bluuebunny/crossref_metadata_2025 huggingface dataset:
NOD-scid IL2rg
null (NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC
Authors: Brehm Michael A. , Wiles Michael , Kenney Laurie Lea , Burzenski Lisa , Keck James , Greiner Dale L. , Shultz Leonard D.
Published: January 2023

Humanized mice are emerging as an exciting platform to study human immuno-oncology and provide preclinical tools to test cutting edge immunotherapies. An effective and simplistic approach to engraft immunodeficient mice with functional human T cells is the injection of human peripheral blood mononuclear cells (PBMC). This PBMC engraftment model (termed Hu-PBL-SCID) has supported studies of human immune system-tumor interactions following injection of human PBMC into immunodeficient mice implanted with patient-derived (PDX) tumors. However, a major limitation of this model is the development of acute xenogeneic GVHD due to human T cell recognition of murine MHC class I and class II. To address this limitation, we created two strains of NSG mice that lack murine MHC antigens, one by crossing NSG-B2Mnullwith NSG-(IA IEnull) mice, the second by knocking out the MHC class II IAb gene in NSG-(KbDb)null mice using TALEN technology. We observed that human IgG clearance was rapid in NSG-B2Mnull(IA IEnull) mice that lack functional FcRn activity whereas clearance in NSG-(KbDb)null(IAnull) mice was comparable to that observed in NSG mice. Injection of human PBMC into both strains led to long term engraftment of human T cells without development of acute GVHD. The engrafted human T cells were functional as documented by their ability to reject human islet allografts. Moreover, NSG-(KbDb)null(IAnull) mice injected with a PDX tumor and PBMC from an allogeneic donor supported significant tumor growth in the absence of acute GVHD. These data document that NSG mice deficient in murine MHC class I and class II support studies of human immune system-tumor interactions in the absence of acute GVHD and provide a model to evaluate human antibody therapeutics.
The ceramide structure of sulfatide-analogues influences the functional activity of type II NKT cells
Authors: Pasquet Lise , Camara Kaddy , Bloom Anja C. , Richardson Stewart K. , Howell Amy R. , Terabe Masaki , Berzofsky Jay A.
Published: January 2023

The immune system plays a dual role in tumor elimination. Tumor antigens activate cytotoxic cells that kill tumor cells, but the autologous origin of tumors also activates regulatory cells supporting tumor escape. The CD1d-restricted sulfatide–reactive type II NKT cells have a regulatory role by increasing the development of lung metastasis after sulfatide injection in a murine model of colon carcinoma. The identification of compounds able to control the activation of type II NKT cells is essential.

Sulfatide (C24:1), a lipid enriched in many tumors, is composed of a galactose head with a sulfate group β-linked to a ceramide moiety. The structure of the ceramide influences the TCR/lipid-CD1d complex interaction and determines the reactivity to the lipid. We synthesized diverse sulfatide-analogues varying in the number of double bonds and hydroxyl groups in the ceramide. Using analogue-loaded-CD1d tetramers, we identified analogue-specific cells in the lung. These cells were also sulfatide-reactive but distinct from α-GalCer-specific type I NKT cells. In vitro, analogues exhibiting a phytosphingosine chain activated both type II and type I NKT cell hybridomas. All analogues induced CD1d-dependent IL-13 production by lung cells but in differing amounts. In vivo, analogues had variable effects on lung metastasis. Interestingly, the analogue C24:2, recognized only by the type II NKT hybridoma, induced significantly more IL-13 production by lung cells than sulfatide while significantly reducing the development of lung metastasis. The distinct effects of sulfatide and C24:2 on the specific activation of type II NKT cells and on tumor elimination could make C24:2 a valuable addition to the armamentarium of anti-tumor immunotherapies.
De novo DNA methylation programs restrain T cell rejuvenation during immune checkpoint blockade therapy
Authors: Youngblood Ben A. , Ghoniem Hazem , Moustaki Ardiana , Abdelsamed Hossam Aly , Fan Yiping , Thomas Paul G. , Federico Sara , Stewart Elizabeth
Published: January 2023

Immune-checkpoint blockade (ICB)-mediated rejuvenation of CD8 T cell effector functions has emerged as one of the most promising frontiers for treating cancer and chronic infections. However, antigen-specific T cells that have experienced prolonged antigen exposure are often terminally differentiated, and have a limited capacity to mount an effector response during ICB treatment. Such exhaustion of effector potential is a major impediment of current T cell based immunotherapy efforts. Using in vivo mouse models of tumor and chronic viral infection, we assessed the role of de novo epigenetic programming in establishing ICB-refractory exhausted T cells. We observed that genetic deletion of Dnmt3a in T cells at the effector stage of an immune response to chronic lymphocytic choriomeningitis virus (LCMV) infection allowed antigen-specific T cells to remain highly functional despite expressing high levels of PD-1 and having prolonged exposure to antigen. Quite strikingly, PD-1 blockade treatment of chronically infected animals resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Whole-genome methylation profiling of WT and Dnmt3a-deficient LCMV-specific CD8 T cells identified de novo DNA methylation programs that are coupled to development of ICB-nonresponsive virus and tumor-specific T cells. Building upon these findings, we have identified de novo epigenetic programs acquired in human tumor-associated PD-1hi CD8 T cells. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key regulator in establishing ICB-refractory exhausted CD8 T cells and highlights epigenetic reprogramming of T cells as a novel approach to enhance T cell-based cancer therapies.
Glycogen synthase kinase-3 (GSK-3) inactivation compensates for the lack of CD28 in the priming of CD8+ cytotoxic T-cells: Implications for anti-PD-1 immunotherapy
Authors: Rudd Christopher E. , Krueger Janna , Taylor Alison
Published: January 2023

