Interdependence of CD3-Ti and CD2 activation pathways in human T lymphocytes.

Human T lymphocytes can be activated through either the antigen/MHC receptor complex T3-Ti (CD3-Ti) or the T11 (CD2) molecule to proliferate via an IL-2 dependent mechanism. To investigate the relationship of these pathways to one another, we generated and characterized Jurkat mutants which selectively express either surface CD3-Ti or CD2. Here we show that CD3-Ti- mutants fail to be stimulated by either pathway to increase phosphoinositide turnover, mobilize calcium or induce the IL-2 gene. The activation capacity of these mutants via CD2 as well as CD3-Ti can be restored following reconstitution of surface CD3-Ti expression upon appropriate DNA transfer (e.g. Ti beta subunit cDNA into Ti beta- Jurkat variants). Collectively, these results demonstrate that CD3-Ti and CD2 pathways are interdependent and that phosphoinositide turnover is linked to the CD3-Ti complex.     

Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody-stimulated T cells

Addition of anti-CD3 mAb 147 (IgG1), 446 (IgG1), or 454 (IgG2a) to cultures of T plus non-T cells can result in both B cell growth and differentiation. To determine whether lymphokines mediating these activities were similar to those described from conventional mitogen-induced T cell activation, normal peripheral blood T cells were stimulated with anti-CD3 mAb for 48 h. The supernatants were assayed for factors inducing B cell growth or differentiation (BCDF). A marked increase in Ig secretion was observed when either EBV-transformed B cell lines or normal B cells, pre-activated with Staphylococcus aureus Cowan I strain, were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies. In contrast, IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446. Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells. To determine whether these effector activities were due to distinct lymphokines, supernatants were pooled and concentrated by ammonium sulfate precipitation. Superose 12 permeation chromatography revealed BCDF activity with an apparent Mr of approximately 30,000 Da. The growth factor activity eluted over a wider and higher molecular weight range which overlapped the differentiation factor activity. Fractions containing BCDF activity were pooled, dialyzed, applied to a Mono Q anion-exchange column, and eluted with a linear NaCl gradient. The growth factor activity came off in a single-peak while BCDF was found divided into two major areas. The growth factor eluted at an ionic strength between the two BCDF activities. BCDF has an apparent isoelectric point (pI) of 6, in contrast to the reported pI 5 for IL-6 and more acidic than the documented basic pI of IFN-gamma. Lastly, peaks with BCDF activity were not active in assays for either IL-2 or IL-4. In addition, a rabbit anti-IL-6 heteroantiserum failed to inhibit the pI 6 BCDF, suggesting non-identity between IL-6 and anti-CD3 induced BCDF. Thus, anti-CD3 activated T cells generate both growth factor activity and BCDF as separate molecular entities distinct from IFN-gamma, IL-2, IL-4, and conventional IL-6.     

Jakmip1 is expressed upon T cell differentiation and has an inhibitory function in cytotoxic T lymphocytes

Jakmip1 belongs to a family of three related genes encoding proteins rich in coiled-coils. Jakmip1 is expressed predominantly in neuronal and lymphoid cells and colocalizes with microtubules. We have studied the expression of Jakmip1 mRNA and protein in distinct subsets of human primary lymphocytes. Jakmip1 is absent in naive CD8(+) and CD4(+) T lymphocytes from peripheral blood but is highly expressed in Ag-experienced T cells. In cord blood T lymphocytes, induction of Jakmip1 occurs upon TCR/CD28 stimulation and parallels induction of effector proteins, such as granzyme B and perforin. Further analysis of CD8(+) and CD4(+) T cell subsets showed a higher expression of Jakmip1 in the effector CCR7(-) and CD27(-) T cell subpopulations. In a gene expression follow-up of the development of CMV-specific CD8(+) response, Jakmip1 emerged as one of the most highly up-regulated genes from primary infection to latent stage. To investigate the relationship between Jakmip1 and effector function, we monitored cytotoxicity of primary CD8(+) T cells silenced for Jakmip1 or transduced with the full-length protein or the N-terminal region. Our findings point to Jakmip1 being a novel effector memory gene restraining T cell-mediated cytotoxicity.     

Impact of cytokines and T lymphocytes upon osteoclast differentiation and function

Historically, the osteoblast has been considered the master cell in the control of osteoclast development and, therefore, bone resorption. Now the interactions between cells of the immune system and bone cells have redefined our thinking on the regulation of bone resorption. Moreover, the crosstalk between these cell types has special significance in inflammatory conditions such as rheumatoid arthritis. This report highlights the contribution that T lymphocytes make in regulating osteoclast formation and bone resorption.     

