bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular selection and initial characterization of a new inducer and a new destabilizer of non-bilayer phospholipid arrangements
Authors: Barbosa Sandra Sánchez , Landa-Saldívar Carla , Nevarez-Lechuga Irene , Wong Carlos , Wong-Baeza Carlos , Baeza Isabel
Published: January 2023

The mouse model of a lupus-like disease developed by our research group is unique, because it’s obtained by the stabilization of lipidic structures called non-bilayer phospholipid arrangements (NPAs), which induce the production of anti-NPA antibodies that trigger the disease in mice, which are in turn detected in patients with systemic lupus erythematosus. NPAs are produced when a lipid bilayer composed of conic and cylindric phospholipids, is modified by certain drugs (procainamide, chlorpromazine) or the manganese cation. Usually, drugs/substances are chosen to be tested as NPA inducers when they are known to trigger a lupus-like disease in humans. However, here we selected a group of molecules only by their structural similarity with the known inducers and the known destabilizer (chloroquine) and experimentally tested their effect on the formation or destabilization of NPAs in liposomes by flow cytometry. Firstly, we identified promethazine and procaine, similar to chlorpromazine and chloroquine, respectively. Promethazine acted as an inducer, with a broader range of concentrations of action in comparison with chlorpromazine, but it was more sensitive to the destabilizer. Procaine was found to be a weaker destabilizer than chloroquine, as it has an effect only at higher concentrations. This strategy showed that molecular characteristics could also be good predictors of their action on lipid bilayers and NPAs formation.
Soluble interleukin-27 receptor alpha is a valuable predictive biomarker for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation
Authors: Ma Shoubao , Liu Shuangzhu , Gong Huanle , Wu Depei
Published: January 2023

Interleukin-27 receptor alpha (IL-27Rα) is a co-receptor of IL-27, which is an inflammatory cytokine and displays extensive immunological functions. It has been reported that IL-27Rα can exist in its soluble form (sIL-27Rα) in human serum and function as a natural IL-27 antagonist. In this study, we examined the serum sIL-27Rα levels and evaluated its predictive and prognosis value in aGVHD. Serum sIL-27Rα levels at the time of neutrophil engraftment were significantly decreased in grade II–IV aGVHD patients (P<0.001) as well as in patients with skin (P<0.01) or liver aGVHD (P<0.05). sIL-27Rα at neutrophil engraftment could be used to predict grade II–IV aGVHD (AUC=0.735, 95% CI 0.618–0.853, P=0.001). Cumulative incidences of grade II–IV aGVHD (P=0.004), recurrence rate (P=0.008), and non-recurrent mortality (P=0.008) in patients with high serum sIL-27Rα (≥59.40 ng/ml) were significantly lower than those in patients with low serum sIL-27Rα (<59.40 ng/ml). Multivariate analysis confirmed that low sIL-27Rα level at neutrophil engraftment (HR=2.83 95%CI 1.29–6.19, P<0.01) was an independent risk factor for predicting grade II–IV aGVHD. In addition, serum sIL-27Rα were positively associated with IL-27 (R=0.27, P=0.029), IL-10 (R=0.37, P=0.0015) and HGF (R=0.27, P=0.0208), whereas negatively associated with TNFR1 (R=−0.365, P=0.0022), Elafin (R=−0.29, P=0.0117), and REG3α (R=−0.417, P=0.0003). Taken together, our findings demonstrated that serum sIL-27Rα was upregulated after allo-HSCT, their levels at neutrophil engraftment maybe a valuable predictive biomarker in predicting the incidence of moderate-to-severe aGVHD after allo-HSCT.
BMT procedure alters the functions of dendritic cells and subsequently causes pathogenic Th responses to gammaherpesvirus infection
Authors: Zhou Xiaofeng , Chadwick Matthew M. , CHan Paul R. , Wilke Carol A. , Podsiad Amy B. , Yanik Gregory A. , Moore Bethany B.
Published: January 2023

Hematopoietic stem cell transplantation is a standard of care for many patients with genetic disorders, autoimmune diseases and malignancies. However, stem cell recipients are often more susceptible to microbial infections even after full hematopoietic reconstitution. We have previously demonstrated that syngeneic bone marrow transplantation (syn-BMT) in mice causes increased Th17 response and decreased Th1 response to murine gamma herpesvirus MHV-68 infection leading to pulmonary fibrosis. In this study, we found that Notch2-dependent conventional dendritic cells (DCs), i.e., a set of CD11b+ DCs, are required for the Th17 response to MHV-68 infection in BMT mice, while Batf3-dependent CD103+ DCs suppress the Th17 response. Furthermore, CD11b+ DCs in BMT lungs are hyper-activated with high expression of CD80 and CD86. When co-cultured with CD4+ T cells, DCs from BMT lungs promote higher productions of both IL-17A and IFN-γ than DCs from non-BMT mice, and addition of antibodies against CD80 and CD86 abolish the productions of both IL-17 and IFN-γ. Although the DCs in the BMT lungs are potent in inducing both Th1 and Th17 responses ex vivo, they are impaired in migrating into the lung draining lymph nodes (dLNs). Surprisingly, migration of DCs into the dLNs is not required for Th17 priming, since transplanting CCR7−/− bone marrow, which gives rise to non-migrating DCs in the recipient mice, does not prevent the Th17 response, but does abolish the Th1 response, suggesting a role for peripheral priming of the Th17 response in the lung post-BMT. In summary, syn-BMT causes a hyper-activated phenotype of CD11b+ DCs that promotes a pathogenic Th17 response and also causes dysfunction in DC trafficking that is required for a maximum Th1 response.
The enigmatic impact of donor T cell subsets on the therapeutic efficacy of tolerogenic protocols for transplant rejection
Authors: Lozano Marcos Iglesias , Chicco Maria , Lee W.P. Andrew , Brandacher Gerald , Raimondi Giorgio
Published: January 2023

Costimulation blockade (CoB) regimens are a promising immunomodulatory strategy to prevent transplant rejection. However, we are learning that their efficacy is affected by multiple factors. Recent reports have started highlighting the unexpected capacity of passenger donor lymphocytes to enhance the recipient’s anti-graft response. New studies are necessary to understand this novel phenomenon and how it can affect the induction of transplant tolerance.

