Identification of a CD2- CD3+ T cell receptor-gamma+ peripheral blood lymphocyte subpopulation

We have identified, in a healthy individual, a sub-population of human peripheral lymphocytes which surface express a CD3-TCR-gamma complex recognized by anti-Ti gamma A mAb, while being unreactive with a phycoerythrin-conjugated anti-CD2 antibody with T11/1 specificity. Further immunofluorescence analyses performed on uncultured cells indicated that such a putative CD2-CD3+ phenotype was restricted to a fraction of those T lymphocytes which carry a surface receptor of the "second family" (gamma/delta). The actual lack of CD2 expression was confirmed by a subsequent series of cloning experiments which showed that none of the three well characterized CD2 epitopic clusters, namely T11/1, T11/2, and T11/3, were detectable on the surface of the relevant cells. The cultured CD2-, CD3+/TCR gamma + lymphocytes were found to display, as well as their CD2+ counterparts, both non-MHC-restricted cytotoxic function and proliferative responses induced via the gamma receptor complex. In contrast, the proliferative capacity of the CD2-, CD3+/TCR-gamma + cells observed in a culture system designed for in vitro expansion of lymphocytes with undefined specificity was extremely limited. This may relate to an impaired interaction of the CD2- cloned lymphocytes with lymphocyte function-associated (LFA)3+ irradiated cells present in the feeder layer. Further characterization of such minor CD2- T lymphocytes subsets may help to better understand the biologic relevance of the CD2/LFA3 pathway of cell-cell interaction.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Functional reactivity of WT31 monoclonal antibody with T cell receptor-gamma expressing CD3+4-8- T cells

About 3% of normal peripheral blood T lymphocytes have the phenotype CD3+4-8-. The vast majority of these cells lack the conventional TCR-alpha-, beta complex but express the recently identified TCR-gamma, delta/CD3 receptor complex. These TCR gamma+/CD3+ cells were initially discovered by using as a criterion the lack of reactivity with WT31 mAb. This mAb has been reported to recognize a "framework" epitope on the TCR-alpha, beta/CD3 complex. However, using high concentrations of WT31 mAb, low levels of reactivity with the cell membrane of TCR-gamma+ cells can be observed. This reactivity was significantly increased upon removal of sialic acid residues by neuraminidase. In addition, WT31 mAb is capable to induce lysis by TCR-gamma+ clones. Moreover, immunoprecipitation with WT31 by using cell lysates prepared with the mild detergent digitonin resulted in the isolation of the intact TCR-gamma/CD3 complex. Thus, in contrast to what was previously assumed, WT31 mAb also reacts with a functional epitope present on gamma, delta/CD3 T cells, and therefore lack of reactivity with WT31 mAb is not always a proper hallmark for TCR-gamma-expressing cells.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

A murine CD4-, CD8- T cell receptor-gamma delta T lymphocyte clone specific for herpes simplex virus glycoprotein I.

The role of TCR-gamma delta T lymphocytes in immune responses is currently not well understood. TCR-gamma delta cells have a limited repertoire suggesting that TCR-gamma delta T a limited number of evolutionarily conserved Ag such as nonpolymorphic MHC and heat shock proteins. TCR-gamma delta T lymphocytes appear in enhanced numbers in skin lesions produced by Mycobacterium leprae and in the synovial fluid of joints affected by rheumatoid arthritis, raising the possibility that this subset of T lymphocytes may play a role in control of infectious processes and in autoimmune diseases. We report the identification of a TCR-gamma delta T cell clone isolated from a HSV-infected mouse that recognizes glycoprotein I of HSV type 1. Clone recognition of glycoprotein I does not appear to require the expression of MHC class I or class II gene products. These data suggest that TCR-gamma delta lymphocytes may play an important role in the immune response to viral infections.     

Functional and phenotypic characterization of CD57+CD4+ T cells and their association with HIV-1-induced T cell dysfunction