Clonal analysis of HLA-restricted virus-specific cytotoxic T lymphocytes from cerebrospinal fluid in mumps meningitis

T lymphocytes were cloned directly from the cerebrospinal fluid of a patient with mumps meningitis by limiting dilution in the presence of irradiated feeder cells and T cell growth factor. Of 84 colonies analyzed, 41 were cytotoxic, as shown by their ability to exert phytohemagglutinin-dependent killing. Of these, 39 showed specificity for the autologous mumps-virus infected target cells. The cytotoxic T cell colonies showed the same pattern of HLA restriction as bulk cultures of CSF lymphocytes. This study shows that it is possible to perform functional assays on inflammatory exudate cells at the clonal level. The data also suggest that recruitment of effector cells from the peripheral compartment into the meningeal spaces in mumps meningitis is highly antigen-specific.     

Suppressor T cell growth and differentiation: evidence for induced receptors on suppressor T cells that bind a suppressor T cell differentiation factor

T suppressor cell differentiation factor (TsDF) induces the differentiation of alloantigen-primed suppressor T cells (MLR-Ts) to expression of their effector function, i.e., to active TsF production. The initial activation stimulus to Ts is provided by alloantigen binding; after this binding, Ts are functionally responsive only for a period of hours to the additional stimulus provided by TsDF. The present studies addressed the possibility that MLR-Ts responsiveness to TsDF reflects the induced and transient display of TsDF-binding receptors. TsDF receptor expression was investigated by determining the capacity of TsDF-responsive MLR-Ts to adsorb TsDF activity and to respond to that TsDF pulse by TsF production. Primed Ts populations that were alloantigen restimulated for 8 hr adsorbed TsDF in a cell dose-dependent fashion and produced TsF in response to that adsorption, whereas alloantigen-stimulated naive cells or primed but nonrestimulated cells neither responded to nor bound TsDF. Primed and restimulated L3T4-Ly-2+ but not L3T4+-Ly-2--enriched T cells bound TsDF. TsDF adsorption was saturable and time and temperature dependent. Glutaraldehyde fixation did not prevent TsDF adsorption by restimulated MLR-Ts, whereas pronase treatment abolished their TsDF-binding capacity. Kinetic analyses demonstrated that the capacity to bind TsDF developed rapidly after alloantigen reexposure, with maximal binding within 8 hr, followed by rapid decay with loss of TsDF binding by 36 hr. The kinetics of TsDF-induced TsF production correlated precisely with those of TsDF binding. These observations provide strong evidence that TsDF affects primed alloantigen-reactive Ts by interaction with antigen-induced and transiently expressed cell surface receptors. TsDF-receptor binding is then the stimulus for expression of Ts effector function.     

B cell stimulatory factor-2 is involved in the differentiation of cytotoxic T lymphocytes

The induction of cytotoxic T lymphocytes (CTL) from precursor T cells requires both antigen and lymphokine signals. Previous work from our laboratory has indicated that three lymphokines are required for the induction of CTL from murine thymocytes; interleukin 2, interferon-gamma (IFN-gamma), and a partially characterized factor referred to as cytotoxic differentiation factor (CDF). While attempting to clone CDF from the human T cell line C10-MJ2, we found that a gene encoding CDF-like activity is identical to the gene encoding the factor known variously as B cell stimulatory factor-2 (BSF-2), IFN-beta 2, and 26-kDa protein. We report here that BSF-2 can induce the differentiation of Ly-2+ CTL from murine thymocytes in the presence of interleukin 2 and that the level of cytotoxicity is augmented by the addition of murine IFN-gamma. Serine esterase, a marker for cytotoxic granules in CTL, was induced only in the presence of BSF-2, and the level of serine esterase activity correlated with the level of serine esterase activity correlated with the level of cytotoxicity. These data suggest that BSF-2 is a differentiation factor for CTL and that it functions in part by inducing proteins required for mediating target cell lysis.     

Human lymphocyte differentiation antigens HB-10 and HB-11. II. Differential production of B cell growth and differentiation factors by distinct helper T cell subpopulations

Two monoclonal antibodies (HB-10 and HB-11), which react with human T, B, and NK cells, identify approximately 50% of the Leu-3+ T helper (TH) cells in adult blood. In the present studies, the functional capabilities of the HB-11+ and HB-11-TH cell subpopulations were examined after purification by fluorescence-activated cell sorting. Both subpopulations proliferated in response to PHA, Con A, PWM, and OKT-3 antibodies. The HB-11+ TH cells gave a minimal proliferative response to soluble tetanus toxoid antigen, whereas HB-11-TH cells responded well. After mitogen activation, both HB-11+ and HB-11-TH cells and to produce soluble factors which induce large B cells to proliferate. However, PWM-stimulated HB-11+TH cells were incapable of inducing B cells to differentiate into antibody-secreting plasma cells, whereas HB-11-TH cells were efficient in this regard. The results suggest that the HB-11 antigen is expressed on a subpopulation of virgin TH cells that can produce B cell growth factors but are deficient in the ability to produce B cell differentiation factors.     

