the COX-2 promoter.
Antiretroviral drugs and tobacco smoke dysregulate human platelets: A novel investigation into the etiology of HIV co-morbid cardiovascular disease.
Authors: Loelius Shannon G , Lannan Katie L , Casey Ann E , Spinelli Sherry L , Phipps Richard P
Published: January 2023

While antiretroviral drugs (ARVs) successfully suppress Human Immunodeficiency Virus (HIV), HIV-infected persons still have a shorter lifespan and are at higher risk of developing cardiovascular disease (CVD) compared to uninfected persons. CVD is the number one cause of death globally, and is now appreciated as an inflammatory disease. HIV-infected persons experience chronic inflammation, thought to be caused by low levels of HIV proteins, daily use of ARVs, and increased rates of smoking. The impact of ARVs on CVD risk remains unclear, with some studies linking ARVs to CVD. Additionally, when controlled for smoking status HIV-infected persons were still at greater risk for CVD. Platelets are important in the development of CVD, but the effects of ARVs on platelets are unstudied. Additionally, cigarette smoke is known to activate platelets, inducing inflammation. Our novel study investigates the effects of ARVs and cigarette smoke, alone and in combination, on human platelet function. Our striking in vitro findings demonstrate that the ARVs Ritonavir and Darunavir, as well as cigarette smoke, dysregulate platelet function. Treatment with the protease inhibitor Ritonavir resulted in a dose-dependent increase in platelet production of proinflammatory mediator prostaglandin E2 (PGE2) (p<0.001). Additionally, when combined with cigarette smoke, Darunavir induced production of PGE2 (p<0.01). Combined doses of Ritonavir and Darunavir at physiological concentrations inhibited platelet spreading, similar to the effect of cigarette smoke. We hypothesize that cigarette smoke and ARVs act in combination to activate platelets and thus contribute to the chronic inflammation which leads to CVD.
Inducing a human atypical memory B cell phenotype in vitro
Authors: Ambegaonkar Abhijit A , Pierce Susan K. , Sohn Haewon
Published: January 2023

Chronic human infectious diseases such as malaria are associated with expansion of an unusual population of memory B cells (MBCs) referred to as atypical MBCs. In malaria, atypical MBCs are CD21−, CD27−, highly express the transcription factor, T-bet, and also express a variety of inhibitory receptors, including FcRL5 and Siglec6. These atypical MBCs signal poorly through their B cell receptors (BCRs) and exhibit poor B cell function. We concluded from these studies that atypical MBCs contribute to the slow, inefficient acquisition of immunity to malaria in African children. However, the mechanisms by which atypical MBCs are induced during chronic infectious diseases are not known. Acute febrile malaria in African children is correlated with expansion of less-functional TH1-type T follicular helper T cells which secrete IFN-γ and are impaired in their helper function, suggesting that they may contribute to the generation of atypical MBCs. We investigated the conditions under which human naïve B cells were induced to express an atypical MBC phenotype in vitro. Studying human tonsillar B cells we showed that BCR cross linking by anti-Ig in the presence of IFN-γ and CpG, induced high levels of T-bet and FcRL5. Moreover, expression of these markers was further enhanced by the persistent engagement of B cells with membrane bound anti-Ig. We also found that atypical MBCs induced in vitro by persistent antigen, CpG and IFN-γ showed severely reduced early BCR signaling when re-challenged with soluble antigen, characteristic of atypical MBCs. Taken together these results suggest that atypical MBCs may be the product of persistent antigen engagement by B cells in the highly inflammatory environments that develop during chronic malaria exposure.
Defining T helper cell immunity in bronchopulmonary dysplasia
Authors: Lao Jason Chun , Pang Merrin A , Malhotra Atul , Tan Kenneth , Beker Friederike , Konig Kai , Collins Clare , Theda Christiane , Kamlin C. Omar , Yeomans Emma , Elsayed Kristy , Noble Elizabeth , Walsh Geraldine , Argus Brenda , Twitchell Emily , Berger Philip , Veldman Alex , Nold-Petry Claudia A , Nold Marcel Friedrich
Published: January 2023

Background

Bronchopulmonary dysplasia (BPD) is a common neonatal lung disease that has been linked to pulmonary inflammation. Previous studies have suggested involvement of various cell types and soluble factors but have thus far failed to reveal a dominant pathway that underpins disease progression. It remains unknown that which T helper (Th) cell polarization, if any, contributes to the pathogenesis of BPD.

