Human CD4 T-cell Research Articles

The immunodominant HLA-A2-restricted MART-1 epitope is not presented on the surface of many melanoma cell lines.

Among the relatively large number of known tumor-associated antigens (TAA) which are recognized by human CD8 T-cells, Melan-A/MART-1 is one of the most-if not the most-frequently used target for anti-cancer vaccines in HLA-A2 + melanoma patients. In this study, we analyzed the killing of a large panel of melanoma cells by a high avidity, MART-1-specific T-cell clone or a MART-1-specific, polyclonal T-cell culture. Strikingly, we observed that the MART-1-specific T-cells only killed around half of the analyzed melanoma cell lines. In contrast a Bcl-2-specific T-cell clone killed all melanoma cell lines, although the T-cell avidity of this clone was significantly lower. The MART-1-specific T-cell clone expressed NKG-2D and was fully capable of releasing both perforin and Granzyme B. Notably, the resistance to killing by the MART-1-specific T-cells could be overcome by pulsing of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy.

FOXP3 inhibits HIV-1 infection of CD4 T-cells via inhibition of LTR transcriptional activity

FOXP3 is a necessary transcription factor for the development and function of CD4+ regulatory T-cells (Tregs). The role of Tregs in HIV-1 infection remains unclear. Here, we show that expression of FOXP3 in primary human CD4 T-cells significantly inhibits HIV-1 infection. Since FOXP3 inhibits NFAT activity, and NFAT proteins contribute to HIV-1 transcription, we explore a transcriptional repressive function of HIV-1 LTR by FOXP3. Over-expression of FOXP3 in primary CD4 T-cells inhibits wild-type HIV-1 LTR reporter activity, and truncation mutants demonstrate that repression of the LTR by FOXP3 requires the dual proximal NFκB/NFAT binding sites. Interestingly, FOXP3 decreases binding of NFAT2 to the HIV-1 LTR in vivo. Furthermore, FOXP3 does not inhibit infection of HIV-1 NL4-3 which is mutated to disrupt transcription factor binding at either proximal NFAT or NFκB binding sites. These data suggest that resistance of Tregs to HIV-1 infection is due to inhibition of HIV-1 LTR transcription by FOXP3.

Induction of Pluripotent Protective Immunity Following Immunisation with a Chimeric Vaccine against Human Cytomegalovirus

Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8+ and CD4+ T-cells which co-expressed IFN-γ and TNF-α, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8+ and CD4+ T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.

Antiretroviral Therapy prior to Acute Viral Replication Preserves CD4 T Cells in the Periphery but Not in Rectal Mucosa during Acute Simian Immunodeficiency Virus Infection

The rectal mucosa is a major site for human immunodeficiency virus entry and CD4 T-cell depletion. The early and near-total loss of these cells from the rectal mucosa severely compromises the ability of the mucosal immune system to control various opportunistic infections. Protecting these cells from infection and destruction can delay disease progression, leading to a better long-term outcome. Here we show that effective suppression of viral infection in memory CD4 T cells from the rectal mucosa and peripheral blood to a very low level with antiretroviral therapy (ART) initiated prior to the peak of infection is associated with opposite outcomes in these tissues. A near-total loss of CD4 T cells in the rectal mucosa contrasted with preservation of most memory CD4 T cells in peripheral blood during the course of treatment. Interestingly, ART significantly reduced viral infection in memory CD4 T cells from both rectal mucosa and peripheral blood. Although early ART was of limited value in protecting the CD4 T cells in the rectal mucosa, the significant preservation of peripheral CD4 T cells could contribute to maintaining immune competence, leading to a better long-term outcome.

C-Cbl expression levels regulate the functional responses of human central and effector memory CD4 T cells

AbstractThe biochemical mechanisms controlling the diverse functional outcomes of human central memory (CM) and effector memory (EM) T-cell responses triggered through the T-cell receptor (TCR) remain poorly understood. We implemented reverse phase protein arrays to profile TCR signaling components in human CD8 and CD4 memory T-cell subsets isolated ex vivo. As compared with CD4 CM cells, EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c-Cbl, Syk, Fyn, and LAT. Moreover, in EM cells reduced expression of negative regulator c-Cbl correlates with expression of c-Cbl kinases (Syk and Fyn), PI3K, and LAT. Importantly, consistent with reduced expression of c-Cbl, EM cells display a lower functional threshold than CM cells. Increasing c-Cbl content of EM cells to the same level as that of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism depends primarily on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Our study reports c-Cbl as a critical regulator of the functional responses of memory T cell subsets and identifies for the first time in humans a mechanism controlling the functional heterogeneity of memory CD4 cells.

