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Background: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4 + , CD8 + , Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads., Methods: Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 10 5 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 10 5  C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4 + , CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively., Results: The IHC results showed that CD4 + , CD8 + , Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4 + levels and low CD8 + expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4 + level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4 + , CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues., Conclusions: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis., (© 2023. BioMed Central Ltd., part of Springer Nature.)

BATF3-deficient mice that lack CD8 + dendritic cells (DCs) showed an exacerbation of chronic graft-versus-host disease (cGVHD), including T follicular helper (Tfh) cell and autoantibody responses, whereas mice carrying the Sle2c2 lupus-suppressive locus with a mutation in the G-CSFR showed an expansion of CD8 + DCs and a poor mobilization of plasmacytoid DCs (pDCs) and responded poorly to cGVHD induction. Here, we investigated the contribution of CD8 + DCs and pDCs to the humoral response to protein immunization, where CD8 neg DCs are thought to represent the major inducers. Both BATF3 -/- and Sle2c2 mice had reduced humoral and germinal center (GC) responses compared with C57BL/6 (B6) controls. We showed that B6-derived CD4 + DCs are the major early producers of IL-6, followed by CD4 - CD8 - DCs. Surprisingly, IL-6 production and CD80 expression also increased in CD8 + DCs after immunization, and B6-derived CD8 + DCs rescued Ag-specific adaptive responses in BATF3 -/- mice. In addition, inflammatory pDCs (ipDCs) produced more IL-6 than all conventional DCs combined. Interestingly, G-CSFR is highly expressed on pDCs. G-CSF expanded pDC and CD8 + DC numbers and IL-6 production by ipDCs and CD4 + DCs, and it improved the quality of Ab response, increasing the localization of Ag-specific T cells to the GC. Finally, G-CSF activated STAT3 in early G-CSFR + common lymphoid progenitors of cDCs/pDCs but not in mature cells. In conclusion, we showed a multilayered role of DC subsets in priming Tfh cells in protein immunization, and we unveiled the importance of G-CSFR signaling in the development and function pDCs., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Abstract: Recent data suggest that programmed cell death -1 (PD-1) and programmed cell death ligand-1 (PD-L1) are involved in the pathogenesis of Langerhans cell histiocytoma (LCH); however, their contributions are not well established. Also, the involvement of PD-1/PD-L1 molecules in musculoskeletal LCH remains particularly unclear. The current study aims to characterize the involvement of PD-1/PD-L1 immune checkpoint system in the pathogenesis of musculoskeletal LCH. PD-1/PD-L1 expression was evaluated in 6 patients, 3 men and 3 women with a mean age of 13.5 years, with musculoskeletal LCH who were treated at Kindai University Hospital and Osaka Women's and Children's Hospital between November 2005 and December 2020. The median follow-up period for all patients with musculoskeletal LCH was 41 months. We surveyed symptoms, number of lesions, treatment modality, and outcomes. Immunostaining for CD4, CD8, PD-1, and PD-L1 was also performed on pathological specimens obtained by biopsy. Multiple lesions were observed in 5 cases, and a single lesion was observed in 1 case. The chief complaint in 5 cases was pain. Four patients underwent spontaneous regression. The other 2 patients received chemotherapy. The outcomes included continuous disease-free (n = 5) and alive with the disease (n = 1). The CD4-, CD8-, PD-1-, and PD-L1-positive rates among all specimens were 100%, 100%, 16.6%, and 83.3%, respectively. The CD4/PD-L1, CD8/PD-L1, and PD-1/PD-L1 positive rates in all the specimens were 83.3%, 83.3%, and 16.6%, respectively. We believe that the PD-1/PD-L1 immune checkpoint molecules may play some role in the microenvironment of musculoskeletal LCH., Competing Interests: The authors have no funding and conflicts of interests to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

The antioxidant MnTBAP was previously shown to down-regulate the surface expression of CD4 molecule in T cells. This observation obviously holds great potential impact in a number of pathological human conditions, including autoimmunity. Three different single doses of MnTBAP reduced the frequency of CD4 high cells. However, the median florescent intensity (MFI) was not different. Initiation of in vivo pharmacotherapy or vehicle control was performed inC57BL/6 mice that were actively immunized for experimental autoimmune encephalomyelitis (EAE). In contrast to published reports, the mean frequency of CD4 high cells, and the median fluorescent intensity (MFI) of CD4 was similar in both treatment groups. 25-day survival following active immunization among the MnTBAP treated animals compared to vehicle controls was16.6 ± 6.9 days vs 23.6 ± 2.7 days; (P value <0.05). We conclude that MnTBAP (Sack and Herzog, 2009 (Sack and Herzog, 2009)) does not effectively downregulate CD4 expression in T cells in vivo, probably due to extensive mechanism that distinguishes it from an in vitro model (Harding, 1993 (Harding, 1993)) possesses toxic properties that may limit its clinic use in possible doses that could deliver the immunomodulation through down regulation of CD4 expression, and (Saizawa et al., 1987 (Saizawa et al., 1987)) has limited availability in specific tissues, including the CNS., (Published by Elsevier B.V.)

