CD4 Downregulation Research Articles

FACS isolation of low percentage human antigen-specific CD8+ T cells based on activation-induced CD3 and CD8 downregulation

As T cell activation leads to downregulation of T cell receptor (TCR) and coreceptor CD8, we developed a novel FACS-based sorting method to enrich activated antigen-specific CD8+ T cells. Using multiple established or low percentage T cell cultures, with either single antigen specificity or multiple influenza A virus antigen specificities, we have optimized the sorting method for T cell activation time and stimulating antigen dose. We have also sorted various numbers of antigen-specific CD8+ T cells into 96-well plates to demonstrate these T cells are capable of expanding into nearly pure CD8+ T cell lines. Our approach has the advantage of sorting antigen-specific T cells without knowing their specific antigenic epitopes or restricting HLA. We believe this method can be very helpful for successfully establishing CD8+ T cell lines for various purpose, including immunotherapy.

Potent Enhancement of HIV-1 Replication by Nef in the Absence of SERINC3 and SERINC5.

It has recently emerged that HIV-1 Nef counteracts the antiviral host proteins SERINC3 and SERINC5. In particular, SERINC5 inhibits the infectivity of progeny virions when incorporated. SERINC3 and SERINC5 are also counteracted by the unrelated murine leukemia virus glycosylated Gag (glycoGag) protein, which possesses a potent Nef-like activity on HIV-1 infectivity. We now report that a minimal glycoGag termed glycoMA can fully substitute for Nef in promoting HIV-1 replication in Jurkat T lymphoid cells, indicating that Nef enhances replication in these cells mainly by counteracting SERINCs. In contrast, the SERINC antagonist glycoMA was unable to substitute for Nef in MOLT-3 T lymphoid cells, in which HIV-1 replication was highly dependent on Nef, and remained so even in the absence of SERINC3 and SERINC5. As in MOLT-3 cells, glycoMA was unable to substitute for Nef in stimulating HIV-1 replication in primary human cells. Although the ability of Nef mutants to promote HIV-1 replication in MOLT-3 cells correlated with the ability to engage endocytic machinery and to downregulate CD4, Nef nevertheless rescued virus replication under conditions where CD4 downregulation did not occur. Taken together, our observations raise the possibility that Nef triggers the endocytosis of a novel antiviral factor that is active against both laboratory-adapted and primary HIV-1 strains.IMPORTANCE The Nef protein of HIV-1 and the unrelated glycoGag protein of a murine leukemia virus similarly prevent the uptake of antiviral host proteins called SERINC3 and SERINC5 into HIV-1 particles, which enhances their infectiousness. We now show that although both SERINC antagonists can in principle similarly enhance HIV-1 replication, glycoGag is unable to substitute for Nef in primary human cells and in a T cell line called MOLT-3. In MOLT-3 cells, Nef remained crucial for HIV-1 replication even in the absence of SERINC3 and SERINC5. The pronounced effect of Nef on HIV-1 spreading in MOLT-3 cells correlated with the ability of Nef to engage cellular endocytic machinery and to downregulate the HIV-1 receptor CD4 but nevertheless persisted in the absence of CD4 downregulation. Collectively, our results provide evidence for a potent novel restriction activity that affects even relatively SERINC-resistant HIV-1 isolates and is counteracted by Nef.

PS1127 SF3B1 MUTATIONS ASSOCIATE WITH LOW CD20 EXPRESSION IN CLL: ANOTHER NOTCH1‐DEPENDENT MECHANISM?