The rescue of exhausted CD8+ cytolytic T-cells (CTLs) by anti-programmed cell death-1 (anti-PD-1) immune checkpoint blockade has been found to require CD28 expression. At the same time, we have shown that the inactivation of the serine/threonine kinase glycogen synthase kinase (GSK)-3α/β with small-interfering RNAs (siRNAs) and small molecule inhibitors (SMIs) specifically down-regulates PD-1 expression for enhanced CD8+ CTL function and clearance of tumors and viral infections (Taylor et al., 2016 Immunity, 44(2):274-86, Taylor et al., 2017 Cancer Research, canres.0491.2017). It has been unclear whether the GSK-3α/β pathway accounts for CD28 costimulation of CD8+ CTL function. In this presentation, we show for the first time that the inactivation of GSK-3α/β through siRNA or by SMIs during priming can substitute CD28 co-stimulation in the potentiation of cytotoxic CD8+ CTL function against the EL-4 lymphoma cells expressing OVA peptide. The effect was seen using several structurally distinct GSK-3 SMIs and was accompanied by an increase in Lamp-1 and GZMB expression. Conversely, CD28 crosslinking obviated the need for GSK-3α/β inhibition in its enhancement of CTL function. Our findings support a model where GSK-3 is the central co-signal for CD28 priming of CD8+ CTLs in anti-PD-1 immunotherapy. Anti-PD-1 and GSK-3 SMIs cooperate synergistically to limit the growth of solid melanoma tumors in mice. We will present data on the altered nature of tumor-infiltrating lymphocytes (TILs) in response to anti-PD-1 versus GSK-3 blockade and combination therapy. Our findings outline a next-generation approach using small molecule inhibitors in combination with check-point blockade therapy for the elimination of tumors.
IL-33 inhibits tumor growth through the activation of anti-tumor CD8+ T cell response in hepatocellular carcinoma
Authors: Jin Ziqi , Liu Haiyan
Published: January 2023

IL-33 secreted by epithelial cells and activated innate immune cells is a member of IL-1 family with dual-immunological effects. It can induce both type 1 and type 2 immune responses and its function as an alarmin has been well demonstrated. However, the role of IL-33 in the development of hepatocellular carcinoma (HCC) is not clear. Here, we examined the function of IL-33 in various murine HCC models by hydrodynamically injecting either IL-33-expressing tumor cells or IL-33-expressing plasmids into the liver of tumor-bearing mice. Tumor growth was greatly inhibited by IL-33 expression. Similarly, in DEN-induced HCC model, we also demonstrated that IL-33 expression inhibited tumor development. This effect was shown to be dependent on extracellular IL-33 and ST2 expression, because it was diminished in ST2 KO mice. Further study showed there were increased percentages and numbers of CD8+ T cells in both spleen and liver in IL-33-expressing tumor-bearing mice. The percentages and numbers of activated and effector CD4+ and CD8+ T cells were also increased in the liver with IL-33 expression. Moreover, IFN-γ production of the CD8+ T cells was upregulated in both spleen and liver by IL-33. The cytotoxicity of CTLs against tumor cells was also enhanced. Depletion of CD8+ and CD4+T cells diminished the antitumor activity of IL-33, suggesting the anti-tumor function of IL-33 was T cell-dependent. In vitro IL-33 treatment could preferentially expand CD8+ T cells and promote their activation and IFN-γ production. Taken together, we demonstrated in murine HCC models that IL-33 could inhibit tumor development through its interaction with ST2 to promote anti-tumor CD8+ T cell response.
Discovery screen platform for co-modulators of T cell effector function against tumors
Authors: Kim Edward , Sekar Keerthana , Sakamoto Doreen , Yen David , Lee Fei , Bailis Julie , Ouyang Wenjun , Zheng Yan
Published: January 2023

A major goal for cancer immunotherapy is to target pathways that drive optimal anti-tumor efficacy. T cell checkpoint therapy aims to enhance T cell responses during priming and/or within tumor tissue, but the key drivers of efficacy in broad patient populations has remained elusive. Here we first sought to determine whether responsiveness to anti-PD1 therapy in established syngeneic murine tumors depends on recruitment of T cells from lymphoid organs during treatment, or if PD1 blockade on T cells already within tumors is sufficient to drive efficacy. We compared anti-PD1 efficacy on established MC38 tumors when FTY720 (S1PR antagonist that blocks lymphocyte egress from lymphoid organs) or vehicle control was administered from the start of treatment. We found that anti-PD1 efficacy did not require additional T cells migrating from lymphoid organs, suggesting that PD1 blockade can act directly on T cells that were already present within tumor tissue. We next examined the functionality of tumor-infiltrating T cell subsets during anti-PD1 treatment and found that cytokine production is enhanced in a population of PD1intermediate T cells, but not on PD1high T cells. Together, this suggests that PD1 checkpoint therapy is driven by amelioration of a fraction of PD1+ tumor-infiltrated T cells, and that additional pathways within established tumors may need to be co-targeted to drive optimal efficacy. We then developed an in vitro discovery screening platform to identify pathways that co-modulate human T cell effector function against PDL1+ cells under conditions that drive high levels of PD1 on T cells. We are currently screening genes of interest that are expressed in patient tumor tissues and highly associated with a T cell-inflamed signature.
TIGIT blockade prevents CTL and NK cell exhaustion and leads to tumor rejection in mice
Authors: Tian Zhigang
Published: January 2023

