The optimized priming effect of FGF-1 and FGF-2 enhances preadipocyte lineage commitment in human adipose-derived mesenchymal stem cells.

The cell-assisted lipotransfer technique, integrating adipose-derived mesenchymal stem cells (ADMSCs), has transformed lipofilling, enhancing fat graft viability. However, the multipotent nature of ADMSCs poses challenges. To improve safety and graft vitality and to reduce unwanted lineage differentiation, this study refines the methodology by priming ADMSCs into preadipocytes-unipotent, self-renewing cells. We explored the impact of fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF), either alone or in combination, on primary human ADMSCs during the proliferative phase. FGF-2 emerged as a robust stimulator of cell proliferation, preserving stemness markers, especially when combined with EGF. Conversely, FGF-1, while not significantly affecting cell growth, influenced cell morphology, transitioning cells to a rounded shape with reduced CD34 expression. Furthermore, co-priming with FGF-1 and FGF-2 enhanced adipogenic potential, limiting osteogenic and chondrogenic tendencies, and possibly promoting preadipocyte commitment. These preadipocytes exhibited unique features: rounded morphology, reduced CD34, decreased preadipocyte factor 1 (Pref-1), and elevated C/EBPα and PPARγ, alongside sustained stemness markers (CD73, CD90, CD105). Mechanistically, FGF-1 and FGF-2 activated key adipogenic transcription factors-C/EBPα and PPARγ-while inhibiting GATA3 and Notch3, which are adipogenesis inhibitors. These findings hold the potential to advance innovative strategies for ADMSC-mediated lipofilling procedures.

Efficacy of Bortezomib for Treating Anti-Interferon-Gamma Autoantibody-Associated Adult-Onset Immunodeficiency Syndrome.

BACKGROUND: Currently, there is no effective treatment for adult-onset immunodeficiency (AOID) syndrome with anti-interferon-gamma autoantibodies (anti-IFN-γ-auto-Abs). This study aimed to investigate the effectiveness of bortezomib (BTZ) for decreasing anti-IFN-γ-auto-Abs. METHODS: A pre- and post-intervention study was conducted from February 2017 through June 2019 at Siriraj Hospital (Bangkok, Thailand). Five patients were invited to receive once-weekly BTZ (1.3 mg/m2 body surface area) subcutaneously for 8 weeks followed by oral cyclophosphamide (1 mg/kg/d) for 4 months. The primary outcomes were the difference in antibody level at 8 and 48 weeks compared with baseline and the incidence of serious adverse events (AEs). The secondary outcome was the occurrence of opportunistic infections (OIs) during the 72 weeks after starting BTZ. RESULTS: The median patient age was 46 years (range, 34-53). All patients had 3-5 OIs prior to enrollment. All patients were receiving antimycobacterial agents for treatment of nontuberculous mycobacterial infection at enrollment. There was no significant difference in the mean optical density of auto-Abs at 8 weeks (3.73 ± 0.72) or 48 weeks (3.74 ± 0.53) compared with baseline (3.84 ± 0.49; P = .336 and P = .555, respectively). However, after serum dilution, the antibody titer nonsignificantly decreased 8-16 weeks after BTZ initiation (P = .345). Ten OIs were observed 24-72 weeks after BTZ initiation. CONCLUSIONS: Treatment with BTZ followed by cyclophosphamide yielded no significant decrease in antibody titer levels, and 10 OIs were observed during 24-72 weeks of BTZ treatment. No serious AEs were observed. Combining rituximab with BTZ is likely necessary to prevent generation of new autoantibody-producing plasma cells. Clinical Trials Registration. NCT03103555.

Synergistic immunosuppressive effect of hispidulin and nepetin mixtures on human T lymphocytes.