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

This study evaluated the biochemical, molecular, and histopathological mechanisms involved in the hypoglycaemic effect of zinc oxide nanoparticles (ZnONPs) in experimental diabetic rats. ZnONPs were prepared by the sol-gel method and characterised by scanning and transmission electron microscopy (SEM and TEM). To explore the possible hypoglycaemic and antioxidant effect of ZnONPs, rats were grouped as follows: control group, ZnONPs treated group, diabetic group, and diabetic + ZnONPs group. Upon treatment with ZnONPs, a significant alteration in the activities of superoxide dismutase, glutathione peroxidase, and the levels of insulin, haemoglobin A1c, and the expression of cluster of differentiation 4+ (CD4+), CD8+ T cells, glucose transporter type-4 (GLUT-4), tumour necrosis factor, and interleukin-6 when compared to diabetic and their control rats. ZnONPs administration to the diabetic group showed eminent blood glucose control and restoration of the biochemical profile. This raises their active role in controlling pancreas functions to improve glycaemic status as well as the inflammatory responses. Histopathological investigations showed the non-toxic and therapeutic effect of ZnONPs on the pancreas. TEM of pancreatic tissues displayed restoration of islets of Langerhans and increased insulin-secreting granules. This shows the therapeutic application of ZnONPs as a safe anti-diabetic agent and to have a potential for the control of diabetes.

Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [ n  = 63], B [ n  = 84], C [ n  = 94], and D [ n  = 59]) plus intersubtype recombinants and uncommon strains ( n  = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P  < 0.0001). Specifically, subtype C clones exhibited the lowest CD4 and tetherin downregulation activities, while subtype D and B clones were most functional for both activities. We also identified Vpu polymorphisms associated with CD4 or tetherin downregulation function and validated six of these using site-directed mutagenesis. Our results highlight the marked extent to which Vpu function varies among global HIV-1 strains, raising the possibility that natural variation in this accessory protein may contribute to viral pathogenesis and/or spread. IMPORTANCE The HIV-1 accessory protein Vpu enhances viral spread by downregulating CD4 and BST-2/tetherin on the surface of infected cells. Natural variability in these Vpu functions may contribute to HIV-1 pathogenesis, but this has not been investigated among the diverse viral subtypes that contribute to the HIV-1 pandemic. In this study, we found that Vpu function differs significantly among HIV-1 subtypes A, B, C, and D. On average, subtype C clones displayed the lowest ability to downregulate both CD4 and tetherin, while subtype B and D clones were more functional. We also identified Vpu polymorphisms that associate with functional differences among HIV-1 isolates and subtypes. Our study suggests that genetic diversity in Vpu may play an important role in the differential pathogenesis and/or spread of HIV-1., (Copyright © 2020 Umviligihozo et al.)

Background: Atopic dermatitis (AD) pathogenesis still needs to be elucidated, but invariant natural killer T (iNKT) cell involvement was already described by several groups. Our group has demonstrated that IgG antibodies purified from AD patients can modulate cytokine production by thymic T cells. Here we aimed to investigate if IgG from AD patients can modulate infant non-atopic thymic iNKT cells cytokine production in order to collaborate with the elucidation of AD development in infancy., Methods: Thymic tissues were obtained from children from non-atopic mothers, and IgG was purified from AD patients diagnosed as moderate or severe and, as controls, from subjects clinically classified as non-atopic individuals. PBMCs from non-atopic individuals were also used in this study., Results: Our results demonstrated that IgG from AD patients could induce non-atopic children thymic iNKT cells to produce higher levels of intracellular IL-4, IL-10, and IL-17 when compared to all control conditions. No effect was observed in non-atopic adults peripheral iNKT. We also observed that IgG from AD patients induces an increase in the expression of CD4 and Rorγt transcription factor in non-atopic children thymic iNKT cells compared to the condition of all controls., Conclusions: These observations suggest that IgG from AD patients can induce a cytokine profile by thymic iNKT cells from non-atopic infants compatible with the observations in AD development, which can collaborate with the elucidation of AD pathogenesis., (© 2019 The International Society of Dermatology.)

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH 4 Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5 + early, Rab7 + late, and Rab11 + recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction. IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection., (Copyright © 2018 American Society for Microbiology.)

Bowman-Birk inhibitor (BBI) is a soybean-derived protease inhibitor that has anti-inflammation and anti-HIV effect. Here, we further investigated the anti-HIV action of BBI in macrophages, focusing on its effect on viral entry. We found that BBI could significantly block HIV entry into macrophages. Investigation of the mechanism(s) of the BBI action on HIV inhibition showed that BBI down-regulated the expression of CD4 receptor (as much as 80%) and induced the production of the CC chemokines (up to 60 folds at protein level) in macrophages. This inhibitory effect of BBI on HIV entry could be blocked by the neutralization antibodies to CC chemokines. These findings indicate that BBI may have therapeutic potential as a viral entry inhibitor for the prevention and treatment of HIV infection., (Copyright © 2017. Published by Elsevier Inc.)

An improved expression protocol is proposed for amino acid type-specific [ 13 C], [ 15 N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449-456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.