HIV-1 replication is associated with reduced or absent HIV-1-specific CD4+ T cell proliferation and skewing of HIV-1-specific CD4+ T cells toward an IFN-gamma-producing, CCR7- phenotype. The CCR7- T cell population is heterogeneous and can be subdivided based on the expression of CD57. Although CD57 expression on CD8+ T cells is associated with proliferation incompetence and replicative senescence, less is known about the function of CD57-expressing CD4+ T cells. In this study, the frequency, phenotype, and function of CD57+CD4+ T cells were evaluated in 25 HIV-1-infected subjects and 10 seronegative controls. CD57+CD4+ T cells were found to be proliferation incompetent, even after strong mitogen stimulation. Percentages of CD4+ T cells that expressed CD57 were significantly higher in untreated HIV-1-infected subjects than in HIV-1-seronegative donors, and CD57 expression did not normalize in subjects receiving at least 6 mo of effective antiretroviral therapy. CD57 was predominately expressed on the CCR7- fraction of the CD4+ T cell compartment and accounted for the majority of cells in the CCR7-CD45RA+ population from untreated HIV-1-infected subjects. HIV-1-specific CD4+ T cells producing only IFN-gamma had the highest expression of CD57, whereas few cells producing IL-2 alone expressed CD57. These findings further define a novel population of proliferation-incompetent CD4+ T cells that are generated in the presence of chronic Ag exposure. A better understanding of the generation and persistence of CD57+ T cells in HIV-1 infection could provide important insights into the immunopathogenesis of this disease.     

Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

EBV-specific CD4+ T cell clones exhibit vigorous allogeneic responses

Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.     

Biochemical differences in the alphabeta T cell receptor.CD3 surface complex between CD8+ and CD4+ human mature T lymphocytes

We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.     

General characterization of human cytomegalovirus-specific proliferative CD4+ T cell clones

Over 80 human T helper cell (Th) clones reactive with human cytomegalovirus (HCMV) were generated using purified whole Towne strain HCMV as the in vitro antigen. These cloned T cells are CD3+, CD4+, CD8- and proliferate specifically to HCMV. All of the clones tested produce interleukin 2 and gamma-interferon and failed to show HCMV-specific cytotoxicity or natural killer (NK) activity. Most of the Th clones recognize multiple laboratory-adapted and wild-type strains of HCMV. The Th clones were also tested for their reactivity to a major envelope glycoprotein complex (gcI) and a 64,000 dalton internal matrix protein. Our results show that both proteins as well as other unidentified protein(s) are involved in Th responses to HCMV.     

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

Summary Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneo ^R gene. The presence of theneo ^R gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneo ^R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4⁺ and CD8⁺ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4⁺ and CD8⁺ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4⁺ or CD8⁺ cells. In other experiments highly purified CD4⁺ and CD8⁺ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneo ^R gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4⁺ and CD8⁺ gene-modified TIL. Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel     

Natural killer (NK)-like cytotoxic activity of allospecific T cell receptor-gamma,delta+ T cell clones. Distinct receptor-ligand interactions mediate NK-like and allospecific cytotoxicity

We recently generated a series of human alloantigen-specific, CD3+,TCR-gamma,delta+ clones by stimulating CD3+,CD4-,CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). As reported previously, these clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Further studies of these and other TCR-gamma,delta+ clones, described in this report, indicate that most but not all of these clones express the NK cell associated marker, NKH-1 or Leu-19, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not an irrelevant LCL or NK-resistant line, Raji. TCR-gamma,delta+ clones which lacked expression of Leu-19 lysed their allospecific targets but had no detectable NK activity. The allospecific cytotoxicity of Leu-19+ and Leu-19- clones was inhibited by mAb to CD3 or the TCR delta-chain. In contrast, the NK-like activity of the Leu-19+ clones was enhanced by these antibodies over a wide range of antibody concentration. Although mAb to LFA-1 markedly inhibited both the allospecific and NK-like activity of these clones, an HLA class I framework specific mAb (W6/32) had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the TCR-gamma,delta+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+,TCR-gamma,delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

Phenotype of T cell clones obtained from bronchial biopsies and peripheral blood from three asthmatics.

Establishment of T cell clones derived from bronchial biopsies and peripheral blood from allergic asthmatics has shown that they secret a different IL-4 and IFN-γ pattern of cytokines, suggesting that the micro-environment of the bronchi may influence the phenotype of the T cells.     

Regulation of apoptosis in mature alphabeta+CD4-CD8- antigen-specific suppressor T cell clones

The regulation of apoptosis in mature CD4+ or CD8+ alphabeta+ T cells has been well studied. How the survival and death is regulated in peripheral CD4-CD8- (double negative, DN) alphabeta+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of alphabeta+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-alpha/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.     

