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A sharp increase in 2005 of pp'DDT and its metabolites was observed in mussels and fish from lakes Como and Iseo, the main glacier-fed southern Alpine lakes. DDTs in zebra mussels (Dreissena polymorpha) were more than 150 times higher than levels in 2003, and concentrations in pelagic fish (0.12 mgkg(-1) w.w.) exceeded the Italian safety threshold for human consumption (0.05 mgkg(-1) w.w.). The histological examination of the ovaries revealed many mussels with oocyte degeneration throughout the studied period. Prior to being banned in Italy in 1978, DDT was used in large amounts for fruit-tree treatment from the 1950s to 1970s in valleys just below the glaciers. Since glacier volume was increasing at that time and then continuously retreated, meltwater should be the main cause of the pollution peak recently observed in biota of downstream lakes. PCBs did not peak in biota tissues to a comparable extent probably because local sources were not as important as for DDTs.

We are reporting on a 7-months-old boy with suspected hyper-IgE syndrome, presenting with a therapy resistant severe eczema and an overall reduction of in vitro cytokine production. Interferon-alpha (IFN-alpha) treatment resulted in a marked and stable clinical improvement and normalization of in vitro T-cell cytokine production, indicating a valid therapeutic potential of IFN-alpha as immunomodulating drug.

Summary: T-regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function of different types of Tregs can lead to autoimmunity, allergy, and graft rejection, whereas an over-abundance of Tregs can inhibit anti-tumor and anti-pathogen immunity. Experimental models in mice have demonstrated that manipulating the numbers and/or function of Tregs can decrease pathology in a wide range of contexts, including transplantation, autoimmunity, and cancer, and it is widely assumed that similar approaches will be possible in humans. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4(+) Tregs: forkhead box protein 3 (FOXP3)(+) Tregs and interleukin-10-producing type 1 Tregs (Tr1 cells). The aim of this review is to highlight current information on the characteristics of human FOXP3(+) Tregs and Tr1 cells that make them an attractive therapeutic target. We discuss the progress and limitations that must be overcome to develop methods to enhance Tregs in vivo, expand or induce them in vitro for adoptive transfer, and/or inhibit their function in vivo. Although many technical and theoretical challenges remain, the next decade will see the first clinical trials testing whether Treg-based therapies are effective in humans.

Embryotoxic effects of Carbaryl (CB), a widely used carbamate insecticide, was evaluated by modified Frog Embryo Teratogenesis Assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. X. laevis embryos were exposed to 1, 2, 4, 8, 16 and 24 mg/L CB from stage 8 to stage 47. From an estimated LC50 of 20.28 mg/L and TC50 of 8.43 mg/L a TI of 2.41 was derived, indicating that CB is to be considered teratogenic for X. laevis embryos. The most characteristic terata, classified as abnormal tail flexure, involved a significant percentage of larvae from 1 mg/L CB onward, reaching 100% at 24 mg/L CB. Histopathological screening revealed tail musculature and notochord as the main targets for CB. Skeletal muscle lesions consisted of myotomes reduced in size, showing myocytes with disorganized contractile systems and irregular myosepta, coupled with disarranged myocyte apexes. Notochords from CB exposed larvae appeared wavy or bent, with irregular connective sheaths and histologically characterized by protrusions of fibrous matrix and inclusions of ectopic cell masses. This axial-skeletal damage was hypothesized to be related both to the inhibition of acetylcholinesterase, with consequent muscular tetanic spasms, and to disorders in the organization of the connective tissue matrix surrounding the notochord.

Forkhead box P3 (FOXP3) is considered a specific marker for CD4(+)CD25(+) regulatory T (Treg) cells, but increasing evidence suggests that human CD4(+)CD25(-) effector T (Teff) cells can transiently express FOXP3 upon activation. We demonstrate that the signal transducer and activator of transcription 5 (STAT5)-signaling cytokines, IL-2, IL-15 and to a lesser extent IL-7, induce FOXP3 up-regulation in vitro in activated human Teff cells. In contrast, cytokines which do not activate STAT5, such as IL-4 or transforming growth factor-beta alone, do not directly induce FOXP3 expression in activated Teff cells. Moreover, expression of a constitutively active form of STAT5a is sufficient to induce FOXP3 expression in Teff cells. Expression of FOXP3 in activated Teff cells requires both TCR-mediated activation and endogenous IL-2, but is not dependent on cell division and does not induce suppressive function. The presence of STAT5-activating cytokines is also required to maintain high FOXP3 expression and suppressive activity of Treg cells in vitro. These data indicate that activation of STAT5 sustains FOXP3 expression in both Treg and Teff cells and contribute to our understanding of how cytokines affect the expression of FOXP3.

Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naïve and memory CD4(+) T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4(+) Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications.

Immune regulation and tolerance are specific functions of the immune system, meaning at prevention or limitation of effector immune responses against inner and external insults. Regulatory T (Treg) cells are crucial players in this immune balance network. Research over the last 10 years has significantly contributed to characterizing Treg cell features, their mechanisms of function, and their role in human pathologies. The discovery of FOXP3 as an essential transcription factor not only for differentiation and function of naturally occurring Treg cells but also for regulation of intracellular molecules related to effector T-cell responses has provided new insights into the pathogenesis of immune-mediated diseases. Interestingly, there is increasing evidence that the individual signature of genes relevant for immune regulation definitely influences the final outcome of an immune response.