In this study we assessed if T lymphocytes contained in the donor specific transfusion (DST) inoculum, used in combination with anti-CD154 (MR1) as CoB regimen, impact the regulation of mouse skin transplant rejection. When T cells were depleted from the DST, transplant survival was almost doubled. T cell subset-depletion studies indicated, unexpectedly, that donor CD8 T cells were responsible for limiting the efficacy of the tolerogenic regimen. Even more surprisingly, the presence of CD4 T cells in DST induced a remarkable improvement in transplant survival, beyond that observed with full T cell depletion. Ongoing experiments are determining the correlation between donor T lymphocytes and variations in humoral and cellular anti-donor responses.

Overall, these data reveal the existence of a novel and unexpected opposing role of donor passenger lymphocytes that significantly modifies the therapeutic efficacy of DST+MR1 based regimens: a deleterious role for donor CD8, and a beneficial one for CD4 T cells. Identification of the specific mechanisms through which these divergent modulations of the anti-donor alloresponse are exerted could suggest significant changes in how we should approach immunoregulation, both experimentally as well as clinically.
Recipient conditioning contributes to IL-33-driven Th1 alloimmune responses following rapid ST2 upregulation on donor CD4+ T cells during lymphopenia-induced proliferation
Authors: Dwyer Gaelen K. , Mathews Lisa R. , Lucas Anna , Blazar Bruce R. , Turnquist Heth R.
Published: January 2023

IL-33 is augmented in recipient tissues by conditioning before allogeneic stem cell transplantation (alloSCT) and is a required signal to donor T cell for GVHD. Targetable mechanisms by which IL-33 supports GVHD are yet undefined. Conditioning causes lymphopenia-induced proliferation (LIP) and releases microbial products. These products promote myeloid cell secretion of IL-12, which induces the IL-33 receptor, ST2, on T cells in vitro. We hypothesized that IL-12 induces ST2 on donor T cells during LIP promoting IL-33 augmentation of the Th1 responses leading to GVHD. To establish the role of IL-12 in T cell ST2 expression and IL-33-mediated GVHD, irradiated BALB/c mice were given B6 alloSCT and T cells. Some mice received IL-12p40 neutralizing Ab or control Ab alone, with or without concurrent IL-33 administration. To test the impact of lymphopenia on T cells, ST2−Foxp3−CD4+ T cells alone or with ST2+ Treg were transferred into B6 Rag2−/−γc−/− mice. Mice received PBS or IL-33 on days 1–8. Neutralizing IL-12 did not reduce ST2 on T cells after alloSCT nor rescue mice from IL-33-mediated acceleration of GVHD. Post-alloSCT, IL-33 worked with IL-12 to expand ST2+Tbet+ Th1 cells. Neutralizing IL-12, but not delivery of IL-33, increased Gata3+ Th2 cells after alloSCT. During LIP, CD4+Foxp3− T cells rapidly upregulated ST2 independent of IL-33, but IL-33 then favored the expansion of Th1 cells, even in the presence of ST2+ Treg. These data suggest that CD4+ T cells rapidly upregulate ST2 during alloSCT conditioning induced lymphopenia. After alloSCT, IL-33 does not support Treg or Th2 cells, but works with IL-12 to augment Th1 responses and GVHD. Blocking stimuli that induces ST2 on proliferating donor CD4+ T cells may be effective for limiting GVHD.
Graft dendritic cell p40 homodimers activate donor-reactive endogenous memory CD8 T cells within higher risk allografts
Authors: Fairchild Robert L. , Tsuda Hidetoshi , Valujskikh Anna
Published: January 2023

Memory T cells with donor-reactivity pose a major barrier to successful transplantation and tolerance induction. Longer cold ischemic storage (CIS) prior to transplantation promotes endogenous donor-reactive memory CD8 T cell infiltration and activation within cardiac allografts to directly mediate CTLA-4Ig-resistant rejection between days 15–22 post-transplant vs. > 60 days for allografts subjected to minimal CIS. Using BrdU labeling, graftinfiltrating memory CD4 and CD8 T cells proliferated much more strongly within allografts subjected to 8 vs. 0.5 hr CIS and proliferation of endogenous memory CD8 T cells within the higher risk allografts required recipient CD4 T cells, graft dendritic cells (DC) and graft expression of class II MHC, CD40 and IL-12p40, but not p35. IL-12p40 but not IL-12 p35 or IL-23p19 mRNA was elevated at 48 hr post-transplant in allografts subjected to 8 vs. 0.5 hr CIS and the increase in p40 mRNA was reduced to the levels seen in 0.5 CIS allografts by recipient CD4 T cell depletion, but was restored by treating with agonist anti-CD40 mAb. p40 homodimers, but not p70 heterodimers, were increased in allografts subjected to 8 hr CIS and were dependent on recipient CD4 T cells. Peri-transplant anti-p40 mAb reversed CTLA-4Ig-resistant rejection of higher risk allografts and peri-transplant p40 homodimers induced endogenous memory CD8 T cell proliferation within allografts subjected to 0.5 hr CIS to the levels observed in allografts subjected to 8 hr CIS. These data indicate that endogenous memory CD8 T cell activation within higher risk cardiac allografts requires CD4 T cell help via CD40-CD154 interactions with graft DC to induce their production of p40 homodimers.
The impact of androgens on neutrophils and melanoma tumor burden.
Authors: Markman Janet , Wakita Daiko , Crother Timothy R. , Arditi Moshe
Published: January 2023

Background

Aged men have increasingly higher incidence of melanoma compared with women and continuously decreasing testosterone (T). Investigation into the mechanism underlying the sex disparity is warranted as there is a low survival rate for metastatic melanoma patients, especially in men.

