Bone-metastatic prostate carcinoma favors mesenchymal stem cell differentiation toward osteoblasts and reduces their osteoclastogenic potential.

Bone homeostasis is achieved by the balance between osteoclast-dependent bone resorption and osteoblastic events involving differentiation of adult mesenchymal stem cells (MSCs). Prostate carcinoma (PC) cells display the propensity to metastasize to bone marrow where they disrupt bone homeostasis as a result of mixed osteolytic and osteoblastic lesions. The PC-dependent activation of osteoclasts represents the initial step of tumor engraftment into bone, followed by an accelerated osteoblastic activity and exaggerated bone formation. However, the interactions between PC cells and MSCs and their participation in the disease progression remain as yet unclear. In this study, we show that bone metastatic PC-3 carcinoma cells release factors that increase the expression by human (h)MSCs of several known pro-osteoblastic commitment factors, such as α5/ß1 integrins, fibronectin, and osteoprotegerin. As a consequence, as shown in an osteogenesis assay, hMSCs treated with conditioned medium (C(ed) M) derived from PC-3 cells have an enhanced potential to differentiate into osteoblasts, as compared to hMSCs treated with control medium or with C(ed) M from non-metastatic 22RV1 cells. We demonstrate that FGF-9, one of the factors produced by PC-3 cells, is involved in this process. Furthermore, we show that PC-3 C(ed) M decreases the pro-osteoclastic activity of hMSCs. Altogether, these findings allow us to propose clues to understand the mechanisms by which PC favors bone synthesis by regulating MSC outcome and properties.

A cloned human T cell line cytotoxic for autologous and allogeneic B lymphoma cells.

A human cytotoxic T cell clone (MWS-14) with auto-tumor reactivity was established in serum-free medium in a mixed tumor cell culture by repetitive stimulation with fresh autologous lymphoma cells. This clone and its subclones are of the T3+ T4+ T8- phenotype. They were strongly cytotoxic for the autologous lymphoma cells, whereas autologous PHA blasts were not killed. Analysis of the specificity of MWS-14, MWS-14-30, and MWS-14-34 indicated that these CTL clones were cytotoxic for 7/7 allogeneic lymphoma cells, whereas only 3/23 of normal and non-lymphoma cells were lysed. Blocking studies with monoclonal antibodies directed at MHC class I and class II antigens showed that this preferential, anti-lymphoma reactivity was not directed at HLA determinants. The anti-lymphoma activity is not due to an aspecific susceptibility of the lymphoma cells to lysis. In contrast to CTL clones specific for HLA antigens present on the lymphoma cells, T3 and T4 were not involved in the cytotoxic reaction of MWS-14 against the autologous lymphoma cells. The reactivity of this clone could be blocked by a monoclonal antibody directed at leukocyte function-associated antigen. It can be concluded from these results that these T4+ CTL clones recognize a determinant, which is preferentially expressed on autologous and allogeneic lymphoma cells.

A distinct wave of human T cell receptor gamma/delta lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production.

The rearrangement and expression of human T cell receptor (TCR)-gamma and -delta gene segments in clonal and polyclonal populations of early fetal and postnatal human TCR-gamma/delta thymocytes were examined. The data suggest that the TCR-gamma and -delta loci rearrange in an ordered and coordinated fashion. Initial rearrangements at the TCR-delta locus join V delta 2 to D delta 3, and initial rearrangements at the TCR-gamma locus join downstream V gamma gene segments (V gamma 1.8 and V gamma 2) to upstream J gamma gene segments associated with C gamma 1. These rearrangements are characterized by minimal junctional diversity. At later times there is a switch at the TCR-delta locus such that V delta 1 is joined to upstream D delta gene segments, and a switch at the TCR-gamma locus such that upstream V gamma gene segments are joined to downstream J gamma gene segments associated with C gamma 2. These rearrangements are characterized by extensive junctional diversity. Programmed rearrangement explains in part the origin of discrete subpopulations of peripheral blood TCR-gamma/delta lymphocytes that have been defined in previous studies. In addition, cytokine production by early fetal and postnatal TCR-gamma/delta thymocyte clones was examined. Fetal thymocyte clones produced significant levels of IL-4 and IL-5 following stimulation, whereas postnatal thymocyte clones did not produce these cytokines. Thus, these cell populations may represent functionally distinct subsets as well.