T-cell Ig and ITIM domain (TIGIT), is a co-inhibitory receptor expressed on both T cells and NK cells. Studies by our group as well as other groups have shown that TIGIT contribute to immune tolerance by inhibiting both T cell and NK cell-mediated immune responses through binding its ligand, CD155, on APCs or target cells. In this study, we showed that TIGIT was up-regulated on CTL and NK cells during tumor progression in several tumor mouse models. TIGIT deficiency delayed inoculated B16/F10 tumor growth in vivo. Blocking TIGIT signaling in vivo using monoclonal antibodies, in combination with Flt3L overexpression in vivo by gene delivery, reversed the exhaustion of anti-tumor effector cells, CTL and NK cells, in B16/F10 tumor-bearing mice and significantly suppressed pre-established B16/F10 tumor growth and metastasis, thereafter improving overall survival. We also showed that TIGIT blockade inhibited colon cancer, breast cancer and MCA-induced fibrosarcoma growth in vivo. These findings demonstrated that TIGIT contributes to tumor-induced immune exhaustion and might be a promising target for cancer immunotherapy.
Immune Checkpoints Acting as Gate-keepers of T Lymphocyte Self-renewal
Authors: Carrera Maria
Published: January 2023

Immune checkpoint inhibition represents a promising therapeutic modality for several previously incurable cancer syndromes. Why successful T cell responses are achieved in some patients but not others is incompletely understood. Because blockade of more than one inhibitory signal is now being investigated as approach to increase the chances of therapeutic success, we investigated how combined loss of PD-1 and LAG-3 signaling in CD8+ T cells would influence a well-characterized immune response. After challenge with Vaccinia virus, T cells from mice lacking both inhibitory pathways underwent a more vigorous primary effector cell response but were severely defective during a secondary challenge. During modeled T cell activation, immune checkpoint inhibition resulted in greater production of irreversibly differentiated effector cell progeny, but this came at the expense of accelerated depletion of self-renewing progenitor cells. Pharmacological interventions that impede anabolism-associated signaling were able to correct progenitor cells loss in the face of checkpoint inhibition. Unleashing greater and greater T cell activation may result in regenerative failure of critical clones. By fostering the expansion of self-renewing progenitors, reversible agents that dampen T cell signal strength might paradoxically improve durability of immune checkpoint inhibition.
CD57+ T cells phenotype and function in non-small cell lung cancer
Authors: Li Lequn , Huang Bing
Published: January 2023

The composition of TILs is heterogeneous and the contribution of various subsets of TILs to anti-tumor effects can be incredibly different. Therefore, understanding the immune function of existing subsets of TILs in particular cancer types is of great significance. Previous studies, conducted by others and ourselves, have shown that CD103+ TILs have good prognostic value and are functionally primed to respond to PD-1 blockade. In this study, we investigated the phenotypic and functional features of CD57+ TILs in non-small cell lung cancer (NSCLC). CD57+T cells have been considered as end-stage, senescent T cells during chronic viral infections. However, the immune functions of CD57+ T cells in NSCLC remain unclear. Using a multicolor staining approach, we analyzed TILs isolated from freshly resected tumor tissues from NSCLC patients and determined that CD57+ T cells were largely CD8+ cells and were a different subset from CD103+T cells. CD28 expression was significantly downregulated in CD57+ T cells compared to CD57−T cells. Functional analysis revealed that CD57+ T cells produced a significant amount of IFN-γ in response to PMA/IONMYCIN stimulation compared to CD57− T cells, suggesting that these cells are functional effector/effector memory cells within the tumor microenvironment. Interestingly, immune response of CD57+ T cells in vitro was significantly impaired as determined by the expression of IFN-γ and Ki-67 in response to anti-CD3/CD28 stimulation. In contrast to CD103+ T cells, additional PD-1 inhibitor did not improve the ability of CD57+ T cells to produce IFN-γ. These results suggest that CD57+ T cells combined with CD103+ T cells might serve as predictive biomarkers for PD-1 based immunotherapy.
PD-1 blockade activates conventional CD4 T cells and the innate immune response during glioblastoma eradication
Authors: Klein Sarah , Ziello Jennifer , Speranza Maria , Gokhale Prafulla , Kirschmeier Paul , Crosby Katherine , Freeman Gordon , Reardon David
Published: January 2023

