Intracellular Antigens Research Articles

Autoimmune Mechanisms in Focal Epilepsy of Unknown Cause

ABSTRACT The manifestation of immunological findings in diverse disorders presenting with seizures points to autoimmunity and inflammation in the etiology of epilepsy. Typical examples of autoimmunity-associated epilepsy are autoimmune encephalitis, Rasmussen encephalitis, and glutamic acid decarboxylase (GAD) antibody-positive temporal lobe epilepsy (TLE). The last entity is a typical example of antibody-positive focal epilepsy of unknown cause (FEUC). One of the most prominent findings emphasizing the coexistence of epilepsy and autoimmunity is the detection of anti-neuronal antibodies in patients manifesting with seizures. Emergence of antibody-producing plasma cells in the early course of GAD-antibody-positive TLE and induction of seizures in rodents upon intracerebral administration of N-methyl-D-aspartate receptor antibodies indicate that anti-neuronal antibodies may play a causal (rather than bystander) role in the induction of seizures. By contrast, innate immunity of the central nervous system (CNS) and infiltrating cytotoxic T-cells appear to participate in Rasmussen encephalitis and autoimmune encephalitis with antibodies to intracellular antigens. In addition, repetitive seizures may activate glial cells through the release of damage-associate molecular pattern mediators and activation of toll-like receptors, which in turn leads to disrupted blood–brain barrier and increased cerebral infiltration of peripheral blood immune cells. In conclusion, complex interactions of humoral and cellular immunity in the CNS appear to cause or at least contribute to seizure induction in FEUC. The nature of these interactions has recently started to be understood. Investigation of these mechanisms is substantial for the discovery of new treatment strategies and biomarkers in epilepsy.

Comparative Transcriptional Signature Analysis of Peripheral Blood Mononuclear Cells in Early Stage of Hepatitis B-related Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is a prevalent and life-threatening tumor with high morbidity and mortality. Proper prediction and prognosis are incredibly stressed to diagnose HCC and increase patient survival. Objectives: This research aims to evaluate gene expression levels of pre-differentiated transcripts for those suffering from chronic hepatitis B (CHB) and HCC. Methods: To examine the previously analyzed peripheral blood mononuclear cells (PBMCs) transcriptomic array data, we selected seven differentially expressed genes (DEGs) in normal versus CHB and CHB versus HCC (CD44, SP3, USP8, E2F2, UFM1, IFN regulative factor binding protein 2 (IRF2BP2), and T-cell intracellular antigen 1 (TIA1)). The study included individuals with treatment-naïve CHB (n = 30) and primary HCC (n = 25) and healthy controls (n = 15). Subsequently, the expression of genes was assayed using qRT-PCR. A phylogenetic evaluation was performed using direct sequencing of HBsAg. Results: In HCC patients, 60% (n = 15) were HBeAg-positive. HBeAg was negative in all CHB patients, but all were anti-HBe-positive. The hepatitis B virus (HBV) load of HCC patients was more than that of CHB subjects. All patients were of the Iranian race and HBV D genotype. The expression of five transcriptional markers (CD44, SP3, USP8, E2F2, and UFM1) was higher in HCC patients than in CHB and healthy subjects, which was similar to the initial microarray data analysis. Conclusions: Transcriptional signatures may be related to the pathogenesis of HCC and used as diagnostic biological markers for the initial monitoring and prediction of HCC.

Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach.

Antibodies that bind peptide-MHC (pMHC) complex in a manner akin to T cell receptor (TCR) have not only helped in understanding the mechanism of TCR-pMHC interactions in the context of T cell biology but also spurred considerable interest in recent years as potential cancer therapeutics. Traditional methods to generate such antibodies using hybridoma and B cell sorting technologies are sometimes inadequate, possibly due to the small contribution of peptide to the overall B cell epitope space on the surface of the pMHC complex (typical peptide MW=1kDa versus MHC MW=45kDa) and to the multiple efficiency limiting steps inherent in these methods. In this chapter we describe phage display approaches, including a cell panning strategy, for the rapid generation of such antibodies with high specificity and affinity.

Immunopotentiation effects of apigenin on NK cell proliferation and killing pancreatic cancer cells.