Functional expression of B7/BB1 on activated T lymphocytes.

B7/BB1 is a membrane differentiation antigen expressed on activated B cells, macrophages, and dendritic cells that binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T cell activation initiated via the CD3/T cell receptor complex. We now report that B7 is also expressed on activated human peripheral blood T cells, CD4 T cell clones, CD8 T cell clones, and natural killer cell clones. B7 appears relatively late after T cell activation, can be detected on both CD4 and CD8 T cell subsets, and is present on antigen-specific, major histocompatibility complex-restricted CD4 and CD8 T cell clones. Expression of B7 on activated T cells was confirmed by immunoprecipitation from 125I-labeled activated T cells and by detection of B7 transcripts. A B7+ CD4+ T cell clone was able to stimulate a primary allogeneic mixed lymphocyte response using small, resting peripheral blood T cells as responders. The alloantigen-induced proliferative response and cytokine production was partially inhibited by anti-B7 monoclonal antibody. Since activated T cells can coexpress both CD28 and its counter-receptor, B7, this suggests that activated T cells may be capable of autocrine costimulation via the CD28 activation pathway.

RANTES induces tyrosine kinase activity of stably complexed p125FAK and ZAP-70 in human T cells.

The chemokine RANTES is a chemoattractant and activating factor for T lymphocytes. Investigation of the signal transduction mechanisms induced by RANTES in T cells revealed tyrosine phosphorylation of multiple protein species with prominent bands at 70-85 and 120-130 kD. Immunoprecipitation and Western analyses revealed that a protein of 125 kD was identical to the focal adhesion kinase (FAK) pp125FAK. RANTES stimulated phosphorylation of FAK as early as 30 seconds and immunoblots using antiphosphotyrosine monoclonal antibodies revealed that there was consistent phosphorylation of a 68-70 kD species in the pp125FAK immunoprecipitates. Immunoblotting and kinase assays showed this to be two separate proteins, the tyrosine kinase zeta-associated protein (ZAP) 70, and the focal adhesion protein paxillin. These results indicate a potentially important role for RANTES in the generation of T cell focal adhesions and subsequent cell activation via a molecular complex containing FAK, ZAP-70, and paxillin.

Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level.

The development of CD4+ T helper (Th) type 1 and 2 cells is essential for the eradication of pathogens, but can also be responsible for various pathological disorders. Therefore, modulation of Th cell differentiation may have clinical utility in the treatment of human disease. Here, we show that interleukin (IL) 12 and IL-4 directly induce human neonatal CD4- T cells, activated via CD3 and CD28, to differentiate into Th1 and Th2 subsets. In contrast, IL-13, which shares many biological activities with IL-4, failed to induce T cell differentiation, consistent with the observation that human T cells do not express IL-13 receptors. Both the IL-12-induced Th1 subset and the IL-4-induced Th2 subset produce large quantities of IL-10, confirming that human IL-10 is not a typical human Th2 cytokine. Interestingly, IL-4-driven Th2 cell differentiation was completely prevented by an IL-4 mutant protein (IL-4.Y124D), indicating that this molecule acts as a strong IL-4 receptor antagonist. Analysis of single T cells producing interferon gamma or IL-4 revealed that induction of Th1 cell differentiation occurred rapidly and required only 4 d of priming of the neonatal CD4+ T cells in the presence of IL-12. The IL-12-induced Th1 cell phenotype was stable and was not significantly affected when repeatedly stimulated in the presence of recombinant IL-4. In contrast, the differentiation of Th2 cells occurred slowly and required not only 6 d of priming, but also additional restimulation of the primed CD4+ T cells in the presence of IL-4. Moreover, IL-4-induced Th2 cell phenotypes were not stable and could rapidly be reverted into a population predominantly containing Th0 and Th1 cells, after a single restimulation in the presence of IL-12. The observed differences in stability of IL-12- and IL-4-induced human Th1 and Th2 subsets, respectively, may have implications for cytokine-based therapies of chronic disease.