Lysophosphatidylcholine upregulates LOX-1, chemokine receptors, and activation-related transcription factors in human T-cell line Jurkat

In arteriosclerosis, activated T cells infiltrating the atherosclerotic lesions are directly involved in the pathogenesis of these vascular disorders. Infiltration and accumulation of T cells into the vascular wall occur at the earliest stage of atherosclerosis. The atherosclerotic lesion also sees the accumulation of modified lipids such as lysophosphatidylcholine (lysoPC), the main phospholipid component of oxidized low-density lipoprotein. However, it remains less clear how lysoPC affects CD4 T cells. Therefore, we assessed the effect of lysoPC on various mRNA expressions in human T cells using real-time quantitative Reverse Transcription-PCR. Exposure of Jurkat cells (a human CD4 T-cell line) to lysoPC resulted in upregulation of CXCR4 and CCR5 chemokine receptors, receptor for oxidized low-density lipoprotein (LOX-1), the transcription factors, nuclear factor-kappa beta (NF-kB) and Yin Yang 1, and target molecules of NF-kB, A1/Bfl1/BCL2A1 and c-IAP1/BIRC2, in a dose-dependent manner. These data indicate that lysoPC is a positive regulator of the inflammatory response in human CD4 T cells. Further, it suggested that lysoPC and CD4 T cells accumulating in atherosclerotic lesions contribute to the development of arteriosclerosis.

Programmed death-1 blockade enhances expansion and functional capacity of human melanoma antigen-specific CTLs

Negative co-stimulatory signaling mediated via cell surface programmed death (PD)-1 expression modulates T and B cell activation and is involved in maintaining peripheral tolerance. In this study, we examined the effects of a fully human PD-1-abrogating antibody on the in vitro expansion and function of human vaccine-induced CD8+ T cells (CTLs) specific for the melanoma-associated antigens glycoprotein 100 (gp100) and melanoma antigen recognized by T cells (MART)-1. PD-1 blockade during peptide stimulation augmented the absolute numbers of CD3+, CD4+, CD8+ and gp100/MART-1 MHC:peptide tetramer+ CTLs. This correlated with increased frequencies of IFN-gamma-secreting antigen-specific cells and augmented lysis of gp100+/MART-1+ melanoma targets. PD-1 blockade also increased the fraction of antigen-specific CTLs that recognized melanoma targets by degranulation, suggesting increased recognition efficiency for cognate peptide. The increased frequencies and absolute numbers of antigen-specific CTLs by PD-1 blockade resulted from augmented proliferation, not decreased apoptosis. Kinetic analysis of cytokine secretion demonstrated that PD-1 blockade increased both type-1 and type-2 cytokine accumulation in culture without any apparent skewing of the cytokine repertoire. These findings have implications for developing new cancer immunotherapy strategies.

Anti-IE1 CD4+ T-cell clones kill peptide-pulsed, but not human cytomegalovirus-infected, target cells

Cellular immunity plays a major role in the control of human cytomegalovirus (HCMV) infection. CD4(+) T lymphocytes have been shown to contribute to this function but their precise role is a matter of debate. Although CD4(+) T cells have been shown to kill target cells through the perforin/granzyme pathway, whether HCMV-specific CD4(+) T cells are capable of killing HCMV-infected targets has not yet been documented. In the present paper, we have taken advantage of well established cellular reagents to address this issue. Human CD4(+) T-cell clones specific for the major immediate-early protein IE1 were shown to perform perforin-based cytotoxicity against peptide-pulsed targets. However, when tested on infected anitgen presenting cell targets, cytotoxicity was not detectable, although gamma interferon (IFN-gamma) production was significant. Furthermore, cytotoxicity against peptide-pulsed targets was inhibited by HCMV infection, whereas IFN-gamma production was not modified, suggesting that antigen processing was not altered. Remarkably, degranulation of CD4(+) T cells in the presence of infected targets was significant. Together, our data suggest that impaired cytotoxicity is not due to failure to recognize infected targets but rather to a mechanism specifically related to cytotoxicity.