Methods

We used B16 and YUMM melanoma lung metastatic models. Neutrophils or NK depletion and ovariectomy or castrations were performed. Neutrophils from prostate cancer patients were analyzed.

Data

Compared with males, female mice with melanoma have higher lung tumor burden, reduced neutrophil infiltration, and decreased NK cell activation. While ovariectomy did not affect tumor burden, castrated males had increased lung metastasis and reduced survival compared with sham, suggesting a protective role for T. Further, T replacement in castrated mice reduced metastasis to sham levels. Depletion of NK cells increased tumor burden in male and female mice in a sex independent manner. In contrast, neutrophil depletion increased tumor burden only in males, suggesting a sex difference in neutrophils. More immature neutrophils with impaired function were observed in both castrated mice and prostate cancer patients on androgen ablation therapy.

Conclusion

Our data indicate that innate immunity is important for the response against melanoma colonization of the lung. Neutrophil depletion and castration data suggest a sex specific response which may contribute to the sex disparity observed in human melanoma incidence and survival. Finally, prostate cancer patients on androgen ablation therapy show a similar decrease in mature neutrophils and impaired neutrophil function, which may put these patients at a higher risk for infection and melanoma.
CD103 expression by DCs: a new player in lung cancer immunity.
Authors: Brassard Julyanne , Bernatchez Emilie , Langlois Anick , Blanchet Marie-Renée
Published: January 2023

Despite the use of various therapies, lung cancer remains one of the world’s leading causes of death. Cancer immunotherapy is a promising therapeutic avenue, however, molecular and cellular mechanisms involved in cancer immunity remain elusive. Dendritic cells (DCs), which play an important role in CD8+ T cell activation in cancer immunity, can be divided into two major populations, namely DC1 and DC2, and respectively identified by XCR1/CD103 and Sirpα/CD11b expression. Importantly, studies have demonstrated that DC1s promote cancer immune responses. Furthermore, CD103+ DC1s specialize in the transport of tumor antigens to draining lymph nodes and cross-presentation. However, the influence of the cancer environment, where inflammatory factors (such as TNF) and various inflammatory cells are found, on CD103 expression by DC has never been studied. To verify whether lung cancer-induced inflammation modulates CD103 DC population, fresh lung and splenic DCs were isolated in wild type (WT) mice and exposed to GM-CSF (a known inducer of CD103 expression in the lung) with or without TNF. Furthermore, to directly assess the impact of lung cancer on CD103 DC expression, WT mice were euthanized 2 and 3 weeks following intravenous injection of Lewis lung carcinoma (LLC) cells and the percentage of CD103+DC1 was analyzed in lungs. Exposure to TNF blocked the GM-CSF-induced CD103 expression by DCs, independently of the timing at which TNF is added to cultures (before or after GM-CSF). Also, the % of CD103+ DCs gradually decreased in time following LLC injection. This suggests a new mechanism by which lung cancer negatively impacts the function of immune cells to promote cancer development, and could lead to new therapeutic avenues in cancer immunity.
Inhaled IL-10 Suppresses Lung Tumorigenesis via Abrogation of the Inflammatory Macrophage - T17-cell Axis
Authors: Li Qingsheng , Egilmez Nejat K.
Published: January 2023

Lung tumorigenesis has been linked to chronic type 17 inflammation in both murine models and patients. Interleukin-10 is a pleiotropic regulatory cytokine with profound inhibitory effects on type 17 responses. Intratracheal administration of a sustained-release IL-10 formulation resulted in suppression of tumor development in the LSL-K-ras spontaneous lung cancer model. Analysis of lung lymphocyte populations demonstrated that the antitumor activity of IL-10 was associated with a rapid 3 to 5-fold decline in IL-17-producing T-cell populations. Type 17 T-cells did not express detectable levels of IL-10RA suggesting that IL-10 was not directly acting on T-cells. In contrast, select myeloid cell populations including macrophages and a small subset of dendritic cells were found to express IL-10RA in the lung. In tumor-bearing control mice macrophages displayed a pro-inflammatory phenotype that reverted to a naive-like state following IL-10 treatment. Importantly, depletion of phagocytes resulted in a 3 to 5-fold decline in type 17 T-cell numbers with a concurrent 7-fold reduction in tumor burden. These findings demonstrate that in the LSL-K-ras model lung tumorigenesis is driven by the inflammatory macrophage - type 17 T-cell axis and that IL-10 abrogates this process via its direct activity on macrophages. Inhaled IL-10 formulations may be of utility in prophylaxis of lung cancer in high-risk patients.
Population dynamics of tumor-specific CD8 T cell differentiation from plastic to fixed dysfunctional states
Authors: Philip Mary , Pilkinton Mark , Ramesh Ram , McDonnell Wyatt J. , Gangula Rama D. , Camara Steven , Chopra Abha , Schietinger Andrea , Mallal Simon Alexander
Published: January 2023