BACKGROUND: Clerodendrum petasites S. Moore predominantly contains hispidulin (His) and nepetin (Nep) which are immunosuppressive potentials. Although the effect of individual compounds was previously confirmed, a cumulative suppression of these flavonoid mixtures is unknown. This study thus investigated their inhibitory effects and cytotoxicity on T cells by using His:Nep ratios following a naturally occurring dose (3:1) and optimized doses (1:1 and 1:3). MATERIALS AND METHODS: Anti-CD3/28 stimulated peripheral blood mononuclear cells were treated with individual compounds and their mixtures. Changes in early cell activation markers in activated T cells and apoptosis were analyzed by a flow cytometer. RESULTS: Mixtures at 3:1 suppressed CD69 and CD25 expression in CD4+ and CD8+ T cells in a dose-dependent manner. At the highest concentration of 200 µM His + 66.7 µM Nep, over 90% inhibition was observed for CD25 expression in CD4+ and CD8+ T cells, whereas a lesser effect was observed for CD69 expression. A dose-dependent inhibition was still observed when using 1:1 and 1:3 ratios. Interestingly, 80-97% inhibition were observed in CD69 and CD25 expression without inducing cell death after treated with the highest doses of each ratio (66.7 µM His + 200 µM Nep and 200 µM His + 200 µM Nep). These mixtures were also exhibited a better suppression than individual compounds. CONCLUSIONS: The optimized mixture of His and Nep at 66.7:200 µM is suggested for further study due to a greater suppressive effect than a single compound or a naturally-occurring dose.

Extensive Characterization of Mesenchymal Stem Cell Marker Expression on Freshly Isolated and In Vitro Expanded Human Adipose-Derived Stem Cells from Breast Cancer Patients.

Variation in numbers and functions of cells in fat tissues may affect therapeutic outcomes and adverse events after autologous fat tissue grafting in postmastectomy breast cancer patients; however, the relevant information regarding cellular components is still incomplete. Phenotypic characterization of heterogeneous cell subsets in stromal vascular fraction (SVF) isolated from fat tissues by flow cytometry was also limited to a combination of few molecules. This study, therefore, developed a polychromatic staining panel for an in-depth characterization of freshly isolated SVF and expanded adipose-derived stem cells (ADSC) from the patients. ADSC were found predominant in SVF (~65% of CD45- cells) with a homogenous phenotype of CD13+CD31-CD34+CD45-CD73+CD90+CD105-CD146- (~94% of total ADSC). Endothelial progenitor cells (EPC) and pericytes were minor (~18% and ~11% of CD45- cells, respectively) with large heterogeneity. Downregulation of CD34 and upregulation of CD105 in ADSC were profound at passage 3, showing a phenotype similar to the classical mesenchymal stem cells from the bone marrow. Results from this study demonstrated that fat tissue collected from patients contains ADSC with a highly homogenous phenotype. The in vitro culture of these cells maintained their homogeneity with modified CD34 and CD105 expression, suggesting the expansion from a single population of ADSC.

Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction.

Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.

Determination of suppressive effect on human T-cell activation by hispidulin, nepetin, and vanillic acid.

Background: Hispidulin, nepetin, and vanillic acid are phenolic compounds potentially possessing immunosuppressive property, however, no information on their pharmacological effect and cytotoxicity has been investigated on human T lymphocytes.Materials and methods: Human peripheral blood mononuclear cells were stimulated with anti-CD3/28 coated beads and treated with individual compound at different concentrations (50-200 µM). Inhibition of early cell activation and induction of apoptosis were analyzed by flow cytometric technique.Results: At 200 µM, frequencies of CD25 and CD69 in CD4+ and CD8+ T lymphocytes were markedly decreased by hispidulin and nepetin. When lowering to 100 and 50 µM, hispidulin had no effect on the expression of CD69 in CD4+ T cells, whereas nepetin selectively suppressed CD25 and CD69 expressions in CD8+ T cells at 100 µM and only inhibited CD69 in CD8+ T cells at 50 µM. For vanillic acid, no inhibitory effect was observed while cell activation was significantly increased for all treated concentrations. None of these compounds disturbed levels of total apoptotic cells in CD4+ and CD8+ populations.Conclusions: Hispidulin and nepetin, therefore, exhibit dose-dependent inhibitory activity of early T-cell activation without inducing cell death, considering feasible immunosuppressants for inflammation-related diseases. However, vanillic acid has no effect on immunosuppression but shows more potential on immunostimulation.HighlightsImmunosuppressive effects of hispidulin and nepetin on human T cells were studied.Dose-dependent activity for T-cell suppression was found in hispidulin and nepetin.Vanillic acid showed immunostimulating potential rather than immunosuppression.All compounds did not induce cell death.