Apigenin is a kind of flavonoid with many beneficial biological effects. It not only has direct cytotoxicity to tumor cells, but also can boost the antitumor effect of immune cells by modulating immune system. The purpose of this study was to investigate the proliferation of NK cells treated with apigenin and its cytotoxicity to pancreatic cancer cells in vitro, and explore its potential molecular mechanism. In this study, the effect of apigenin on NK cell proliferation and killing pancreatic cancer cells were measured by CCK-8 assay. Perforin, granzyme B (Gran B), CD107a, and NKG2D expressions of NK cells induced with apigenin were detected by flow cytometry (FCM). The mRNA expression of Bcl-2, Bax and protein expression of Bcl-2, Bax, p-ERK, and p-JNK in NK cells were evaluated by qRT-PCR and western blotting analysis, respectively. The results showed that appropriate concentration of apigenin could significantly promote the proliferation of NK cells in vitro and enhance the killing activity of NK cells against pancreatic cancer cells. The expressions of surface antigen NKG2D and intracellular antigen perforin and Gran B of NK cells were upregulated after treating with apigenin. Bcl-2 mRNA expression was increased, while Bax mRNA expression was decreased. Similarly, the expression of Bcl-2, p-JNK, and p-ERK protein was upregulated, and the expression of Bax protein was downregulated. The molecular mechanism of the immunopotentiation effects of apigenin may be that it up-regulates Bcl-2 and down-regulates Bax expression at the gene and protein levels to facilitate NK cell proliferation, and up-regulates the expression of perforin, Gran B, and NKG2D through the activation of JNK and ERK pathways to enhance NK cell cytotoxicity.

Frequency of Respiratory Syncytial Virus Among Hospitalized Children with Acute Lower Respiratory Tract Infections

Objective: To determine the frequency of respiratory syncytial virus (RSV) among hospitalized children with acute lower respiratory tract infections (ALRI) and associated risk factors.
 Study Design: Comparative cross-sectional study.
 Place and Duration of Study: Pak Emirates Military Hospital, Rawalpindi Pakistan, from Jul 2019 to Aug 2020.
 Methodology: After approval from the Ethical Review Board, 126 children were included in the study. Nasopharyngeal aspirates were collected from children. The direct immunofluorescence method was utilized to detect intracellular viral antigens of the respiratory syncytial virus. In addition, the presence of symptoms and history for identification of the presence or absence of risk factors was recorded.
 Results: 57(45.2%) patients were found to be respiratory syncytial virus positive, and 69(54.8%) were respiratory syncytial virus negative. Significant differences were found in symptoms between RSV-positive and negative children (p=0.001).
 Conclusion: Respiratory syncytial virus was among the leading causes of children hospitalized with acute lower respiratory tract infections, with fever and recurrent wheezing as the most common symptoms.

Seizures, Epilepsy, and NORSE Secondary to Autoimmune Encephalitis: A Practical Guide for Clinicians.

The most recent International League Against Epilepsy (ILAE) classification has included "immune etiology" along with other well-known causes of epilepsy. This was possible thanks to the progress in detection of pathogenic neural antibodies (Abs) in a subset of patients, and resulted in an increased interest in identifying potentially treatable causes of otherwise refractory seizures. Most autoimmune encephalitides (AE) present with seizures, but only a minority of cases evolve to long-term epilepsy. The risk of epilepsy is higher for patients harboring Abs targeting intracellular antigens (T cell-mediated and mostly paraneoplastic, such as Hu, CV2/CRMP5, Ma2, GAD65 Abs), compared with patients with neuronal surface Abs (antibody-mediated and less frequently paraneoplastic, such as NMDAR, GABAbR, LGI1, CASPR2 Abs). To consider these aspects, conceptual definitions for two entities were provided: acute symptomatic seizures secondary to AE, and autoimmune-associated epilepsy, which reflect the different pathophysiology and prognoses. Through this manuscript, we provide an up-to-date review on the current state of knowledge concerning diagnosis and management of patients with Ab-mediated encephalitis and associated epilepsy. Special emphasis is placed on clinical aspects, such as brain magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) specificities, electroencephalographic (EEG) findings, cancer screening and suggestions for a rational therapeutic approach.