Background:Chronic lymphocytic leukemia (CLL) is characterized by a CD20 expression lower than other lymphoproliferative disorders, hampering treatment efficacy with anti‐CD20 antibodies. NOTCH1 mutations determine a decrease of CD20 expression via a NOTCH1‐driven repression of CD20 transcription by an epigenetic HDAC‐mediated mechanism (Pozzo et al. Leukemia 2016). This may explain the association between NOTCH1 mutations and resistance to anti‐CD20 immunotherapy in CLL clinical trials (Stilgenbauer et al. Blood 2014). SF3B1 mutations, which are found in 10% CLL at diagnosis, induce transcriptome‐wide alterations of splicing patterns, thus affecting several pathways, including the NOTCH1 pathway (Wang et al. Cancer Cell 2016). In this context, one of the most aberrantly‐spliced genes is DVL2, a key component of the Wnt pathway and a known negative regulator of the NOTCH1 pathway (Collu et al. Development 2012).Aims:To define the association between CD20 expression and SF3B1‐mutation in CLL, by investigating NOTCH1‐mutation‐independent activation mechanisms of the NOTCH1 pathway through loss‐of‐function splicing alterations of the NOTCH1 negative regulator DVL2.Methods:Mutational status of NOTCH1 and SF3B1 was evaluated by NGS. CD20 expression was evaluated by flow cytometry by computing mean fluorescence intensities (MFI) and/or the percentages of the CD20dim population. Transcript levels of spliced DVL2 were evaluated by qRT‐PCR and NGS. Gene expression profile (GEP) was performed by a one‐color labeling strategy using the Agilent 8x60K platform.Results:In a cohort of 537 unselected CLL, 89 cases carried NOTCH1 mutations (NOTCH1‐mut); 48 cases carried SF3B1 mutations (SF3B1‐mut), the K700 hotspot being the most frequent (50%) followed by the G742 (30%) hotspot; 6 cases were both NOTCH1/SF3B1‐ mut. Confirming previous findings, NOTCH1‐mut cases were characterized by lower CD20 MFI compared to wild‐type (WT) cases (p = 0.0154). CD20 expression was found diminished also in SF3B1‐mut cases (p = 0.0059) and, consistently, NOTCH1 or SF3B1 mutations were also characterized by a greater proportion of CD20dim CLL cells (Fig.1A). Alternatively spliced DVL2 (altDVL2) was detected only in SF3B1‐mut CLL and its expression correlated with the SF3B1 mutational burden (Fig.1B). As DVL2 can act as negative regulator of NOTCH1, we speculated that loss of wild‐type DVL2 due to alternative splicing may result in a more active NOTCH1 pathway. The NOTCH1 target gene DTX1, more expressed in NOTCH1‐mut cases compared to WT, was also increased SF3B1‐mut cases (Fig.1C), where it positively correlated with altDVL2 levels and with a greater proportion of CD20dim CLL cells. In a GEP carried out on selected NOTCH1‐mut versus WT cases (6 vs 7), 760 probes were differentially expressed (272 probes were up‐regulated, including the NOTCH1‐related genes DTX1 and HNRNPH3 and the NF‐kB‐related gene RELA; 488 probes were down‐regulated, including the CD20 gene, MS4A1, and DUSP22). When applying this NOTCH1‐related signature to SF3B1‐mut cases (5 cases), the latter segregated with NOTCH1‐mut cases (Fig.1D), suggesting a GEP relationship between NOTCH1‐mut and SF3B1‐mut CLL cells. Finally, loss of wild‐type DVL2 in the CLL‐like MEC1 cell line by siRNA transfection resulted, after 24 hours, in a downregulation of CD20 at both protein (p = 0.0062) and transcript (p = 0.0129) level (Fig.1E).Summary/Conclusion:CLL cases bearing SF3B1 mutations are characterized by a lower CD20 expression, allegedly through a more active NOTCH1 pathway, potentially resulting in clinical resistance to anti‐CD20 monoclonal antibodies.image

CD4- and Time-Dependent Susceptibility of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity.

HIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) antibodies within HIV-1-positive (HIV-1+) individuals predominantly target CD4-induced (CD4i) epitopes on HIV-1 envelope glycoprotein (Env). These CD4i epitopes are usually concealed on the surface of infected cells due to CD4 downregulation by the HIV-1 accessory proteins Nef and Vpu. We hypothesized that early-stage infected cells in the process of downregulating CD4 could be more susceptible to ADCC than late-stage infected cells that have fully downregulated CD4. There was significantly higher binding of antibodies within plasma from HIV-1-infected individuals to early-stage infected cells expressing intermediate levels of CD4 (CD4-intermediate cells) than in late-stage infected cells expressing low levels of CD4 (CD4-low cells). However, we noted that HIV-1-uninfected bystander cells and HIV-1-infected cells, at various stages of downregulating CD4, were all susceptible to NK cell-mediated ADCC. Importantly, we observed that the cytolysis of bystander cells and early infected cells in this culture system was driven by sensitization of target cells by inoculum-derived HIV-1 Env or virions. This phenomenon provided Env to target cells prior to de novo Env expression, resulting in artifactual ADCC measurements. Future studies should take into consideration the inherent caveats of in vitro infection systems and develop improved models to address the potential role for ADCC against cells with nascent HIV-1 infection.IMPORTANCE An increasing body of evidence suggests that ADCC contributes to protection against HIV-1 acquisition and slower HIV-1 disease progression. Targeting cells early during the infection cycle would be most effective in limiting virus production and spread. We hypothesized that there could be a time-dependent susceptibility of HIV-1-infected cells to ADCC in regard to CD4 expression. We observed NK cell-mediated ADCC of HIV-1-infected cells at multiple stages of CD4 downregulation. Importantly, ADCC of early infected cells appeared to be driven by a previously unappreciated problem of soluble Env and virions from the viral inoculum sensitizing uninfected cells to ADCC prior to de novo Env expression. These results have implications for studies examining ADCC against cells with nascent HIV-1 infection.

CD4-Dependent Modulation of HIV-1 Entry by LY6E.

Lymphocyte antigen 6E (LY6E) is a GPI-anchored, interferon-inducible protein that has been shown to modulate viral infection in a cell type-dependent manner. Our recent work showed that LY6E promotes HIV-1 infection in some high-CD4-expressing cells, including human peripheral blood mononuclear cells (PBMCs) and the SupT1 cell line. In this work, we provide evidence that LY6E inhibits HIV-1 entry and spread in low-CD4-expressing Jurkat cells and human monocyte-derived macrophages (MDMs) through downregulation of the viral receptor CD4. We found that knockdown of LY6E in Jurkat cells and MDMs increases HIV-1 infection, yet overexpression of LY6E in Jurkat cells inhibits HIV-1 entry and replication. LY6E was found to be colocalized with CD4 on the plasma membrane of Jurkat cells and MDMs and enhances CD4 internalization. We artificially manipulated the CD4 level in Jurkat and SupT1 cells and found that overexpression of CD4 in Jurkat cells overcomes the inhibitory effect of LY6E; conversely, blocking the function of CD4 in SupT1 with a neutralizing antibody eliminates the enhancement of LY6E on HIV-1 entry. The CD4-dependent inhibitory phenotype of LY6E in low-CD4-expressing human MDMs can be recapitulated for a panel of transmitted founder viruses and laboratory-adapted HIV-1 strains. Given that HIV-1 can target low-CD4-expressing cells during acute infection yet replicates efficiently in high-CD4-expressing T cells at the late stage of disease, our observation that LY6E differentially modulates HIV-1 replication in a CD4-dependent manner has implications for understanding the complex roles of interferon (IFN)-induced proteins in AIDS pathogenesis.IMPORTANCE The role of IFN-induced genes (ISGs) in viral infection remains incompletely understood. While most ISGs are antiviral, some ISGs have been shown to promote viral infection, including HIV-1 infection. We previously showed that IFN-inducible LY6E protein promotes HIV-1 infection in human PMBCs and high-CD4-expressing SupT1 cells. Here we found that LY6E inhibits HIV-1 entry and replication in low-CD4-expressing MDMs and Jurkat cells. Mechanistically, we demonstrated that LY6E downregulates the cell surface receptor CD4, thus impairing the virus binding to target cells. This is in contrast to the situation of high-CD4-expressing cells, where LY6E predominantly promotes viral membrane fusion. The opposing role of IFN-inducible LY6E in modulating HIV-1 infection highlights the complex roles of ISGs in viral infection and viral pathogenesis.