Tumor-specific CD8 T cells (TST) encountering cognate antigen in tumors differentiate to a dysfunctional state characterized by expression of multiple inhibitory receptors and failure to make effector cytokines. We demonstrated that TST differentiate through two discrete chromatin states; TST were initially in a plastic chromatin state and could be functionally rescued but then transitioned to a fixed chromatin state resistant to therapeutic reprogramming. T cells harboring these discrete chromatin states could be identified with distinct, novel membrane protein expression profiles, permitting prospective identification of reprogrammable tumor-specific T cells from bulk tumor-infiltrating T cell populations. Importantly, human tumor-infiltrating PD1-high CD8 T cells were in a similar chromatin accessibility state as murine fixed dysfunctional T cells. Based on our studies, two models could describe TST differentiation: (i) TST progress en-masse from the plastic to fixed state, potentially through a transitional state or (ii) individual TST within a population exist in either the plastic or fixed dysfunctional state; initially, most TST are in the plastic state, however over time plastic T cells fail to proliferate or die while the fixed dysfunctional TST preferentially proliferate and/or survive. To test these models, we carried out single-cell RNA-Sequencing on TST at various time points during tumor progression. Determining the population dynamics of TST differentiation through dysfunctional states in tumors could inform our strategies for TST rescue, for example, by selecting rare plastic TST from bulk populations versus novel strategies to reprogram the epigenome of fixed dysfunctional TST.
Blockade of CD25 reduces regulatory T cell expansion and some manifestations of IL-2 mediated toxicity but does not improve anti-tumor efficacy in animal models of IL-2 immunotherapy
Authors: Harding Fiona A. , Alvarez-Jares Hamsell , Belmar Nicole , Choi Donghee , Forsyth Charles , Fox Melvin , Sheridan James , Stickler Marcia , Tang Anthony , Hollenbaugh Diane
Published: January 2023

High dose IL-2 (HD IL-2) is an approved treatment for melanoma and RCC that can result in long term disease remission, but is associated with severe adverse side effects. Adverse effects may be mediated by non-specific activation of cells expressing the high affinity CD25-containing IL-2 receptor. HD IL-2 also results in the expansion of regulatory T cells (Treg) that may negatively impact tumor immunotherapy. Blocking IL-2 binding to CD25 was explored as a mechanism to reduce expansion of Tregs and adverse effects. We created a high affinity anti-mouse CD25 mAb that effectively blocks the interaction of IL-2 with CD25 and carries the N297A Fc region modification to reduce FcgR binding. Saturation of CD25 ameliorates some but not all biomarkers of IL-2 toxicity. These include behavioral signs and serum ALT elevation. However, lung and spleen weight increase induced by IL-2 was not affected by CD25 blockade. CD25 blockade reduced IL-2 mediated expansion of Tregs. A saturating dose of CD25 antibody prior to 5-6 days of IL-2 therapy resulted in a complete loss of IL-2-mediated tumor efficacy in the CT26 model. CD25 blockade and IL-2 expanded CD8+ and NK cells while reducing blood and TIL Tregs, but did not impact primary tumor growth in the 4T1 or B16F10 tumor models. The loss of anti-tumor activity in the CT26 model correlated with reduced CD8+ CD25+ expansion in the blood and the TIL populations. Expansion of CD25 negative CD8+ and NK cells with concomitant Treg cell suppression in three different syngeneic tumor models did not result in improved anti-tumor efficacy. This result has implications for IL-2 and IL-15 engineering, and suggests that IL-2 mediated toxicity cannot be completely controlled by re-directing IL-2 to the CD122/CD132 receptor.
Prediction of hosts who are more likely to respond to PD-1 inhibitors/blocking antibodies
Authors: Daftarian Pirouz M. , George Marybeth , Tleugabulova Mayra Cruz , Magcase Dennie , Ouerkaxi Wushouer , Delcommenne Marc C.
Published: January 2023

A major challenge in the development of immune checkpoint blockades (ICB) is the variability in outcome of such therapeutics. This leads to a large number of non-responder patients receiving costly therapies that are also associated with some toxicities. Surrogates of efficacy are needed both to select the right patient population for the clinical trials and in later stages, to identify the right patients for the right ICB, ideally with assays that are rapid, high throughput and noninvasive. T cell clonal expansions caused by ICB are mainly local. As a result, the expanded T cells are mostly found in the tumor deposit and hard to detect in the peripheral blood. We optimized an antigen recall assay that assesses the ability of PD-1 blocking antibody to further enhance antigen specific CD8 T cell expansion and thereby discriminates responders from non-responders. A simple version of this assay has been used for functional screening of ICBs. By improving each component of the assay and its readout, we have increased the “signal to noise” window allowing us to rapidly detect PD-1 blockades’ effects on the host. We use a cocktail of optimized recall peptides, a characterized conditioned media and 96-well plates to support rapid (7 days) expansion of memory T cells. Finally, as a readout system, cocktail of MHC class I tetramers generated by peptide exchange was used to detect the overall expanded clonotypes. Currently, we have optimized the assay with commercially available anti-PD-1 and anti-PD-L1 antibodies. This assay system has the potential to assist in predicting those who are more likely to respond to ICB therapies. We have initiated blinded studies using clinical samples for clinical validation.
Changes of immune profile following nanosecond electric pulse treatment for pancreatic cancer
Authors: Guo Siqi , Burcus Niculina I. , Hornef James , Jing Yu , Jiang Chunqi , Heller Richard , Beebe Stephen J.
Published: January 2023