The Vif Accessory Protein Alters the Cell Cycle of Human Immunodeficiency Virus Type 1 Infected Cells (45.15)

Abstract The viral infectivity factor gene (vif) of HIV-1 increases the infectivity of viral particles by inactivation of cellular anti-viral factors, and supports productive viral replication in primary human CD4 T-cells and in certain non-permissive T cell lines. Here, we demonstrate that Vif also contributes to the arrest of HIV-1 infected cells in the G2 phase of the cell cycle. Viruses deleted in Vif or Vpr induce less cell cycle arrest than wild-type virus, while cells infected with HIV-1 deleted in both Vif and Vpr have a cell cycle profile equivalent to that of uninfected cells. Furthermore, expression of Vif alone induces accumulation of cells in the G2 phase of the cell cycle. These data demonstrate a novel role for Vif in cell cycle regulation and suggest that Vif and Vpr independently drive G2 arrest in HIV-1 infected cells. Our results may have implications for the actions and interactions of key HIV-1 accessory proteins in AIDS pathogenesis.

Silencing of HALP Decreases HIV-1 Infection of Primary CD4+ T-cells (45.4)

Abstract HIV/AIDS is an ideal candidate for a novel gene therapy approach since it is an incurable and terminal disease. We have identified a set of candidate host cell genes, associated with survival of HIV-infected T-cells. We named one of these genes, “HALP”, for “HIV-Associated Life Preserver”. Recently, we used a lentiviral vector to stably express HALP shRNA in primary human CD4 T-cells. In the absence of HIV infection, silencing of HALP appeared to have no effect on viability of these cells. As predicted, silencing of HALP markedly reduced HIV infection of primary CD4+ T-cells, as measured by the percent of p24+ cells and by supernatant p24. Published data suggest that hypoxia induces HALP expression by increasing the activity of the transcription factor, Hif-1α. HIF-1 binding sites have been identified within the HALP promoter and HALP is responsive to HIF-1 in non-immune cells. We show here that HIV-1 infection of primary CD4+ T-cells increases Hif-1α mRNA expression, as measured by real time RT-PCR. Transfection of T-cells with wild-type Hif-1α or with a mutated form of Hif that is resistant to proteolysis (Hif-DPA) also increased expression of HALP mRNA. These data are consistent with a pathway of Hif-induced HALP expression in HIV-1 infected cells, leading to a hypoxia-like stress response and, ultimately, to cell survival. Our work represents one of the first attempts to search for novel cellular genes that contribute to viral inhibition of host cell apoptosis and, thus, to viral latency. HALP represents a candidate for a host cell anti-apoptotic protein, which is triggered by HIV infection, and may provide a novel molecular target for drug use and design.

Identification of novel consensus CD4 T-cell epitopes from clade B HIV-1 whole genome that are frequently recognized by HIV-1 infected patients

To identify promiscuous and potentially protective human CD4 T-cell epitopes in most conserved regions within the protein-coding genome of HIV-1 clade B consensus sequence. We used the TEPITOPE algorithm to screen the most conserved regions of the whole genome of the HIV-1 subtype B consensus sequence to identify promiscuous human CD4 T-cell epitopes in HIV-1. The actual promiscuity of HLA binding of the 18 selected peptides was assessed by binding assays to nine prevalent HLA-DR molecules. Synthetic peptides were tested with interferon-gamma ELISPOT assays on peripheral blood mononuclear cells (PBMC) from 38 HIV-1 infected patients and eight uninfected controls. Most peptides bound to multiple HLA-DR molecules. PBMC from 91% of chronically HIV-1 infected patients recognized at least one of the promiscuous peptides, while none of the healthy controls recognized peptides. All 18 peptides were recognized, and each peptide was recognized by at least 18% of patients; 44% of the patients recognized five or more peptides. This response was not associated to particular HLA-DR alleles. Similar responses were obtained in CD8 T-cell-depleted PBMC. In silico prediction of promiscuous epitopes led to the identification of naturally immunodominant CD4 T-cell epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to HIV-1. This combination of CD4 T-cell epitopes - 11 of them not described before - may have the potential for inclusion in a vaccine against HIV-1, allowing the immunization of genetically distinct populations.