T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napa hyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napa hyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP] i . Depletion of [ATP] i inhibited mTORC2 dependent NFκB activation in Napa hyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napa hyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function.

A low-cost rodent model of HIV infection and which presents high application value is an effective tool to investigate HIV infection and pathogenesis. However, development of such a small animal model has been hampered by the unsuitability of rodent cells for HIV-1 replication given that the retrovirus HIV-1 has high selectivity to its host cell. Our study used the mouse leukemia cell lines L615 and L1210 that were induced by murine leukemia virus and transfected with hCD4/CCR5 loaded-lentiviral vector. Lentiviral vectors containing the genes hCD4/CCR5 under the transcriptional control of cytomegalovirus promoter were designed. Transfection efficiencies of human CD4 and CCR5 in L615 and L1210 cells were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot assay. Results showed that hCD4 and CCR5 proteins were expressed on the cell surface, demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells. Moreover, the sensitivity of human CD4/CCR5 transgenic mouse cells to HIV infection was confirmed by RT-PCR and ELISA. Mouse leukemia cell lines that could express hCD4 and CCR5 were thus established to facilitate normal entry of HIV-1 so that a human CD4/CCR5 transgenic mice cell model can be used to investigate the transmission and pathogenesis of HIV/AIDS and potential antiviral drugs against this disease.

Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: HIV-1 Vpu and Nef proteins downregulate cell surface levels of natural killer (NK) cell ligands but functional consequences of individual downregulation events are unclear. We tested how well-conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients., Methods: Proviral vpu and nef sequences were amplified from 27 chronic HIV patients, subcloned, and tested for their ability to downregulate cell surface receptors., Results: Cell surface downregulation of CD4, CD317/tetherin, and major histocompatibility complex class 1 that exert biological functions other than NK cell activation were well conserved among patient-derived Vpu and Nef variants. Among NK cell ligands, NK-T-B-antigen, poliovirus receptor, and UL16-binding protein were identified as main targets for Vpu and Nef, the downregulation of which by at least 1 viral protein was highly conserved. NK cell ligands displayed specific sensitivity to Vpu (NK-T-B-antigen) or Nef (poliovirus receptor), and downregulation of cell surface UL16-binding protein was identified as a novel and highly conserved activity of HIV-1 Vpu but not Nef., Conclusions: The conservation of downregulation of major NK cell ligands by either HIV-1 Vpu or Nef suggests an important pathophysiological role of this activity, which may impact the acute but not the chronic phase of HIV infection.

HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, β-COP, and ACOT8 for CD4 downmodulation in HIV-1-infected short hairpin RNA (shRNA)-expressing CD4(+) T cells and characterized direct interaction with Nef by Förster resonance energy transfer (FRET). Binding of lentiviral Nefs to CD4 and AP-2 was conserved, and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current antiretroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV-infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage-tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages., Importance: Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage-targeted therapy for HIV infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In the present study was evaluated the ontogenic of immunocompetent organs of cobia up to 53 days after hatching (dah) through histology and immunohistochemistry techniques. The kidney was the first lymphohematopoietic organ to appear, at 1 dah, followed by the spleen at 5 dah and the thymus at 7 dah. The first CD3 receptors on the lymphocytes were observed in 27% of the thymic tissue at 7 dah and in 99% at 53 dah. The phenotypic expression of CD3 receptors was registered in 10% of the kidney at 8 dah and in 32% at 53 dah. CD4 receptors were observed in 5% and 63% of the thymic area at 7 and 53 dah, respectively. In the kidney, T4 lymphocytes were first observed at 13 dah in 9% of the organ and in 28% at 53 dah, defining the functional development of the specific system associated with immunological memory capacity.

Th-inducing Pox virus and zinc finger/Krüppel-like factor (ThPOK) is a key commitment factor for CD4(+) lineage T cells and is essential for the maintenance of CD4 lineage integrity; thus, the expression of ThPOK has to be tightly controlled. In this article, we demonstrate that Myc-associated zinc finger-related factor (MAZR) and Runt-related transcription factor 1 (Runx1) together repressed ThPOK in preselection double-positive thymocytes, whereas MAZR acted in synergy with Runx3 in the repression of ThPOK in CD8(+) T cells. Furthermore, MAZR-Runx1 and MAZR-Runx3 double-mutant mice showed enhanced derepression of Cd4 in double-negative thymocytes and in CD8(+) T cells in comparison with Runx1 or Runx3 single-deficient mice, respectively, indicating that MAZR modulates Cd4 silencing. Thus, our data demonstrate developmental stage-specific synergistic activities between MAZR and Runx/core-binding factor β (CBFβ) complexes. Finally, retroviral Cre-mediated conditional deletion of MAZR in peripheral CD8(+) T cells led to the derepression of ThPOK, thus showing that MAZR is also part of the molecular machinery that maintains a repressed state of ThPOK in CD8(+) T cells., (Copyright © 2015 by The American Association of Immunologists, Inc.)