Clerodendrum petasites S. Moore: The therapeutic potential of phytochemicals, hispidulin, vanillic acid, verbascoside, and apigenin.

Clerodendrum petasites S. Moore has been prescribed in Thai traditional medicine for over 30 years for the treatment of ailments including asthma, inflammation, fever, cough, vomiting, and skin disorders. The phytochemicals from this plant have been identified as phenolic acids, flavones, flavone glycosides, glycosides, phenylpropanoid, and diterpenoid. The pharmacological activities both in vitro and in vivo have mostly been reported from crude extracts and not from pure compounds. This review, therefore, brings together information on the specific phytochemicals found in C. petasites in order to provide a guide to the natural bioactive compounds that are potentially used in medicines together with mechanisms underlying their pharmacological uses. All relevant information was searched for the terms of plant name, naturally-occurring compounds, and traditional uses from reliable databases, such as PubMed, Science Direct and Google Scholar, along with Thai traditional medicine textbooks. There was no specific timeline set for the search and this review selected to report only mechanisms studied by using standard compounds for their biological activities. Four dominant compounds comprising hispidulin, vanillic acid, verbascoside, and apigenin, have robust evidence to support their medical effects. Hispidulin was discovered to be possibly responsible for the treatment of cancer, osteolytic bone diseases, and neurological diseases. Other compounds were also found to tentatively support the uses in inflammation and neurological diseases. C. petasites extracts may provide an option as complimentary medicine, and or for the pharmacological development of new drugs derived from the phytochemicals found within.

Determination of cell expansion and surface molecule expression on anti-CD3/28 expanded CD4+ T cells.

CD4+ T cell immunotherapy has potential for treatment in HIV-infected patients. A large number of expanded CD4+ T cells and confirmation of functional-related phenotypes are required for ensuring the successful outcomes of treatment. Freshly isolated CD4+ T cells from healthy donors were activated with anti-CD3/28-coated magnetic beads at different bead-to-cell ratios and cultured in the absence and presence of IL-2 supplementation for 3 weeks. Fold expansion, cell viability, growth kinetic and lymphocyte subset identities were determined. Data demonstrated that a 1:1 bead-to-cell ratio rendered the highest expansion of 1044-fold with 88% viability and 99.5% purity followed by the 2:1 and 0.5:1 ratios. No significant difference in proliferation and phenotypes was found between non-IL-2 and IL-2 supplementation groups. Several specific surface molecule expressions of the expanded cells including chemokine receptors, adhesion molecules, co-stimulatory molecules, activation molecules, maturation markers, cytokine receptors and other molecules were altered when compared to the unexpanded cells. This optimized expansion protocol using the 1:1 bead-to-cell ratio of anti-CD3/28-coated magnetic beads and culture condition without IL-2 supplementation provided the satisfactory yield with good reproducibility. Specific surface molecule expressions of the expanded cells presented potential roles in proliferation, differentiation, homeostasis, apoptosis and organ homing.

Tregitope-linked Refined Allergen Vaccines for Immunotherapy in Cockroach Allergy.

Allergen-specific immunotherapy (AIT) facilitates long-term resolution of allergic morbidity resulting in reduced drug use and increased refractoriness to new sensitization. AIT effectiveness has been demonstrated in seasonal and perennial allergies, and insect stings. However, data and studies in AIT relative to cockroach (CR) allergy are relatively scarce. In this study, mice allergic to American CR (Periplaneta americana) were treated with a liposome (L)-entrapped vaccine made of mouse Tregitope289-Per a 9 of the CR, Tregitope167-Per a 9, or Per a 9 alone - or placebo. Allergic mice that received an individual vaccine intranasally had reduced Th2 response, reduced lung inflammation, and reduced respiratory tissue remodeling. However, only L-Tregitope289-Per a 9 and L-Tregitope167-Per a 9 induced expression of immunosuppressive cytokine genes (IL-10, TGF-ß, and IL-35 for L-Tregitope289-Per a 9, and IL-10 and TGF-ß for L-Tregitope167-Per a 9) and increment of idoleamine-2,3-dioxygenase 1 (IDO1), indicating that these vaccines caused allergic disease suppression and reversal of respiratory tissue remodeling via generation of regulatory lymphocytes. Liposome entrapped-recombinant Per a 9 (L-Per a 9) did not cause upregulation of immunosuppressive cytokine genes and IDO1 increment; rather, L-Per a 9 induced high expression of IFN-γ in lungs of treated mice, which resulted in mitigation of allergic manifestations. This study provides compelling evidence that both liposome-entrapped vaccines made of single refined major allergen alone and single refined major allergen linked with Tregitopes are effective for reducing allergen-mediated respiratory tissue inflammation and remodeling, but through different mechanisms.