The Novel HDAC Inhibitor Chidamide Synergizes with Rituximab to Inhibit DLBCL Tumor Growth in Vitro and In Vivo By up-Regulating CD20 Expression

Background: Histone deacetylases (HDACs) are crucial proteins for supporting tumorigenesis. HDACs reverse chromatin acetylation and alter transcription of oncogenes and tumor suppressor genes by removing acetyl groups from histones. HDAC inhibitors are considered as promising anti-cancer drugs, particularly in combination with other standard treatment regimens. Chidamide is the world first oral HDAC inhibitor which selectively inhibits class I HDAC1, HDAC2, and HDAC3 as well as class IIb HDAC10. Chidamide has been approved by China FDA in 2015 for the treatment of relapsed or refractory peripheral T-cell lymphoma.Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of B-cell lymphoma. Treatment with R-CHOP i.e. Rituximab (the anti-CD20 monoclonal antibody) plus CHOP (Cyclophosphamide, doxorubicin, vincristine, and prednisone) has significantly improved clinical outcome for DLBCL patients. However, treatment-induced deacetylation of CD20 gene and consequently down-regulation of CD20 protein expression causes an acquired resistance to further treatment with R-CHOP.We hypothesize that inhibition of HDACs by Chidamide could overcome Rituximab-mediated down-regulation of CD20 and facilitate Rituximab-induced DLBCL tumor growth inhibition. The aim of this study is to determine the synergistic effect of Chidamide and Rituximab in the treatment of DLBCL in vitro and in vivo.Methods: The levels of CD20 (MS4A1) mRNA expression and clinical outcomes in patients with DLBCL treated either with R-CHOP or CHOP were obtained from the Gene Expression Omnibus (GEO) repository (NCBI GSE 10846). The association of CD20 expression with overall survival (OS) was analyzed by Cox regression analysis and the cut-off point was calculated by the X-tile software. CD20 protein surface expression and Rituximab-induced cell death were analyzed by flow cytometry. The IC50s of Chidamide and the synergisms with Rituximab (10 µg/ml) on five DLBCB cell lines (OCI-LY3, OCI-LY7, Su-DHL6, Su-DHL8, and Su-DLH10) were determined by MTT test after cells were treated with a range of concentrations of Chidamide with or without Rituximab for 24 hours. The synergism was calculated using ComboSyn software to obtain the combination index (CI). For in vivo experiments, the human DLBCL cell line OCI-LY7 were injected to 6 weeks BALB/C nude mice to develop xenograft DLBCL mice models. After tumors were palpable, mice were divided into four groups and injected with NaCl (control), Rituximab, Chidamide and Rituximab plus Chidamide daily for three weeks. The tumor volumes were monitored frequently during the treatment.Results: In R-CHOP treated cohort (n=233), higher expression of CD20 expression (n=137) is significantly associated with superior clinical outcomes compared with lower CD20 expression (n=96) with P=0.0038, HR=0.4753, 95% CI=0.274-0.779. However, the levels of CD20 have no effect on clinical outcome in DLBCL patients treated with CHOP (n=183). The levels of CD20 protein surface expression on five DLBCL cell lines were significantly and positively correlated with the sensitivities of cells to Rituximab-induced cell death (P=0.0018, R=0.88). HDAC1, HDCA2 and HDCA3 proteins were detected in these DLBCL cell lines. Treatment with Rituximab significantly reduced CD20 surface expression but treatment with Chidamide significantly increased CD20 surface expression in DLBCL cells. The CI numbers for combined treatment with Chidamide and Rituximab were either <0.01 (very strong synergism) or <0.3 (strong synergism), indicating that Chidamide significantly synergized Rituximab-induced cell death. For in vivo assay, treatment with either Rituximab or Chidamide alone slightly but not significantly reduced tumor volume. Combination with Chidamide and Rituximab significantly inhibited tumor growth in DLBCL xenograft mice (P<0.0001). Mice with combined treatment showed significantly prolonged survival compared with other groups.Conclusions: our data demonstrate for the first time that inhibition of HDACs by Chidamide significantly synergized Rituximab-induced tumor growth inhibition in vitro and in vivo. We propose that CD20 surface expression should be used clinically to evaluate treatment response in patients with DLBCL. Chidamide is a promising sensitizer for the treatment of DLBCL with R-CHOP. DisclosuresNo relevant conflicts of interest to declare.