Interleukin-7 Receptor Expression on CD8 T-Cells Is Downregulated by the HIV Tat Protein

We have previously shown decreased expression of the interleukin (IL)-7 receptor alpha-chain (CD127) on CD8 T-cells in HIV-infected patients and an apparent recovery of this receptor in those receiving antiretroviral therapy with sustained viral suppression. Here, we demonstrate that the HIV Tat protein specifically downregulates cell surface expression of CD127 on human CD8 T-cells in a dose- and time-dependent manner. The effects of Tat on CD127 expression could be blocked with anti-Tat monoclonal antibodies or by preincubating Tat with heparin. Tat had no effect on the expression of other cell surface proteins examined, including CD132, or on cell viability over 72 hours. Further, CD127 expression was not altered by other HIV proteins, including gp160 or Nef. Preincubation of purified CD8 T-cells with Tat protein inhibited CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Because IL-7 signaling is essential for optimal CD8 T-cell proliferation and function, the downregulation of CD127 and apparent inhibition of cytotoxic activity by Tat may play an important role in HIV-induced immune dysregulation and impaired cell-mediated immunity.

Human Bone Marrow Stromal Cells Hamper Specific Interactions of CD4 and CD8 T Lymphocytes with Antigen-Presenting Cells

Bone marrow stromal cells (BMSCs) may inhibit T-cell functions in vitro and thus have been proposed as immunoregulators to control in vivo graft-versus-host disease (GVHD) in haploidentical hemopoietic stem cell transplants. To better investigate this phenomenon, we used a defined experimental system in which responding T cells are antigen-specific and devoid of alloreactivity against BMSC from a different subject. Thus, we established antigen-specific human CD4 and CD8 T-cell lines as the readout system. Antigen-dependent proliferation was reduced with both T-cell subsets cultured on confluent BMSCs, and also on confluent human skin fibroblasts (HSF) inhibited T-cell proliferation with similar efficiency. Morphological observations of the cocultures showed impairment of physical interactions between T-cell and antigen-presenting cells in the presence of BMSC, with lack of formation of antigen-dependent clusters of T cells and antigen-presenting cells (APCs). In contrast, no effects were seen with BMSC-conditioned medium. Since suppression was seen only with confluent mesenchymal cells, this phenomenon may not be relevant in vivo, where BMSCs are at low frequency. In addition, if the reported suppressive effect of BMSCs on GVHD in vivo is confirmed, a different in vitro system should be envisaged to better understand and exploit the underlying mechanism.

Sa.13. Function of TIM3 May Be Compromised in Human CD4 T-Cells of Untreated Patients with Multiple Sclerosis

Sa.13. Function of TIM3 May Be Compromised in Human CD4 T-Cells of Untreated Patients with Multiple Sclerosis

Ligation of CD28 Alone by its Natural Ligand, CD86, Induces Lipid Raft Polarization in Human CD4 T-cells

Results Ligation of CD28 by natural ligand, but not antibody, induced polarization of LRs at the cell-bead interface, in absence of TCR ligation. This correlated with activation of Vav-1, increased IC calcium and translocation of NFkB p65, but did not result in proliferation or cytokine production. Using DNA microarrays, we detected induction of a subset of genes, including the Egr1 family of transcription factors. Engagement of CTLA-4 blocked CD86Ig induction of LR polarization and new transcription.

Human Naive CD4 T-Cell Clones Specific for HIV Envelope Persist for Years In Vivo in the Absence of Antigenic Challenge

To study the persistence of HIV-specific human naive CD4-lymphocytes in vivo in the absence of antigenic stimulation, we identified 2 HIV-seronegative low-risk subjects carrying CD4-cells specific for gp120 that could be expanded in vitro. CD4 T-cell lines specific for gp120 were generated by stimulation cycles with antigen-pulsed antigen-presenting cells. Clonal analysis was performed by spectratyping and by sequencing of the CDR3 regions of the BV and AV-T-cell receptor (TCR) genes. HIV-specific T cells were expanded in vitro in 1989 and 2004. These lines were generated from naive precursors. Analysis of TCR-BV gene family use and sequencing of the TCR-BV22 hypervariable region revealed a BV22 clonotype in the 1989 line. The BV22-CDR3-based polymerase chain reaction primer confirmed that the 1989 and 2004 T-cell lines contained the same clonotype. In addition, the 1989 and 2004 T cells used the same TCR-AV38 gene family and identical CDR3-AV regions, confirming clonal identity. Similar data for a persistent clonotype defined by BV CDR3 sequencing were obtained from the second subject. In conclusion, naive CD4-cells specific for an HIV antigen not encountered in vivo persisted for more than 10 to 15 years. An extended lifespan, homeostatic proliferation, or the ability of the thymus to issue the same CD4 T-cell clone reiteratively might account for the phenomenon.