B cell subset alteration and the expression of tissue homing molecules in dengue infected patients.

BACKGROUND: B cells play an essential role during dengue viral infection. While a major expansion of antibody secreting cells (ASCs) was observed, the importance of these increased frequencies of ASCs remains unclear. The alteration of B cell subsets may result from the expression of tissue specific homing molecules leading to their mobilization and distribution to different target organs during acute dengue viral infection. METHODS: In this study, whole blood samples were obtained from thirty pediatric dengue-infected patients and ten healthy children and then stained with fluorochrome-conjugated monoclonal antibodies against CD3, CD14, CD19, CD20, CD21, CD27, CD38, CD45, CD138 and homing molecules of interest before analyzed by polychromatic flow cytometry. B cell subsets were characterized throughout acute infection period. RESULTS: Data shows that there were no detectable differences in frequencies of resting, activated and tissue memory cells, whereas the frequency of ASCs was significantly increased and associated with the lower frequency of naïve cells. These results were found from patients with both dengue fever and dengue hemorrhagic fever, suggesting that such change or alteration of B cells was not associated with disease severity. Moreover, several homing molecules (e.g., CXCR3 and CCR2) were found in ASCs, indicating that ASCs may distribute to inflamed tissues and various organs. CONCLUSIONS: Findings from this study provide insight into B cell subset distribution. Furthermore, organ mobilization according to homing molecule expression on different B cell subsets during the course of dengue viral infection also suggests they are distributed to inflamed tissues and various organs.

Identification of changes in dendritic cell subsets that correlate with disease severity in dengue infection.

Dengue virus (DENV) is the most prevalent arthropod-borne viral disease in humans. DENV causes a spectrum of illness ranging from mild to potentially severe complications. Dendritic cells (DCs) play a critical role in initiating and regulating highly effective antiviral immune response that include linking innate and adaptive immune responses. This study was conducted to comparatively characterize in detail the relative proportion, phenotypic changes, and maturation profile of subsets of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in children with dengue fever (DF), dengue hemorrhagic fever (DHF) and for purposes of control healthy individuals. The mDCs (Lin-CD11c+CD123lo), the pDCs (Lin-CD11c-CD123+) and the double negative (DN) subset (Lin-/HLA-DR+/CD11c-CD123-) were analyzed by polychromatic flow cytometry. The data were first analyzed on blood samples collected from DENV-infected patients at various times post-infection. Results showed that the relative proportion of mDCs were significantly decreased which was associated with an increase in disease severity in samples from DENV-infected patients. While there was no significant difference in the relative proportion of pDCs between healthy and DENV-infected patients, there was a marked increase in the DN subset. Analysis of the kinetics of changes of pDCs showed that there was an increase but only during the early febrile phase. Additionally, samples from patients during acute disease showed marked decreases in the relative proportion of CD141+ and CD16+ mDC subsets that were the major mDC subsets in healthy individuals. In addition, there was a significant decrease in the level of CD33-expressing mDCs in DENV patients. While the pDCs showed an up-regulation of maturation profile during acute DENV infection, the mDCs showed an alteration of maturation status. This study suggests that different relative proportion and phenotypic changes as well as alteration of maturation profile of DC subsets may play a critical role in the dengue pathogenesis and disease outcome.

A closed-culture system using a GMP-grade culture bag and anti-CD3/28 coated bead stimulation for CD4+ T cell expansion from healthy and HIV-infected donors.