Cutting Edge: The Histone Methyltransferase G9a Is Required for Silencing of Helper T Lineage-Associated Genes in Proliferating CD8 T Cells.

Helper versus cytotoxic T lineage decision in the thymus has been studied as a model for silencing of alternative lineage genes. Although the transcription factor RUNX3 is required for the initiation of Cd4 silencing in developing CD8 T cells, it is unknown how silencing of Cd4 and other helper T lineage genes is maintained. We show that the histone methyltransferase G9a is necessary for silencing helper T lineage genes in proliferating mouse CD8 T cells. Despite normal initial Cd4 downregulation, G9a-deficient CD8 T cells derepress Cd4 and other helper lineage genes during repeated division in lymphopenia or in response to tumor Ag. However, G9a was dispensable for continued silencing of those genes in CD8 T cells that respond to infection by Listeria monocytogenes These results demonstrate that G9a facilitates maintenance of cellular identity of CD8 T cells during cell division, which is further reinforced by inflammatory signals.

Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses.

ABSTRACTHIV-1-infected cells expressing envelope glycoproteins (Env) in the CD4-bound conformation on their surfaces are targeted by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) antibodies and sera from HIV-1-infected individuals (HIV+ sera). By downregulating the surface expression of CD4, Nef prevents Env-CD4 interaction, thus protecting HIV-1-infected cells from ADCC. HIV-1 infectious molecular clones (IMCs) are widely used to measure ADCC. In order to facilitate the identification of infected cells and high-throughput ADCC analysis, reporter genes (e.g., the Renilla luciferase [LucR] gene) are often introduced into IMC constructs. We evaluated the susceptibility of HIV-1-infected CD4+ T lymphocytes to ADCC using a panel of parental IMCs and derivatives that expressed the LucR reporter gene, utilizing different molecular strategies, including one specifically designed to retain Nef expression. We found that in some of these constructs, Nef expression in CD4+ T cells was suboptimal, and consequently, CD4 downregulation was incomplete. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Strikingly, protection from ADCC was observed when cells were infected with the parental IMC, which exhibited strong CD4 downregulation. This discrepancy between the parental and Nef-impaired viruses was independent of the strains of Env expressed, but rather, it was correlated with the levels of CD4 surface expression. Overall, our results indicate that caution should be taken when selecting IMCs for ADCC measurements and that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.IMPORTANCE In-depth understanding of the susceptibility of HIV-1-infected cells to ADCC might help establish correlates of vaccine protection and guide the development of HIV-1 vaccine strategies. Different ADCC assays have been developed, including those using infectious molecular clones (IMCs) carrying a LucR reporter gene that greatly facilitates large-scale quantitative analysis. We previously reported different molecular strategies for introducing LucR while maintaining Nef expression and function and, consequently, CD4 surface downregulation. Here, we demonstrate that utilizing IMCs that exhibit impaired Nef expression can have undesirable consequences due to incomplete CD4 downregulation. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Overall, our results indicate that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

The Effects of Anti-LAP Monoclonal Antibody Down-regulation of CD4+LAP+ T Cells on Allogeneic Corneal Transplantation in Mice

CD4+latency-associated peptide (LAP)+ T cells are a newly discovered T cell subset with suppressive function on immune responses. In this study, we investigate the role of CD4+LAP+ T cells on mice corneal allograft survival by down-regulating their expression using anti-LAP mAb. We show that a blockage of LAP leads to a decrease in the percentage of T cells expressing CD4+Foxp3+, CD4+GARP+, CD4+LAP+ and CD4+IL-10+ in the lymph nodes and spleens of mice undergoing orthotopic penetrating transplantation of corneal allograft, without affecting corneal graft survival. In addition, higher percentages of CD4+IFN-γ+ and CD4+IL-17A+ T cells in the lymph nodes and spleens, as well as TNF, IFN-γ, IL-17A and IL-6 levels in the aqueous humor, significantly increase in mice with rejected corneal grafts. The expression of TGF-β1 decreases in corneal grafts during corneal rejection period. It is therefore possible that anti-LAP mAb can down-regulate the regulatory T cell subsets with its immunosuppressive effects. The rejection of corneal grafts seems to mainly be associated with the up-regulation of Th1 and Th17 cell subsets in peripheral lymph nodes.

HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System.

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 NH4Cl 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.

Peripheral blood T lymphocytes are downregulated by the PD-1/PD-L1 axis in advanced gastric cancer.

IntroductionProgrammed death-1 (PD-1) and programmed death ligand-1 (PD-L1) function as an immune checkpoint pathway that can be exploited by tumor cells to evade immuno-surveillance. The precise role of PD-1/PD-L1 inhibition of the immune response in GC is unknown. The study investigated PD-1 and PD-L1 expression on peripheral T-cells and its potential association with clinicopathological features in gastric cancer (GC) patients.Material and methodsPD-1/PD-L1 expression on CD4(+) and CD8(+) T-cells from peripheral blood of 40 patients primarily diagnosed with advanced GC was evaluated by multicolor flow cytometry.ResultsThe frequency of CD4(+)PD-1(+) and CD8(+)PD-1(+) cells in GC patients was higher than in the control group (p < 0.0001 and p < 0.01, respectively). Expression of PD-1 on CD8(+) cells in GC was higher than in the control group (p < 0.0001). The frequency of CD4(+)PD-L1(+) and CD8(+)PD-L1(+) cells was higher than in the control group (p < 0.0001). Expression of PD-L1 on CD4(+) and CD8(+) cells in GC was higher than in the control group (p < 0.0001). A higher frequency of CD4(+)PD-1(+) cells was found in diffuse-type compared to intestinal tumors (p < 0.029). A higher frequency of CD8(+)PD-1(+) cells was found in patients with poorly differentiated compared to well/moderately differentiated tumors (p < 0.019).ConclusionsDownregulation of peripheral blood CD4(+) and CD8(+) lymphocytes can be associated with PD-1/PD-L1 expression. This can lead to attenuation of the general immune response in GC.

Leukemic phase of primary cutaneous anaplastic large-cell lymphoma (ALK-negative), with downregulation of CD30

Leukemic phase of anaplastic large cell lymphoma (ALCL) is extremely rare. We present a case of primary cutaneous-ALCL (C-ALCL), who developed nodal involvement 4 years later. The patient was treated with combination chemotherapy and autologous stem cell transplantation; however, she relapsed 5 months later. At that time, the patient was given brentuximab vedotin (BV) with initial response. One year later, the patient demonstrated progressive adenopathy and leukemic involvement by her ALCL (comparative T cell clonality showed PCR products of the same size in the diagnostic skin and relapsed leukemic blood). Interestingly, the circulating tumor cells showed only subset expression of CD30 by flow cytometry (performed on peripheral blood). This patient demonstrates an unusual manifestation of ALCL (leukemic phase), with the additional confounder of CD30 downregulation, perhaps secondary to the BV therapy, or possible CD30-negative tumor escape.

Maintenance of AP-2-Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses.

