Lianxian Cui

Generation of Human Regulatory γδ T Cells by TCRγδ Stimulation in the Presence of TGF-β and Their Involvement in the Pathogenesis of Systemic Lupus Erythematosus

As a component of the innate immune cell population, γδ T cells are involved in tumor immunosurveillance and host defense against viral invasion. In this study, we demonstrated a novel function of human γδ T cells as regulatory cells by detecting their suppressive effect on the proliferation of autologous naive CD4+ T cells. These regulatory γδ T cells (γδ Tregs) could be generated in vitro by stimulating with anti-TCRγδ in the presence of TGF-β and IL-2. Similar to CD4+Foxp3+ Tregs, γδ Tregs also expressed Foxp3. Additionally, they primarily belonged to the Vδ1 subset with a CD27+CD25high phenotype. Furthermore, these γδ Tregs showed an immunoregulatory activity mainly through cell-to-cell contact. Importantly, this γδ regulatory population decreased in the peripheral blood of systemic lupus erythematosus patients, suggesting a potential mechanism in understanding the pathogenesis of systemic lupus erythematosus.

Identification and characterization of Foxp3+ γδ T cells in mouse and human

Regulatory T cells (Tregs) expressing TCRalphabeta play a critical role in the maintenance of the immune system homeostasis. Tregs express the cell surface markers CD4 and CD25 as well as the transcription factor Foxp3. Foxp3(+)CD4(+)CD25(+)TCRalphabeta(+) Tregs can be generated from mouse and human CD4(+)CD25(-) T cells in vitro via TGF-beta induction. As growing evidences suggest that gammadelta T cells also have immunoregulatory function, we have attempted to identify and characterize Foxp3(+) cells in mouse and human gammadelta T cells. We found that freshly isolated mouse splenic gammadelta T cells did not express Foxp3. When mouse splenocytes were stimulated with anti-TCRgammadelta in the presence of TGF-beta, a population of Foxp3(+) gammadelta T cells appeared, in most of which expressed CD25 as well. Compared with CD25(-) gammadelta T cells, TGF-beta induced CD25(+) gammadelta T cells not only expressed Foxp3, but also had increased TGF-beta and GITR expression. Furthermore, the TGF-beta induced gammadelta T cells mediated a potent immunosuppressive effect on anti-CD3 stimulated T cell activation and proliferation. In contrast, although a small fraction of human peripheral blood and tumor infiltrating gammadelta T cells expressed Foxp3, similar culture condition with anti-TCRgammadelta plus TGF-beta failed to generate functional human Foxp3(+) gammadelta T cells. In conclusion, our results suggest that mouse splenic Foxp3(+) gammadelta T cells with suppressive function can be induced by TCR and TGF-beta costimulation, whereas functional human Foxp3(+) gammadelta T cells in peripheral blood could not be generated under the same condition.

Membrane HSP70: The Molecule Triggering γδ T Cells in the Early Stage of Tumorigenesis

Many studies support the supposition that HSPs expressed on the cell membrane play an important role in cancer immunity. In the present study, we demonstrated that HSP60 and HSP70 are markedly increased on the cell membrane of human Epstein-Barr virus (EBV) transformed B cells. In order to investigate whether these molecules were involved in the response of human γδ T cells to transformed cells, the cytotoxicities of γδ T cells to transformed cells with or without an HSP60/70 gene knockdown were evaluated. γδ T cells showed marked cytotoxities to transformed cells. Down-regulation of HSP70 expression could inhibit the reactions, whereas down-regulation of HSP60 expression had little such effect. Moreover, HSP72 could significantly induce human γδ T cells to proliferate in vitro. Taken together, our data indicated that HSP60 and HSP70 could be valuable biomarkers for the prediction of early stage in tumorigenesis. Additionally, HSP72 might be a potential candidate of the adjuvant for γδ T cells in tumor immunotherapy.

Anti-γδ TCR antibody-expanded γδ T cells: a better choice for the adoptive immunotherapy of lymphoid malignancies.

