Potentiating Functional Antigen-specific CD8+ T Cell Immunity by a Novel PD1 Isoform-based Fusion DNA Vaccine

Understanding and identifying new ways of mounting an effective CD8⁺ T cell immune response is important for eliminating infectious pathogens. Although upregulated programmed death-1 (PD1) in chronic infections (such as HIV-1 and tuberculosis) impedes T cell responses, blocking this PD1/PD-L pathway could functionally rescue the “exhausted” T cells. However, there exists a number of PD1 spliced variants with unknown biological function. Here, we identified a new isoform of human PD1 (Δ42PD1) that contains a 42-nucleotide in-frame deletion located at exon 2 domain found expressed in peripheral blood mononuclear cells (PBMCs). Δ42PD1 appears to function distinctly from PD1, as it does not engage PD-L1/PD-L2 but its recombinant form could induce proinflammatory cytokines. We utilized Δ42PD1 as an intramolecular adjuvant to develop a fusion DNA vaccine with HIV-1 Gag p24 antigen to immunize mice, which elicited a significantly enhanced level of anti-p24 IgG1/IgG2a antibody titers, and important p24-specific and tetramer⁺CD8⁺ T cells responses that lasted for ≥7.5 months. Furthermore, p24-specific CD8⁺ T cells remain functionally improved in proliferative and cytolytic capacities. Importantly, the enhanced antigen-specific immunity protected mice against pathogenic viral challenge and tumor growth. Thus, this newly identified PD1 variant (Δ42PD1) amplifies the generation of antigen-specific CD8⁺ T cell immunity when used in a DNA vaccine.     

Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4⁺ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4⁺ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4⁺ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4⁺ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4⁺ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4⁺ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4⁺ T cells during HIV-1 infection.     

Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

Type 1 regulatory T (Tr1) cells are an inducible subset of CD4⁺ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4⁺ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP⁺ LV-IL-10–transduced human CD4⁺ T (CD4^LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4^LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4^LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4⁺ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.     

CD161 (NKR-P1A) Costimulation of CD1d-dependent Activation of Human T Cells Expressing Invariant Vα24JαQ T Cell Receptor α Chains

A population of human T cells expressing an invariant Vα24JαQ T cell antigen receptor (TCR) α chain and high levels of CD161 (NKR-P1A) appears to play an immunoregulatory role through production of both T helper (Th) type 1 and Th2 cytokines. Unlike other CD161⁺ T cells, the major histocompatibility complex–like nonpolymorphic CD1d molecule is the target for the TCR expressed by these T cells (Vα24^invt T cells) and by the homologous murine NK1 (NKR-P1C)⁺ T cell population. In this report, CD161 was shown to act as a specific costimulatory molecule for TCR-mediated proliferation and cytokine secretion by Vα24^invt T cells. However, in contrast to results in the mouse, ligation of CD161 in the absence of TCR stimulation did not result in Vα24^invt T cell activation, and costimulation through CD161 did not cause polarization of the cytokine secretion pattern. CD161 monoclonal antibodies specifically inhibited Vα24^invt T cell proliferation and cytokine secretion in response to CD1d⁺ target cells, demonstrating a physiological accessory molecule function for CD161. However, CD1d-restricted target cell lysis by activated Vα24^invt T cells, which involved a granule-mediated exocytotic mechanism, was CD161-independent. In further contrast to the mouse, the signaling pathway involved in Vα24^invt T cell costimulation through CD161 did not appear to involve stable association with tyrosine kinase p56^Lck. These results demonstrate a role for CD161 as a novel costimulatory molecule for TCR-mediated recognition of CD1d by human Vα24^invt T cells.     

Phenotypic and Functional Separation of Memory and Effector Human CD8+ T Cells

Human CD8⁺ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8⁺ T cell subset. First, CD45RA⁻CD45R0⁺ cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8⁺ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon γ, tumor necrosis factor α, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8⁺CD45RA⁺CD27⁻ population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-γ and TNF-α. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8⁺CD45RA⁺CD27⁻ cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8⁺ T cell subset.     