Blockade of immune cell co-inhibitory receptor PD-1 using monoclonal antibodies enables anti-tumor immune responses in various solid tumors and lymphoid malignancies. Our laboratory previously demonstrated PD-1 blockade elicits an anti-tumor immune response resulting in tumor rejection and long-term survival in approximately 50% of mice with an orthotopic GL261 glioblastoma, despite lacking a corresponding accumulation of CD8+ cytotoxic T cell in the tumor or draining lymph nodes. In this investigation, we evaluated the role of conventional CD4+ T cells and the innate immune response in PD-1 mediated anti-glioma immunity using multiplex technologies for immunohistochemistry and flow cytometry. In response to PD-1 monotherapy, intratumoral CD4 T cells expressed significantly elevated levels of proteins required for T cell proliferation, activation, and effector function. CD4 T cell activation was accompanied by the classical activation and M1 polarization of resident microglia and tumor-infiltrating macrophages, including down-regulation of PD-L1 and up-regulation of MHC class II surface expression. We also demonstrated that depletion of either CD4 or CD8 T cells was sufficient to completely ablate anti-PD-1-mediated tumor eradication and long-term survival. Our data suggests CD4 T cells and myeloid cells may play a prominent role in the eradication of glioblastoma by PD-1 blockade.
Particulate Delivery of Innate Immune Agonist for Cancer Immunotherapy
Authors: Watkins Rebekah , Tiet Pamela , Junkins Robert , Gallovic Matthew , Chen Ning , Johnson Brandon , Bachelder Eric , Ainslie Kristy , Ting Jenny P.-Y.
Published: January 2023

Immunotherapies have dramatically improved cancer patient survival; however, certain forms of cancer still rely on conventional chemotherapies and radiation. We have designed a novel cancer treatment that utilizes an immunotherapeutic to augment the adaptive immune system. Many FDA-approved immunotherapies have several drawbacks including high cost, short half-life, and flu-like side effects leading to patient noncompliance. To overcome these difficulties, we have utilized pathogen-associated molecular patterns (PAMP) to stimulate an innate immune response and induce a potent cytokine response. However, the receptor for the PAMP is located in the cytosol, and the PAMP cannot transverse the cell membrane. To overcome this, we have designed a polymeric microparticle (MP) to deliver the PAMP (PAMP MPs) into the cytosol. The polymeric MP is composed of a dextran-based, biodegradable polymer. It has several critical advantages over other FDA-approved delivery vehicles, such as liposomes, including greater stability, higher encapsulation efficiency (EE) of hydrophilic cargo, ease and reproducibility of production, and ease of end point sterilization. Treatment with PAMP MPs resulted in reduced tumor burden compared to mice treated with the soluble PAMP in a model of melanoma, suggesting that the MPs increase the biological activity. Intratumoral treatment with PAMP MPs resulted in increased natural killer cells and professional antigen presenting cells in the tumor environment. In conclusion, our PAMP MPs resulted in significant anti-tumor efficacy in an aggressive murine model of melanoma, and the data suggest that this anti-tumor function is due to an increased immune response within the tumor.
Expression optimization and functional testing of a scorpion toxin/antibody fragment fusion protein, ACDClx, for the immunotherapeutic treatment of glioblastoma
Authors: Cook Rebecca , Diamos Andrew , Mason Hugh , Sirianni Rachael , Mor Tsafrir , Blattman Joseph
Published: January 2023

The prognosis for patients with glioblastoma (GBM), a deadly form of brain tumor with a median survival of <15 months, has not substantially improved since the 1970s. To overcome the myriad of treatment obstacles for GBM, we sought to design a new therapeutic capable of selectively enhancing T cell recognition and killing of all GBM cells while preserving healthy tissue. For this purpose, we have designed, expressed, and performed initial testing of the novel bispecific fusion protein, anti-CD3/chlorotoxin (ACDClx). ACDClx is a fusion of anti-CD3 heavy and light variable regions with a non-toxic, GBM-selective scorpion-derived peptide, chlorotoxin, which has been shown to bind to 100% of GBM cells with no selectivity for any healthy tissue types, including brain. ACDClx was expressed in leaves of N. benthamiana, purified, and tested for the capacity to selectively activate T cells. Initial experiments showed that ACDClx activates T cells, measured via calcium flux and CD69 expression, only if T cells, GBM cells, and ACDClx were all present. Mock purified protein in the presence GBM cells and T cells did not result in T cell activation, nor did ACDClx alone. Before further testing in vitro and in vivo, we sought to increase the production of ACDClx, the expression of which is significantly hampered due to its six disulfide bonds. To this end, we have produced 7 new constructs (5 for N. benthamiana, 2 for E. coli), 5 of which include a unique fusion partner for ACDClx to enhance solubility. Ongoing work includes evaluation of the most efficient construct, followed by evaluation in an immunocompetent tumor model of GBM.
Priming with percutaneous bacillus Calmette-Guerin (BCG) prior to intravesical BCG treatment safely improves BCG-specific response in patients with bladder cancer
Authors: Ji Niannian , Mukherjee Neelam , Morales Edwin E. , Tomasini Maggie E. , Hurez Vincent , Curiel Tyler J. , Abate Getahun , Hoft Daniel F. , Zhao Xiang-Ru , Sourindra Maiti , Cooper Laurence J.N. , Svatek Robert S.
Published: January 2023

Bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, responses to BCG are heterogeneous and limited options exist when BCG therapy fails. Preclinical data in animal models of bladder cancer (BC) suggests that priming with percutaneous BCG vaccine prior to intravesical BCG instillation can enhance BCG-specific immunity and improve outcome. To study the safety, immunogenicity and preliminary efficacy of this approach, we administered percutaneous BCG 21 days prior to intravesical BCG instillation in “prime patients” with NMIBC (n=13) in a prospective single-arm clinical trial. Immune responses and clinical outcomes were monitored and compared to a contemporaneous cohort of “control patients” (n=9) receiving intravesical BCG without prior percutaneous BCG. Priming was well tolerated and no grade ≥3 adverse events were observed. Compared to control, prime patients had improvement in both local and systemic measures of BCG-specific immunity, scored by increased post-BCG urinary IL-8 and IL-17A and increased circulating CD4, CD8, and γδ T cell proliferation and effector function in response to BCG. Furthermore, ex vivo cytotoxicity of circulating NK and γδ T cells against RT4 BC was significantly increased in prime patient after BCG treatment and was mediated in part by NKG2D, suggesting an important role of innate effector immune mechanisms underlying BCG’s antitumor activity. Remarkably, no prime patients progressed whereas 3 out of 9 control patients progressed to muscle-invasive disease and underwent cystectomy. Thus, BCG priming safely enhances BCG-specific immunity and could improve outcome for patients with NMIBC.
Agonist Redirected Checkpoint (ARC), TIM3-Fc-OX40L, for Cancer Immunotherapy
Authors: Fromm George , de Silva Suresh , Patel Arpita , Johannes Kellsey , Hornblower Josiah , Schreiber Taylor H.
Published: January 2023

Combination immunotherapy with bispecific abs, linked scFv’s or T cell engagers have not shown that both checkpoint blockade and TNFR activation can be achieved with a single molecule, likely due to a loss of target avidity. Fusion proteins incorporating the extracellular domain of type I membrane proteins (eg. Enbrel) or type II membrane proteins (eg. OX40L-Fc), linked via an antibody Fc domain, are both functional despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain.

The TIM3 end of the ARC binds GAL9 and phosphatidylserine (PS) in vitro and in vivo, and OX40L binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in the absence of Fc receptor cross-linking, stimulates IL2/TNFa secretion with the superantigen SEB, and increases the in vitro killing of GAL9+ tumor cells. In vivo, TIM3-Fc-OX40L stimulated expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells (+ova/alum). Live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L tethering T cells to tumor cells and eliciting cytotoxic activity (tumor killing). Finally, the therapeutic activity of TIM3-Fc-OX40L in established murine MC38, B16.F10 and CT26 tumors was superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. These data demonstrate feasibility of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is unique with this molecule compared to antibody approaches, since all TIM3 ligands can be targeted simultaneously.
Progress in the development of a cell culture system to study the immune responses of reef-building corals.
Authors: Garrison Keith E. , Kuo Catherine
Published: January 2023

Previous work in the laboratory has focused on using fragments of aquaculture-raised specimens of the stony coral Acropora and Montipora. We are interested in these species because Acropora has an expanded immune gene repertoire in comparison to the closely-related anemone species Nematostella, especially in the number of TLR and IL-1R genes. However, space restrictions and maintaining acceptable water chemistry parameters pose a challenge to working with samples of stony corals in this form. In addition, coral fragments are complex holobionts that contain a mixture of coral cells, photosynthetic zooxanthelle, and bacteria, all on a calcium carbonate skeletal matrix. We seek a simplified model system of coral cells growing in isolation to facilitate performing the virus isolation, propagation, and controlled infection experiments that we think are necessary to advance our understanding of coral disease and immune responses. A number of protocols exist to propagate coral cells in a mixture of artificial seawater and typical mammalian cell growth medium. We compared the results of mechanical versus enzymatic isolation of coral tissues from the underlying skeletal matrix. We performed microscopic assessments of the coral and endosymbiont cells immediately following isolation and again after one week in culture. The endosymbiont zooxanthelle are easily visually distinguishable, and appear to persist longer under our culture conditions than the coral cells. It is unclear whether this is due to an improved ability of the zooxanthelle to live independently, or increased susceptibility of the coral cells to pathogen infections not eliminated by antibiotics in the culture medium.
CD8+ single positive and CD4+CD8+ double positive cells constitute a significant proportion of the total lymphocytes in the anterior chamber of the normal porcine eye
Authors: Maslanka Tomasz , Socha Piotr , Dąbrowski Michał , Ziółkowska Natalia , Ziółkowski Hubert
Published: January 2023

This study investigated whether CD4− and CD8-expressing cells are present in the anterior chamber of the normal porcine eye. Intact porcine globes were obtained from local slaughterhouses. They did not show any abnormalities or signs of disease. The cornea was incised and then aqueous humor (AH) from both globes of the same animal was collected. The lymphocyte population in peripheral blood was gated on the basis of forward and side scatter properties. The positioning of this gate served as a reference for setting the lymphocyte gate in AH samples. The number of lymphocytes in the anterior chamber was extremely low; only about 5000 events in the lymphocyte gate were collected for the entire volume of AH samples. Single positive (SP) CD8+ cells constituted almost 40% of the total AH lymphocyte population. In contrast, the mean percentage of SP CD4+ cells in the lymphocyte pool was only 1.77. It is well known that the peripheral blood lymphocyte (PBL) population in pigs contains a large share of CD4+(high)CD8+(low) double positive (DP) cells. Likewise, a large contribution of these cells was found in the anterior chamber, namely their mean percentage was 17.45. However, in contrast to their PBL counterpart, the majority of AH DP cells exhibited low expression of CD4. The results suggest that the anterior chamber in swine is an element in the normal migratory pathway of SP CD4+, SP CD8+ and DP cells. However, the trafficking of these cells into this compartment seems to be relatively poor. Moreover, the study demonstrated a limited presence of SP CD4+ cells in the anterior chamber compared to SP CD8+ and DP cells. Such selective homing of SP CD8+ and CD4+ cells to the anterior chamber may be involved in the induction and maintenance of ocular immune privilege.
Classification the WC1 gene family in Sus scrofa and evaluation of individual SRCR domain affinity for Mycobacterium bovis and Leptospira spp.
Authors: Le Page Lauren Ann , Buck Jessica , Boisvert Natalie , Gillespie Alexandria , Hudgens Edward , Hsu Haoting , Baldwin Cynthia L. , Telfer Janice C.
Published: January 2023