Cell-based immunotherapy for lymphoid malignancies has gained increasing attention as patients develop resistance to conventional treatments. γδ T cells, which have major histocompatibility complex (MHC)-unrestricted lytic activity, have become a promising candidate population for adoptive cell transfer therapy. We previously established a stable condition for expanding γδ T cells by using anti-γδ T-cell receptor (TCR) antibody. In this study, we found that adoptive transfer of the expanded γδ T cells to Daudi lymphoma-bearing nude mice significantly prolonged the survival time of the mice and improved their living status. We further investigated the characteristics of these antibody-expanded γδ T cells compared to the more commonly used phosphoantigen-expanded γδ T cells and evaluated the feasibility of employing them in the treatment of lymphoid malignancies. Slow but sustained proliferation of human peripheral blood γδ T cells was observed upon stimulation with anti-γδ TCR antibody. Compared to phosphoantigen-stimulated γδ T cells, the antibody-expanded cells manifested similar functional phenotypes and cytotoxic activity towards lymphoma cell lines. It is noteworthy that the anti-γδ TCR antibody could expand both the Vδ1 and Vδ2 subsets of γδ T cells. The in vitro-expanded Vδ1 T cells displayed comparable tumour cell-killing activity to Vδ2 T cells. Importantly, owing to higher C–C chemokine receptor 4 (CCR4) and CCR8 expression, the Vδ1 T cells were more prone to infiltrate CCL17- or CCL22-expressing lymphomas than the Vδ2 T cells. Characterizing the peripheral blood γδ T cells from lymphoma patients further confirmed that the anti-γδ TCR antibody-expanded γδ T cells could be a more efficacious choice for the treatment of lymphoid malignancies than phosphoantigen-expanded γδ T cells.

Immobilized MICA Could Expand Human Vδ1 γδ T Cells In Vitro that Displayed Major Histocompatibility Complex Class I Chain‐Related A‐Dependent Cytotoxicity to Human Epithelial Carcinomas

Human major histocompatibility complex class I chain‐related A (MICA) is a human leucocyte antigen‐related polymorphic molecule, which is expressed on many kinds of epithelial tumours and can be recognized by the Vδ1 subset of γδ T cells. In the present study, monoclonal antibodies (MoAbs) were produced in mice immunized with recombinant MICA (rMICA)*008. It was found that MICA was expressed on ovarian and colonic tumour tissues and could be detected by these anti‐MICA MoAbs. The immobilized rMICA could induce the proliferation of human ovarian epithelial carcinoma‐ or colonic carcinoma‐derived γδ T cells of the Vδ1 phenotype in vitro. These Vδ1 T cells displayed a strong, broad‐range cytolytic activity towards tumour cell lines positive for MICA. The efficiency of this cytolytic activity depended greatly on the level of MICA expressed on the cell surface and could be inhibited by anti‐MICA MoAbs. Therefore, MICA may play an important role in immune responses against epithelial tumours and function as a stimulating factor for the growth of Vδ1 γδ T cells, whereas MICA‐reactive Vδ1 γδ T cells might serve as a new candidate for adoptive cellular therapy of tumours.

Four novel ULBP splice variants are ligands for human NKG2D

UL16-binding proteins [ULBPs, also termed as retinoic acid early transcripts (RAET1) molecules] are frequently expressed by malignant transformed cells and stimulate anti-tumor immune responses mediated by NKG2D-positive NK cells, CD8(+) alphabeta T cells and gammadelta T cells in vitro and in vivo. In this study, we identified four novel functional splice variants of ULBPs including ULBP4-I, ULBP4-II, ULBP4-III and RAET1G3 in HepG2 liver carcinoma cells, WISH human amnion cells, Hep-2 larynx carcinoma cells and K562 leukemia cells, respectively, by reverse transcription-PCR and T vector cloning strategy. Analysis of alignments of amino acid sequences of the splice variants illustrated that there were important modifications between splice variants and their individual parental ULBP. All ULBP4 splice variants (ULBP4-I, ULBP4-II and ULBP4-III) were type 1 membrane-spanning molecules and had the ability to bind with human NKG2D receptor in vitro. Ectopic expressions of ULBP4 and ULBP4 splice variants resulted in the enhanced cytotoxic sensitivity of target cells against NK cells, which could be blocked by anti-NKG2D mAb. Moreover, co-culture-free soluble forms of ULBP4 splice variants (their alpha1 + alpha2 ectodomains) and RAET1G3 (soluble splice variant of RAET1G2) with NK cells down-regulated the cell surface expression of NKG2D. Finally, immobilized in a plate-bound form of RAET1G3 stimulated NK cells to secrete IFN-gamma. Taken together, all the identified functional splice variants will help to advance our knowledge regarding the overall functions of ULBP gene family.