Neuronal Uptake of Paraneoplastic and Other IgGs is Mediated by the Fc Portion of the IgG Molecule and Involves Previously Uncharacterized Neuronal FcγRI Receptors: Implications for Antibody-Mediated Neuronal Injury

Objective To investigate the mechanisms by which neurons take up paraneoplastic and other antibodies. Background Our laboratory has previously demonstrated that neurons can take up both normal and paraneoplastic IgGs and that paraneoplastic autoantibodies such as anti-Yo and anti-Hu can bind to their intracellular target antigens to produce neuronal death. In this study we investigated how neuronal antibody uptake occurs. Design/Methods We first compared neuronal uptake of normal and paraneoplastic Fab fragments with that of normal IgG Fc fragments or whole paraneoplastic IgGs. To determine whether neurons expressed receptors capable of binding the Fc portion of the IgG molecule, paraformaldehyde-fixed mouse and rat brains sections were probed with antibodies for the three major types of Fc receptors: FcγRI (CD64), FcγRII, (CD32) and FcγRIII (CD16). Neuronal uptake of antineuronal IgGs was compared between wild type mice and knockout mice lacking the FcγRI receptor. We also investigated whether neuronal IgG uptake could be blocked by normal IgG. Results Neurons incorporated the Fc fragment of normal IgG but not the Fab fragment. Intact paraneoplastic IgGs were taken up by neurons, but immunospecific Fab fragments were excluded. Neurons throughout cerebrum, cerebellum, and brainstem showed immunolabeling for FcγRI, but only rare neurons expressed FcγRII or FcγRIII. Uptake of paraneoplastic IgG and neuronal death were not observed in cultures from FcγRI knockout mice but were extensive in cultures from wild type controls. Paraneoplastic antibody uptake could be inhibited by normal IgG. Conclusions Neuronal uptake of normal and paraneoplastic IgGs requires the interaction of the Fc portion of the IgG molecule with previously uncharacterized neuronal FcγRI receptors. Our study provides a mechanism through which antibodies reactive with intracellular neuronal proteins could gain access to their target antigens to cause neuronal injury and neurological disease. The observation that neuronal antibody uptake can be blocked by normal IgG has possible implications for patient treatment.

The splicing regulators TIA1 and TIAL1 are required for the expression of the DNA damage repair machinery during B cell lymphopoiesis.

B cell lymphopoiesis requires dynamic modulation of the B cell transcriptome for timely coordination of somatic mutagenesis and DNA repair in progenitor B (pro-B) cells. Here, we show that, in pro-B cells, the RNA-binding proteins Tcell intracellular antigen 1 (TIA1) and TIA1-like protein (TIAL1) act redundantly to enable developmental progression. They are global splicing regulators that control the expression of hundreds of mRNAs, including those involved in DNA damage repair. Mechanistically, TIA1 and TIAL1 bind to 5' splice sites for exon definition, splicing, and expression of DNA damage sensors, such as Chek2 and Rif1. In their absence, pro-B cells show exacerbated DNA damage, altered P53 expression, and increased cell death. Our study uncovers the importance of tight regulation of RNA splicing by TIA1 and TIAL1 for the expression of integrative transcriptional programs that control DNA damage sensing and repair during B cell development.

Major histocompatibility complex I-induced endoplasmic reticulum stress mediates the secretion of pro-inflammatory muscle-derived cytokines.

Major histocompatibility complex (MHC) I is an important component of intracellular antigen presentation. However, improper expression of MHC I upon the cell surface has been associated with several autoimmune diseases. Myositis is a rare acquired autoimmune disease which targets skeletal muscle, and MHC I overexpression on the surface of muscle fibres and immune cell infiltration are clinical hallmarks. MHC I overexpression may have an important pathogenic role, mediated by the activation of the endoplasmic reticulum (ER) stress response. Given the evidence that muscle is a diverse source of cytokines, we aimed to investigate whether MHC I overexpression can modify the profile of muscle-derived cytokines and what role the ER stress pathway may play. Using C2C12 myoblasts we overexpressed MHC I with a H-2kb vector in the presence or absence of salubrinal an ER stress pathway modifying compound. MHC I overexpression induced ER stress pathway activation and elevated cytokine gene expression. MHC I overexpression caused significant release of cytokines and chemokines, which was attenuated in the presence of salubrinal. Conditioned media from MHC I overexpressing cells induced in vitro T-cell chemotaxis, atrophy of healthy myotubes and modified mitochondrial function, features which were attenuated in the presence of salubrinal. Collectively, these data suggest that MHC I overexpression can induce pro-inflammatory cytokine/chemokine release from C2C12 myoblasts, a process which appears to be mediated in-part by the ER stress pathway.