Human skin is colonized by T cells that recognize CD1a independently of lipid

CD1a-autoreactive T cells contribute to skin disease, but the identity of immunodominant self-lipid antigens and their mode of recognition are not yet solved. In most models, MHC and CD1 proteins serve as display platforms for smaller antigens. Here, we showed that CD1a tetramers without added antigen stained large T cell pools in every subject tested, accounting for approximately 1% of skin T cells. The mechanism of tetramer binding to T cells did not require any defined antigen. Binding occurred with approximately 100 lipid ligands carried by CD1a proteins, but could be tuned upward or downward with certain natural self-lipids. TCR recognition mapped to the outer A′ roof of CD1a at sites remote from the antigen exit portal, explaining how TCRs can bind CD1a rather than carried lipids. Thus, a major antigenic target of CD1a T cell autoreactivity in vivo is CD1a itself. Based on their high frequency and prevalence among donors, we conclude that CD1a-specific, lipid-independent T cells are a normal component of the human skin T cell repertoire. Bypassing the need to select antigens and effector molecules, CD1a tetramers represent a simple method to track such CD1a-specific T cells from tissues and in any clinical disease.     

LPS刺激来自人单核细胞的树突状细胞在调节自体CD4+CD25+T细胞活性作用研究

树突状细胞（DC）是现今被认为最具潜能的专职抗原呈递细胞.应用不同方法在体内诱导细胞毒T细胞（CTL）来识别肿瘤相关抗原的肿瘤免疫治疗研究已有报告.然而,在体内免疫治疗的有效性可能仅限制在局部或系统抑制CTL产生和功能.为了检测LPS刺激人单核细胞的DC来抑制自体CD4^＋CD25^＋T细胞能力,使用HLA-A2限制性p53264-272肽作为肿瘤抗原,用LPS（DC-LPS^＋）或不用LPS（DC-LPS^-）产生的DC分别与自体T细胞共同培养.结果显示：在DC-LPS^＋活化的T细胞的CD4^＋CD25^＋T细胞群比DC-LPS^-活化的T细胞要低.这个结果提示,DC-LPS^＋与CD4^＋CD25^＋T细胞群有关联,而且这种特性可能是由于T细胞对肿瘤相关抗原的调节作用.     

CCL20抑制小鼠胸腺CD4^＋CD25^＋天然调节性T细胞的发育

本研究探讨趋化因子CCL20对小鼠胸腺CD4^＋CD25^＋双阳性调节性T细胞发育的影响,为天然调节性T细胞调控的研究提供基础数据。采用14.5天龄胚鼠胸腺进行体外培养,以FACS检测不同时间点胸腺CD4^＋CD25^＋细胞的改变,同时计数每个胸腺小叶的细胞数变化。结果表明：体外胸腺培养的第1-6天胸腺细胞比例、细胞数量的变化趋势与胸腺体内发育的第14.5、15、16、17、18、19天CD4^＋CD25^＋双阳性T细胞的比例、细胞数量的变化趋势相似;在胸腺体外培养的第1-6天,CD4＋CD25^＋T细胞占CD4^＋T细胞的比例分别为58.29%、12.14%、6.08%、17.78%、9.06%、4.04%,占CD25^＋T细胞的比例分别为3.75%、10.81%、17.20%、51.93%、61.64%、80.06%,这一发育趋势与体内结果具有一致性。在4μg/ml的CCL20干预下,胸腺体外培养的第3、6天CD4^＋CD25^＋胸腺细胞分别从3.24±0.18、3.96±0.24下降至1.27±0.11、1.76±0.22（p值均〈0.001）。结论：体外培养的CD4^＋CD25^＋双阳性胸腺细胞数量和比例变化与体内发育变化趋势一致,CCL20明显下调胸腺CD4^＋CD25^＋的表达,这将为天然调节性T细胞的调控研究提供有效的参考依据。     

Human Peripheral Blood Eosinophils Express a Functional c-kit Receptor for Stem Cell Factor that Stimulates Very Late Antigen 4 (VLA-4)–mediated Cell Adhesion to Fibronectin and Vascular Cell Adhesion Molecule 1 (VCAM-1)

We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell–derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0– 3.6-fold, average 2.8 ± 0.6-fold, greater than the negative control). The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of 100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 μg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in 15 min by 7.7 ± 1.4-, 5.3 ± 3.3-, and 5.4 ± 0.2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 μg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 ± 9.2-, 3.8 ± 2.5-, and 1.7 ± 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml, 5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the α⁴ and β₁ integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4–mediated adhesion, which could contribute to the exit of these cells from the blood, their tissue localization, and their prominence in inflammatory lesions.     