Unlike αβ T cells, γδ T cells respond to a variety non-proteinaceous molecules independent of MHC presentation. γδ T cells are less susceptible to immune evasion, making them an attractive target for next generation vaccines, however, little is known about the best way to prime them. WC1, a member of the group B Scavenger Receptor Cysteine Rich (SRCR) superfamily, is expressed exclusively on γδ T cells in swine and ruminants. Previous work in our lab has determined that there are 13 genes encoding for WC1 in cattle. We have also shown that WC1 functions as hybrid co-receptor and pattern recognition receptor for the γδ TCR. WC1+ γδ T cells share a restriction in TCR gene usage, yet respond to different pathogens based on which WC1 molecule(s) they express. This can be attributed to the ability of the expressed WC1 molecule to recognize and directly bind whole pathogens via its SRCR domains. Because WC1 is expressed as a multigene array, we hypothesize that each WC1 gene has co-evolved with a different set of pathogens. Swine belong to the same order as cattle, Artiodactyl, and are susceptible to infection with many of the same pathogens. The current swine assembly contains two predicted WC1 proteins, neither of which had been confirmed with cDNA evidence. Prior to this study only one full-length cDNA transcript had been successfully amplified. Using 5′/3′ RACE PCR and RT-PCR, we have obtained full length cDNA transcripts for seven WC1 genes with the SRCR domain patterns of a1-[b-c-d-e-d′] or d1-[b-c-d-e-d′]. Through bacterial pull-down assays, we have shown that multiple SRCR domains from different swine WC1 genes bind to vaccine strain Leptospira spp, and freshly grown Pasteur and Danish strains of Mycobacterium bovis.
Evolutionary modification of the VLR-based adaptive immune system in jawless vertebrates: Functional implications
Authors: Das Sabyasachi , Li Jianxu , Hirano Masayuki , Rast Jonathan , Cooper Max D.
Published: January 2023

The three types of variable lymphocyte receptor genes, VLRA, VLRB and VLRC, in the extant jawless vertebrates, encode antigen receptors, the remarkably diverse repertoire of which is generated by insertion of neighboring leucine rich repeat (LRR) sequences into the incomplete germline genes. In both lampreys and hagfish, the B-cell like VLRB+ cells differentiate into VLRB-secreting plasma cells, whereas the αβ and γδ T cell-like VLRA+ and VLRC+ cells express their VLR products solely as cell surface proteins. However, in comparative studies of lampreys and hagfish, we find that remarkable functional differences have evolved in these lymphocyte lineages. In contrast with their striking predominance in lampreys, the VLRB+ cells constitute a minor lymphocyte population in hagfish, and the VLRC+ cells are predominate. Notably the germline VLRB gene in hagfish contains a short non-coding intervening sequence, whereas the VLRB genes in sea lampreys and Japanese lampreys have very long intervening sequences which contain multiple transposable elements that may influence VLRB expression. In keeping with the relative low numbers of hagfish VLRB+ cells, antibody responses to a model immunogen, sheep erythrocytes, are much less robust in hagfish than in lampreys. Thus, even though the fundamental genetic program for differentiation of two prototypic T-like lymphocyte lineages and one B-like lineage is conserved in both jawless and jawed vertebrates, the genetic programs used for fine tuning of the VLR-based immunity have undergone notable independent evolutionary changes in lampreys and hagfish over the past ~480 million years.
Probiotic microbes evoke different responses from bovine BAL in vitro
Authors: Eicher Susan D. , Chitko-McKown Carol G. , Bryan Keith A.
Published: January 2023

Probiotics are used for enteric health. How they interact with upper respiratory immune cells is not known. We aimed to determine how microbes affect functions and markers of bronchoalveolar lung lavage cells (BAL). BAL were isolated from lungs of calves at slaughter. Treatments were a Lactobacillus probiotic (US), a commercially available probiotic (PB), Lactobacillus animalis (LA-51), Propionibacterium freudenreichii (PF-24), Enterococcus faecium (CH-212), E. faecium (SF-273), E. faecium (M-74), Bifidobacterium animalis ssp lactis (BB-12), Bacillus subtilis (EB-15 ), and Bacillus amyloliquifaciens (ZM-16). Microbial cells and BAL were incubated 1:1 for 1 h at 37° C and 5% CO2, then control (CNT), CD14 (FITC) and CD205 (RPE), CD18 (Alexa Fluor 647), and E. coli bioparticles (Molecular Probes) were added. Differences = P ≤ 0.05. Mean fluorescence of phagocytosis of E. coli bioparticles was less than CNT for BB-12 and EB-15, but percentage (%) of cells phagocytizing was not different. Oxidative burst of M-74 was less than CH-212, BB-12, EB-15, PB, and US, but none were different than CNT. The % of cells with oxidative burst was greater for PF-24 than for LA-51, BB-12, ZM-16, PB, and CNT. CD 14 mean fluorescence was least for M-74 compared with all other microbes and CNT, but % of cells expressing CD14 was greatest on PF-24 and US compared with EB-15 and BB-12, but not CNT. DC-205 mean fluorescence was greatest for M-74 compared with LA-51, PF-24, CH-212, SF-273, CNT, and US. Percentage of cell expressing DC-205 and CD18 was not different from controls. PF-24 and M-74 were most immunomodulatory compared with controls. M-74 appeared to suppress and PF-24 to increase leukocyte functions, showing that probiotic bacteria differentially modulate BAL.
Decoding the Feline Leukocyte Antigen MHC class I system via SMRT sequencing
Authors: Hess Paul R. , Holmes Jennifer C. , Dickey Allison N. , Scholl Elizabeth H. , Thorne Jeffrey L.
Published: January 2023