Human leukocyte antigen system associations in Malassezia-related skin diseases.

The human leukocyte antigen system (HLA) is divided into two classes involved in antigen presentation: class I presenting intracellular antigens and class II presenting extracellular antigens. While susceptibility to infections is correlated with the HLA system, data on associations between HLA genotypes and Malassezia-related skin diseases (MRSD) are lacking. Thus, the objective of this study was to investigate associations between HLA alleles and MRSD. Participants in The Danish Blood Donor Study (2010-2018) provided questionnaire data on life style, anthropometric measures, and registry data on filled prescriptions. Genotyping was done using Illumina Infinium Global Screening Array, and HLA alleles were imputed using the HIBAG algorithm. Cases and controls were defined using filled prescriptions on topical ketoconazole 2% as a proxy of MRSD. Logistic regressions assessed associations between HLA alleles and MRSD adjusted for confounders and Bonferroni corrected for multiple tests. A total of 9455 participants were considered MRSD cases and 24,144 participants as controls. We identified four risk alleles B*57:01, OR 1.19 (95% CI: 1.09-1.31), C*01:02, OR 1.19 (95% CI: 1.08-1.32), C*06:02, OR 1.14 (95% CI: 1.08-1.22), and DRB1*01:01, OR 1.10 (95% CI: 1.04-1.17), and two protective alleles, DQB1*02:01, OR 0.89 (95% CI: 0.85-0.94), and DRB1*03:01, OR 0.89 (95% CI: 0.85-0.94). Five novel associations between HLA alleles and MRSD were identified in our cohort, and one previous association was confirmed. Future studies should assess the correlation between Malassezia antigens and antigen-binding properties of the associated HLA alleles.

Inhibiting Activated Protein C Cleavage of Factor VIII for Hemophilia a Gene Therapy