Clonal and frequency analyses of tumor-infiltrating T lymphocytes from human solid tumors

A limiting dilution analysis (LDA) was used to assess the functional profiles of tumor-infiltrating lymphocytes (TIL) recovered from 15 human solid tumors. The microculture system applied in this study has been shown to allow virtually all normal peripheral blood T lymphocytes (PBL-T) to undergo clonal proliferation and was applied to obtain estimates of the frequency of both proliferating and cytolytic cells among the TIL population. A total of 624 microcultures proliferating in the presence of irradiated allogeneic spleen cells and interleukin 2 (IL 2) were expanded for clonal analysis. These TIL microcultures were assessed for surface antigen phenotype, IL 2 production (helper function) and for their cytolytic capabilities against the human erythroleukemic line K562 (natural killer (NK)-like activity) and P815, a mouse mastocytoma line, in the presence of phytohemagglutinin (PHA), i.e., lectin-dependent cell cytotoxicity (LDCC) which allows the detection of cytolytic activity irrespective of the antigenic specificity of the effector cells. Whenever feasible, cytolytic activity against autologous and allogeneic tumor cells was tested. LDA first demonstrated that the proliferative potential was decreased in T lymphocytes infiltrating human solid tumors (approximately 1 in 50 to 1 in 2 proliferating T lymphocyte precursors (PTL-P) in this series) as compared to normal PBL-T (1 in 2 to 1 in 1 PTL-P). The growth pattern in the titration cultures showed a remarkable agreement with the single-hit Poisson model implying that third party cells are unlikely to be involved in the reduced proliferative potential. Quantitative estimates of functional precursors showed that, in spite of reduced proliferative potential, cytolytic T lymphocyte precursors (CTL-P) against unknown antigens (LDCC-reactive) accounted for a considerable part of the microcultures in many cases. The precursor frequency of T lymphocytes with NK-like activity was usually low in situ (with the exception of glioma), whereas it was in the normal range in the patient's autologous PBL-T. In four evaluable cases, quantitative assessment showed that 1 in 200 to 1 in 1000 T lymphocytes from TIL was cytolytic against allogeneic tumor cells, which is in the range of alloreactive cytolytic T lymphocytes (CTL) generated in the mixed lymphocyte culture from normal PBL. Cytolytic activity against autologous target cells could not be quantitatively estimated but out of 88 clones from 4 patients, 3 clones originating from 2 glioma patients showed high lytic values against autologous tumor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Directed differentiation of induced pluripotent stem cells towards T lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.     

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma

Summary This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11^– T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11⁺ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.     

Proliferation of human malignant lymphocytes induced by anti-IgM independent of B cell growth factor

Human malignant B lymphocytes were identified that proliferate in response to small doses of anti-immunoglobulin. Proliferation was induced by monoclonal mouse anti-HIgM, polyclonal goat anti-HIgM, and F(ab')2 fragments thereof, in vitro, and was not accompanied by immunoglobulin secretion. Proliferation was found to be unaffected by T cell depletion and was not enhanced by supplementation with B cell growth factor. Culture fluids from unstimulated malignant lymphocytes as well as from malignant lymphocytes stimulated with anti-HIgM contained no measurable B cell growth factor activity. Thus, proliferation of these malignant lymphocytes was not dependent on the presence of T lymphocytes and was independent of the presence of B cell growth factor. These results imply that B cell stimulatory factors may not be required for proliferation of all human B lymphocytes. Moreover, these results imply that treatment with anti-immunoglobulin reagents may be inappropriate for some B lymphocyte malignancies.     

Signal requirements for growth and differentiation of activated murine B lymphocytes

Mouse B lymphocytes were stimulated at high cell concentrations with goat anti-IgM antibodies, which leads to the induction of B cell proliferation without the addition of any growth factors. After 48 hr, blast cells were purified and cultured at low cell concentrations. Proliferation and differentiation of purified B lymphocyte blasts is then dependent on the addition of either mitogens (e.g., LPS) or certain lymphokines derived from activated T cells or macrophages. One such lymphokine was isolated from supernatants of various activated T cells and characterized by gel filtration as a material with an apparent m.w. of 40,000 to 50,000, similar to BCGF II. It supports the proliferation of the B cell blasts and induces their differentiation into plaque-forming cells. Lymphokines such as BCGF I, interleukin 2, and BCDF gamma could neither maintain growth nor induce differentiation of B lymphocytes preactivated by goat anti-IgM.     