CD4 immunotherapy is potentially useful in immune reconstitution of CD4+ T cells for HIV-infected patients. Transfusion of anti-CD3/28 expanded CD4+ T cells is also proved to be safe and effective in both SIV-infected macaques and HIV-infected patients. However, there is no such standardized and practical protocol available for cell production in order to use in clinics. This study thus aimed to develop a closed-culture system for in vitro CD4+ T lymphocyte expansion by using a commercially available GMP-grade culture bag and anti-CD3/28 activation. Freshly isolated CD4+ T cells by immunorosette formation from healthy donors and cryopreserved CD4+ T cells from HIV-infected patients with CD4 count over 500 cells/µL were stimulated with anti-CD3/28 coated beads. The activated cells were then expanded in conventional culture flasks and GMP-grade culture bags for three weeks. Fold expansion, cell viability, growth kinetic and phenotypic characters were observed. Results revealed that purified CD4+ T cells from healthy individuals cultured in flasks showed better expansion than those cultured in bags (797-fold and 331-fold, respectively), whereas, their cell viability, growth kinetic and expanded CD4+ T cell purity were almost similar. A large-scale production was also conducted and supported consistency of cell proliferation in the closed-culture system. Frozen CD4+ T lymphocytes from the patients were able to remain their growth function and well expanded with a good yield of 415-fold, 85% viability and 96% purity of CD4+ T cells at the end of a 3-week culture in bags. This developed closed-culture system using culture bags and anti-CD3/28 coated beads, therefore, can achieve a large number of expanded CD4+ T lymphocytes with good reproducibility, suggesting a promising protocol required for adoptive immunotherapy.

Differences in activation and tissue homing markers of natural killer cell subsets during acute dengue infection.

Dengue virus (DENV) infection is considered one of the most important mosquito-borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV-infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56hi CD16- and CD56lo CD16+ NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56hi CD16- subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56lo CD16+ subset compared with those in the CD56hi CD16- subset. Moreover, although the CD56lo CD16+ subset contained a high frequency of cells expressing skin-homing markers, the CD56hi CD16- subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection.

Impact of Vaccination on Distribution of T Cell Subsets in Antiretroviral-Treated HIV-Infected Children.

Antiretroviral therapy (ART) is generally prescribed to patients with human immunodeficiency virus (HIV) infection with vaccination introduced to prevent disease complications. However, little is known about the influence of immunization on T cell subsets' distribution during the course of infection. This study aims to identify the impact of viral replication and immunization on naïve, effector, effector memory, and central memory T cell subpopulations in ART-treated HIV-infected children. Fifty patients were recruited and injected intramuscularly with influenza A (H1N1) 2009 vaccine on the day of enrollment (day 0) and day 28. Blood samples were collected for pre- and postvaccination on days 0 and 56 for analyzing T cell phenotypes by flow cytometry. Phenotypes of all T cell subsets remained the same after vaccination, except for a reduction in effector CD8+ T cells. Moreover, T cell subsets from patients with controllable viral load showed similar patterns to those with virological failure. Absolute CD4 count was also found to have a positive relationship with naïve CD4+ and CD8+ T cells. In conclusion, vaccination and viral replication have a little effect on the distribution of T cell subpopulations. The CD4 count can be used for prediction of naïve T cell level in HIV-infected patients responding to ART.

Human scFvs That Counteract Bioactivities of Staphylococcus aureus TSST-1.

Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vß region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application.

IgG antibodies to dengue enhanced for FcγRIIIA binding determine disease severity.

Dengue virus (DENV) infection in the presence of reactive, non-neutralizing immunoglobulin G (IgG) (RNNIg) is the greatest risk factor for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Progression to DHF/DSS is attributed to antibody-dependent enhancement (ADE); however, because only a fraction of infections occurring in the presence of RNNIg advance to DHF/DSS, the presence of RNNIg alone cannot account for disease severity. We discovered that DHF/DSS patients respond to infection by producing IgGs with enhanced affinity for the activating Fc receptor FcγRIIIA due to afucosylated Fc glycans and IgG1 subclass. RNNIg enriched for afucosylated IgG1 triggered platelet reduction in vivo and was a significant risk factor for thrombocytopenia. Thus, therapeutics and vaccines restricting production of afucosylated, IgG1 RNNIg during infection may prevent ADE of DENV disease.