Background Despite progress in adeno-associated virus (AAV) mediated factor VIII (FVIII) gene transfer for hemophilia A (HA), durable and sustained FVIII expression sufficient to ameliorate bleeding in all participants is an unrealized goal. Following the success of using the gain of function FIX-Padua variant in hemophilia B gene therapy, we hypothesized that use of an enhanced hemostatic function FVIII would similarly improve HA gene therapy by: 1) permitting use of lower vector doses, thereby overcoming dose-dependent safety and efficacy limitations and 2) achieving efficacy with lower FVIII antigen. We previously published a FVIII variant resistant to activated protein C (APC) cleavage (FVIII-R336Q/R562Q or FVIII-QQ) with normal specific activity and 4-5 fold improved hemostatic function in recombinant protein experiments (Wilhelm et al. Blood 2021). Aims We evaluated the possible clinical translation of FVIII-QQ for HA AAV-mediated gene therapy by determining the hemostatic benefit and potential prothrombotic and immunological risks in mice. Additionally, FVIII-QQ steady-state expression was 2.5 to 5-fold higher than FVIII-WT. We hypothesized this was due to disrupted cleavage by APC or other serine proteases at R336 and/or R562. Results Survival studies in HA/CD4KO and WT/CD4KO mice treated with AAV vectors to express (h)uman FVIII-WT and hFVIII-QQ in the range of mild HA, normal, and supratherapeutic FVIII levels thus far demonstrate no significant differences in survival at 7-11 month follow up or D-dimer levels after 8 months. Additionally, FVIII-QQ mice were generated using CRISPR/Cas9 to introduce R562Q (WT mice are Q336) for endogenous (m)ouse FVIII-QQ expression. Endogenous mFVIII-QQ mice were viable, fertile, normal weight, and D-dimers did not significantly differ from WT mice, while FVIII activity was ~2-fold higher than WT mice. HA immune competent mice treated with either 6 weekly doses of hFVIII-WT or hFVIII-QQ protein (0.2 μg) or AAV vector to express hFVIII-QQ or hFVIII-WT had no significant differences in inhibitor titers. To evaluate hemostasis, HA/CD4KO mice were treated with AAV vector only differing by 2 amino acids (R336, R362) at 3 different doses (n=6-12 mice/cohort) to express hFVIII-QQ or hFVIII-WT and underwent tail clip challenge. Estimated EC50 and EC80 of vector doses required to normalize blood loss to WT mice were 5 and 10-fold lower, respectively, for hFVIII-QQ versus hFVIII-WT supporting a hemostatic benefit to using the hFVIII-QQ transgene. Interestingly, across 3 different vector constructs differing only by F8 codon optimization, we observed hFVIII-QQ antigen values 2.5 to 5-fold higher than in mice expressing hFVIII-WT. Analysis of liver tissue from HA/CD4KO mice treated with the same vector construct and dose to express hFVIII-WT or hFVIII-QQ demonstrated approximately the same mRNA values, but plasma hFVIII-QQ antigen was 4-fold higher than hFVIII-WT and intracellular hFVIII-QQ antigen was the same or lower than hFVIII-WT antigen. Consistent with our in vivo AAV experiments, preliminary studies of intracellular versus secreted hFVIII antigen in BHK stable cell lines expressing hFVIII-WT or hFVIII-QQ demonstrated similar mRNA values with higher relative secreted hFVIII-QQ protein. To further probe the mechanism of higher hFVIII-QQ steady-state expression, we evaluated a panel of serine proteases (APC, FIXa, FXa, plasmin, PACE/furin and thrombin) for cleavage of FVIII-QQ relative to FVIII-WT protein. As expected, hFVIII-QQ was resistant to APC cleavage. Surprisingly, all other serine proteases tested demonstrated cleavage of hFVIII-WT, but not hFVIII-QQ, at R336 and/or R562 suggesting protease attack at these cleavage sites may occur more than previously appreciated, though the physiologic significance remains to be determined. Conclusion Our studies outline the use of hFVIII-QQ transgene expression thus far does not demonstrate safety concerns and confers hemostatic benefit over hFVIII-WT via: 1) improved hemostatic function and 2) higher steady-state expression. Enhanced steady state hFVIII-QQ expression relative to hFVIII-WT was observed across multiple model systems. Preliminary data demonstrate multiple serine proteases can cleave at the described FVIII APC cleavage sites (R336, R562) suggesting hFVIII-QQ may generally be more proteolytically stable thereby permitting improved steady-state expression.

Generation of antigen-specific T lymphocytes targeting Wilms tumor 1 using activated B cells

In order to develop a therapeutic target for T cells, it is necessary to amplify T cells and increase activity through antigen-presenting cells (APCs) expressing an intracellular cancer antigen. Although dendritic cells are frequently used as APCs, producing dendritic cells is costly and time-consuming. In addition, as dendritic cells are attached cells, they are not suitable for mass production for use as immune cell therapy. On the other hand, B cells are non-adherent floating cells, and thus can easily be cultured in suspension systems. As such, B cells can be considered as suitable substance cells for the development of immune cell therapeutics.B cells lack the antigen-presenting ability of dendritic cells. Therefore, to use B cells as APCs, we previously reported a technology that can be used which simply and effectively produces anti-viral T cells in vitro by activating B cells with α-galactosylceramide (α-GalCer). To apply this technology to anti-cancer treatment, Wilms tumor 1, the most representative cancer antigen expressed in various cancers, was selected. Wilms tumor 1 (WT1) was used to produce anti-cancer (anti-WT1) T cells using active B cells as APCs, and their respective activities were investigated.

A tetrahedral framework nucleic acid based multifunctional nanocapsule for tumor prophylactic mRNA vaccination

Synthetic antigen-encoding mRNA plays an increasingly significant role in tumor vaccine technology owing to its antigen-specific immune-activation. However, its immune efficacy is challenged by inferior delivery efficiency and demand for suitable adjuvants. Here, we develop a novel mRNA nanovaccine based on a multifunctional nanocapsule, which is a dual-adjuvant formulation composed of cytosine-phosphate-guanine motifs loaded tetrahedral framework nucleic acid (CpG-tFNA) and an immunopeptide murine β-defensin 2 (mDF2β). This mRNA nanovaccine successfully achieves intracellular delivery, antigen expression and presentation of dendritic cells, and proliferation of antigen-specific T cells. In a tumor prophylactic vaccination model, it exerts an excellent inhibitory effect on lymphoma occurrence through cellular immunity. This mRNA nanovaccine has promising prophylactic applications in tumors and many other diseases.