Pancreatic cancer is a notorious malignancy with a very poor prognosis. Without significant advance in the treatment and early diagnosis, pancreatic cancer will continue to be a serious global health issue. Nanosecond electric pulses (nsEPs) or nanosecond pulsed electric fields (nsPEFs) have been demonstrated to result in completely local ablation and protection from the second tumor challenge in several cancer models. Here, we assessed the potential advantages of nsEPs for pancreatic cancer in a syngeneic mouse Pan02 pancreatic cancer model and characterized its immune profile. We found that a single nsEP treatment could achieve complete tumor regression without any mortality. Animals with partial regression prolonged survival in those treated with nsEPs more than those treated with irreversible electroporation (IRE). Low rate of abscopal effect (10%) and vaccine-like protective effect (1/15) was observed in animals treated with nsEPs but not with surgery or IRE. Significant reduction of both T regulatory cells and myeloid derived suppressor cells (MDSCs) was seen in the blood of tumor animals after nsEP treatment. Heterogeneous T cell responses following nsEP treatment were found. Very few T cells were infiltrated in the tumor but large amount of MDSCs existed in tumor. The number of dendritic cells in the tumor was increased and activated with upregulation of multiple activation markers following nsEP treatment. Our findings could help design a more effective combinational nsEP therapeutic strategy for pancreatic cancer.
Tissue specific CD91+ APCs determine effectiveness of immunosurveillance mediated by HSPs
Authors: Sedlacek Abigail L. , Kinner Lauren B. , Wang Yifei , Mizes Alicia P. , Binder Robert J.
Published: January 2023

Gp96, an immunogenic Heat Shock Protein, is capable of eliciting potent immune responses against its chaperoned antigens and is being investigated as a potential cancer immunotherapy. Gp96 induces immune responses by activating antigen presenting cells (APCs) through CD91 which elicits downstream T and NK cell activation. T cell activation following gp96 is well understood, including the necessary APCs. Conversely, NK cell activation is less understood, even though NK cells are required for full T cell activation. We have determined that macrophages (MΦ) are capable of inducing IFNγ production by NK cells but, inversely, dendritic cells (DC) are not, despite similar expression of CD91. Thus, we investigated MΦ and DC production of soluble mediators of NK cell activation. IP10 was identified as a chemokine which is more greatly induced in MΦ than DC in response to gp96. Blocking the IP10 receptor, CXCR3, prevents IFNγ production by NK cells when cultured with gp96 activated MΦ. IP10 is STAT1 induced, thus we examined STAT1 phosphorylation (STAT1p) in MΦ and DC in response to gp96. STAT1 is phosphorylated in MΦ, but gp96 fails to induce STAT1p of tyrosine in DC. In STAT1 signaling, p38 is proximally upstream of STAT1p and interestingly, both MΦ and DC phosphorylate p38 to similar levels, suggesting that the difference between MΦ and DC is downstream of p38. There are several phosphatases downstream of CD91 which could be inhibiting STAT1p. To further support this, we inhibited tyrosine phosphatases using pervanadate, resulting in DC STAT1p in response to gp96. Collectively, these data indicate that gp96 activation of MΦ may result in a more robust immune response by activating NK cells which is a critical consideration for clinical applications.
The prevalent Boxer MHC class Ia allele Dog Leukocyte Antigen (DLA)-88*034:01 presents 9-mer peptides with a defined binding motif
Authors: Hess Paul R. , Nemec Paige S. , Kapatos Alexander , Holmes Jennifer C.
Published: January 2023

Adoptive cell transfer (ACT) for chemoresistant malignancies can induce dramatic remissions and prolong life, but factors that determine T-cell efficacy and off-target/-tumor events associated with therapy remain incompletely understood. Modeling ACT in spontaneous canine cancer models could provide critical insights and propel development; however, relevant peptides from tumor-specific antigens (TSAs) have yet to be reported in dogs. Discovery of TSA epitopes restricted by a high-prevalence MHC allele can be facilitated by in silico analysis using a defined binding motif and a large peptide dataset to train prediction software. We sought to characterize the binding preferences of the DLA-88*034:01 allele, carried by 82% of Boxers, an inbred, popular breed with increased risks for glioma and lymphoma, important ACT targets. We hypothesized that DLA-88*034:01 presents diverse self-peptides with conserved residue preferences at ≥1 position(s) that can be inferred by pattern analysis. To test this prediction, DLA-88*034:01-FLAG was immunoprecipitated from large-scale cultures of a canine cell clone, PN62, and alkaline-eluted peptides were sequenced by liquid chromatography-tandem mass spectrometry. DLA-88*034:01 prefers peptides that are 9 amino acids (AAs) in length. Analysis of 640 9-mers showed a preference for acidic AAs at position(P)1, non-bulky hydrophobic AAs at P2, and basic AAs at P4. At P6 and P9, H and F were highly favored, respectively. Bulky hydrophobic and charged AAs were unfavorable at P2, as were W, M and Y at P8. This dataset will allow more accurate prediction of TSA-derived peptides recognized by CTL in a majority of Boxers with glioma or lymphoma, and should foster development of an ACT model in this breed.
PD-1/PD-L1 Monoclonal Antibody Development for Canine Cancer Therapy
Authors: Choi Jin Wook , Withers Sita S. , Sciammas Roger , Rebhun Robert B. , McSorley Stephen J.
Published: January 2023