CD4 T-cell responses are functionally complex and regulate many aspects of innate and adaptive immunity. Follicular helper (Tfh) cells are CD4 T cells specialized to support B-cell production of isotype-switched, high-affinity antibody. So far, studies of Tfh cells in humans have focused on their differentiation requirements, with little research devoted to their antigen specificity. Here, after separating circulating human memory CD4 T cells based on expression of CXCR5, a signature marker of Tfh, we have quantified and assayed the influenza protein antigen specificity of blood Tfh cells and CD4 T cells lacking this marker. Through the use of peptide pools derived from nucleoprotein (NP) or haemagglutinin (HA) and a panel of human donors, we have discovered that circulating Tfh cells preferentially recognize peptide epitopes from HA while cells lacking CXCR5 are enriched for specificity toward NP. These studies suggest that reactive CD4 T cells specific for distinct viral antigens may have generalized differences in their functional potential due to their previous stimulation history., (© 2015 John Wiley & Sons Ltd.)

Viral protein U (Vpu) is a lentiviral viroporin encoded by human immunodeficiency virus type 1 (HIV-1) and some simian immunodeficiency virus (SIV) strains. This small protein of 81 amino acids contains a single transmembrane domain that allows for supramolecular organization via homoligomerization or interaction with other proteins. The topology and trafficking of Vpu through subcellular compartments result in pleiotropic effects in host cells. Notwithstanding the high variability of its amino acid sequence, the functionality of Vpu is well conserved in pandemic virus isolates. This review outlines our current knowledge on the interactions of Vpu with the host cell. The regulation of cellular physiology by Vpu and the validity of this viroporin as a therapeutic target are also discussed.

Objective: To analyze the frequencies of the expression of major histocompatibility complex class I (MHC-I) antigens, and CD4 and CD8 cells in skeletal muscle in polymyositis (PM) and dermatomyositis (DM)., Methods: This was a retrospective study of 34 PM cases, 8 DM cases, and 29 control patients with non-inflammatory myopathies., Results: MHC-I antigens were expressed in the sarcolemma and/or sarcoplasm in 79.4% of PM cases, 62.5% of DM cases, and 27.6% of controls (CD4 expression was observed in 76.5%, 75%, and 13.8%, respectively). There was a high suspicion of PM/DM (mainly PM) in patients in whom MHC-I antigens and CD4 were co-expressed. In 14.3% of PM/DM cases, we observed MHC-I antigens expression alone, without inflammatory cells., Conclusion: MCH-I antigens expression and CD4 positivity might add to strong diagnostic suspicion of PM/DM. No cellular infiltration was observed in 14.3% of such cases., (Copyright © 2014 Elsevier Editora Ltda. All rights reserved.)

This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units., (© 2015 International Society for Advancement of Cytometry.)

Background: Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation., Results: To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells., Conclusions: We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.

Chronic inflammation is a contributing factor to most life-shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood, making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a(-/-) mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice, we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells, and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155(fl/fl) mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counterregulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.

The application of fluorescently-labeled antibodies for flow cytometric identification and characterization of specific cell types within heterogeneous populations by their protein expression profile is well established. While detection of proteins is informative, concomitant transcript analysis in the same cells would provide a more complete and comprehensive view of intracellular signaling events. We recently reported on the efficient detection of RNA in suspension cells for flow cytometric analysis. The improved RNA flow cytometry procedure described here allows for the specific labeling of multiple RNA species, and is compatible with antibody-based targeting of extracellular and intracellular antigens for multiplexing purposes. To show proof of concept, human peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for a maximum of 5 h, during which their CD4 and interferon-gamma (IFN-γ) transcript and protein levels were monitored. Substantial and increasing numbers of IFN-γ mRNA+ cells were detected within 30 min after initiation of induction, while IFN-γ protein+ cells could only be discerned at 1 h and beyond. Surprisingly, resting lymphocytes contained less CD4 mRNA but more of the protein per cell compared with monocytes, revealing a difference in the relationship of transcript and protein levels in these two cell types. We additionally applied monensin, which is commonly used to block cytokine secretion, and found that IFN-γ mRNA can still be analyzed consistently using the improved RNA flow cytometry staining method. Notably, a subset of IFN-γ mRNA(-)/protein+ cells that were not observed in the absence of monensin became apparent at the 5-h mark. This subset probably represents cells that have accumulated IFN-γ protein, but no longer transcribe mRNA. Collectively, the results described here exemplify how the improved RNA flow cytometry labeling procedure can be applied to simultaneously assess mRNA and protein dynamics to gain insight into the regulation of gene transcription and translation in individual cells., (© 2014 International Society for Advancement of Cytometry.)