CD8+ T-cell cytotoxicity is an important component of anti-viral immune responses and is restricted by MHC class I peptide presentation. Investigating virus-specific CTL in cats is impeded by the rudimentary understanding of the Feline Leukocyte Antigen class I (FLAI) system. As polygeny and polymorphisms are inherent MHC features, defining functional loci and cataloging alleles are the first steps in characterizing FLAI. Additionally, identification of prevalent alleles will permit discovery of immunodominant CTL responses. Previously, using conventional sequencing of class I hypervariable regions, we defined three class Ia loci, FLA-E, -H and -K, and found allele sharing, even in our small sample of cats. Because of high FLAI homology, partial-length sequences did not allow unambiguous assignment of all alleles to their originating locus. We hypothesized that the PacBio SMRT sequencing platform would allow us to efficiently obtain full-length genotyping of all class Ia loci in multi-cat cohorts. Consensus building of FLAI contigs provided complete MHC sequences with high accuracy, greater depth and sensitivity, and in a fraction of the time of traditional cloning and Sanger techniques. In sequencing 17 cats (including 2 reference individuals), we clarified locus assignments, identified >40 novel classical class I alleles and discovered several common allele supergroups (FLA-E*003, -E*009, -K*002, and -K*003). Also, for the first time, we confirmed the identify and expression of 2 class Ib loci (FLA-J, -L). This work brings the current total of known FLAI alleles to ~100 across 3 class Ia and ~35 across 2 class Ib loci. This new dataset should accelerate feline anti-viral research and facilitate allotransplantation in cats.
Development and Characterization of Chicken CD127-Specific Antibodies
Authors: Sun Zhifeng , Zhao Hongyan , Kim Woohyun , Panebra Alfredo , Lillehoj Hyun S. , Xianyu Zhezi , Gu Changqin , Yan Xianghe , Li Charles
Published: January 2023

Research in avian immunology has been significantly hampered by lack of effective immunological reagents in birds cross-reactive with mammalian orthologs and lack of sensitive assays for a long time. To better serve the avian immunology community, monoclonal and polyclonal antibodies specific for avian molecules of Clusters of Differentiation (CD) are being developed to promote thorough research on avian infection and immunity. In this study, chicken CD127 antibodies were developed and characterized. CD127, also named interleukin-7 receptor (IL-7R), is expressed on various cell types, including naive and memory T cells as well as many other types of immune cells, and plays a critical role in differentiation and activation of T lymphocytes. An extracellular region of chicken CD127 gene of 678 bp was cloned in pET28a(+) vector and expressed in BL21-AI™ E. coli. The expressed CD127 protein was around 30 kDa and crossed-reacted with monoclonal antibodies specific for human CD127 (Clone G-11). The protein was then immunized in BALB/c mice. After 3 immunizations, murine sera were found to detect a protein of 30 kDa expressed in E. coli, and react to the protein expressed on chicken T cell line (CU205) by flow cytometry as well. Flow cytometric analysis of cells from two-week-old chickens showed that CD127-positive populations were detected in spleen and thymus with such murine sera as polyclonal antibodies, and detected in thymus when using anti-human Clone G-11. Availability of these antibodies for CD127 and other CD molecules will dramatically facilitate the commercialization of poultry immune reagents, greatly benefit the avian immunology community and promote poultry health and production.
Immunomediator expression profiling in two beluga whale (Delphinapterus leucas) clinical cases
Authors: Hofstetter Amelia R. , Van Bonn William , Sacco Randy E.
Published: January 2023

Cytokines and other immunomediators can be biomarkers of inflammation. Quantitative real-time PCR (qPCR) has been used to examine cytokine gene expression in beluga whale (Delphinapterus leucas) peripheral blood mononuclear cells (PBMC). Thus, qPCR-based immunomediator assays could supplement clinical data from routine blood draws, revealing altered immune status. We compared immunomediator expression between beluga whales facing immune challenges and healthy companions at two aquaria. In both case studies, mitogens stimulated increased IL-1β, Cox2 and TNF-α message in PBMC from the abnormal whale over the control, but decreased IL-10 mRNA. This supports further elucidation of beluga PBMC immunomediator profiles for use as immune biomarkers.
Bioactive lipids serve as biomarkers of host immune response during severe influenza infection in pediatric patients
Authors: Anania Veronica G. , Randolph Adrienne , McBride Jacqueline M. , Yang Xiaoying , Rosenberger Carrie , Mathews Rodney
Published: January 2023