Methods

Citrated whole cord and peripheral blood were collected from infants born between 24+0–28+6 gestational weeks at five different time points, namely at birth, on days 1, 7 and 14 and at 36 weeks corrected gestational age (CGA). Cells were stimulated with either PMA, ionomycin and brefeldin A or vehicle and brefeldin A overnight. Cells were then stained for multi-color flow cytometry to enumerate Th cell subsets including Th1, Th2 and Th17 and regulatory T cells. Results were analyzed against BPD disease status at 36 weeks CGA and corrected for confounders.

Results

50 infants were enrolled and 228 unique samples were collected. 36 enrolled infants (72%) had BPD (mild to severe) at 36 weeks CGA. When compared to infants that did not have the disease, the frequency of interleukin(IL)-4 in CD4+ Th cells in peripheral blood was increased in infants with BPD on days 1 (p=0.0449), 7 (p=0.0423), 14 (p=0.0489) and 36 weeks CGA but not within cord blood. There was no difference in the frequency of cells positive for interferon-gamma (IFNγ), IL-17A or FOXP3 among CD4+ cells in blood from BPD and non-BPD infants across all time points.

Conclusions

T helper cells are polarized towards a Th2 phenotype in infants with BPD based on elevated levels of IL-4 in CD4+ cells. We hypothesize that BPD infants may benefit from therapies that target the Th2 pathway.
Culturally appropriate advisory and governance oversight for the study of rheumatic fever and rheumatic heart disease in New Zealand
Authors: Sika-Paotonu Dianne , Azevedo Pedro , Faatoese Allamanda , Wihongi Helen , Nuku Hiueni , Hudson Maui , Havea Palatasa , Parks Tom , Merriman Tony , Lennon Diana
Published: January 2023

Acute rheumatic fever (ARF) is an autoimmune condition caused by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause cardiac damage, known as Rheumatic Heart Disease (RHD). RHD remains a significant cause of morbidity and mortality is rare in developed countries. In New Zealand, the disease burden of ARF and RHD amongst Indigenous Maori and Pasifika communities is one of the highest in the world, usually affecting children and young adults. Previous work carried out in New Zealand sought to explore the immunogenetics of ARF and RHD and involved candidate gene analysis techniques utilizing samples from individuals diagnosed with RHD where a functional variant in the gene encoding the interleukin IL-6 cytokine known as IL-6 rs1800797 was found to be significantly associated with RHD. Current work seeks to explore the genetic susceptibility to ARF/RHD in greater depth using more advanced genome-wide technology. An international genome wide association study (GWAS) is currently underway to further understand the genetics of ARF/RHD using data obtained from global sources. To undertake the proposed GWAS work and amend the original New Zealand study, an Advisory team comprised of Pasifika and Māori representatives comprised of community leaders, researchers, scientists, and academics is providing the appropriate governance, cultural support and guidance essential for the project work.
Transcriptomic profiles of NK cell function indicate impaired responsiveness in chronic HIV-1 infection and enhanced functional activity post-vaccination
Authors: Costanzo Margaret C , Kim Dohoon , Creegan Matthew , Lal Kerri G , Ake Julie , Currier Jeffrey R , Streeck Hendrik , Robb Merlin L , Michael Nelson L , Bolton Diane L , Steers Nicholas James Robert , Eller Michael A.
Published: January 2023

Natural Killer (NK) cells limit viral replication by direct recognition and killing of infected cells, performing Fc mediated ADCC and releasing cytokines to modulate other effector activity. Despite growing evidence that supports the importance of NK cells in HIV-1 disease progression, understanding how these cells engage in vivo remains unclear. Here, we combined flow cytometry and gene expression analysis of NK cells responding to three in vitro models of NK stimulation to reveal distinct transcriptional profiles. While 76 gene transcripts were commonly upregulated following stimulation by direct cellular recognition, ADCC, and cytokine activation, 276, 36, and 293 genes were uniquely upregulated, respectively. Subsequently, a targeted gene expression panel was used to assess NK cell transcriptional content, directly ex vivo, in HIV-1 chronic infection. Transcriptional profiles in HIV-1 chronically infected individuals showed higher expression of genes associated with global activation and lower expression of genes associated with direct cell recognition and cytokine activation, compared to healthy individuals. We next looked at the transcriptional content of NK cells in response to MVA-CMDR vaccination, a live-attenuated viral vectored HIV-1 vaccine. NK cell transcriptional profiles of vaccinated individuals revealed upregulation of genes associated with direct cell recognition. These findings demonstrate that targeted transcriptional profiling provides a sensitive assessment of NK cell activity directly ex vivo that will improve our understanding of how NK cells respond to viral infections and after vaccination.