Lymphocyte activation gene-3 (LAG-3; CD223), a structural homolog of CD4, binds to MHC class II molecules. Recent research indicated that signaling mediated by LAG-3 inhibits T cell proliferation, and LAG-3 serves as a key surface molecule for the function of regulatory T cells. Previous reports demonstrated that the majority of LAG-3 is retained in the intracellular compartments and is rapidly translocated to the cell surface upon stimulation. However, the mechanism by which LAG-3 translocates to the cell surface was unclear. In this study, we examined the trafficking of human LAG-3 under unstimulated as well as stimulated conditions of T cells. Under the unstimulated condition, the majority of LAG-3 did not reach the cell surface, but rather degraded within the lysosomal compartments. After stimulation, the majority of LAG-3 translocated to the cell surface without degradation in the lysosomal compartments. Results indicated that the cytoplasmic domain without Glu-Pro repetitive sequence is critical for the translocation of LAG-3 from lysosomal compartments to the cell surface. Moreover, protein kinase C signaling leads to the translocation of LAG-3 to the cell surface. However, two potential serine phosphorylation sites from the LAG-3 cytoplasmic domain are not involved in the translocation of LAG-3. These results clearly indicate that LAG-3 trafficking from lysosomal compartments to the cell surface is dependent on the cytoplasmic domain through protein kinase C signaling in activated T cells., (Copyright © 2014 by The American Association of Immunologists, Inc.)

C/EBPα is expressed preferentially in myeloid compared with lymphoid or erythroid cells and directs myeloid lineage specification. C/EBPα is also expressed at lower levels in HSCs and in several nonhematopoietic tissues. The Cebpa gene has a conserved, 450-bp segment at +37 kb that harbors enhancer-specific epigenetic marks and is activate in a myeloid cell line. Herein, we characterize transgenic C57BL/6 mice, in which the Cebpa enhancer and 845-bp promoter regulate a hCD4 reporter. FACS analysis, in vitro colony assays, and in vivo competitive and secondary transplantation revealed that myeloid but not MEPs or lymphoid progenitors and also functional LT-HSCs are found almost exclusively in the Cebpa-hCD4(+) compared with hCD4(-) marrow population. hCD4(+) CMP yielded predominantly myeloid, whereas hCD4(-) CMP generated mainly Meg/E colonies. Providing insight into control of CMP maturation, Cebpa and Pu.1 RNAs were preferentially expressed in hCD4(+) CMP, Scl, Gata2, Gata1, Klf1, Ets1, and Fli1 predominated in hCD4(-) CMP, and Runx1, Myb, HoxA9, and Erg levels were similar in both. Cebpa-hCD4 transgene expression was lacking in multiple nonhematopoietic tissues. In summary, the +37-kb Cebpa enhancer and promoter are sufficient for marrow myeloid progenitor and LT-HSC-specific expression., (© 2014 Society for Leukocyte Biology.)

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

The effects of virus-like double-stranded RNA (dsRNA, PolyI:C) and DNA methyltransferase inhibitor (Aza-CdR) on CD4 gene expression were investigated in a porcine kidney cell line (PK15). We found that expression levels of TLR3 and IFNαwere significantly upregulated by PolyI:C, compared to the untreated PK15 cells, which shows that PolyI:C successfully mimics viral infection in PK15 cells. We also found that PolyI:C (10 μg/ml) and/or Aza-CdR (5 μM) significantly induces DNA demethylation of porcine CD4, promoting the binding of NF-κB to the CpG site on the CD4 promoter and activating expression of CD4. These data help clarify the regulatory mechanism of DNA methylation of the CD4 gene in non-immune cell response to virus replication. Further study is warranted to identify CD4 gene expression regulated by DNA methylation and live virus infection.

Cell-based immunotherapy has been gaining interest as an improved means to treat human immunodeficiency virus (HIV)/AIDS. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) could become a potential resource. Our previous studies have shown hESC and iPSC-derived natural killer (NK) cells can inhibit HIV-infected targets in vitro. Here, we advance those studies by expressing a HIV chimeric receptor combining the extracellular portion of CD4 to the CD3ζ intracellular signaling chain. We hypothesized that expression of this CD4ζ receptor would more efficiently direct hESC- and iPSC-derived NK cells to target HIV-infected cells. In vitro studies showed the CD4ζ expressing hESC- and iPSC-NK cells inhibited HIV replication in CD4+ T-cells more efficiently than their unmodified counterparts. We then evaluated CD4ζ expressing hESC (CD4ζ-hESC)- and iPSC-NK cells in vivo anti-HIV activity using a humanized mouse model. We demonstrated significant suppression of HIV replication in mice treated with both CD4ζ-modified and -unmodified hESC-/iPSC-NK cells compared with control mice. However, we did not observe significantly increased efficacy of CD4ζ expression in suppression of HIV infection. These studies indicate that hESC/iPSC-based immunotherapy can be used as a unique resource to target HIV/AIDS., (© 2013 AlphaMed Press.)

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4(+)CD25(-)EGFP(-) T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4(+)FOXP3(-) T cells into functional CD4(+)FOXP3(+) Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.