PD-1 is an inhibitory receptor that binds to PD-L1 on the surface of tumor and antigen presenting cells in the tumor microenvironment, causing suppression of T-cell activity and tumor progression. Human clinical trials with anti PD-1 monoclonal antibodies, nivolumab and pembrolizumab, showed an objective response rate (ORR) of 30% to 40% with melanoma and 87% with relapsed or refractory Hodgkin’s lymphoma. Here, we have explored the development of canine PD-1/PD-L1 reagents for potential veterinary applications. An S2 insect cell expression system was utilized to produce recombinant canine PD-1Ig and PD-L1Ig and antigen-specific B cells were enriched using PD-1 and PD-L1 tetramers. Enriched B cells were fused and resulting hybridomas were screened for specific reactivity to PD-1 and PD-L1. Then, antibodies capable of blocking PD-1Ig and PD-L1Ig were selected. A total of 5 monoclonal antibodies were generated from the two rounds of immunizations and are being studied for in vitro activity in T cell assays. These canine reagents have multiple diagnostic and therapeutic applications in the context of canine oncology.
Porcine macrophage-like cells permit viral replication, produce inflammatory mediators, and undergo apoptosis following infection with Rift Valley fever virus MP-12
Authors: Noronha Leela E. , Smolensky Dmitriy , Cox Victoria , Schirtzinger Erin E. , Chitko-McKown Carol G. , Fawver Zachary , Wilson William C.
Published: January 2023

Rift Valley fever virus (RVFV) is a mosquito-borne virus that can cause severe disease in a variety of domestic animal species as well as in humans. Outbreaks occur primarily in Sub-Saharan Africa with ruminant livestock most affected and outcomes that include abortion storms and high mortality among young animals. Clinical disease is not known to occur in domestic pigs (Sus scrofa); however, a limited number of studies have detected circulating antibodies to RVFV among wild and domestic members of the pig family (Suidae). Experimentally, RVFV has been shown to replicate in some porcine epithelial and neuronal cells in vitro, and induce a viremia in a fraction of pigs inoculated in vivo. No studies to date have investigated the behavior of RVFV in porcine cells of immune origin, whereas in humans and mice, macrophages and dendritic cells have been implicated as having roles in RVFV pathogenicity. Here, we use a porcine monocyte-derived macrophage-like cell line, CΔ2+, to investigate porcine innate immune cell permissiveness to MP-12, an attenuated form of RVFV. MP-12 caused observable cytopathic effects in CΔ2+ cells and reduced cell viability in dose- and time-dependent manners. At 24 hours, the reduced viability was coincident with apoptosis, with necrosis also occurring at a higher multiplicity of infection. At early time points, MP-12 replicated at levels comparable to cells derived from sheep kidney—a target organ of a highly susceptible host species. Infected CΔ2+ cells also upregulated transcription of inflammatory mediators. This work provides the first evidence that porcine immune cells are permissive to RVFV, and indicates that additional studies are warranted to better understand the dynamics of RVFV in suids.
Leukocyte immune-type receptor expressing cells increase in response to Edwardsiella ictaluri infection in Channel Catfish, Ictalurus punctatus
Authors: Blackmon Laura Elizabeth , Quiniou Sylvie , Wilson Melanie , Bengten Eva
Published: January 2023

Unique to teleosts, leukocyte immune-type receptors (LITRs) represent a large polygenic, polymorphic family of immunoregulatory receptors. Monoclonal antibodies (mAb), CC34 and CC41, were developed against a catfish clonal CTL line and a catfish NK cell line, respectively. Both recognize members of the LITR family. One subset of LITRs, defined by reactivity with mAb CC41, is a distinct marker of a cytotoxic cell population, which expands during the antiviral response to Channel Catfish Virus (CCV). It is hypothesized that these mAb CC41-reactive LITRs function as NK-like receptors and recognize stress induced ligands on target cells. Edwardsiella ictaluri, an intracellular pathogen, primarily establishes infection in pronephros (head kidney) monocytes and may elicit a strong cytotoxic response. To explore this, small fish (<1kg) were infected with E. ictaluri via bath immersion (1×107cfu/ml). Following infection, whole blood was collected and analyzed daily by flow cytometry for mAb CC34 and CC41 staining. In parallel, an in vitro model was developed to monitor the number of CC34 and CC41-reactive cells in response to E. ictaluri infection. Briefly, head kidney monocytes and peripheral blood lymphocytes (PBL) were isolated from catfish of either susceptible or resistant background. Head kidney cells were infected with E. ictaluri (MOI 10) and then mixed with PBL from the same individual catfish and analyzed daily by flow cytometry. There was an increase in staining with both mAbs CC34 and CC41 by day 3 after infection both in vivo and in vitro. This study highlights the utilization of these mAb reagents to monitor cytotoxic cell response to E. ictaluri infection.
Priming of CD8 T cells in established chronic viral infection directs a distinct differentiation and functional program for long-term immunity
Authors: Snell Laura M. , Brooks David G.
Published: January 2023

CD8 T cell exhaustion impedes control of chronic virus infection, yet how CD8 T cell fate decisions and function are instilled and progressively controlled throughout infection is unclear. We demonstrate that in contrast to T cells primed at the onset of persistent viral infection, CD8 T cells primed after the chronic infection is established preferentially generate into memory-like cells that resist contraction, are less exhausted and have heightened responsiveness to immunotherapy. Mechanistically, viral persistence down-regulates APC activation machinery, decreasing T cell intrinsic TCR and CD28 signaling upon priming. The lower signal intensity provides the required threshold for activation and proliferation, but alters transcriptional programming and cellular differentiation to enable long-term retention of antiviral functions despite high levels of virus replication. In the absence of strong antigenic signaling, IL-21 acquires the ability to induce functional effectors, while preserving the memory-like reservoir. Analogous mechanisms also govern differentiation of anti-tumor CD8 T cells in the tumor microenvironment during chronic viral infection, undermining tumor control. Thus, dynamic changes in signal intensity and IL-21 usage during chronic infection function as fate-assigning rheostats to resist exhaustion and program long-term immunity.
MHC class II antigen-processing chaperone H2-O shapes CD4 T cell receptor repertoire
Authors: Song Nianbin , Welsh Robin A. , Boronina Tatiana , Cole Robert , Foss Catherine , Sadegh-Nasseri Scheherazade
Published: January 2023