Tire debris (TD) and its organic components were identified as a main source of PM10 atmospheric and water pollution. Because few data are available on the embryotoxic effects of TD organic components, the lethal and teratogenic potential of tire debris organic extract (TDOE) was evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. From stage 8 to stage 47, Xenopus laevis embryos were exposed to TDOE at concentrations of 50, 80, 100, 120 and 140 mg/L. The results showed 50 mg/L TDOE to be the non-observable effect concentration (NOEC). TDOE mortality at 80 mg/L was significantly higher than the control, but did not increase further with higher concentrations. A good concentration-response was observed for percentages of malformed larva and from 80 mg/L on these percentages were significantly higher than the control. Therefore, probit analysis gave a 144.6 mg/L TC50. At 120 and 140 mg/L, many larvae were plurimalformed. The most frequent alterations observed were abnormal gut coiling, microphthalmia, monolateral anophthalmia, and narrowing eyes. The histological screening mainly revealed ocular malformations such as double retina, retina nervous cell layer coiling, and altered lens. Moreover severe vacuolisation and necrosis were scored in liver and axial musculature. These results strongly support the assumption that TDOE is a powerful teratogen for X. laevis.

Forkhead box P3 (FOXP3) is currently thought to be the most specific marker for naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs). In mice, expression of FoxP3 is strictly correlated with regulatory activity, whereas increasing evidence suggests that in humans, activated T effector cells (Teffs) may also express FOXP3. In order to better define the role of FOXP3 in human Teff cells, we investigated the intensity and kinetics of expression in ex vivo Teff cells, suppressed Teff cells and Teff cell lines. We found that all dividing Teff cells expressed FOXP3, but only transiently, and at levels that were significantly lower than those in suppressive nTregs. This temporary expression in Teff cells was insufficient to suppress expression of reported targets of FOXP3 repressor activity, including CD127, IL-2 and IFN-gamma, and was not correlated with induction of a nTreg phenotype. Thus expression of FOXP3 is a normal consequence of CD4(+) T cell activation and, in humans, it can no longer be used as an exclusive marker of nTregs. These data indicate that our current understanding of how FOXP3 contributes to immune tolerance in humans needs to be re-evaluated.

Fully HLA-mismatched stem cells from human fetal livers were transplanted into 17 infants and two fetuses to treat severe combined immunodeficiency disease in 1976-2000. Donor cell engraftment and immunological reconstitution were obtained in 14/19 patients, three of whom have been extensively and repeatedly studied immunologically during prolonged follow-up. T-cells were derived totally from donor cells; B-cells and antigen-presenting cells (APC) remained mainly of host origin. Due to class I and II mismatches between T-cells and all other cells (APC, B-cells, virus-infected target cells), limitations in the defense against infections in vivo and in T-cell functions in vitro (helper and cytotoxic activities) were predicted; however, these did not occur. Anti-tetanus toxoid responses (including specific antibody production) developed despite HLA disparities between T-cells and B-cells or APC in the chimeric children. Similarly, cytotoxic T-cells (of donor HLA phenotype) recognized host Epstein-Barr virus-infected target cells. Recognition of antigenic peptide by T-cells under these conditions involved presentation by host allogeneic HLA molecules and not by self HLA antigens. Tolerance to donor antigens was acquired by clonal deletion; tolerance to host antigens existed despite the presence of many host-reactive T-cells and involved clonal anergy.

Regulatory T cells play a central role in controlling homeostasis, and in inducing and maintaining tolerance to both foreign and self-antigens. Several types of T cells with regulatory activity have been described both in mice and humans, and those within the CD4+ subset have been extensively studied. Among them, the best characterized are the naturally occurring CD4+CD25+ regulatory T (Treg) cells, and the adaptive type 1 regulatory T (Tr1) cells. Natural Treg cells can arise directly from the thymus, are characterized by the constitutive expression of the transcription factor Foxp3, and suppress T cell responses in a cell-cell contact mediated mechanism. On the contrary, adaptive Tr1 cells arise in the periphery upon encountering antigen in a tolerogenic environment, produce high levels of interleukin (IL)-10 and mediate suppression via IL-10. During the last decade, much effort has been placed on developing protocols to generate regulatory T-cell lines and clones, to further define the similarities and differences between various regulatory T-cell subsets. In this chapter, we will outline protocols to expand naturally occurring Treg cells, to differentiate homogeneous population of Tr1 cells in vitro, and to generate natural Treg and Tr1 cell clones and cell lines.

Interleukin-10 (IL-10)-secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL-10 and transforming growth factor-beta(TGF-beta), and by their ability to suppress antigen-specific effector T-cell responses via a cytokine-dependent mechanism. In contrast to the naturally occurring CD4+ CD25+ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL-10-dependent process in vitro and in vivo. Specialized IL-10-producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL-10-producing T-cell populations that are induced by IL-10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4+ CD25+ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance.

Background: Since MYS is a microtubular poison with a reversible activity, Xenopus blastulae were exposed to MYS to verify the eventual drug-related developmental suspension and the reversibility of this effect., Methods: Lethal and teratogenic effects of myoseverin (MYS) were evaluated using the FETAX. Embryos were exposed to different MYS concentrations from stage 8 to stage 47., Results: Probit analysis gave 12.14 microM LC50 and 7.67 microM TC50 from which 1.58 T.I. is derived. Several malformations were observed such as facial abnormalities, abnormal tail flexure, heart ventricle chamber enlargement and external appendix. MYS led to an arrest of living embryo development. Before MYS removing, exposed blastulae showed the lack of mitotic spindles along with different nuclei alterations. Living embryos, moved in control solution, mainly died around the hatching showing severe malformations likely ascribable to the altered planes of newly occurring mitosis., Conclusion: In spite of the low T.I, MYS has to be considered a highly teratogenic compound., (Copyright 2006 Wiley-Liss, Inc.)

The herbicide paraquat (PQ), still widely used in developing countries, represents a serious risk factor for human and environmental health. To test the sublethal effects of PQ on the freshwater bivalve Dreissena polymorpha, mussels were exposed to 0.125, 0.250, 0.500 mg/L for 7 and 14 days and histologically screened. PQ's genotoxic potential was also determined in haemocytes by the micronucleus, MN, assay. At concentrations > or = 0.250 mg/L, severe lesions, such as cellular vacuolation, lysis and thinness of the germinative epithelia were observed in the digestive gland and testis. A positive trend between the number of granulocytes and all PQ concentrations was observed in both gonads and digestive glands, addressing the inflammatory capacity of this herbicide on these tissues. Mussels exposed to PQ also exhibited a significant MN induction. The spontaneous MN frequencies ranged from 2.75 to 0.425 per cent, while PQ-induced MN rates in treated mussels were between 3.50 and 1.250 per cent. The histopathological effects on the digestive and reproductive systems, as well as the MN induction in the haemocytes, confirmed the cytotoxic and genotoxic effects of PQ also in D. polymorpha.