Borrelia burgdorferi activates a T helper type 1-like T cell subset in Lyme arthritis.

18 cloned T cell lines reactive with Borrelia burgdorferi proteins, all CD3+4+8-TCR-alpha/beta+ and restricted by HLA class II proteins, were isolated from four patients with chronic Lyme arthritis. Analysis of these T cell clones indicated that the T cell response to the Lyme disease spirochete is not oligoclonally restricted; yet all produced the same pattern of lymphokines, resembling that of murine type 1 T helper cells, after antigen-specific or nonspecific stimulation. Therefore, a subset of human CD4+ T cells, with a distinct profile of lymphokine secretion, is selectively activated by the pathogen inciting this chronic inflammatory disease.

Autoreactive T cell clones specific for class I and class II HLA antigens isolated from a human chimera.

T cell clones of donor origin that specifically react with recipient cells were obtained from a SCID patient successfully reconstituted by allogeneic fetal liver and thymus transplantation performed 10 yr ago. The majority of these clones displayed both cytotoxic and proliferative responses towards PBL and an EBV-transformed B cell line derived from the patient. In addition, these T cell clones had proliferative and cytotoxic responses towards the parental PBL, EBV cell lines, and PHA blasts. Blocking studies with anti-class I and anti-class II HLA mAbs indicated that the activity of the CD4+ T cell clones was specifically directed against class II HLA antigens of the recipient. On the other hand, the cytotoxic and proliferative responses of the CD8+ T cell clones were specific for class I HLA antigens which are ubiquitously expressed on the recipient cells. Thus, the establishment of transplantation tolerance observed in this stable human chimera is not due to the elimination of host-reactive T cells from the repertoire and suggests the presence of a peripheral autoregulatory suppressor mechanism.

Antigen recognition by MHC-incompatible cells of a human mismatched chimera.

Tetanus toxin (TT)-specific T cell clones of donor origin were obtained from a patient with severe combined immunodeficiency (SCID) successfully reconstituted by transplantation of allogeneic fetal liver and thymus cells from two different donors performed 10 yr ago. A series of these clones recognized TT in the context of "allo" class II HLA determinants expressed by recipient APC. The restriction element of two T cell clones with the HLA phenotype of the first donor (HLA-DR1,8) and one T cell clone with the HLA phenotype of the second transplant (HLA-DR3,9) was HLA-DR4 of the recipient, whereas other T cell clones derived from the second transplant recognized TT in the context of HLA-DR5 of the recipient's APC. These latter T cell clones were not able to proliferate in response to TT when autologous APC were used. These data demonstrate that recipient and donor cells having different HLA phenotypes could cooperate across the allogeneic barrier and that MHC restriction of antigen (Ag) recognition is independent from the MHC genotype of the T cells but is influenced by the environment in which the T cells mature. We also isolated T cell clones that were able to recognize processed TT presented by all allogeneic EBV cell lines tested, indicating that the Ag specificity of these clones was not restricted by a particular class II MHC molecule. The Ag-specific proliferative response of one of these clones could be blocked by anti-class II MHC mAbs. These results demonstrate that in addition to Ag recognition in the context of specific class II MHC Ags, other types of Ag-specific responses may occur in this human chimera. It is not clear whether this "allo" plus Ag recognition is the result of education of transplanted fetal cells in the host thymus. Taking into consideration our previous findings indicating that alloreactive T cell clones specific for the recipient cells could be isolated in vitro from the PBL of the same patient, our data suggest that the mechanism for deletion of self-reactive clones and the generation of MHC-restricted responses are different.

