Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-stimulatory Signal Domain Exhibits High Functional Potency.

Chimeric antigen receptor (CAR) is a hybrid molecule consisting of an antigen-binding domain and a signal transduction domain. The artificial T cells expressing CAR (CAR-T cells) are expected to be a useful tool for treatment of various diseases, such as cancer. The addition of a co-stimulatory signal domain (CSSD) to CAR is shown to be critical for modulating CAR-T cell activities. However, the interplay among types of CSSDs, effector functions, and characteristics of CAR-T cells is largely unknown. To elucidate the interplay, we analyzed effector functions, differentiation to memory T cell subsets, exhaustion, and energy metabolism of the CAR-T cells with different CSSDs. Comparing to the CAR-T cells bearing a CD28- or 4-1BB-derived CSSD, which are currently used for CAR-T cell development, we found that the CAR-T cells with a herpes virus entry mediator (HVEM)-derived CSSD exhibited enhanced effector functions and efficient and balanced differentiation to both central and effector memory subsets, associated with an elevated energy metabolism and a reduced level of exhaustion. Thus, we developed the CAR-T cells bearing the CSSD derived from HVEM with high functional potency. The HVEM-derived CSSD may be useful for developing effective CAR-T cells.

Regulatory T Cells Contribute to HIV-1 Reservoir Persistence in CD4+ T Cells Through Cyclic Adenosine Monophosphate-Dependent Mechanisms in Humanized Mice In Vivo.

Background: Regulatory T cells (Tregs) suppress T-cell immune activation and human immunodeficiency virus type 1 (HIV-1) replication, but the role of Tregs in HIV-1 reservoir persistence is poorly defined. Methods: Tregs were depleted by denileukin diftitox in humanized mice with chronic HIV-1 infection. Viral replication in lineage cells was determined by p24 expression. Levels of HIV-1 RNA and DNA in human cells, as well as replication-competent-virus-producing cells, were measured to quantified viral replication and reservoirs. Results: Treg depletion resulted in a blip of HIV-1 replication in T cells but not in myeloid cells. The major activated reservoir cells were memory CD4+ T cells in vivo. Interestingly, the transient activation of viral replication led to HIV-1 reservoir reduction after viremia resuppression, as indicated by the quantity of HIV-1 DNA and replication-competent-virus-producing cells. Furthermore, we demonstrated that Tregs use cyclic adenosine monophosphate (cAMP)-dependent protein kinase A pathway to inhibit HIV-1 activation and replication in resting conventional T cells in vitro. Conclusion: Tregs suppress HIV-1 replication in T cells and contribute to HIV-1 reservoir persistence. cAMP produced in Tregs is involved in their suppression of viral gene activation and expression. Treg depletion combined with combination antiretroviral therapy provides a novel strategy for HIV-1 cure.

Plasmacytoid dendritic cells suppress HIV-1 replication but contribute to HIV-1 induced immunopathogenesis in humanized mice.

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.

Regulatory T cells prevent liver fibrosis during HIV type 1 infection in a humanized mouse model.

Human immunodeficiency virus type 1 (HIV-1) disease is associated with aberrant immune activation, and coinfection with hepatitis C virus (HCV) exacerbates hepatic inflammation and fibrosis. However, the role of HIV-1 infection or host immune modulation in liver pathogenesis is not clearly defined. Here, we report that regulatory T (Treg) cells prevent liver immunopathogenesis during HIV-1 infection in a humanized mouse model. In the absence of Treg cells, HIV-1 infection induced liver fibrosis associated with hepatic stellate cell activation, hepatitis, and liver injury. Our findings provide new insight linking Treg cells and liver immunopathogenesis during HIV-1 infection.

Short communication: generation of recombinant monoclonal antibodies against an immunodominant HLA-A*2402-restricted HIV type 1 CTL epitope.