Background/aims: H. pylori persists for the virtual life of its host. Recent studies suggested that CD4 CD25+ regulatory T cells may be involved in this process. However, the alteration of CD4+ CD25+ regulatory T cells after eradication of H. pylori remains a question., Methodology: By using biopsies from 45 H. pylori-positive patients and the ones after eradication of H. pylori and 35 H. pylori-negative adults, real-time PCR and general PCR were used to quantify the expression of Foxp3 mRNA. IHC was used to semi- quantify the number of CD4+ CD25+ T cells in gastric mucosa., Results: We found that proportion ofCD25+ T cell in CD4+ T cells accounted for 0.739% in H. pylori-negative individuals, while it was accounted for 5.012% in H. pylori-positive patients. After eradication of H. pylori, proportion of CD25+ T cell in CD4+ T cells declined (P mRNA significantly decreased (P < 0.01) in gastric mucosa of patients after eradication of H. pylori., Conclusions: CD4+ CD25+ regulatory T cells decreased in gastric mucosa when patients received eradication of H. pylori. Eradication of H. pylori results in the significant decrease of Foxp3 mRNA in gastric mucosal, or using the drugs of anti-H. pylori induce the reduction of gastric mucosal Foxp3 mRNA expression, which is the a key regulatory gene for the development and function of CD4+ CD25+ regulatory T cells, thus contributing to the eradication of H. pylori. All the data offer new possibilities that Foxp3 gene may be the new target of immunization intervention strategies for eradication of H. pylori.

The current antiretroviral therapy (ART) can effectively reduce plasma HIV loads to undetectable levels, but cannot eliminate latently infected resting memory CD4 T cells that persist for the lifetime of infected patients. Therefore, designing new therapeutic approaches to eliminate these latently infected cells or the cells that produce HIV upon reactivation from latency is a priority in the ART era in order to progress to a cure of HIV. Here, we show that "designer" T cells expressing chimeric antigen receptor (CAR), CD4-CD28-CD3ζ, can target and kill HIV Env-expressing cells. Further, they secrete effector cytokines upon contact with HIV Env+ target cells that can reactivate latent HIV in a cell line model, thereby exposing those cells to recognition and killing by anti-HIV CAR+ T cells. Taken to the limit, this process could form the basis for an eventual functional or sterilizing cure for HIV in patients., (© 2013 Elsevier Inc. All rights reserved.)

Optical neuritis (ON) is characterized by inflammation of the optic nerve, and is one of the first clinical signs of multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is the animal model used to study MS and ON. The present study evaluated the induction, development and progression of ON using an EAE model induced by 100 μg or 300 μg of MOG35-55. An EAE model was induced in C57BL/6 mice by tail base injection of 100 μg or 300 μg of MOG35-55 in complete Freund's adjuvant, supplemented with Mycobacterium tuberculosis. On the day of injection and 48 h later, animals received intraperitoneally 300 ng of pertussis toxin. On days 7, 10, 14, 21 and 58 the optic nerve was dissected for histological analysis, production of CCL5 and immunohistochemical detection of CD4 and CD8. The histological changes observed in the optic nerves consisted of inflammatory cell infiltrates showing varying degrees of ON in the two groups. The onset of ON in the 300 μg of MOG35-55 group was coincident with higher production of CCL5, on day 10 after induction. However, the 100 μg MOG35-55 group showed more intense inflammatory infiltrate on day 14 after induction, with higher amounts of CD4 and CD8, reaching an excessive demyelination process on days 21 and 58 after induction. The results suggest that two different concentrations of MOG35-55 lead to different forms of evolution of optic neuritis.

Mice that are transgenic (Tg) for T cell receptor (TCR) expression are used extensively to analyze longitudinal T cell responses during effector and memory phases of the T cell response. Generation of TCR Tg mice generally requires T cell stimulation and cloning in vitro prior to amplification, processes which introduce biases into selection of the TCR that is ultimately chosen for TCR Tg mouse generation. Here we describe an alternative approach that involves no T cell stimulation or propagation in vitro. We generated mice that were transgenic for a TCR responding to a CD4 T cell epitope (epitope M133) that is immunodominant in mice infected with a neurotropic coronavirus, the JHM strain of mouse hepatitis virus. The CD4 T cell response to epitope M133 is of particular interest because it may be pathogenic, protective or regulatory, depending upon the physiological setting. We applied an iterative process in which we identified a TCR-β chain expressed by all mice that were examined ('public sequence'). This TCR-β chain was introduced into bone marrow cells with a lentivirus vector, generating TCR-β retrogenic mice. A TCR-α chain that paired with this TCR-β was then identified and used to generate a second set of TCR (α/β) retrogenic mice. After demonstrating that these cells were functional and responded to epitope M133, these TCR chains were used to generate an epitope M133-specific TCR Tg mouse. This method should be generally useful for engineering TCR Tg mice without introduction of bias caused by in vitro manipulation and propagation., (© 2013.)