HLA-DO (DO) is a MHC class II-encoded, nonpolymorphic heterodimeric protein in humans (known as H2-O in mice). It is expressed only in thymic medulla, B cells and some subsets of dendritic cells. Although DO is found to form a stable complex with HLA—DM, its physical function remains unknown. To solve this mystery, our lab previously proposed a model suggesting that the DO facilitates the binding of DM-resistant peptides and inhibits the binding of DM-sensitive peptides to the HLA-DR1 molecules in vitro. Based on this model, we expected to see lower density of pMHC expression on mTEC cells in thymus of DO-KO mice, leading to faulty negative selection and different CD4 TCR repertoires as compared to DO-WT mice. By developing a “Prime-Restim” strategy based on the principle of mixed lymphocyte reaction, we observed the proliferation of primed DO-WT CD4 T cells in responding to repeated in vitro stimulations by antigen presenting cells from DO-KO mice and vice versa. This strategy led to enrichment of some clonal populations in DO-KO mice reactive to self-peptides presented by B cells from DO-WT mice. To identify the self-antigens these T cell clones recognize, we developed CD4 T cell hybridomas from them and screened their specificities with peptides eluted from B cells of DO-KO and DO-WT mice. We also identified a peptide from an autoimmunity-associated protein that eluted only from DO-KO B cells but not seen from DO-WT B cells. These results demonstrate that in the absence of DO, the peptide repertoire of MHC II in the DO-KO mice is different from the peptide repertoire of MHC II in the DO-WT mice. These findings support the idea that the expression of DO in thymic medulla might lead to better thymic deletion of self-reactive CD4 T cells.
Critical role of STAT5 tetramerization in the pathogenesis of experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis
Authors: Monaghan Kelly Lynn , Farris Breanne Y , Amend Courtney D , Lin Jianxin , Leonard Warren J , Wan Edwin C.K.
Published: January 2023

Multiple sclerosis (MS) is an immune mediated disease that predominantly impacts the central nervous system (CNS). Current MS treatments have demonstrated the ability to reduce the rate of MS relapses; however, they are not effective at preventing disease progression. Elucidating the pathways that govern the pathogenesis of MS may identify molecular mediators, which can act as novel therapeutic targets to treat MS progression. STAT5 is a transcription factor critical for the generation, activation, and function of different immune cell types. Upon activation, STAT5 can form dimers or tetramers, which have distinct functions. Using a Stat5a-Stat5b double knock-in N-domain mutant mouse strain (DKI mice), in which STAT5 dimers are functional, but STAT5 tetramers cannot be formed, we demonstrated that DKI mice developed less severe experimental autoimmune encephalomyelitis (EAE), a model of MS, compared to WT littermates. Moreover, a portion of DKI mice did not develop EAE symptoms. Flow cytometric analysis of peripheral blood revealed that during the early phase of EAE, WT and DKI mice exhibited similar levels of immune cell expansion in the periphery. However, at the peak of EAE, DKI mice had fewer immune cells in the CNS. This suggests that STAT5 tetramers promote immune cell egress to the CNS or/and immune cell expansion within the CNS. In addition, multiplex protein assays established that the expression of a pro-inflammatory cytokine and several chemokines, including IL-1α, CCL3, CCL17, and CCL22, are reduced in DKI mice compared to WT mice, which may account for the reduced disease severity observed in DKI mice. Taken together, the data suggest that STAT5 tetramers plays a multifaceted role in promoting inflammation resulting in EAE.
Distinct waves of CD8 T cell affinity during demyelinating disease progression
Authors: Jacobs Jesica R , Blanchfield Lori , Liu Baoyu , Evavold Brian D
Published: January 2023

The animal model of Multiple Sclerosis, Experimental Autoimmune Encephalomyelitis (EAE), requires CD4 T cells to induce demyelinating disease. However, CD8 T cells are ever present during EAE and have been hypothesized to have both effector and suppressor functions. To understand autoimmune disease progression, we analyzed the affinity and genetic profile of the CD4 and CD8 T cell response over time. Affinity analysis of CD8 T cells during the autoimmune disease course revealed the greatest affinity for myelin at peak disease (d21-23), whereas affinities were significantly lower at onset (d14) and chronic (d36-38) timepoints. Conversely, affinity analysis of CD4 T cells revealed no significant changes at the same time points. Additionally, CD8 T cells displayed a smaller, 10-fold range in affinity, suggestive of a restricted TCR clonal response. CD4 affinities covered a much wider 10,000-fold range, consisting of low and high affinity clones. To further understand the dynamics of the T cell population during disease, we performed single cell RNA-sequencing. Heterogenous populations of CD4 and CD8 T cells were observed at peak EAE disease, including a genetically distinct population of FoxP3+, CD25+ CD8 T cells which accounted for 3.33% the total CD8 population. These genomic data paired with the known associations between affinity and T cell fate suggest phenotypically and functionally distinct waves of CD8 T cells with limited clonality infiltrate the CNS during EAE progression.
Modulation of liver protein sialylation drives spontaneous generation of fatty liver and modulates multiple inflammatory models
Authors: Oswald Douglas M , Jones Mark B , Cobb Brian A
Published: January 2023