The Effects of Anti-CD3/CD28 Coated Beads and IL-2 on Expanded T Cell for Immunotherapy.

BACKGROUND: The activation of peripheral blood mononucleated cells (PBMCs) with anti-CD3/CD28-coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion. OBJECTIVES: The aim of this study was to define an optimal cell isolation protocol for the expansion of PBMCs using anti-CD3/CD28-coated bead stimulation, with the ultimate goal of using these cells for adoptive therapy. MATERIAL AND METHODS: PBMCs were isolated from healthy donor blood samples. To determine the effect of varying the bead-to-cell ratios on the expansion rate and phenotypic characterization of the expanded cells, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at bead-to-cell ratios of 0.1 : 1, 0.5 : 1 and 1.0 : 1 in the presence of 100 U/mL exogenous IL-2; also, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at a bead-to-cell ratio of 0.5 : 1 in the presence of varying concentrations of IL-2 (20, 100 and 1000 U/mL). Cell expansion was carried out for three weeks. The cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry. RESULTS: The initial experiments showed no difference in the expansion rate from cells grown with the three different bead-to-cell ratios. PBMCs can be expanded up to 1000-fold at a 0.5 : 1 bead-to-cell ratio after three weeks of cell expansion with a high viability (90%). Furthermore, in the presence of 100 U/mL IL-2, the percentages of CD3-CD16+CD56+ cells showed marked increases. CONCLUSIONS: The results demonstrate that PBMCs were stimulated with anti-CD3/CD28-coated beads. This method may provide an alternative for driving T cell expansion, which may be very useful in adoptive immunotherapy.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India.

Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR- CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE: Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCR-confirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations.

B Cell Responses during Secondary Dengue Virus Infection Are Dominated by Highly Cross-Reactive, Memory-Derived Plasmablasts.

UNLABELLED: Dengue virus (DENV) infection results in the production of both type-specific and cross-neutralizing antibodies. While immunity to the infecting serotype is long-lived, heterotypic immunity wanes a few months after infection. Epidemiological studies link secondary heterotypic infections with more severe symptoms, and cross-reactive, poorly neutralizing antibodies have been implicated in this increased disease severity. To understand the cellular and functional properties of the acute dengue virus B cell response and its role in protection and immunopathology, we characterized the plasmablast response in four secondary DENV type 2 (DENV2) patients. Dengue plasmablasts had high degrees of somatic hypermutation, with a clear preference for replacement mutations. Clonal expansions were also present in each donor, strongly supporting a memory origin for these acutely induced cells. We generated 53 monoclonal antibodies (MAbs) from sorted patient plasmablasts and found that DENV-reactive MAbs were largely envelope specific and cross neutralizing. Many more MAbs neutralized DENV than reacted to envelope protein, emphasizing the significance of virion-dependent B cell epitopes and the limitations of envelope protein-based antibody screening. A majority of DENV-reactive MAbs, irrespective of neutralization potency, enhanced infection by antibody-dependent enhancement (ADE). Interestingly, even though DENV2 was the infecting serotype in all four patients, several MAbs from two patients neutralized DENV1 more potently than DENV2. Further, half of all type-specific neutralizing MAbs were also DENV1 biased in binding. Taken together, these findings are reminiscent of original antigenic sin (OAS), given that the patients had prior dengue virus exposures. These data describe the ongoing B cell response in secondary patients and may further our understanding of the impact of antibodies in dengue virus pathogenesis. IMPORTANCE: In addition to their role in protection, antibody responses have been hypothesized to contribute to the pathology of dengue. Recent studies characterizing memory B cell (MBC)-derived MAbs have provided valuable insight into the targets and functions of B cell responses generated after DENV exposure. However, in the case of secondary infections, such MBC-based approaches fail to distinguish acutely induced cells from the preexisting MBC pool. Our characterization of plasmablasts and plasmablast-derived MAbs provides a focused analysis of B cell responses activated during ongoing infection. Additionally, our studies provide evidence of OAS in the acute-phase dengue virus immune response, providing a basis for future work examining the impact of OAS phenotype antibodies on protective immunity and disease severity in secondary infections.