Potential regulatory role of in vitro-expanded Vδ1 T cells from human peripheral blood.

γδ T cells represent a relevant proportion of T lymphocytes that express T cell receptors (TCRs) encoded by the γ and δ T cell receptor genes. Whereas the most circulating γδ T cell type, Vδ2 T cells, has been described and studied intensively, the potential role of Vδ1 T cells remains largely unclear. Here, we identified that expanded peripheral blood Vδ1 T cells stimulated with anti-human TCR Vδ1 monoclonal antibody (mAb) in vitro predominantly expressed forkhead box p3 (Foxp3). In addition, these cells also expressed other regulatory T cell-related molecules, such as CD25, glucocorticoid-induced TNFR family-related protein and cytotoxic T lymphocyte-associated protein-4 (CTLA-4), and held the potent capacity for the production of transforming growth factor beta 1 (TGF-ß1). These autocrine and/or paracrine TGF-ß1 could bind TGF-ß1 receptors on Vδ1 T cells and induce sustained Foxp3 expression. Moreover, Foxp3 expression coincided with high CD25 expression. CD25(high) Vδ1 T cells exhibited stronger suppression on CD4(+) T cell proliferation compared with TGF-ß1-induced CD25(high) CD4(+) regulatory T cells. Therefore, our phenotypic and functional analyses first demonstrate the potential regulatory property of anti-human TCR Vδ1 mAb-activated Vδ1 T cells. These results will broaden our understanding about the role of peripheral blood Vδ1 T cells under physical and pathological conditions.

[Selection of culture media for the mass production of gamma delta T cells used in adoptive immunotherapy].

OBJECTIVE: To select the optimal culture media for the mass production of gamma delta T cells used in adoptive immunotherapy. METHODS: Three different culture media (RPMI-1640, AIM-V, and OpTmizer, with or without autologous serum) were used to culture gamma delta T cells. The survival rate, purity, proliferation efficiency, and biological functions of the expanded gamma delta T cells were examined and compared. RESULTS: The survival rate of gamma delta T cells expanded in RPMI-1640 decreased over culture time. The purities of gamma delta T cells cultured in AIM-V or OpTmizer with or without serum were higher than those cultured in RPMI-1640. After two weeks of culture in the absence of serum, the purity and proliferation efficiency of gamma delta T cells cultured in OpTmizer were significantly higher than those cultured in RPMI-1640 (P < 0.05) and AIM-V (P < 0.05). gamma delta T cells in different culture media had similar CD107a expression and tumor necrosis factor-alpha production (P > 0.05). However, cells expanded in RPMI-1640 exhibited significantly weaker cytotoxicity against Daudi lymphoma cells than those expanded in OpTmizer (P < 0.05) and AIM-V (P < 0.05). CONCLUSION: Due to low serum-dependence, high proliferation efficiency, and favorable biology function of expanded cells, OpTmizer is the most suitable medium for the mass production of gamma delta T cells used in adoptive immunotherapy.

Activity of recombinant human interleukin-15 against tumor recurrence and metastasis in mice.

Transplantable experimental tumor models were constructed to study the activities of recombinant human interleukin-15 (rhIL-15) against tumor recurrence and metastasis. The results showed that tumor nodule formation was retarded and tumor growth was inhibited in the subcutaneous tumor model of LA795 lung adenocarcinoma after treatment with rhIL-15, and the survival rate of T739 tumor-bearing mice treated with rhIL-15 was much higher than that of mice treated with either saline or with the same dose of rhIL-2. This indicats that rhIL-15 had better antitumor effect than rhIL-2 at the same dose level. In some rhIL-15 treated mice, the tumor cells inoculated subcutaneously were eradicated and there was no tumor formation even 138 days after tumor cell inoculation. The tumor-free mice were rechallenged with live tumor cells and no tumor reoccurred in the following two months in all of these mice, indicating that long-lasting antitumor systemic immunity developed. It was also shown that tumor recurrence and metastasis were inhibited markedly after treatment with rhIL-15, but not with the same dose of rhIL-2, in both subcutaneously and intravenously disseminated tumor models of LA795 lung adenocarcinoma. Simultaneously, the CTL and NK cell activities of the splenocytes obtained from tumor-bearing mice that had been treated with either rhIL-15 or rhIL-2 were both markedly enhanced. However, the enhancement of CTL and NK cell activities was more significant in rhIL-15 treated mice than that in rhIL-2 treated mice. This suggests that the anti-tumor effect of rhIL-15 in vivo was achieved by enhancing the CTL and NK cell activities in tumor immune response.

[Expression and biological function of ULBP4 in Rosetta-gamiTMB(DE3) and mAb preparation].