Human B cell clones can be induced to proliferate and to switch to IgE and IgG4 synthesis by interleukin 4 and a signal provided by activated CD4+ T cell clones.

In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.

Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.

Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.

Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the Fc epsilonRI, CD40L, IL-4, and IL-13, and can induce IgE synthesis in B cells.

Cross-linking of allergen specific IgE bound to the high affinity IgE receptor (FC epsilonRI) on the surface of mast cells with multivalent allergens results in the release of both pre-formed and newly generated mediators, and in the manifestation of allergic symptoms. The expression of Fc epsilonRI, and the synthesis of IgE are therefore critical for the development of allergic diseases. In this study, we report that nasal mast cells (NMC) from patients with perennial allergic rhinitis (PAR) expressed significantly greater levels of the Fc epsilonRI, CD40L, IL-4, and IL-13 as compared to NMC from patients with chronic infective rhinitis (CIR). The level of Fc epsilonRI expression in NMC of PAR patients strongly correlated with the levels of serum total (r = 0.8, P < 0.003) and specific IgE (r = 0.89, P < 0.0004) antibodies. In addition, stimulation of NMC with IL-4, upregulated the Fc epsilonRIalpha chain expression both at the protein and mRNA levels, as detected by flow cytometry and reverse transcriptase-polymerase chain reaction. Furthermore, NMC from PAR, but not CIR, patients induced IgE synthesis by purified B cells in the presence of Der fII (mite antigen). These results suggest novel and critical roles for mast cells in promoting the allergic reaction through the increased expression of Fc epsilonRI and by enhancing and amplifying the IgE production, within the local microenvironment.

Induction of non-responsiveness in human allergen-specific type 2 T helper cells.

Activation of allergen-reactive human T helper (Th)2 cells in the absence of professional antigen-presenting cells, induces non-responsiveness or anergy in these cells in vitro. This induction of anergy is accompanied by phenotypic modulation and altered cytokine production. Furthermore, peptide-treated Th2 cells fail to provide B-cell help for IgE synthesis. Recent studies indicate that impaired signal transduction via the T-cell receptor may account for the lack of responsiveness to antigenic stimulation. Here, we review present knowledge on the cell biology of non-responsive or anergic Th2 cells.

Peptide modulation of allergen-specific immune responses.

In vitro peptide stimulation of allergen-reactive T-helper type 1 and type 0 cells, in the absence of costimulatory signals, induces anergy that is accompanied by the modulation of cell surface phenotype and changes in cytokine production. In experimental animal models, the administration of allergen-derived peptides may result in the downregulation of cytokine and antibody production, which is preceded by transient activation of CD4+ T cells, without the induction of effector immunity. Preliminary results of clinical trials using allergen-derived peptides for desensitization are becoming available and should provide some insight into the efficacy of peptide therapy in man.

Antagonizing the differentiation and functions of human T helper type 2 cells.

Allergen-specific T cells from atopic patients generally belong to the T-helper type 2 subset. IL-4 and IL-13 produced by these cells induce IgE synthesis by B cells and play a major role in allergic disease mediated by IgE. Recent advances in our understanding of the differentiation and IgE-inducing activities of T-helper type 2 cells suggest that targeting allergen-specific T cells may provide a novel way to intervene in allergy.

Interleukin-10.

Despite the short history of interleukin-10, accumulated evidence indicates that this interleukin plays a major role in suppressing immune and inflammatory responses. Yet interleukin-10 also maintains cell viability and acts as a cofactor to promote the growth of lymphoid and myeloid cells in vitro. Here we review the present knowledge on the structure and function of interleukin-10.

Regulation of IgE synthesis by cytokines.

Considerable progress has been made in our understanding of the mechanisms underlying regulation of human IgE synthesis. Interleukin-4 induces IgE production specifically, but costimulatory signals provided by T cells are required. Other cytokines modulate interleukin-4-induced IgE synthesis. The roles of T cells and cytokines in regulating IgE switching are discussed.

A role for T cell-derived interleukin 22 in psoriatic skin inflammation.