Induction of graft versus host-associated immunodeficiency by CD4+ T cell clones

Previous studies have shown that the injection of parental T cells into MHC class II mismatched F1 recipient mice can lead to graft-vs-host (GvH) reaction that manifests itself by multiple symptoms. The objective of our study was to analyze GvH reactivity induced by a single T cell clone specific for host I-A or I-E alloantigen. The T cell clones tested for GvH potential were CD4+, with or without cytolytic activity in vitro and with a lymphokine pattern that classifies them as Th1 cells. The inoculation of a single T cell clone induced a severe, but transient immunodeficiency in the host that was independent of its cytolytic activity, as demonstrated by the failure to generate a CTL response to third party allogeneic cells in vitro. Induction of immunodeficiency in the recipients required preactivation of the clones in vitro by rIl-2 and the presence of the stimulator class II alloantigen in the host. Spleen cells from these mice lacked suppressor cells, they were deficient in Il-2 secretion and exhibited a decrease in the number of CD4+ T cells. In addition, I-E expression was reduced, however, without any changes in the macrophage population and an increase in surface Ig and the B cell marker B220. Simultaneous to the immunodeficiency, the clone-injected mice produced elevated antibody titers to ssDNA.     

人类疱疹病毒6型特异性CD4+ T细胞的初步克隆

目的 克隆HHV-6特异性CD4⁺ T细胞,并分析其基本特征。方法 微孔有限稀释法克隆HHV-6特异性CD4⁺ T细胞;3H-TdR掺入法细胞增殖试验筛选HHV-6 特异性CD4+ T细胞克隆;FACS分析HHV-6特异性CD4⁺ T细胞克隆的表面标志;ELISA检测HHV-6特异性CD4⁺ T细胞克隆的细胞因子分泌水平。结果 获得的细胞克隆中有4株以HHV-6感染细胞裂解物特异的方式增殖,并且其增殖水平与HHV-6感染细胞裂解物呈剂量依赖性;细胞克隆表面标志为CD3⁺CD4⁺;W-2和W-4细胞克隆的细胞因子分泌以IL-10为主,W-1细胞克隆的细胞因子分泌以IL-10及IFN-γ为主,W-3细胞克隆的细胞因子分泌以IFN-γ为主。结论 初步建立HHV-6特异性CD4⁺ T细胞克隆,并分析其表面标志和细胞因子分泌水平,为HHV-6特异性Treg细胞的克隆、筛选及其功能的研究奠定基础。     

Establishment of cytotoxic CD4+ T cell clones from cancer patients treated by local immunotherapy

We have previously reported that the antitumor effect of OK-432, aStreptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (Cancer, 69: 636–642, 1992). In order to elucidate the mechanism of the antitumor effects, we established T cell clones from regional lymph nodes of colorectal cancer patients who received this local immunotherapy. By culture of lymph node lymphocytes, in the presence of IL-2 and OK-432, 4 clones of T cells were established from 4 patients treated by local immunotherapy. These clones had a helper T cell phenotype (CD3⁺, CD4⁺, CD8^–, CD56^–, WT31⁺) and were successfully maintained for several months. The cells strongly expressed CD25 when stimulated with OK-432 and exhibited a high level of cytotoxic activity in part explained by the increased expression of ICAM-1 and LFA-1, and the release of TNF. These results suggest that the CD4⁺ T cells play a role in the antitumor mechanism of local immunotherapy.     

Herpes simplex virus glycoprotein D is recognized as antigen by CD4+ and CD8+ T lymphocytes from infected mice. Characterization of T cell clones

Several previous reports have described the surprising inability to detect murine CTL specific for glycoprotein D (gD), one of the important protective immunogens of HSV. Using slight variations of published procedures, we were able to show that the immune response to HSV in infected mice includes the generation of CTL specific for gD. C3H/OuJ (H-2k) mice were infected by injection in the hind footpads with purified HSV-1. Lymphocytes from draining lymph nodes were then isolated and shown to proliferate in response to, and to kill, transformed fibroblasts (H-2k) expressing HSV-1 gD. Two gD-specific T cell clones were isolated. One clone, designated CGD1, was shwon to be CD8+. This clone recognizes HSV-1 gD, but not HSV-2 gD, in the context of class I MHC molecules and kills the appropriate MHC-matched fibroblasts expressing HSV-1 gD. Unusual features of this cytolytic clone include augmentation by IL-4 of proliferative responses to Ag, inhibition of its lytic activity by a mAb specific for Thy-1 and recognition of infected fibroblasts in preference to infected lymphoblasts. The other clone, designated CGD3, was shown to be CD4+. This clone recognizes both HSV-1 gD and HSV-2 gD in the context of class II MHC molecules and has cytolytic potential.