A new effect of IL-4 on human γδ T cells: promoting regulatory Vδ1 T cells via IL-10 production and inhibiting function of Vδ2 T cells

Interleukin 4 (IL-4) has a variety of immune functions, including helper T-cell (Th-cell) differentiation and innate immune-response processes. However, the impact of IL-4 on gamma delta (γδ) T cells remains unclear. In this study, we investigate the effects of IL-4 on the activation and proliferation of γδ T cells and the balance between variable delta 1 (Vδ1) and Vδ2 T cells in humans. The results show that IL-4 inhibits the activation of γδ T cells in the presence of γδ T-cell receptor (TCR) stimulation in a STAT6-dependent manner. IL-4 promoted the growth of activated γδ T cells and increased the levels of Vδ1 T cells, which in turn inhibited Vδ2 T-cell growth via significant IL-10 secretion. Vδ1 T cells secreted significantly less interferon gamma (IFNγ) and more IL-10 relative to Vδ2. Furthermore, Vδ1 T cells showed relatively low levels of Natural Killer Group 2D (NKG2D) expression in the presence of IL-4, suggesting that Vδ1 T cells weaken the γδ T cell-mediated anti-tumor immune response. For the first time, our findings demonstrate a negative regulatory role of IL-4 in γδ T cell-mediated anti-tumor immunity.

IL-15 increases the frequency of effector memory CD8+ T cells in rhesus monkeys immunized with HIV vaccine.

Several studies have suggested that interleukin (IL)-15 is a promising adjuvant that promotes cellular immunity when administered with human immunodeficiency virus (HIV) vaccine. Here we evaluated the effect of IL-15 plasmid on HIV-specific immune responses, especially cellular immunity, in eight rhesus monkeys. These monkeys were immunized three times with HIV DNA vaccine with or without IL-15 plasmid and boosted with recombinant Tiantan strain vaccinia virus-based HIV vaccine (rTV) 22 weeks after the first immunization. Although we did not detect any significant differences in the HIV-specific CD8+ T-cell response between monkeys with IL-15 coimmunization and monkeys with HIV vaccine alone, our results showed that the frequency of effector CD8+ memory T cells in the peripheral blood was significantly higher in monkeys with IL-15 coimmunization than those with HIV vaccine alone at almost all of the time points examined. Furthermore, the titers of anti-HIV antibodies were higher in Group T than those in Group C after rTV boosting. These findings in rhesus monkeys suggest that IL-15 may be useful as a cytokine adjuvant for HIV vaccine.

Antigen Specificity of γδ T Cells Depends Primarily on the Flanking Sequences of CDR3δ

The structural basis that determines the specificity of γδ T cell receptor (TCR) recognition remains undefined. Our previous data show that the complementary determining region of human TCRδ (CDR3δ) is critical to ligand binding. Here we used linear and configurational approaches to examine the roles of V, N-D-N, or J regions in CDR3δ-mediated antigen recognition. Surprisingly, we found that the binding activities of CDR3δ from different γδ TCRs to their target tissues and ligands depend on the conserved flanking sequences (V and J) but not as much on the D region of CDR3δ fragment. We further defined the key residues in the V and J regions of CDR3δ fragments, including the cysteine residue in the V fragment and the leucine residue in the J fragment that determine their ligand binding specificity. Our results demonstrate that TCRδ primarily uses conserved flanking regions to bind ligands. This finding may provide an explanation for the limited number of γδ TCR ligands that have as yet been identified.

Efficient augmentation of a long-lasting immune responses in HIV-1 gag DNA vaccination by IL-15 plasmid boosting.

Cytokines are major regulators of the immune response, and have been used as adjuvants to improve vaccine potency. In this study, we investigated the adjuvant effects of interleukin (IL)-15 on improving the immunogenicity of human immunodeficiency virus (HIV)-1 gag DNA vaccine in Balb/c mice. During a 370-day follow-up, cellular and humoral immune responses in three separate cohorts of mice were monitored. These results were exemplified through: lymphocyte proliferation, induction of antigen-specific CD8(+) T lymphocytes, long-term production of specific antibodies, and proportion of differentiated memory CD8(+) T cells. These data revealed that just boost of IL-15 at day 8 after co-immunization induced more homeostatic cell proliferation, augmented proliferation frequency of IFN-gamma-secreting antigen-specific CD8(+) T lymphocytes, maintained the long-lasting humoral immune response and promoted the turnover of memory T cell precursors into central memory T cells. Taken together, our data demonstrated that a single IL-15 boosting can enhance both the humoral and cellular immune responses of the HIV-1 gag DNA vaccination. This novel boosting strategy may facilitate the application of IL-15 as an adjuvant for HIV vaccination.