Interleukin (IL)-22 is a T cell-derived cytokine that has been reported recently to induce cutaneous inflammation in an experimental murine model of psoriasis, and to induce in vitro an inflammatory-like phenotype. In the present study, we assessed the presence of IL-22 and the IL-22 receptor 1 (IL-22R1) in skin lesions, skin-derived T cells, as well as IL-22 levels in sera from patients with psoriasis. IL-22R1 and IL-10R2 transcripts are expressed at a similar level in psoriatic and healthy skin. In contrast, IL-22 mRNA expression was up-regulated in psoriatic skin lesions compared to normal skin, whereas IL-22 mRNA levels in peripheral blood mononuclear cells from psoriatic patients and normal subjects were similar. Circulating IL-22 levels were significantly higher in psoriatic patients than in normal subjects. T cells isolated from psoriatic skin produced higher levels of IL-22 in comparison to peripheral T cells isolated from the same patients. IL-10 was expressed at similar levels in skin biopsies and peripheral blood mononuclear cells of psoriatic patients and normal subjects. Finally, we show here that supernatants of lesional psoriatic skin-infiltrating T cells induce an inflammatory response by normal human epidermal keratinocytes, resembling that observed in psoriatic lesions. Taken together, the results reported in this study indicate that IL-22 is a cytokine produced by skin-infiltrating lymphocytes that is potentially involved in initiation and/or maintenance of the pathogenesis of psoriasis.

Cloning of Human T and Natural Killer Cells

The discovery of the lymphokine interleukin 2 (IL-2), which induces growth of T cells, set the stage for establishing methods for in vitro cloning of human T cells. More recently, it has become clear that in addition to IL-2, other T-cell growth factors, such as IL-4 and IL-7, can be used for generation and expansion of T-cell clones. In the past it was shown that it is possible to grow T cells differing in function and phenotype. In vitro-expanded T-cell clones have been instrumental in studies on the antigen specificities and biological properties of these cells. Cloned lines of natural killer (NK) cells can also be established using IL-2, permitting detailed studies on the biology of these cells. Here we describe the methods we use to generate and to expand human T-cell and NK cell clones. We discuss the differences between IL-2 and IL-4 as growth factors to expand T-cell clones. In addition, we summarize some recent findings of studies using T- and NK cell clones.

Phenotypic characterization of human CD4+ regulatory T cells obtained from cutaneous dinitrochlorobenzene-induced delayed type hypersensitivity reactions.

In this study, we describe the generation and characterization of cloned human CD4+ T lymphocyte populations that have infiltrated into cutaneous, 2,4-dinitrochlorobenzene-induced delayed type hypersensitivity reactions in healthy human subjects. It is shown that, in addition to T helper type 1 clones, elevated numbers of regulatory T clones, producing high levels of interleukin-10 and interleukin-5, but no measurable interleukin-4, were isolated from delayed type hypersensitivity reactions in four of six donors. A subsequent challenge with 2,4-dinitrochlorobenzene of two donors from whom only few interleukin-10-producing T cell clones had been generated after primary challenge, resulted in a decrease in the frequency of T helper type 1 clones and a strong increase in the number of interleukin-10-producing T helper type 2 and regulatory T clones. Culture supernatants from the latter cells, activated with anti-CD3 and anti-CD28 monoclonal antibody, inhibited alloantigen-mediated T cell proliferation which was, partly dependent on interleukin-10, and independent of transforming growth factor-beta. In addition, dendritic cells generated in vitro in the presence of these culture supernatants were impaired in their ability to induce alloantigen-induced proliferative responses. Differential expression of transcripts for the T1/ST2 molecule enabled a phenotypic distinction between resting regulatory T cells and T helper type 2 cells, but not between regulatory T cells and T helper type 1 cells. This experimental model provides a useful tool to isolate human inflammatory and anti-inflammatory T cell subpopulations and, furthermore, enables the study of the kinetics of their appearance into delayed type hypersensitivity reactions.