The oxidative agent paraquat induced tail abnormalities during Xenopus laevis development. Specimens exposed from blastula to the tadpole stage revealed pear-shaped myocytes and irregular intersomitic boundaries. The histological feature of the axial musculature was evaluated in embryos sampled at significant stages of the primary myogenesis. During the somitogenesis PQ-treated embryos showed normal appearing myotomes, but reduced PAS activity in the post-rotating myotomal cells, and myoblasts with slight vacuolations. Once etched from the vitelline envelope, embryos showed severely altered myoblasts with irregular cellular apexes, heavy sarcoplasmic vacuolations, pyknotic nuclei and disorganizing intersomitic boundaries. Myotomes with many necrotic myocytes containing disorganized contractile material and heavily malformed intersomitic boundaries characterized the late myogenic stages. Our results evidence the heaviest PQ histopathological effects to affect myogenesis of post-etched embryos, suggesting a possible linkage between the swimming activity and the oxidative damage to muscle tissue.

Background: As previously shown, Paraquat (PQ) treatments of Xenopus developing embryos mainly induce a characteristic developmental alteration we named "abnormal tail flexure." PQ oxidative activity has been indicated as the cause of this malformation. Since PQ evokes reactive oxygen species (ROS), among which hydroxyl radicals (OH(*)), and H(2)O(2) can be converted to (OH(*)) via Fenton reaction, we compared here the lethal and teratogenic potentials of both oxidants by using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), in order to grasp eventual similarities in their teratogenic activity., Methods: Xenopus embryos were exposed, from stage 8 to stage 47, at 368, 491, 612, and 735 microM H(2)O(2) and 0.388 microM PQ. The probit analysis of H(2)O(2) mortality and malformed larva percents gave a 598.82 microM Lethal Concentration 50% (LC(50)) and 536.04 microM Teratogenic Concentration 50% (TC(50)) from which a 1.11 Teratogenic Index (T.I.) has been calculated. This T.I. value should allow the classification of H(2)O(2) as a non-teratogenic compound., Results: A comparison of H(2)O(2) mortality and malformed larva percents with those obtained from PQ exposure showed the higher embryotoxicity of PQ, but, markedly, both compounds mainly induced the "abnormal tail flexure." Histological analysis of both H(2)O(2) and PQ malformed embryo tails showed a similar distorted morphology of both somites and myocytes. Some of muscle cells were necrotic and affected by an apical enlargement as well as a detachment from the connective tissue of intersomitic boundaries., Conclusions: In our opinion, both of the tested chemicals likely weaken the mechanical bridge connecting the myocyte contractile apparatus to the extracellular matrix, therefore causing the detachment of some of tail myocytes from their connectival septum as well as their apical enlargement. This could lead to the unbalance of tail tensional forces and, in turn, to the appearance of the "abnormal tail flexure."

The autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by mutations in the forkhead box protein P3 (FOXP3) gene. In the mouse model of FOXP3 deficiency, the lack of CD4+ CD25+ Tregs is responsible for lethal autoimmunity, indicating that FOXP3 is required for the differentiation of this Treg subset. We show that the number and phenotype of CD4+ CD25+ T cells from IPEX patients are comparable to those of normal donors. CD4+ CD25high T cells from IPEX patients who express FOXP3 protein suppressed the in vitro proliferation of effector T cells from normal donors, when activated by "weak" TCR stimuli. In contrast, the suppressive function of CD4+ CD25high T cells from IPEX patients who do not express FOXP3 protein was profoundly impaired. Importantly, CD4+ CD25high T cells from either FOXP3+ or FOXP3- IPEX patients showed altered suppression toward autologous effector T cells. Interestingly, IL-2 and IFN-gamma production by PBMCs from IPEX patients was significantly decreased. These findings indicate that FOXP3 mutations in IPEX patients result in heterogeneous biological abnormalities, leading not necessarily to a lack of differentiation of CD4+ CD25high Tregs but rather to a dysfunction in these cells and in effector T cells.

Peripheral tolerance is mediated by multiple mechanisms such as anergy and/or active suppression of effector T cells by T regulatory (Tr) cells. Among the CD4(+) Tr cells, T regulatory type 1 cells (Tr1) have been shown to down-modulate immune responses through production of the immunosuppressive cytokines IL-10 and TGF-beta. Tr1 cells maintain peripheral tolerance, control autoimmunity, and prevent allograft rejection and graft versus host disease (GvHD). Cellular therapy with ex vivo generated Tr1 cells has been proven to be effective in several preclinical models of T cell-mediated pathologies and therefore, represents a promising approach for clinical application. This review will summarize the new findings on Tr1 cells, the recent development of methods for their ex vivo expansion, and their potential clinical relevance as cellular therapy.

Little is known about the molecules that control the development and function of CD4+ CD25+ Tregs. Recently, it was shown that the transcription factor FOXP3 is necessary and sufficient for the generation of CD4+ CD25+ Tregs in mice. We investigated the capacity of FOXP3 to drive the generation of suppressive CD4+ CD25+ Tregs in humans. Surprisingly, although ectopic expression of FOXP3 in human CD4+ T cells resulted in induction of hyporesponsiveness and suppression of IL-2 production, it did not lead to acquisition of significant suppressor activity in vitro. Similarly, ectopic expression of FOXP3delta2, an isoform found in human CD4+ CD25+ Tregs that lacks exon 2, also failed to induce the development of suppressor T cells. Moreover, when FOXP3 and FOXP3delta2 were simultaneously overexpressed, although the expression of several Treg-associated cell surface markers was significantly increased, only a modest suppressive activity was induced. These data indicate that in humans, overexpression of FOXP3 alone or together with FOXP3delta2 is not an effective method to generate potent suppressor T cells in vitro and suggest that factors in addition to FOXP3 are required during the process of activation and/or differentiation for the development of bona fide Tregs.