Nef-specific CD8+ T lymphocytes (CD8TL) are linked to extraordinary control of primate lentiviral replication, but the mechanisms underlying their efficacy remain largely unknown. The immunodominant, Mamu-B*017:01+-restricted Nef195-203MW9 epitope in SIVmac239 partially overlaps a sorting motif important for interactions with host AP-2 proteins and, hence, downmodulation of several host proteins, including Tetherin (CD317/BST-2), CD28, CD4, SERINC3, and SERINC5. We reasoned that CD8TL-driven evolution in this epitope might compromise Nef's ability to modulate these important molecules. Here, we used deep sequencing of SIV from nine B*017:01+ macaques throughout infection with SIVmac239 to characterize the patterns of viral escape in this epitope and then assayed the impacts of these variants on Nef-mediated modulation of multiple host molecules. Acute variation in multiple Nef195-203MW9 residues significantly compromised Nef's ability to downregulate surface Tetherin, CD4, and CD28 and reduced its ability to prevent SERINC5-mediated reduction in viral infectivity but did not impact downregulation of CD3 or major histocompatibility complex class I, suggesting the selective disruption of immunomodulatory pathways involving Nef AP-2 interactions. Together, our data illuminate a pattern of viral escape dictated by a selective balance to maintain AP-2-mediated downregulation while evading epitope-specific CD8TL responses. These data could shed light on mechanisms of both CD8TL-driven viral control generally and on Mamu-B*017:01-mediated viral control specifically.IMPORTANCE A rare subset of humans infected with HIV-1 and macaques infected with SIV can control the virus without aid of antiviral medications. A common feature of these individuals is the ability to mount unusually effective CD8 T lymphocyte responses against the virus. One of the most formidable aspects of HIV is its ability to evolve to evade immune responses, particularly CD8 T lymphocytes. We show that macaques that target a specific peptide in the SIV Nef protein are capable of better control of the virus and that, as the virus evolves to escape this response, it does so at a cost to specific functions performed by the Nef protein. Our results help show how the virus can be controlled by an immune response, which could help in designing effective vaccines.

A single β-octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of partner protein binding.

The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners. In cell-based fluorescence complementation assays, Nef forms homodimers at the plasma membrane. However, recombinant Nef proteins often exist as monomers in solution, suggesting that membrane interaction may also trigger monomer to dimer transitions. In this study, we show that monomeric Nef core proteins can be induced to form dimers in the presence of low concentrations of the non-ionic surfactant, β-octyl glucoside (βOG). X-ray crystallography revealed that a single βOG molecule is present in the Nef dimer, with the 8-carbon acyl chain of the ligand binding to a hydrophobic pocket formed by the dimer interface. This Nef-βOG dimer interface involves helix αB, as observed in previous dimer structures, as well as a helix formed by N-terminal residues 54–66. Nef dimer formation is stabilized in solution by the addition of βOG, providing biochemical validation for the crystal structure. These observations together suggest that the interaction with host cell lipid mediators or other hydrophobic ligands may play a role in Nef dimerization, which has been previously linked to multiple Nef functions including host cell protein kinase activation, CD4 downregulation, and enhancement of HIV-1 replication.

The HIV-1 accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+ T cells

BackgroundThe HIV-1 accessory proteins Nef and Vpu alter cell surface levels of multiple host proteins to modify the immune response and increase viral persistence. Nef and Vpu can downregulate cell surface levels of the co-stimulatory molecule CD28, however the mechanism of this function has not been completely elucidated.ResultsHere, we provide evidence that Nef and Vpu decrease cell surface and total cellular levels of CD28. Moreover, using inhibitors we implicate the cellular degradation machinery in the downregulation of CD28. We shed light on the mechanisms of CD28 downregulation by implicating the Nef LL165 and DD175 motifs in decreasing cell surface CD28 and Nef DD175 in decreasing total cellular CD28. Moreover, the Vpu LV64 and S52/56 motifs were required for cell surface CD28 downregulation, while, unlike for CD4 downregulation, Vpu W22 was dispensable. The Vpu S52/56 motif was also critical for Vpu-mediated decreases in total CD28 protein level. Finally, the ability of Vpu to downregulate CD28 is conserved between multiple group M Vpu proteins and infection with viruses encoding or lacking Nef and Vpu have differential effects on activation upon stimulation.ConclusionsWe report that Nef and Vpu downregulate cell surface and total cellular CD28 levels. We identified inhibitors and mutations within Nef and Vpu that disrupt downregulation, shedding light on the mechanisms utilized to downregulate CD28. The conservation and redundancy between the abilities of two HIV-1 proteins to downregulate CD28 highlight the importance of this function, which may contribute to the development of latently infected cells.

Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.

Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24+ HIV iCD4s that maintained cell surface CD4 (iCD4+), did not maintain CD4 (iCD4−) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4+ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4−) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3

Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. While Nef-mediated CD4 downmodulation is conserved, the ability to internalize CD3 was lost in HIV-1 and its precursors. Although both functions play key roles in lentiviral replication and pathogenicity, the underlying structural requirements are poorly defined. Here, we determine the structure of SIVmac239 Nef bound to the ExxxLM motif of another Nef molecule at 2.5 Å resolution. This provides a basis for a structural model, where a hydrophobic crevice in simian immunodeficiency virus (SIV) Nef targets a dileucine motif in CD4 and a tyrosine-based motif in CD3. Introducing key residues into this crevice of HIV-1 Nef enables CD3 binding but an additional N-terminal tyrosine motif is required for internalization. Our resolution of the CD4/Nef/AP2 complex and generation of HIV-1 Nefs capable of CD3 downregulation provide insights into sorting motif interactions and target discrimination of Nef.

Amino acids at positions 3, 168, and 169 are associated with the ability of Nef proteins from HIV-1 CRF01_AE to downmodulate CD4.

Several HIV-1 subtypes are co-circulating among various high-risk groups in China, and an increasing prevalence of CRF01_AE was observed among MSM (men who have sex with men) within recent years. Patients infected with CRF01_AE may experience a more rapid disease progression than patients infected with non-CRF01_AE; however, the underlying mechanisms remains elusive. HIV-1 Nef is a multifunctional protein and plays critical roles in viral pathogenesis. Nef downregulates CD4 and human leukocyte antigen (HLA) to promote viral transmission and escape from the host immune response. In this study, we investigated the CD4 downmodulation activity of Nef proteins isolated from HIV-1 CRF01_AE and analyzed a potential relationship of Nef's capacity to downregulate CD4 with disease progression. We found that the majority of these Nefs from HIV-1 CRF01_AE efficiently downregulated CD4; Nefs with weaker CD4 downmodulation activity tended to be associated with higher CD4 levels and lower viral loads. Further elucidation revealed that amino acid residues at positions 3, 168, and 169 of CRF01_AE Nefs were associated with the capacity to downregulate CD4. Our data suggest that the capacity of Nef-mediated CD4 downregulation is not the only determinant for controlling disease progression, and other host and viral factors should be considered to explain the rapid disease progression of patients infected with HIV-1 CRF01_AE.

An upregulation of CD8+CD25+Foxp3+ T cells with suppressive function through interleukin 2 pathway in pulmonary arterial hypertension

Accumulating evidence suggests that abnormal inflammation plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH). CD8+CD25+Foxp3+ T cell is a novel cell subtype, and its role in PAH is not yet investigated. Here, we observed that PAH patients presented a significant upregulation of CD8+CD25+Foxp3+ T cells and a downregulation of CD4+CD25+Foxp3+ T cells compared to healthy controls. Regardless, the total number of CD25+Foxp3+ T cells in PAH patients was still smaller than that in healthy controls. Compared to CD8+CD25- T cells, CD8+CD25+ T cells presented higher Foxp3 expression, lower interferon (IFN)-γ expression and higher transforming growth factor (TGF)-β expression, in both healthy and PAH individuals. The CD8+CD25+ T cells in PAH patients also demonstrated regulatory function by suppressing the proliferation of CD4+CD25- and CD8+CD25- effector T cells, albeit at lower efficiency than CD4+CD25+ T cells from PAH patients and healthy volunteers. CD8+CD25+ T cells from PAH responded to interleukin (IL)−2 supplement by expansion and upregulating Foxp3 expression. In PAH patients, IL-2-treated CD8+CD25+ T cells were more potent at inhibiting CD4+CD25- effector T cell proliferation than IL-2-untreated CD8+CD25+ T cells. Together, we found an upregulation of CD8+CD25+Foxp3+ T cells in PAH patients, and this T cell population presented suppressive activity that could be enhanced by IL-2 treatment.