Background: The HIV-1 accessory protein, Nef, is decisive for progression to AIDS. In vitro characterization of the protein has described many Nef activities of unknown in vivo significance including CD4 downregulation and a number of activities that depend on Nef interacting with host SH3 domain proteins. Here, we use the BLT humanized mouse model of HIV-1 infection to assess their impact on viral replication and pathogenesis and the selection pressure to restore these activities using enforced in vivo evolution., Results: We followed the evolution of HIV-1LAI (LAI) with a frame-shifted nef (LAINeffs) during infection of BLT mice. LAINeffs was rapidly replaced in blood by virus with short deletions in nef that restored the open reading frame (LAINeffs∆-1 and LAINeffs∆-13). Subsequently, LAINeffs∆-1 was often replaced by wild type LAI. Unexpectedly, LAINeffs∆-1 and LAINeffs∆-13 Nefs were specifically defective for CD4 downregulation activity. Viruses with these mutant nefs were used to infect BLT mice. LAINeffs∆-1 and LAINeffs∆-13 exhibited three-fold reduced viral replication (compared to LAI) and a 50% reduction of systemic CD4+ T cells (>90% for LAI) demonstrating the importance of CD4 downregulation. These results also demonstrate that functions other than CD4 downregulation enhanced viral replication and pathogenesis of LAINeffs∆-1 and LAINeffs∆-13 compared to LAINeffs. To gain insight into the nature of these activities, we constructed the double mutant P72A/P75A. Multiple Nef activities can be negated by mutating the SH3 domain binding site (P72Q73V74P75L76R77) to P72A/P75A and this mutation does not affect CD4 downregulation. Virus with nef mutated to P72A/P75A closely resembled the wild-type virus in vivo as viral replication and pathogenesis was not significantly altered. Unlike LAINeffs described above, the P72A/P75A mutation had a very weak tendency to revert to wild type sequence., Conclusions: The in vivo phenotype of Nef is significantly dependent on CD4 downregulation but minimally on the numerous Nef activities that require an intact SH3 domain binding motif. These results suggest that CD4 downregulation plus one or more unknown Nef activities contribute to enhanced viral replication and pathogenesis and are suitable targets for anti-HIV therapy. Enforced evolution studies in BLT mice will greatly facilitate identification of these critical activities.

Background: The highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef's most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D., Results: Single HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function., Conclusions: Results support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.

The development of an effective vaccine preventing HIV-1 infection remains elusive. Thus, the development of novel approaches capable of preventing HIV-1 transmission is of paramount importance. However, this is partly hindered by the lack of an easily accessible small-animal model to rapidly measure viral entry. Here, we report the generation of a human CD4- and human CCR5-expressing transgenic luciferase reporter mouse that facilitates measurement of peritoneal and genitomucosal HIV-1 pseudovirus entry in vivo. We show that antibodies and antiretrovirals mediate preexposure protection in this mouse model and that the serum antibody concentration required for protection from cervicovaginal infection is comparable to that required to protect macaques. Our results suggest that this system represents a model for the preclinical evaluation of prophylactic or vaccine candidates. It further supports the idea that broadly neutralizing antibodies should be evaluated for use as preexposure prophylaxis in clinical trials.

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are "plastic", and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1β, but not IL-6. "IL-17 potential" is restricted to population III (CD4(+) CD25(hi) CD127(lo) CD45RA(-) ) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary., (© 2013 The Authors. European Journal of Immunology published by Wiley-VCH Verlag GmbH & Co. KGaA Weinheim.)

Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.

Ptpn22 encodes PEST domain-enriched tyrosine phosphatase (Pep), which negatively regulates TCR proximal signaling and is strongly associated with a variety of autoimmune diseases in humans. The net effect of Pep on the balance of immunity and tolerance is uncertain because of the simultaneous inhibition of TCR-mediated signaling of effector and regulatory T cells (T(regs)). In this study, we generated transgenic NOD mice that overexpressed Pep in T cells. The transgenic mice had a significantly lower incidence of spontaneous autoimmune diabetes, which was accompanied by fewer IFN-γ-producing T cells, and an increased ratio of CD4(+)Foxp3(+) T(regs)to CD4(+)IFN-γ(+) or to CD8(+)IFN-γ(+) T cells, respectively, in pancreatic islets. Transgenic T cells showed markedly decreased TCR-mediated effector cell responses such as proliferation and Th1 differentiation. By contrast, the inhibitory effect of transgenic Pep on TCR signaling did not affect the differentiation of T(regs) or their suppressive activity. Adoptive transfer experiments showed that transgenic splenocytes exhibited attenuated diabetogenic ability. To examine further the pathogenic features of transgenic T cells, we generated Ptpn22/BDC2.5 doubly transgenic mice and found reduced proliferation and Th1 differentiation in CD4(+) T lymphocytes with additional Pep in pancreatic lymph nodes but not in inguinal lymph nodes of NOD/SCID recipients. This finding indicates that transgenic Pep attenuates T cell functions in an islet Ag-driven manner. Taken together, our results demonstrate that Pep overexpression in T cells attenuates autoimmune diabetes in NOD mice by preferentially modulating TCR signaling-mediated functions in diabetogenic T cells but not in T(regs).