The main subsets of CD4+ regulatory T (Tr) cells are the CD4+ CD25+ Tr cells and the type 1 regulatory (Tr1) cells. Both subsets are essential for the maintenance of peripheral tolerance. CD4+ CD25+ Tr cells control immune homeostasis (e.g., in autoimmunity) mainly by cell contact-dependent interactions. In contrast, Tr1 cells regulate immune responses in transplantation, allergy, and autoimmune diseases, via an IL-10- and TGF-beta-dependent mechanism. Identification of the mechanisms responsible for the induction of the different Tr subsets in vivo is a matter of intense investigation. Studies on dendritic cells (DC), performed in the last years by several groups, have significantly contributed to clarify this point. Indeed, it is now clear that the role of DCs is not only to sense danger, but also to tolerize the immune system to antigens (Ags) encountered in the absence of maturation/inflammatory stimuli. Therefore, if a naïve T cell encounters the antigen on immature DCs (iDCs), it differentiates into a Tr cell rather than an effector T cell. A better understanding of the mechanisms underlying the induction and functions of Tr cells in controlling the immune system is critical in view of a future cellular therapy to modulate immune-mediated pathologies.

Purpose of Review: Regulatory T cells exert a dominant effect in controlling autoimmunity and maintaining peripheral tolerance. Regulatory T cells are also involved in preventing allograft rejection and graft versus host disease. Cellular therapy with expanded regulatory T cells represents a promising approach to control T-cell mediated pathology. In this review we will summarize the efforts to design new methods for expanding regulatory T cells and exploit their regulatory function as cellular therapy for the treatment of graft versus host disease after hematopoietic stem cell transplantation., Recent Findings: Among CD4+ T cells, the best described are the naturally occurring CD4+CD25+ regulatory T cells and type 1 regulatory T cells. Recent progress has been made in the characterization of both subsets in terms of isolation and induction, respectively. However, a clear definition of their mechanisms of action has still to be achieved., Summary: Better understanding of the mechanisms of suppression mediated by regulatory T cells might enable their use to modulate specific immune responses. Moreover, the recent development of methods allowing the ex-vivo expansion of regulatory T cells, to provide sufficient number of cells for in-vivo infusion, represents the first step toward the use of these cells as cellular therapy for the treatment of immunologic and hematological diseases.

The effects of a chimeric monoclonal antibody (chA6 mAb) that recognizes both the RO and RB isoforms of the transmembrane protein tyrosine phosphatase CD45 on human T cells were investigated. Chimeric A6 (chA6) mAb potently inhibited antigen-specific and polyclonal T cell responses. ChA6 mAb induced activation-independent apoptosis in CD4(+)CD45RO/RB(high) T cells but not in CD8(+) T cells. In addition, CD4(+) T cell lines specific for tetanus toxoid (TT) generated in the presence of chA6 mAb were anergic and suppressed the proliferation and interferon (IFN)-gamma production by TT-specific effector T cells by an interleukin-10-dependent mechanism, indicating that these cells were equivalent to type 1 regulatory T cells. Similarly, CD8(+) T cell lines specific for the influenza A matrix protein-derived peptide (MP.58-66) generated in the presence of chA6 mAb were anergic and suppressed IFN-gamma production by MP.58-66-specific effector CD8(+) T cells. Furthermore, chA6 mAb significantly prolonged human pancreatic islet allograft survival in nonobese diabetic/severe combined immunodeficiency mice injected with human peripheral blood lymphocytes (hu-PBL-NOD/SCID). Together, these results demonstrate that the chA6 mAb is a new immunomodulatory agent with multiple modes of action, including deletion of preexisting memory and recently activated T cells and induction of anergic CD4(+) and CD8(+) regulatory T cells.

Effective resetting of the immune system cannot be achieved by non-specific immunosuppression. Instead, novel strategies aim at harnessing the body's natural tolerance mechanisms to rectify an Ag-specific response without disturbing other immune functions. Fine-tuning of the balance between Ag-specific effector and regulatory T (Tr) cells is a promising strategy that requires detailed understanding of the differentiation and expansion pathways of the relevant Tr cell subsets. Here we review recent developments regarding the control of alloreactivity by induction and expansion of Tr cells. T-cell activation in the presence of tolerogenic APC and cytokines leads to the induction of Tr cells, which can mediate tolerance through cytokine-dependent and/or contact-dependent mechanisms. Better understanding of the mechanisms of immune regulation mediated by Tr cells may enable fine-tuning of specific immune responses and pave the way for novel therapeutic approaches.

When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.

The embryotoxic potential of chlorpyrifos (CPF) and malathion (MTN), two organophosphorus insecticides (OPs), was evaluated by modified Frog Embryo Teratogenesis Assay-Xenopus (FETAX). CPF and MTN were not embryolethal even at the highest concentration tested (6000 microg/l), but both exhibited a powerful teratogenicity. The probit analysis of malformed larva percentages showed a TC(50) of 161.54mug/l for CPF, and a TC(50) of 2394.01 microg/l for MTN. Therefore, CPF teratogenicity was about 15 times higher than MTN. Larvae of both exposed groups were mainly affected by ventral and/or lateral tail flexure coupled with abnormal gut coiling. Histopathological diagnosis displayed abnormal myotomes and myocytes with marked hypertrophies localized at the cell extremity, probably due to a break away of myofibril extremities at the intersomitic junction level. We speculate that this muscular damage was related to inhibition of acetylcholinesterase that showed a clear concentration-response in CPF and MTN exposed larvae. The teratogenic effects of these anti-cholinesterase compounds on Xenopus laevis myogenesis suggest a possible role played by OPs on induction of congenital muscular dystrophy.