Chimeric antigen receptor T-cell therapy targeting a MAGE A4 peptide and HLA-A*02:01 complex for unresectable advanced or recurrent solid cancer: protocol for a multi-institutional phase 1 clinical trial

IntroductionAdoptive cell transfer of genetically engineered T cells is a promising treatment for malignancies; however, there are few ideal cancer antigens expressed on the cell surface, and the development of...

Modern breast cancer therapy: from tamoxifen to T-cell engineering

The purpose of the study was to conduct a systematic literature review of high-technology methods in breast cancer treatment. Material and methods. To select information sources, a global search was used using the Web of Science, Scopus, PubMed, and RSCI databases. The search included the analysis of metadata by keywords, and relevant publications were used for full-text search. The review used 55 publications from 2001 to 2021. Most of the articles were published over the past 7 years. Results. Modern literature data presented in this review prove that long-term studies based on histological and immunological features of tumor development are very important for improving survival in breast cancer. Clinical treatment protocols that were based primarily on the anatomical characteristics of the disease are now switching to the biological mechanisms underlying carcinogenesis. Drugs targeting estrogen receptors play an important role in systemic therapy and make it possible to correct the mechanisms responsible for endocrine resistance. Targeted therapy targeting the HER2 receptor, especially in an antibody-drug conjugate combination, has associated cytotoxic therapy with anti-HER2 antibodies. Modern methods of biological therapy and cell engineering make it possible to develop methods for treating triple-negative breast cancer based on the regulation of the microenvironment, mechanisms of repair, immunosuppression, and the creation of a target from a larger repertoire of both surface and intracellular antigens. Conclusion. Promising strategies based on the use of signaling and metabolic pathways, cell surface molecules, and cell engineering increase the effectiveness of treatment and improve the progression-free and overall survival in breast cancer patients.

Nanobody-based CAR T cells targeting intracellular tumor antigens

Chimeric antigen receptor (CAR) T-cell immunotherapy has become one of the research hotspots in the treatment of malignant tumors nowadays. However, the available tumor surface antigens are limited in number. Most tumor-associated antigens are intracellular molecules that can’t be targeted by conventional CAR T cells. As the major histocompatibility complex (MHC)/peptide complex is a presentation form of intracellular proteins on the surface of tumor cells, here, we chose the Glypican-3 (GPC3) oncoprotein and Wilms tumor 1 (WT1) oncoprotein as examples to explore whether nanobody (Nb)-based T cell receptor (TCR)-like CAR T cells could kill tumor cells by targeting the MHC/peptide complexes. Using the immune nanobody phage display library, we developed human leukocyte antigen (HLA)-A2/GPC3- and HLA-A2/WT1-specific nanobodies for the first time and then incorporated these nanobodies in two TCR-like CARs, targeting HLA-A2/GPC3 and HLA-A2/WT1 respectively. These TCR-like Nb CAR-redirected T cells could selectively recognize and lyse MHC/peptide complex-expressing tumor cells in vitro assays and subcutaneous mouse tumor models. This study offers a possible strategy for targeting intracellular antigens and widening the application of CAR T-cell therapy.