CD30+间变性大细胞淋巴瘤细胞系的建立及其生物学特性

由1例非霍奇金淋巴瘤患者的胸水标本通过液体培养法建立了1株CD30^ 间变性大细胞淋巴瘤细胞后，并对其生物学特性进行研究。所建立的细胞系可以在含10%小牛血清的RPMI 1640培养液中持久增殖，倍增时间约为36小时，目前已传代120余次，生长良好，暂命名为SH-1。光镜下细胞形态与典型的CD30^ 间变性大细胞淋巴瘤（anaplastic large cell lymphoma,ALCL）细胞的形态十分类似，电镜下可观察到胸浆内存在较多病毒颗粒。细胞组织化学染色：光镜下过氧化物酶POX阴性，糖原染色PAS阳性，酸性磷酸酶（ACP）呈强阳性；电镜下髓过氧化物酶（MPO）阴性，血小板过氧化物酶（PPO）阴性。EB病毒核心抗原-1（EBNA-1）呈阳性。细胞免疫表型为CD30^ 、CD45^ 、HLA-DR^ ，而EMA、CD34、CD38、CD2、CD3、CD4、CD7、CD8、CD10、CD15、CD19、CD20均呈阴性。染色体分析示SH-1细胞系核型为二倍体或四倍体核型，并具有明显的结构和数量异常，但未见典型t(2;5)。结论：所建立的细胞系为CD30^ ALCL细胞系。     

Disturbed CD4+ T Cell Homeostasis and In Vitro HIV-1 Susceptibility in Transgenic Mice Expressing T Cell Line–tropic HIV-1 Receptors

T cell line–tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4⁺ T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4⁺ T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4⁺ T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4⁺ T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4⁺ T cell decline in patients.     

Identification of a Committed T Cell Precursor Population in Adult Human Peripheral Blood

Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4⁺CD3⁺αβ⁺ mature T cells. These cells, which represent 0.1–0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor α (pTα) gene, CD3-γ, CD-δ and CD-ε, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-β locus (D–J). Moreover, low levels of CD3ε protein, but not of TCR-β chain, can be detected in their cytoplasm. Our results suggest that CD4⁺CD3⁻ cells identified in peripheral blood are different from CD3⁻CD4⁺CD8⁻ thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.     

B7-1 Engagement of Cytotoxic T Lymphocyte Antigen 4 Inhibits T Cell Activation in the Absence of CD28

Ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) appears to inhibit T cell responses. Four mechanisms have been proposed to explain the inhibitory activity of CTLA4: competition for B7-1 and B7-2 binding by CD28; sequestration of signaling molecules away from CD28 via endocytosis; delivery of a signal that antagonizes a CD28 signal; and delivery of a signal that antagonizes a T cell receptor (TCR) signal. As three of these potential mechanisms involve functional antagonism of CD28, an experimental model was designed to determine whether CTLA4 could inhibit T cell function in the absence of CD28. TCR transgenic/recombinase activating gene 2–deficient/CD28–wild-type or CD28-deficient mice were generated and immunized with an antigen-expressing tumor. Primed T cells from both types of mice produced cytokines and proliferated in response to stimulator cells lacking B7 expression. However, whereas the response of CD28^+/+ T cells was augmented by costimulation with B7-1, the response of the CD28^−/− T cells was strongly inhibited. This inhibition was reversed by monoclonal antibody against B7-1 or CTLA4. Thus, CTLA4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA4.     

Circulating Exosomes Control CD4+ T Cell Immunometabolic Functions via the Transfer of miR-142 as a Novel Mediator in Myocarditis

1. Download : Download high-res image (114KB)
2. Download : Download full-size image