The DDT contamination of Lake Maggiore (Northern Italy) has been monitored since a serious pollution event occurred in 1996. To assess the environmental risk associated with this contamination, bioaccumulation data coupled with histopathological markers were evaluated on zebra mussel populations from two different contaminated sites from April 2001 to April 2002. Biomonitoring results showed high DDT pollution in 2001 because of a flood which transported DDTs still contained in the sediments of a polluted river to the lake. DDT concentrations reached values of 4-5 microg/g lipids, higher than those recorded in other industrialized countries but comparable to levels measured in developing ones. In the ovaries of the most highly polluted mussels, histological analyses showed a delay in oocyte maturation and a high incidence of pathological pictures mainly referable to oocyte degeneration and haemocytic infiltration. Moreover, despite the presence of mature sperms, in 2001 first male gamete release occurred about 2 months later than in females. These results indicated a neuroendocrine interference of DDT on Dreissena polymorpha reproduction and also showed that these invertebrates can be successfully used to evaluate ecological implications due to exposure to endocrine disruptors in freshwater environments.

Cloned T regulatory type 1 (Tr1) cells produce IL-10, TGF-beta, IFN-gamma, and very low or non-detectable levels of IL-2 and IL-4, following TCR-mediated activation. In addition, upon TCR stimulation, Tr1 cell clones up-regulate activation markers but show low proliferative responses, partially due to the suppressive effect of autocrine IL-10 and TGF-beta. Here we show that Tr1 cells have growth requirements different from those of Th1 and Th2 cells. Exogenous IL-15, and to a lesser extent IL-2, induce and support the proliferation of Tr1 cells in the absence of TCR activation. This strong cytokine response correlates with high constitutive levels of the IL-2/15Rbeta and common gamma chains expressed by Tr1 cell clones. Furthermore, suboptimal doses of IL-15, in combination with IL-2, induce a significant growth (median value: 25-fold increase in cell number) of Tr1 cell clones during a culture period of 11 days, which leads to an in vitro expansion of Tr1 cell clones comparable to that of Th1 and Th2 cell clones. Tr1 cell clones cultured in IL-15 continue to secrete immunosuppressive cytokines and to proliferate poorly upon reactivation via TCR. These findings indicate that Tr1 cells are constitutively capable of responding to cytokines and mainly to IL-15. This growth factor enables a significant in vitro expansion of Tr1 cells facilitating further biological and biochemical characterization of this unique T cell subset.

The freshwater snail Physa fontinalis was used as a bioindicator to study the effects of the herbicide Paraquat (PQ) in laboratory assays. The test solutions used, 0.125, 0.25, and 0.5 mg/l PQ, were in the range of the concentrations recommended for aquatic weed control. The study was carried out in two stages to determine the influence of PQ on the ovipository activity of Physa fontinalis, and the histological effects on these snails. Specimens exposed to PQ continued to be reproductively active, but the number of egg masses and eggs laid decreased significantly. Mortality was almost the same in all the experimental lots, but was significantly related to the production of egg masses only in the controls. The histological analysis showed a clear trend among PQ concentrations and degenerating oocytes, but no visible effects on the male sex-line were observed. By interfering with fertility, PQ has an action that may go well beyond its lethal effect on individuals, suggesting that this herbicide should be strictly regulated in weed control programmes. Moreover, since PQ was observed to interfere with the reproductive process, its endocrine disrupting action must not be excluded.

The high Paraquat (PQ, 1-1'-dimethyl-4,4'bipyridylium dichloride) embryotoxicity in Xenopus laevis has been shown to be due to its rapid reduction and instantaneous re-oxidation which produces a reactive oxygen species, ROS. Nevertheless, PQ did not show any effects before hatching, stage 32, which showed a resistance, in early X. laevis development, to oxidative damage. Moreover, in view of its genotoxic properties in several experimental models, we studied PQ in the X. laevis cleavage phase that, characterized by a series of rapid mitotic divisions, might be damaged by genotoxic compounds. Embryos were exposed to 20, 40, 60, and 80 mg/l PQ concentrations from stage 2 to stage 9, and then left to develop in control FETAX solution until stage 47. The 80 mg/l PQ concentration gave 19% embryo mortality at the end of the exposure time, and 16.7% larvae mortality at the end of the test; both values were statistically different from the control, 5 and 6.8% respectively. These results confirmed the high resistance in early X. laevis development to PQ oxidative damage. The malformed larva percentages in the PQ exposed groups were higher as regards the control value but did not show any concentration-response; the most frequent malformed larvae found were affected by abnormal tail flexure coupled with abnormal gut coiling. A further experiment was carried out using the same methodology, but exposing embryos only to the 80 mg/l PQ concentration. The surviving blastulae were embedded in Paraplast, then the slides were stained with 4',6-diamidino-2-phenylindole (DAPI) and the nuclei were examined with a confocal microscope. This new preliminary procedure did not reveal any significant presence of micronucleated micromeres in PQ exposed blastulae with respect to the control. Nevertheless, the mechanism by which PQ induced abnormal tail flexure after cleavage exposure remained unknown. PQ seemed to pass through the jelly coats and vitelline membrane, but it expressed teratogenicity between the 2nd and 3rd day. PQ might be accumulated in the embryos during the exposure, and might express teratogenicity later, but it did not seem to induce genotoxicity during the cleavage phase of X. laevis even at very high concentrations.