The cytokine tumor necrosis factor (TNF) has pleiotropic functions both in normal physiology and disease. TNF signals by the virtue of two cell surface receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Exogenous TNF promotes experimental metastasis in some models, yet the underlying mechanisms are poorly understood. To study the contribution of host TNFR1 and TNFR2 on tumor cell progression and metastasis, we employed a syngeneic B16F10 melanoma mouse model of lung metastasis combined with in vivo bioluminescence imaging. Treatment of tumor-bearing mice with recombinant human TNF resulted in a significant increase in tumor burden and metastatic foci. This correlated with an increase in pulmonary regulatory CD4(+)/Foxp3(+) T cells. TNF caused an expansion of regulatory T (Treg) cells in vitro in a TNFR2-dependent manner. To assess the contribution of immune cell expression of endogenous TNF and its two receptors on B16F10 metastasis, we generated bone marrow chimeras by reconstituting wild-type mice with bone marrow from different knockout mice. Loss of either TNF or TNFR2 on immune cells resulted in decreased B16F10 metastasis and lower numbers of Treg cells within the lungs of these animals. Selective depletion of Treg cells attenuated metastasis even in conjunction with TNF treatment. We propose a novel mechanism in which TNF activates TNFR2 on Treg cells and thereby expands this immunosuppressive immune cell population. Loss of either TNF or TNFR2 prevents the accumulation of Treg cells and results in a less tolerogenic environment, enabling the immune system to control B16F10 tumor metastasis and growth.

The suppressor function of regulatory T cells (Tregs) is impaired in multiple sclerosis (MS), but the mechanisms underlying this deficiency are not fully understood. As Tregs counteract the sustained elevation of intracellular calcium, which is indispensable for full activation of conventional T cells (Tcons), we hypothesized that interference with this pathway might prompt MS-related Treg dysfunction. Using single-cell live imaging, we observed that Tregs rapidly reduce Ca(2+) influx and downstream signals in Tcons upon cell contact, yet differ in their potency to efficiently suppress several target cells at the same time. Strikingly, individual Tregs harboring a CD4(+)CD25(+)FOXP3(+)CD45RA(+) naive phenotype suppressed significantly more adjacent Tcons than did CD4(+)CD25(+)FOXP3(+)CD45RA(-) memory Tregs. Some constituents even completely failed to dampen Tcon Ca(2+) influx and were contained exclusively in the memory subset. In accordance with their more powerful suppressive performance, the Ca(2+) signature was considerably enhanced in naive Tregs in response to TCR triggering, compared with the memory counterparts. MS Tregs displayed a significantly diminished suppression of mean Ca(2+) influx in the sum of individual Tcons recorded. This reduced inhibitory activity was closely linked to decreased numbers of individual Tcons becoming suppressed by adjacent Tregs and, in turn, correlated with a marked reduction of naive subtypes and concomitant expansion of nonsuppressive memory phenotypes. We conclude that the superior achievement of naive Tregs is pivotal in maintaining Treg efficiency. As a consequence, MS Tregs become defective because they lack naive subtypes and are disproportionately enriched in memory cells that have lost their inherent downregulatory activity.

Nuclear factor (NF)-κB is a transcription factor implicated in the pathogenesis of autoimmune disorders such as rheumatoid arthritis (RA). Here we have examined the effect of intra-articular administration of the IKK inhibitor, NEMO-binding domain peptide (NBD), on the severity of collagen-induced arthritis (CIA). NBD peptides were injected intra-articularly into the knee joints of DBA/1J mice after the onset of disease. Collagen-injected mice given a scrambled peptide served as controls. Arthritis severity was determined by visual examination of paws. Intra-articular NBD injection reduced the arthritis score and ameliorated morphological signs of bone destruction compared to the controls. Serum levels of type-II collagen-specific immunoglobulin (Ig)G2a antibodies were lower in NBD-treated mice versus the control mice, whereas the levels of type-II collagen-specific IgG1 antibodies were increased by NBD treatment. NBD treatment diminished the proinflammatory cytokines interleukin (IL)-17 and interferon (IFN)-γ in serum, but increased the regulatory cytokine IL-10. NBD-treated CIA mice exhibited significantly higher percentages and numbers of forkhead box protein 3 (FoxP3(+)) CD4(+) CD25(+) regulatory T cells than controls. Immunofluorescence analysis of NBD-treated mice revealed that FoxP3 and Ym1, a marker of alternatively activated macrophages, were juxtaposed to each other within draining inguinal lymph nodes. Intra-articular administration of NBD peptide is effective as an experimental therapy in a murine model of RA. Nevertheless, the intra-articular treatment modality is still associated with systemic effects on the immune system., (© 2013 British Society for Immunology.)

Porcine CD4 proximal promoter and enhancer sequences were cloned and aligned with the corresponding human and murine sequences. The alignment showed nucleotide homology between porcine and human sequences was 62.4 % for the CD4 promoter and 56.6 % for the CD4 enhancer. The nucleotide homology between porcine and murine sequences was 42.5 % for the CD4 promoter and 25.4 % for the CD4 enhancer. The proximal enhancer and promoter regions of the CD4 gene from porcine, murine and human cells were compared for their ability to direct foreign gene expression in transiently transfected human cell lines. The results indicated the porcine CD4 promoters and enhancers could effectively direct expression of a foreign gene in human cells. The porcine promoter was equally efficient as CMV and EF-1α in directing gene expression.

Expression of clusters determination of T-cells immune links (CD4, CD8, CD95) and level of interferon-alpha at newborns with multiorgan failure syndrome were investigated. It were shown supressive character of immune reactions and reduction the level of interferon-alpha in this contingent of patients.