Influenza virus infects 5–20% of the population annually, leading to considerable morbidity and mortality especially in children. Robust biomarkers, reflecting the host immune response, are needed to identify patients at risk of prolonged illness or death. Eicosanoids, a family of bioactive arachidonic acid metabolites produced by the host in response to influenza infection, have pro- and anti-inflammatory properties. This study aimed to assess the relationship between the host lipid response at ICU admission with clinical outcomes in a multicenter cohort of 105 children with influenza-related illness (PICFLU study). We developed a mass spectrometry-based method to quantify >100 lipid metabolites from nasopharyngeal swabs and endotracheal aspirates. The bioactive lipid profile in influenza infected children was impacted by multiple factors including patient age and bacterial coinfection. Patients coinfected with methicillin-resistant Staphylococcus aureus and influenza had significantly elevated levels of pro-inflammatory lipids and concomitantly lower levels of anti-inflammatory 15-lipoxygenase metabolites. Interestingly, children that presented with high levels of lipids including prostaglandin E2 and 12-hydroxyeicosatetraenoic acid were more likely to have fatal outcomes despite reports indicating these lipids have opposing immunomodulatory activity. These results suggest children at risk of mortality have an amplified bioactive lipid immune response that may negatively impact disease resolution. Our study provides evidence that bioactive lipids are potential prognostic biomarkers of patient immune response phenotype, and may help to identify children at risk of influenza-related death.
Toll-like receptor signalling protects against IL-33 led virus-induced asthmatic response in a Type I Interferon independent manner
Authors: Wali Shradha , Flores Jose R. , Goldblatt David , Tuvim Michael J. , Dickey Burton F. , Evans Scott E.
Published: January 2023

Respiratory viruses are frequent causes of asthma development and exacerbations. However, there are gaps in understanding how viral infections cause immuno-pathology in the host leading to asthma progression. We have previously shown that mice treated with a combination of Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are protected against broad range of respiratory infections induced by viruses, fungi and bacteria. Pam2-ODN treatment one day prior to virus infection leads to enhanced survival with reduced lung viral burden in mice. Further, Pam2-ODN pretreatment also inhibits acute lung pathology caused by CD8+ T cells and prevents chronic asthmatic phenotype of eosinophilia, airway hyperreactivity and mucus metaplasia. This protective effect was accompanied by reduced virus-driven lung IL-33, which is necessary to induce chronic asthmatic phenotype. Interestingly, Pam2-ODN does not induce increased production of Type I or III Interferons (IFN), nor does the protective response require intact interferon signaling. Transcriptome profiling of the mouse lungs in the chronic phase of infection led to identification of genes involved in airway remodelling, epithelial cell differentiation and chemokine signalling. More importantly, we identified genes from the Pam2-ODN treatment group that are involved in reversing the viral infection effect on day 49. Taken together, our findings reveal novel type I IFN independent mechanisms to prevent virus-induced asthma, and may provide an opportunity to protect vulnerable populations.
Induction and isolation of protective influenza neuraminidase specific antibodies in humans following seasonal vaccination.
Authors: Kobie James , Piepenbrink Michael , Nogales Aitor , Martinez-Sobrido Luis
Published: January 2023

Influenza’s propensity for antigenic drift and shift, and to elicit predominantly strain specific antibodies (Abs) leaves humanity susceptible to waves of new strains with pandemic potential for which limited or no immunity may exist. Subsequently new clinical interventions are needed, particularly those with broad activity against diverse strains. Although hemagglutinin (HA) specificity dominates the humoral response to seasonal inactivated influenza vaccines and infection, Abs targeting neuraminidase (NA) are also generated. NA-specific Abs are suggested to act primarily through inhibiting its enzymatic activity and preventing release of virus from infected cells. Relative to HA, there is substantially less diversity among NA from different influenza types and subtypes, suggesting it is a valuable target for inducing broad protective immunity. To determine if humans generate NA-specific Abs with protective activity we examined plasmablasts from subjects that were immunized with the seasonal influenza inactivated vaccine, and isolated several NA-specific human monoclonal Abs (hmAbs), including those with potent in vitro viral neutralizing activity. One of the NA B–specific hmAbs, KPF2 recognized NA from both the Yamagata and Victoria lineages, and when administered prophylactically to mice resulted in sterilizing immunity. These results suggest seasonal influenza vaccine induces protective NA-specific mAbs in humans. Analysis of other NA-specific hmAbs is ongoing.
Viral MHC class I inhibition evades protective tissue-resident memory CD8+ T cells (TRM) and influences TRM immunodominance within specific niches
Authors: Lauron Elvin James , Yang Liping , Bern Michael D. , Williams Graham D. , Boon Adrianus C.M. , Yokoyama Wayne M.
Published: January 2023

Tissue-resident memory CD8+ T cells (TRM) confer rapid protection and immunity against viral infections. Many viruses have evolved mechanisms to inhibit MHCI presentation in order to evade effector CD8+ T cells, suggesting that these mechanisms may also apply to TRM-mediated protection. However, the effects of viral MHCI inhibition on the function and generation of TRM is unknown. We show that viral MHCI inhibition dampens immunodominance within the lung TRM pool, indicating an effect on the TRM repertoire, but this effect was not observed in the skin. In the presence of viral MHCI inhibition, the overall development of TRM is unaffected, yet is unexpectedly enhanced with local cognate antigen, while local cognate antigen is not required for TRM maintenance. Moreover, viral MHCI inhibition efficiently evades TRM effector functions. These findings suggest that vaccine strategies involving pure T-cell approaches may provide poor efficacy against viruses that target the MHCI pathway.