Zebra mussel (Dreissena polymorpha) is commonly used as contaminant bioindicator in Europe and North America. We used the zebra mussel to follow DDT pollution trends from 1996 to 1997 in Pallanza bay, Lake Maggiore, near the inlet of the River Toce, after a DDT-manufacturing plant discharging residues into a tributary of the River Toce had closed down. DDT contamination fell off sharply outside the bay, but tissue concentrations of the parent compound and residues remained high in molluscs sampled within the bay a year later. Molluscs collected in Pallanza bay in June 1997 released gametes earlier than those sampled at a nearby reference station. Histological studies showed that a significant percentage of these specimens showed marked oocyte degeneration suggesting that DDTs have endocrine-disrupting effects in this species.

Suppression by T regulatory (Tr) cells is essential for induction of tolerance. Many types of Tr cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which these cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell-to-cell interactions and/or the production of interleukin (IL)-10 and transforming growth factor (TGF)-beta. Type-1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-beta. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL-15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL-10 and TGF-beta. Further characterisation of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen.

The toxicity of herbicide Paraquat (PQ, 1-1'-dimethyl-4,4'bipyridylium dichloride) in animal cells is related to its rapid reduction and instantaneous reoxidation to produce the reactive oxygen species. Recently, the PQ evaluation with the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) showed its high embryotoxicity. Supposing that the embryos' death was due to PQ-related oxidative damage, we used ascorbic acid (AA), a well known antioxidant, to reduce the PQ embryotoxicity in Xenopus laevis. Embryos were exposed from stage 8 to 47 to 0.1 mg/l PQ alone, and to PQ with AA concentrations ranging from 20 to 200 mg/l, using the FETAX procedure. PQ caused 72.2% mortality, while 17.1% of surviving larvae were affected by abnormal tail flexure. The PQ mortality percentages were reduced in a clear concentration-response by up to 15.2% in the group exposed to PQ with 200 mg/l AA. The histopathologic diagnoses revealed abnormal notochord flexure coupled with vesiculated, pear-shaped myocytes only in the PQ group. After embryo exposure to PQ with 200 mg/l AA, restoration of normal axial tail structures was evident. In conclusion, PQ embryotoxicity in X. laevis was most likely due to oxidative damage that was drastically reduced by AA.

Paraquat (PQ, 1-1'-dimethyl-4,4'-bipyridylium dichloride) is an effective herbicide widely used in agriculture with a rate of application for aquatic weed control ranging from 0.1 to 2 parts per million. Considering its wide-spread presence in Italian wetlands, we studied its embryotoxic effects with the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The percentage of mortality as well as the percentage of malformed larvae was investigated by probit analysis. The results showed that PQ was highly embryolethal. From a LC(50) of 0.138 mg/l and TC(50) of 0.267 mg/l, a TI(50) of 0.52 was derived; indicating that PQ is to be considered a non-teratogenic compound. Remarkable was the presence of a specific malformation, classified as ventral tail flexure, in the 29% of living larvae exposed to 0.125 mg/l PQ concentration. Their histological examination showed several zones of abnormal somites containing severely affected myocytes. This confirmed the molecular mechanism of PQ toxicity in cell microfilaments. Even at the lowest concentration of 0.0625 mg/l, the difference between the mean head-tail length of control and exposed larvae was statistically significant, a sign of growth retardation. All our data emphasize that PQ must be consider highly embryotoxic on amphibian development, and suggest that this herbicide should be strictly regulated in weed control programs.

We report on a patient with multiple sclerosis (MS) in which we documented an elevated percentage of activated CD56+ natural killer (NK) cells in peripheral blood lymphocytes. NK cells from the patient lysed preferentially glioblastoma but not neuroblastoma cells. Killing of glial cells was not inhibited by a monoclonal antibody against a monomorphic determinant of MHC class I gene products. Lymphokine activated killer (LAK) cell function in the MS patient was comparable to that of controls. Analysis of cytokine production during resting or activated states demonstrated that this patient had a deficit in the ability to secrete T cell derived cytokines associated with increased production of TNFalpha, a product of NK cells. Taken together, these data indicate a possible involvement of NK cells in the pathogenesis of MS.

We have investigated the mechanism of tolerance in a patient with severe combined immunodeficiency (SCID) transplanted with HLA-haploidentical, T cell-depleted bone marrow cells obtained from the mother. At 4 years after transplantation, T cells, natural killer (NK) cells, and a small percentage (2%) of B cells were found to be of donor origin, whereas monocytes and the majority of B cells remained of host origin. In primary mixed lymphocyte cultures (MLC), the engrafted T cells of the donor did not proliferate in response to the host cells, whereas untransplanted donor T cells showed good proliferative responses. However, CD4+ and CD8+ T-cell clones of donor origin with specificity for class II and class I HLA determinants of the host were isolated. CD8+, host-reactive T-cell clones displayed normal cytotoxic activity after stimulation with the host cells, but proliferative responses of CD4+, host-reactive T-cell clones were considerably reduced. In addition, both CD8+ and CD4+, host-reactive T-cell clones produced very low to undetectable levels of interleukin-2 (IL-2), IL-4, IL-5, IL-10, interferon-gamma, and granulocyte-macrophage colony-stimulating factor after specific antigenic activation, which may be responsible for their nonresponsive state in vivo. Expression of the CD3 zeta subunit of the T-cell receptor (TcR) was normal, and after stimulation via CD3, Raf-1 and p42 mitogen activated protein (MAP) kinase were phosphorylated, indicating that this part of the signaling pathway after triggering of the TcR/CD3 complex is present. These results, together with our previous observation that dysfunctional, host-reactive T-cell clones can be isolated in SCID patients transplanted with fetal liver stem cells, demonstrate that lack of clonal deletion of host-reactive T cells is a general phenomenon after HLA-mismatched stem cell transplantation.