Induction of T-Cell Immunity to Antiretroviral Drug-Resistant Human Immunodeficiency Virus Type 1

Antiretroviral drug-resistant human immunodeficiency virus type 1 (HIV-1) is a major, growing, public health problem. Immune responses targeting epitopes spanning drug resistance sites could ameliorate development of drug resistance. We studied 25 individuals harboring multidrug-resistant HIV-1 for T-cell immunity to HIV-1 proteins and peptides spanning all common drug resistance mutations. CD8 T cells targeting epitopes spanning drug-induced mutations were detected but only in the 3 individuals with robust HIV-specific T-cell activity. Novel CD8 T-cell responses were detected against the common L63P and L10I protease inhibitor fitness mutations. Induction of T-cell immunity to drug-resistant variants was demonstrated in simian human immunodeficiency virus-infected macaques, where both CD8 and CD4 T-cell immune responses to reverse transcriptase and protease antiretroviral mutations were elicited using a novel peptide-based immunotherapy. T-cell responses to antiretroviral resistance mutations were strongest in the most immunocompetent animals. This study suggests feasible strategies to further evaluate the potential of limiting antiretroviral drug resistance through induction of T-cell immunity.

Checkpoint Kinase Inhibitors: SAR and Radioprotective Properties of a Series of 2-Arylbenzimidazoles

The discovery of a series of novel, potent, and highly selective inhibitors of the DNA damage control kinase chk2 is disclosed. Here we report the first SAR study around inhibitors of this kinase. High-throughput screening of purified human chk2 led to the identification of a novel series of 2-arylbenzimidazole inhibitors of the kinase. Optimization was facilitated using homology models of chk2 and docking of inhibitors, leading to the highly potent 2-arylbenzimidazole 2h (IC(50) 15 nM). Compound 2h is an ATP-competitive inhibitor of chk2 that dose dependently protects human CD4(+) and CD8(+) T-cells from apoptosis due to ionizing radiation. This work suggests that a selective small molecule inhibitor of chk2 could be a useful adjuvant to radiotherapy, increasing the therapeutic window of such treatment.

Recognition of cmv pp65 protein antigen by human cd4 t-cell lines induced with an immunodominant peptide pool

Cellular immunity against cytomegalovirus (CMV) is essential for recovery from infection and control of viral latency. In immunocompromised hosts, this balance between CMV and cellular immunity is lost. Accordingly, restoration of the CD8 compartment specific for CMV is beneficial for immunocompromised patients. It is clear that CMV-specific CD4 cells provide helper functions facilitating long-term persistence of CD8 cells. Considering the dearth of data on CMV-specific T-helper cells, we investigated the CD4 responses to the immunodominant protein pp65 to define antigenic peptides. Such peptides were pooled and used to generate long-term T-cell lines. The lines were responsive to CMV and pp65. T cells were selected with individual peptides to produce monospecific lines for accurate definition of fine epitope specificity and to confirm human leukocyte antigen HLA-DR restriction. Furthermore, these lines lost alloreactivity, suggesting that they can be generated from the allodonor for adoptive immunoreconstitution of stem cell graft recipients.

The HLA-DR2 haplotype is associated with an increased proliferative response to the immunodominant CD4(+) T-cell epitope in human interferon-beta.

Human CD4(+) T-cell epitopes were identified in interferon-beta (IFN-beta)-1b. A prominent peptide epitope region was found that induced a proliferative response in 16% of all donors tested. Responses corresponded to the presence of the HLA-DR2 haplotype. Responsive donors expressing the HLA-DQ6 allele showed an increased level of proliferation to the epitope as compared to peptide-responsive HLA-DQ6 negative donors. A similar result was found for HLA-DR15-expressing donors. PBMC from donors expressing HLA-DR15 were more likely to proliferate in response to IFN-beta in a whole-protein in vitro assay than donors who did not carry this haplotype. It is striking that the common DQ6 allele HLA-DQB1(*)0602 is found in linkage disequilibrium with HLA-DRB1(*)1501, and this combination defines the HLA genotype associated with the development of multiple sclerosis. The HLA association between a response to IFN-beta and MS might explain the prevalence of neutralizing antibody development, and may underlie the etiology of the disease.