Circulatory protein glycosylation has long been recognized as a marker of multiple disease states and has been applied clinically to gauge diseases including liver dysfunction, heart disease, and diabetes. The etiology and mechanism of this relationship, including whether glycosylation changes are causative in or only indicative of inflammatory shifts, has not yet been elucidated. We present here a model in which the α 2,6 sialyltransferase (ST6Gal1) was selectively knocked out of hepatocytes (H-cKO) using a Cre-lox recombination model driven by albumin expression. We confirm the loss of circulatory glycoprotein α 2,6 sialylation and note no obvious dysfunction in young (8–20 week old) H-cKO mice at baseline. Upon aging, the H-cKO mice spontaneously generate fatty liver disease, visible by gross observation and histologic examination, that is indicative of underlying inflammatory dysfunction. Further, H-cKO mice demonstrate exacerbated disease in models of thioglycolate-driven peritoneal inflammation, dextran sodium sulfate-driven colitis, and house dust mite-driven asthma. Our work positions liver protein secretion and glycosylation as not only indicative of inflammatory disease, but also as a causative factor in disease incidence and severity.
Loss of local innervation induces lymph node expansion
Authors: Chen Chien-Sin , Ince Louise , de Juan Alba , Scheiermann Christoph
Published: January 2023

The nervous system directly innervates both lymphoid and non-lymphoid tissues, but the influence of neural input on regional immune responses remains unclear. Local, unilateral deprivation of nerves to the popliteal lymph node (popLN) and its draining area by surgical denervation led to ipsilateral paw thickening and dramatic nodal expansion of all leukocyte subsets investigated. However, denervation surgeries on nerves bypassing the popLN, such as cutting the femoral nerve, the sciatic nerve below the popliteal fossa or individual branches of the sciatic nerve, failed to increase lymph node cellularity. Expansion was associated with increased expression of Cxcl13, Il1a, Il1b, Il6, Il10, Il17a and Il17f levels in denervated popLNs and paws. Adoptive cell transfer revealed enhanced cellular trafficking of lymphocytes to the lymph node, accounting for increased cellularity as this was ablated in mice receiving antibodies blocking lymph node homing. Additionally, immunofluorescence analyses revealed high levels of B cell proliferation and germinal center formation, which was mirrored by enhanced antibody titers in serum. Blockade of antigen presentation using an MHC class II-neutralizing antibody or surgically ablating drainage of afferent lymphatics to the popLN inhibited denervation-induced nodal expansion. Together, these data provide evidence for an important regulatory role of direct neural innervation in local immune responses.
Induced hyperglycemia promotes sensitization and exacerbates allergic inflammation in a mouse model of asthma
Authors: Queener Ashley , Jeong Brian M , Doan Ton C , Chiarella Sergio E , Walker Matthew T , Coden Mackenzie E , Nagasaka Reina , Berdnikovs Sergejs
Published: January 2023

Glucose is a key source of energy in systemic and cellular metabolism and has known pro-inflammatory properties. It has recently been acknowledged that associations exist between metabolic disorders (diabetes, insulin resistance, obesity), asthma and other allergic diseases. As the prevalence of both metabolic and allergic disorders increases, it is important to understand how metabolism can influence the development of allergy. First, we injected fasted wild type Balbc/J mice intraperitoneally (i.p.) with 2g/kg dextrose (standard hyperglycemic challenge) to test whether hyperglycemia promotes inflammation favoring antigen sensitization. Within one hour of injection, we detected a rapid increase in blood glucose levels (up to 130 mg/dL) followed by an increase in Il1β, Tslp, Cxcl9, and Siglec5 gene expression in the peritoneal tissue compared to vehicle controls. Within 2 hours post injection, IL-1β protein was detected in peritoneal cellular infiltrate. In order to determine whether glucose-induced inflammation promotes allergic response, we sensitized mice by i.p. to chicken egg ovalbumin (OVA) using either alum (standard adjuvant), dextrose or vehicle control. Both alum/OVA and dextrose/OVA mounted lung allergic inflammation in response to inhaled OVA antigen. Dextrose/OVA mice had OVA-specific IgE production similar to alum/OVA group, demonstrating ability to promote sensitization. Remarkably, total cellular infiltrates, bronchoalveolar lavage eosinophils, lung expression of IL-4, IL-13 and IL-33 in dextrose-sensitized mice were equal or surpassing allergic inflammatory responses in mice given standard adjuvant. These results demonstrate a potentially critical role for glucose in promotion of allergy.
ICOS signaling in visceral adipose tissue regulatory T cell homeostasis and function
Authors: Mittelsteadt Kristen L , Campbell Daniel J.
Published: January 2023

Obesity and insulin resistance/type 2 diabetes are linked by chronic inflammation in visceral adipose tissue (VAT). Foxp3+ regulatory T cells (TR) are critical for maintaining immune tolerance and are recently appreciated for their role in tissue repair and homeostasis. Indeed, a unique population of TR reside in the VAT of mice and humans, and these TR regulate adipose inflammation and preserve insulin sensitivity and glucose tolerance. ICOS is a costimulatory molecule expressed at high levels by TR. Surprisingly, the abundance of TR in VAT is elevated by nearly 2-fold in IcosKO mice compared to WT controls, and IcosKO TR preferentially reconstitute the VAT of WT:IcosKO mixed bone marrow chimeras. TR in the VAT of IcosKO mice express high levels of VAT-TR markers, like ST2, KLRG1, and IL-10, compared to WT mice, suggesting a potent ability to prevent insulin resistance and diet-induced metabolic syndrome. Ongoing studies aim to determine whether IcosKO mice are more resistant to diet-induced metabolic syndrome by assessing insulin sensitivity/glucose tolerance, indices of inflammation, and VAT immune cell phenotype and function, and to mechanistically elucidate how ICOS signaling controls the accumulation and phenotype of VAT-TR. These studies will yield important information on the molecular pathways involved in regulating the onset of metabolic syndrome and provide new potential therapeutic targets.