Transplantation of HLA mismatched hematopoietic stem cells in patients with severe combined immunodeficiency (SCID) can result in a selective engraftment of T cells of donor origin with complete immunologic reconstitution and in vivo tolerance. The latter may occur in the absence of clonal deletion of donor T lymphocytes able to recognize the host HLA antigens. The activity of these host-reactive T cells is suppressed in vivo, since no graft-vs. -host disease is observed in these human chimeras. Here it is shown that the CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with fetal liver stem cells produce unusually high quantities of interleukin 10 (IL-10) and very low amounts of IL-2 after antigen-specific stimulation in vitro. The specific proliferative responses of the host-reactive T cell clones were considerably enhanced in the presence of neutralizing concentrations of an anti-IL-10 monoclonal antibody, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. These in vitro data correlate with observations made in vivo. Semi-quantitative polymerase chain reaction analysis carried out on freshly isolated peripheral blood mononuclear cells (PBMC) of the patient indicated that the levels of IL-10 messenger RNA (mRNA) expression were strongly enhanced, whereas IL-2 mRNA expression was much lower than that in PBMC of healthy donors. In vivo IL-10 mRNA expression was not only high in the T cells, but also in the non-T cell fraction, indicating that host cells also contributed to the high levels of IL-10 in vivo. Patient-derived monocytes were found to be major IL-10 producers. Although no circulating IL-10 could be detected, freshly isolated monocytes of the patient showed a reduced expression of class II HLA antigens. However, their capacity to stimulate T cells of normal donors in primary mixed lymphocyte cultures was within the normal range. Interestingly, similar high in vivo IL-10 mRNA expressions in the T and non-T cell compartment were also observed in three SCID patients transplanted with fetal liver stem cells and in four SCID patients transplanted with T cell-depleted haploidentical bone marrow stem cells. Taken together, these data indicate that high endogenous IL-10 production is a general phenomenon in SCID patients in whom allogenic stem cell transplantation results in immunologic reconstitution and induction of tolerance. Both donor T cells and host accessory cells contribute to these high levels of IL-10, which would suppress the activity of host-reactive T cell in vivo.

We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion.

Severe combined immunodeficient (SCID) mice were transplanted with different human fetal organs (SCID-hu mice), including thymus, liver, spleen, and omentum, and the serum levels of human IgM, IgG, IgE, and IgA were measured. In all SCID-hu mice significant levels (up to 590 ng/ml) of IgM were detected, irrespective of the organs transplanted. In contrast, IgG was present (up to 530 ng/ml) only when the fetal thymus was transplanted together with the fetal liver, indicating that the presence of human T cell is a prerequisite for in vivo isotypes switching by human B cells in SCID-hu mice. Additional transplantation of fetal spleen did not significantly increase IgG levels. was observed 4 months after transplantation. At that time, analysis by IEF showed that human IgG present in SCID-hu serum was at least oligoclonal. Furthermore, all IgG subclasses were represented in the human IgG pool. Human B cells were undetectable in the peripheral blood, spleen, and bone marrow of these SCID-hu mice; in contrast, B cells expressing CD19 could be isolated from the SCID-hu thymus. Considerable proportions of the CD19+ B cells coexpressed CD5, CD7, CD10, CD40, and CD2. These B cells spontaneously produced IgM and IgG in vitro and could be induced to switch to IgE-producing cells when cocultured with cloned activated CD4+ T cells in the presence of IL-4. Collectively, these data demonstrate that functionally mature B cells able to produce IgM and IgG in vivo, and IgE in vitro, are present in the SCID-hu human thymus.

We have studied the peripheral T cell repertoire of two patients with severe combined immunodeficiency who were successfully treated with human histocompatibility leukocyte antigen (HLA)-mismatched fetal liver stem cell transplantation. The patients presented a split chimerism. T cells were of donor origin, whereas the B cells/monocytes were of the host phenotype. Interestingly, the natural killer (NK) cells in one patient were donor derived and in the other patient of host origin. The NK cells were functional but did not have antihost or donor reactivity. Despite the HLA mismatch between donor and host cells, complete tolerance was achieved in vivo, and a specific unresponsiveness of peripheral blood mononuclear cells from both patients toward the host cells was demonstrated in vitro. Nevertheless, we could isolate T cell receptor (TCR)alpha beta, CD4+ or CD8+, T cell clones specifically reacting with HLA class I and II molecules of the host. The CD4+ host-reactive T cell clones from both patients produced interleukins 2 and 5, interferon-gamma, granulocyte/macrophage colony-stimulating factor but are specifically defective in interleukin 4 production. The frequencies of CD8+ host-reactive T cells were high, and were in the same range as those observed for CD8+ alloreactive T cells. In contrast, no donor-reactive CD8+ T cells or host or donor-reactive TCR gamma delta + T cells were detected. These data indicate that, after fetal stem cell transplantation, donor-reactive, but not host-reactive cells, are deleted from the T cell repertoire. Therefore, a peripheral mechanism of suppression or clonal anergy, rather than clonal deletion, is involved in maintaining in vivo tolerance toward the host.

Over the last 18 years, we have developed the transplantation of fetal liver cells to treat severe immunodeficiencies, hematological disorders and inborn errors of metabolism. Post-natally, this treatment is successful in two-third of patients and it is therefore very valuable, especially when there is no perfectly matched donor for a bone marrow transplant. Since 1988 we have carried out these fetal liver transplants (FLTs) in utero, immediately after prenatal diagnosis. Engraftment and reconstitution have been obtained, and several advantages appear to be associated with in utero FLT: increased probability of graft take, ideal isolation of the patient (in the maternal uterus) and optimal environment for the differentiation of the transplanted fetal liver cells (in the fetal host).