AIM: To express human ULBP4 in Rosetta-gami(TM) B(DE3) and to prepare monoclonal antibody against ULBP4 for the research of gamma deltaT cells recognition mechanism. METHODS: DNA fragments of ULBP4 were derived from HO-8910 RNA by reverse-transcriptase polymerase chain reaction(RT-PCR). The fragments encoding the former 225 amino acids of ULBP4 were cloned into Histag fusion protein expression vector pET22b(+). The C-Histag fusion ULBP4(225a) protein was expressed in inclusion body and purified step by step according to manufactory's protocol and renatured in bag filter. It's functional effect on NK cells was evaluated by NKG2D binding assay and IFN-gamma secretion experiments. Prokaryotic expressed human ULBP4 was used as an antigen to prepare monoclonal antibodies by means of the B lymphocyte hybridoma technique. RESULTS: ULBP4 recombinant protein can stimulate NK cells to secrete IFN-gamma. Through PEG fusion and screening by limited dilution, we obtained four strains of hybridoma cells secreting anti-ULBP4 antibodies. CONCLUSION: The fusion protein was expressed successfully and functional. At the same time, the anti-ULBP4 mAbs were prepared successfully. Both of them provide a platform for further research.

[Construction of a large human scFv library against SARS virus].

AIM: To construct a large human scFv library against SARS virus by using in vivo recombination. METHODS: Total RNA was isolated from the lymphocytes of 6 patients recovered from SARS. mRNA was isolated and reverse transcribed into cDNA. The V(H) and V(L) fragments were amplified from the cDNA and then assembled into scFv genes. The scFv genes were amplified and ligated into phagemid pDAN5. The primary library was constructed by transforming the recombinant phagemid into E.coli TG1. The secondary library was generated by in vivo recombination in E.coli BS1365 following the infection of BS1365 by primary library phages. RESULTS: A primary library of 3x10(9) and a second library of 3x10(11) were constructed. CONCLUSION: A large human scFv library against SARS virus with good diversity was constructed, which may be used for screening antibodies to SARS virus antigens.

[Cytotoxicity of MICA-reactive V delta 1 gamma delta T cells towards epithelial tumor cells].

OBJECTIVE: To confirm whether human MHC class I chain-related A (MICA) induces the amplification of V delta 1 gamma delta tumor-infiltrating lymphocytes (TILs) in vitro and to identify the cytotoxicity of MICA-reactive V delta 1 gamma delta TILs towards epithelial tumor cells. METHODS: MICA protein was prokaryoticly expressed and purified by molecular cloning technology. The purified recombined MICA (rMICA) was used to induce V delta 1 gamma delta T cells from tumor tissues in vitro and the cytotoxicity of these V delta 1 gamma delta TILs were tested by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). RESULTS: The rMICA was expressed in prokaryocyte with pET30 as a vector. The immobilized rMICA protein could markedly induce the amplification of V delta 1 gamma delta T cells from tumor tissue in vitro. These V delta 1 gamma delta T cells showed strong cytolytic activities towards tumor cell lines expressing MICA. CONCLUSION: The MICA-reactive V delta 1 gamma delta T cell may be a candidate for adoptive cellular therapy of tumors.

PKC epsilon is a unique regulator for hsp90 beta gene in heat shock response.

An early event in cellular heat shock response is the transmittance of stress signals from the cell surface into the nuclei, resulting in the induction of heat shock proteins (Hsps). Protein kinase C (PKC) has been implicated as a key player in transducing stress signals. However, mechanism(s) by which PKC regulates heat shock-induced events remains largely unknown. Here we present data that pan-PKC inhibitor GF109203X, but not classic PKC inhibitor Gö6976, specifically repressed heat shock-induced accumulation of mRNA as well as promoter activity of hsp90 beta, but not hsp90 alpha, in Jurkat cells. Subcellular fractionation studies revealed that heat shock exclusively induced PKC-epsilon membrane translocation. Consistently, expression of a constitutively active PKC-epsilon(A159E) resulted in an enhanced promoter activity of hsp90 beta upon heat shock, whereas a dominant-negative PKC-epsilon(K437R) abolished this effect. In contrast, constitutively active-PKC-alpha or dominant-negative-PKC-alpha had no effects on heat shock induction of the gene. The effect of PKC-epsilon on hsp90 beta expression seems to be stimuli-specific, as phorbol myristate acetate-mediated hsp90 beta expression was PKC-epsilon-independent. We conclude that PKC-epsilon is specifically required in the signaling pathway leading to the induction of hsp90 beta gene in response to heat shock.

[Model of B immunoblastic lymphomas in the Hu-PBL-SCID mice].

OBJECTIVE: To constitute a model of B immunoblastic lymphomas in the Hu-PBL-SCID mice. METHODS: The SCID mice were reconstituted by intraperitoneal injection (i.p.) of 5 x 10(7) human lymphocytes from Epstein-Barr virus (EBV) seronegative individuals. After one week, the SCID mice were inoculated with EBV by i.p. injection, and subjected to the investigation of whether there was any tumor in the abdomen of such SCID mice four weeks later. The characteristics of the found tumor was observed by the methods of Hematoxylin-eosin (HE) stain, immunohistochemical staining and polymerase chain reaction (PCR). RESULTS: Compared with the control groups, all the EBV-infected Hu-PBL-SCID mice had abdominal solid tumors [(32 +/- 12.5) mm3] developed, often located in the liver. HE staining and immunohistochemical staining showed the tumors were human B cell lymphomas. EBV DNA could be detected in the tumors by the PCR. CONCLUSIONS: The model of B immunoblastic lymphomas in the Hu-PBL-SCID mice is successfully constituted, and may well be useful to the human tumor immunological study.