Differentiation of human T and B peripheral blood lymphocytes by high-resolution cell image analysis

Automated cell image analysis of light and electron microscopic pictures was used for differentiation of nonlabeled lymphocytes in blood smears and in smears of purified lymphocyte suspensions. The percentages of T and B lymphocytes were determined by a two-step rosette assay with sheep red blood cells (T cells) and an immunofluorescence assay with FITC-labeled antihuman globulin (B cells). Images from 1,400 Feulgen-stained and 12,000 Pappenheim-stained cells were analyzed. Various classification methods allowed two lymphocyte subpopulations to be discriminated at the light and electron microscopic levels on the basis of different visual and subvisual morphologic features. As found by immunologic methods, morphologically determined subpopulations corresponded to T and non-T cells, with no further differentiation of non-T cells into B or null cells possible. The results allow the conclusion that there are morphologic differences between human T and non-T cells, with the differences distinguishable from individual variations as well as from alterations induced by sample preparation.     

NF-kappa B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes

Ontogenetic, homeostatic, and functional deficiencies within immunoregulatory natural T (iNKT) lymphocytes underlie various inflammatory immune disorders including autoimmunity. Signaling events that control cell fate specification and molecular differentiation of iNKT cells are only partly understood. Here we demonstrate that these processes within iNKT cells require classical NF-kappaB signaling. Inhibition of NF-kappaB signaling blocks iNKT cell ontogeny at an immature stage and reveals an apparent, novel precursor in which negative selection occurs. Most importantly, this block occurs due to a lack of survival signals, as Bcl-x(L) overexpression rescues iNKT cell ontogeny. Maturation of immature iNKT cell precursors induces Bcl-2 expression, which is defective in the absence of NF-kappaB signaling. Bcl-x(L) overexpression also rescues this maturation-induced Bcl-2 expression. Thus, antiapoptotic signals relayed by NF-kappaB critically control cell fate specification and molecular differentiation of iNKT cells and, hence, reveal a novel role for such signals within the immune system.     

Induction of T cell-dependent B cell differentiation by anti-CD3 monoclonal antibodies

Three monoclonal antibodies (mAb) recognizing the CD3 (T3) surface complex each induced B cell differentiation (as measured by PFC generation) in cultures containing T + non-T cells. Irradiation of the T cells before culture usually augmented the PFC response. An IgG2a mAb (454) induced PFC in all donors tested, whereas two IgG1 mAb (147 and 446) induced PFC in only 80% of the donors tested. This heterogeneity in PFC response to IgG1 anti-CD3 mAb strictly paralleled the heterogeneity in proliferative response to IgG1 anti-CD3 mAb and was governed by cells within the non-T population. In IgG1 anti-CD3 high responders (HR), all anti-CD3 mAb tested induced Tac expression. In IgG1 anti-CD3 low responders (LR), mAb 454 induced Tac expression, but mAb 147 did not. However, when the cultures were supplemented with exogenous interleukin 2, Tac expression and PFC generation in response to mAb 147 was similar to the response to mAb 454 in both HR and LR. The addition of anti-Tac to the cultures partially inhibited anti-CD3-induced PFC generation. These studies indicate that anti-CD3 mAb can lead to B cell differentiation under appropriate experimental conditions and may be valuable in studying polyclonal T cell-dependent B cell differentiation in normal and disease states.     

Recombinant T cell replacing factor (interleukin 5) acts with antigen to promote the growth and differentiation of single hapten-specific B lymphocytes

The role of murine recombinant T cell replacing factor (rTRF) (interleukin 5) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein (FLU)-specific B cells was examined in vitro. FLU-specific B cells were selected by their adherence to FLU-gelatin and then cultured in 10-microliters wells with or without rTRF in the presence or absence of the T-independent antigen FLU-polymerized flagellin (FLU-POL). rTRF acting alone was unable to induce early B cell activation as assessed by significant cell enlargement after 24 hr in culture. When acting in the presence of FLU-POL, however, a greater number of B cells were induced to enlarge than with FLU-POL alone. When FLU-specific B cells were cultured in the presence of FLU-POL, the addition of rTRF markedly increased the frequencies of both proliferating clones and AFC clones above that induced by FLU-POL alone. Furthermore, in the presence of FLU-POL, the activity of rTRF was comparable to that seen with the mixture of B cell growth and differentiation factors contained within the supernatant from concanavalin A-stimulated EL4 cells. However, rTRF exerted little activity when acting alone in contrast to the medium conditioned by concanavalin A-stimulated EL4 cells which showed some activity in the absence of FLU-POL. rTRF acting with FLU-POL also promoted AFC clone development among single B cells stimulated in the presence of 3T3 fibroblast filler cells. Thus rTRF can be added to the list of B cell active factors (including recombinant murine interleukin 1 and recombinant human interleukin 2) that act in the concomitant presence of antigen to induce both growth and differentiation among single hapten-specific murine splenic B cells. This stands in contrast to the activity seen with interleukin 4 (formerly termed B cell stimulatory factor 1) which acts to promote early activation and proliferation but not IgM secretion.