The effects of IL-10 on the generation of alloreactivity in primary mixed lymphocyte cultures (MLCs) were investigated. IL-10 inhibited in a dose-dependent fashion the alloantigen-induced proliferative responses. The suppressive effect was maximal when IL-10 was added at the beginning of the cultures, suggesting that it acts on the early stages of T cell activation. The proliferative responses were enhanced in the presence of a neutralizing anti-IL-10 mAb, indicating that endogenously produced IL-10 suppresses proliferation in primary MLC. The inhibitory effects of IL-10 were observed irrespective of whether irradiated allogeneic peripheral blood mononuclear cells, purified monocytes or freshly isolated B cells were used as stimulator cells. The proliferation of both the CD4+ and CD8+ T cell subsets was inhibited to a similar extent. The reduced proliferative responses were only minimally restored by high concentrations of exogenous IL-2, indicating that the effects of IL-10 are not exclusively due to inhibition of IL-2 synthesis. Furthermore, the production of IL-2, interferon (IFN)-gamma, IL-6, granulocyte macrophage colony stimulating factor, and tumor necrosis factor-alpha in primary MLCs was diminished by IL-10 and enhanced in the presence of anti-IL-10 mAb. The strongest effects were observed on the production of IFN-gamma. Although IL-10 reduces the proliferative responses, the ratios of CD3+CD4+ and CD3+CD8+ T cells remained the same in IL-10 treated and control cultures, yet the percentages of activated CD3+ T cells, as judged by CD25 and HLA-DR expression, were consistently reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the T cell repertoire and the mechanism of tolerance in two patients with severe combined immunodeficiency transplanted with HLA mismatched fetal liver stem cells. They are 17 and 5 years old now, healthy, and show normal immunoresponses to recall antigens. Their T cells are of donor origin, whereas monocytes and B cells remained of the host. The NK cells have different sources since in one patient they derive from the donor and in the other one from the host. Despite the HLA mismatch between donor and host cells, no acute or chronic graft-versus-host disease was observed. In vitro experiments with PBMC showed specific nonresponsiveness for the HLA antigens expressed by the host cells. However, an extensive clonal analysis showed that CD4+ and CD8+ host-reactive T cell clones recognizing class II and class I HLA molecules of the host, respectively, were present in the peripheral blood of both patients. Limiting dilution experiments indicated that the frequency of CD8+ host-reactive cells was in the same range as that observed for alloreactive T cells. In contrast, no donor reactive CD8+ T cells could be isolated. Host-reactive CD4+ and CD8+ T cell clones were normal in their capacity to produce IL-2, IFN-gamma, GM-CSF and IL-5, but they failed completely to synthesize IL-4. In addition, CD4+ T cell clones from patient RV secreted very high levels of IL-10. Interestingly, exogenous IL-10 was able to inhibit the proliferative responses of the CD4+ host-reactive T cell clones. Our data demonstrate that host-reactive cells are not deleted from the donor T cell repertoire following allogenic fetal liver stem cell transplantation. Therefore, in vivo tolerance between the host and the donor is maintained by a peripheral autoregulatory mechanism in which cytokines may play a role.

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.

We studied a severe combined immunodeficiency (SCID) patient who received transplantations with completely HLA-mismatched fetal liver and thymus from two different donors. The patient is now 14 years old, healthy and shows normal immunoresponses to recall antigens. His T cells are of donor origin, whereas the monocytes, B cells, and natural killer (NK) cells are of the recipient. The successful immunological reconstitution raised questions as to how T and B cells could collaborate across an HLA barrier and how tolerance was achieved. We have shown that tetanus toxin-specific T cell clones isolated from this patient recognized this antigen in the context of host and not of donor HLA-DR, indicating that those cells were educated in the host environment, presumably the thymus. Despite this, an unexpectedly high frequency of host-reactive clones was found that could recognize MHC antigens of the host. It was particularly striking that CD8+ CTL clones were obtained that recognized class I MHC antigens on the host cells. Nevertheless, the patient did not show any sign of acute or chronic graft-versus-host disease (GVHD). These data indicated that no or only incomplete clonal deletion had taken place in this patient and suggest the presence of a peripheral suppressor mechanism. Thus far, we have no indication for the existence of suppressor T cells. Inasmuch as it was found that host-reactive T cells fail to produce IL-4, which is exceptional for CD4+ T cells, we are exploring the possibility that abnormal cytokine production patterns of host-reactive T cells are associated with suppression of these cells in vivo.

In the present study, we investigated the lymphokine production patterns in a series of CD4+ and CD8+ host-reactive T cell clones isolated from PBL of a SCID patient, who was immunologically reconstituted by two allogeneic fetal liver and thymus transplantations 13 years ago. We demonstrate that these donor-derived T cell clones, specifically reacting with the MHC Ag expressed on the recipient cells, do not produce IL-4 and do not express IL-4 mRNA upon Ag or polyclonal stimulations. In contrast, CD4+ tetanus toxin-specific T cell clones isolated from the same patient and having the same HLA phenotype produced normal amounts of IL-4 upon activation. These data suggest that the failure to produce IL-4 is a specific characteristic of these host-reactive clones and is not due to a genetic defect of the transplanted cells. Furthermore, different modes of activation resulted in simultaneous production of IL-5, IL-2, IFN-gamma, granulocyte/macrophage-CSF, and transcription of the TNF-beta gene by the host-reactive clones, indicating that the lack of IL-4 production is not related to the mode of activation. The finding that some of these clones produce significant levels of IL-5 but no IL-4 indicates that the IL-4 and IL-5 genes are not always coexpressed in activated human T cells.

In the present study, we investigated the interleukin-2 (IL-2) production and the proliferative responses by peripheral blood mononuclear cells (PBMNC) of 23 children suffering from insulin-dependent diabetes mellitus (IDDM). In addition, the presence of circulating activated T lymphocytes expressing the interleukin-2 receptor (IL-2 R) and HLA-DR antigens was evaluated. The patients were tested at hospital admittance, before starting insulin treatment. Decreased IL-2 production by phytohemagglutinin (PHA)-activated PBMNC of IDDM patients was observed when compared to normal donors. In contrast, the proliferative responses of PBMNC to PHA and Con A were in the normal range. The expression of IL-2 R on patient's lymphocytes was not different from that observed in normal donors, whereas the relative and absolute number of HLA-DR+ T cells was increased. These results confirm the presence in IDDM patients of an imbalanced cellular immune response and demonstrate that the IL-2 deficiency is already present at the diagnosis and is not correlated with insulin administration.

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.