Molecular interaction between the peptide/MHC class I complexes (pMHCs) and T cell receptor (TCR) is fundamental to the effector function of cytotoxic T lymphocytes (CTLs). Monoclonal antibody against pMHC with TCR-like specificity is a possible research tool for the antigen presentation. However, it is notoriously difficult to isolate monoclonal antibodies against pMHCs by the conventional hybridoma technique. To isolate monoclonal antibodies against an immunodominant HIV-1-derived CTL epitope in the nef gene, we panned phage clones from a human scFv phage display library. Eight Nef138-10/HLA-A*24(A24)-specific scFv clones were isolated and two of them (scFv#3 and scFv#27) were selected for further analysis. The clones stained A24-positive cells pulsed with Nef138-10 peptides specifically. We reconstituted humanized immunoglobulin Gs (IgGs) using a baculovirus expression system. Reconstituted IgGs kept the original specificities of the parental scFvs. The dissociation constants were 23 microM and 20 microM by Biacore, respectively. This is the first report of a successful generation of monoclonal antibodies against an HIV-1 CTL epitope loaded on an MHC class I molecule.

Highly restricted T-cell receptor repertoire in the CD8+ T-cell response against an HIV-1 epitope with a stereotypic amino acid substitution.

OBJECTIVE: In peripheral blood mononuclear cells (PBMCs) from HIV-1-positive patients, we sought to identify CD8+ T-cell populations and the corresponding T-cell receptor (TCR) repertoires that react to an immunogenic cytotoxic T lymphocyte (CTL) epitope with or without an escape mutation. METHODS: PBMCs from HLA-A*2402(A24)-positive patients were stimulated with peptides representing a wild-type CTL epitope in the HIV-1 Nef protein [Nef138-10(wt)] or an escape mutant with a Y to F (Y139F) substitution at the second position [Nef138-10(2F)]. Cultured PBMCs were stained with peptide-major histocompatibility complex tetramers containing Nef138-10(wt) or Nef138-10(2F) sequences. After in-vitro stimulation of PBMCs with cognate peptides, the CD8+ T-cell population was sorted into different fractions: positive only to the wild-type tetramer (wt-positive), positive only to the mutant tetramer (2F-positive), and positive to both wt-tetramers and mutant-tetramers (dual-positive). TCR repertoires of sorted epitope-specific CD8+ T-cell populations were determined by sequencing. RESULTS: A 2F-positive population was rarely observed under our culture and staining conditions. The wt-positive CD8+ T-cell populations had a diverse TCR repertoire, but the TCR repertoires in dual-positive CD8+ populations were highly restricted. In the dual-positive CD8+ T-cell populations, most clonotypes used the TRBV4-1 and TRBJ2-7 gene segments for the TCR beta-chain and the TRAV8-3 and TRAJ40-1 for the TCR alpha-chain. The CDR3 region of the TCR beta-chain showed little variation. CONCLUSION: These results provide an example of restricted TCR repertoire in a specific CTL response against the escaping epitope. We speculate that impairment of antigen presentation in escaping viruses may underlie the restricted repertoire.

An efficient and versatile mammalian viral vector system for major histocompatibility complex class I/peptide complexes.

We report a Sendai virus (SeV) vector system for expression of major histocompatibility complex (MHC) class I/peptide complexes. We cloned the extracellular domain of a human MHC class I heavy chain, HLA-A*2402, and human beta-2 microglobulin (beta2m) fused with HLA-A*2402-restricted human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte (CTL) epitopes (e-beta2m) in separate SeV vectors. When we coinfected nonhuman mammalian cells with the SeVs, naturally folded human MHC class I/peptide complexes were secreted in the culture supernatants. Biotin binding peptide sequences on the C terminus of the heavy chain were used to tetramerize the complexes. These tetramers made in the SeV system recognized specific CD8-positive T cells in peripheral blood mononuclear cells of HIV-1-positive patients with a specificity and sensitivity similar to those of MHC class I tetramers made in an Escherichia coli system. Solo infection of e-beta2m/SeV produced soluble e-beta2m in the culture supernatant, and cells pulsed with the soluble protein were recognized by specific CTLs. Furthermore, when cells were infected with e-beta2m/SeV, these cells were recognized by the specific CTLs more efficiently than the protein pulse per se. SeV is nonpathogenic for humans, can transduce foreign genes into nondividing cells, and may be useful for immunotherapy to enhance antigen-specific immune responses. Our system can be used not only to detect but also to stimulate antigen-specific cellular immune responses.