USA EXPERIENCE: IN-HOUSE PREPARATION: PROSPECTS AND PROBLEMS

Adoptive Cellular Therapy (ACT) is transitioning from experimental to standard of care. Limited specificities of chimeric antigen receptor T cells (CAR-T) are licensed drugs, with commercial products selling for ∼400,000 USD. Tumor infiltrating lymphocytes (TIL) can target multiple cell surface and intracellular tumor antigens. TIL have not been commercialized, due to complex logistics and cost. Our objective is to leverage a single standardized platform (Miltenyi Prodigy) for in-house ACT. We currently manufacture CD19 CAR-T under an FDA IND and are developing a virtually identical TIL protocol minus the lentiviral vector. TIL are manufactured from malignant pleural effusions rich in immune infiltrates. CD4+ and CD8+ cells are enriched and activated with anti-CD3/CD28 beads. CAR-T are transduced, and both CAR-T and TIL are expanded (IL-7/IL-15) for 5 days. The product is infused fresh, avoiding losses associated with cryopreservation and thawing. Developing and validating release tests (absence of replication competent virus, vector copy number, chimeric antigen receptor expression, endotoxin testing) posed an initial challenge. Having completed assay development, our manufacturing process, including release testing, can be performed for less than 1/10 the cost of commercial CAR-T. We expect that the platform that we have validated will be easily transitioned to new chimeric antigen receptor designs and specificities and will likewise be adaptable to TIL manufacture for the wide variety of cancers that metastasize to the pleura of peritoneum. Standardized in-house ACT manufacture may greatly reduce the cost of cellular immunotherapies, making it more widely available to patients.

Intraepithelial cytotoxic lymphocytes are associated with a poor prognosis in feline intestinal T-cell lymphoma.

The expression of cytotoxic molecules in feline intestinal T-cell lymphoma cells was examined immunohistochemically using endoscopic samples of 50 cases. Cases included 14 large-cell lymphomas (LCLs) and 36 small-cell lymphomas (SCLs). Most LCL and some SCL exhibited marked erosion and villous atrophy. Clonal T-cell receptor (TCR) gene rearrangement was detected in 10/14 (71%) LCL cases and 33/36 (92%) SCL cases. No clonal immunoglobulin heavy chain (IgH) gene rearrangement was detected. Immunohistochemically, all cases were positive for CD3 and negative for CD79α, CD30, CD56, and Foxp3. LCLs were positive for CD8 in 13/14 cases (93%), T-cell intracellular antigen 1 (TIA1) in 14/14 cases (100%), and granzyme B in 6/14 cases (43%). SCLs were positive for CD8 in 28/36 cases (78%), TIA1 in 33/36 cases (92%), and granzyme B in 2/36 cases (6%). TIA1- and granzyme B-positive neoplastic lymphocytes were predominantly observed in the mucosal epithelium of 10/50 cases (20%) and 6/50 cases (12%), respectively. No significant differences in survival time were found based on cell size or epitheliotropism. However, cases with TIA1+ and/or granzyme B+ neoplastic lymphocytes predominantly in the mucosal epithelium had significantly shorter survival times (P < .05), suggesting that mucosal epithelium infiltration of neoplastic cells with a cytotoxic immunophenotype is a negative prognostic factor. Therefore, intraepithelial cytotoxic lymphocytes may be associated with mucosal injury and impaired intestinal function, leading to a poor prognosis in cats with intestinal T-cell lymphoma.

HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection.

HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for an accurate interpretation of in vitro data. However, variations in the HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell-cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than four-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated the subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.

Enhanced immunogenicity of a positively supercharged archaeonthioredoxin scaffold as acell-penetratingantigen carrier for peptide vaccines.

Polycationic resurfaced proteins hold great promise as cell-penetrating bioreagents but their use as carriers for the intracellular delivery of peptide immuno-epitopes has not thus far been explored. Here, we report on the construction and functional characterization of a positively supercharged derivative of Pyrococcus furiosus thioredoxin (PfTrx), a thermally hyperstable protein we have previously validated as a peptide epitope display and immunogenicity enhancing scaffold. Genetic conversion of 13 selected amino acids to lysine residues conferred to PfTrx a net charge of +21 (starting from the -1 charge of the wild-type protein), along with the ability to bind nucleic acids. In its unfused form, +21 PfTrx was readily internalized by HeLa cells and displayed a predominantly cytosolic localization. A different intracellular distribution was observed for a +21 PfTrx-eGFP fusion protein, which although still capable of cell penetration was predominantly localized within endosomes. A mixed cytosolic/endosomal partitioning was observed for a +21 PfTrx derivative harboring three tandemly repeated copies of a previously validated HPV16-L2 (aa 20-38) B-cell epitope grafted to the display site of thioredoxin. Compared to its wild-type counterpart, the positively supercharged antigen induced a faster immune response and displayed an overall superior immunogenicity, including a substantial degree of self-adjuvancy. Altogether, the present data point to +21 PfTrx as a promising novel carrier for intracellular antigen delivery and the construction of potentiated recombinant subunit vaccines.