H Spits

Functional Characterization of Human IL-10

Rapid progress has been made over the past two years in the characterization of the biological activities of interleukin-10. Interleukin-10, produced by T cells, B cells, macrophages/monocytes and keratinocytes, alters profoundly the morphology, the expression of MHC class II antigens and the production of cytokines by monocytes which in turn can affect a variety of immunological responses including antigen specific proliferation and cytokine production of both soluble and allo-antigens by T cells, cytokine production by natural killer cells and immunoglobulin production by B cells. IL-10 also directly affects the function and growth of T cells, B cells and mast cells. These characteristics indicate that IL-10 has strong anti-inflammatory activities and may act as a general suppressor factor of immune reactions with consequences for transplantation, tolerance, cancer therapy and infectious diseases.

Deficiency of a leukocyte surface glycoprotein (LFA-1) in two patients with Mo1 deficiency. Effects of cell activation on Mo1/LFA-1 surface expression in normal and deficient leukocytes.

Mo1, a phagocyte surface glycoprotein heterodimer, is involved in a number of phagocyte adhesion functions such as binding and ingestion of serum-opsonized particles, zymosan-induced degranulation, and superoxide generation. Deficiency of this antigen in humans has been associated with increased susceptibility to recurrent bacterial infections. The beta subunit of Mo1 is shared by another surface glycoprotein named LFA-1, which is involved in lymphocyte proliferation, cytolytic T cell, and natural killing activities. Two unrelated patients with Mo1 deficiency were found to be deficient in LFA-1 as well as in the common beta subunit. Investigation of lymphocyte functions in these two patients revealed normal mixed leukocyte culture-generated cytolytic T cell and natural killing activities and significantly reduced proliferative response to phytohemagglutinin. LFA-1-deficient cells also proliferated in response to soluble antigen and different alloantigens. These responses were partially blocked by anti-LFA-1 antibody. Whereas LFA-1 was undetectable by immunofluorescence and immunoprecipitation on the patients resting T cells, significantly reduced (approximately 5% of normal) but detectable amounts of the heterodimeric LFA-1 antigen were found on mitogen and alloantigen-activated T cells. On granulocytes, Mo1 surface expression was also dependent on the state of cellular activation. The amount of surface Mo1 present on resting normal granulocytes increased by 3-10-fold following exposure to stimuli that induced degranulation, suggesting the presence of a major intracellular pool for this antigen. Analysis of subcellular fractions from granulocytes showed that intracellular Mo1 is located primarily in the specific granule fraction. Activated granulocytes had little or no increase in their surface expression of LFA-1 antigen. Deficient granulocytes had significantly increased numbers of Mo1 antigen expressed on the surface following stimulation with calcium ionophore (1 microM). However, the amount expressed continued to be significantly reduced compared with normal cells. Quantitation of surface Mo1 on granulocytes exposed to calcium ionophore (1 microM) showed that both parents in one family but only the mother in the other family had significantly reduced levels of Mo1, suggesting heterogeneity in the inheritance of this disorder. Whereas LFA-1 deficiency on lymphocytes was associated with normal alloantigen-induced cytolytic T cell and natural killing activities in these two patients, functions which were in part dependent on small amounts of detectable LFA-1 antigen, the Mo1 deficiency state led to significant defects in phagocyte adhesion functions. Hence, the clinical symptoms associated with this combined deficiency state reflect a more profound phagocyte than lymphocyte disorder.

Tracing the Expression of CD7 and other Antigens during T- and Myeloid-cell Differentiation in the Human Fetal Liver and Thymus

During the last decade, the function/s of the cell membrane CD7 antigen have been investigated in human mature T and NK cells, showing the direct involvement of this molecule in multiple effector functions related with activation, proliferation, production of cytokines and modification of adhesion properties. The CD7 glycoprotein is not only expressed by mature lymphoid cells, but also by early hematopoietic progenitors and several types of leukemias, suggesting a role of CD7 during hematopoiesis. However, the function of CD7 in the early stages of hematopoietic development has not yet been elucidated. CD7 has been classically considered the earliest T-cell specific marker. This assumption was based on data indicating the presence of CD45+CD7+CD3-CD4-CD8- cells in the human embryonic/fetal liver at the gestational age at which the thymic rudiment is colonized by T-cell progenitors. In the present article, we review recent results obtained by several groups concerning the expression of CD7 and various other cell surface antigens by T-, B- and myeloid-cell progenitors generated in the adult bone marrow and fetal liver. In addition, we present an hypothetical model of hematopoiesis in the fetal liver and thymus.

Chimerism and tolerance to host and donor in severe combined immunodeficiencies transplanted with fetal liver stem cells.

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.

Comparison of lymphokine secretion and responsiveness of human T cell clones isolated in IL-4 and in IL-2

Interleukin (IL)-4 has been shown to be secreted simultaneously with IL-2 and interferon (IFN)-gamma by the majority of CD4+ human T cell clones isolated and cultured using IL-2 as a growth factor. Moreover, IL-4 was found to be as efficient as IL-2 to promote the outgrowth of human T cell clones. In this study we have investigated the pattern of lymphokine production by human T cell clones isolated and cultured in IL-4. Most of the CD4+ T cell clones isolated in IL-4 were found to have the ability to simultaneously secrete IL-2, IL-4, and IFN-gamma upon activation. The T cell clones isolated in IL-4 produced, in general, more IL-4 and less IL-2 than the clones isolated and cultured in IL-2. This tendency did not appear to be a stable feature inasmuch as when representative CD4+ T cell clones were split and cultured in either IL-2 or IL-4, the clones in IL-2 secreted more IL-2 and less IL-4 than the same cells cultured in IL-4. These results indicate that the isolation and culture of human CD4+ T cells in IL-4 did not lead to an irreversible development of these cells into Th-1- or Th-2-like cells. Clones isolated in IL-4 responded better to IL-4 than they did to IL-2. On the other hand, T cell clones from the same donor isolated in IL-2 showed the reverse pattern since these latter cells were found to respond better to IL-2 than to IL-4. Furthermore, nonresponsiveness of a T cell clones in a [3H]TdR assay to either IL-2 or IL-4 is not a stable feature since clones, unresponsive to a particular lymphokine, could be adapted to become responsive.

Presence of Host-Reactive and MHC-Restricted T Cells in a Transplanted Severe Combined Immunodeficient (SCID) Patient Suggest Positive Selection and Absence of Clonal Deletion

Mature T cells in the periphery are derived from progenitor cells that originate in the fetal liver or bone marrow and migrate to the thymus during ontogeny. These pre-T cells undergo a series of complex maturational events that result in the expression of clonotypic TCRs on these cells. During these maturation processes the T cells learn to distinguish self from non-self. The mechanisms of this selection that occurs in the thymus are incompletely understood. It is believed that T cells carrying aP T-cell receptors with potential to recognize foreign peptide sequences associated vnth self MHC are selected by interactions with self MHC antigens expressed on thymic cortical epithelial cells (Zinkernagel et al. 1978, Bevan & Fink 1978, Kruisbeek et al. 1985, Farr et al. 1986), while those able to recognize self peptide are eliminated when they interact with MHC on bone marrow-derived cells present in the thymic medulla (Jenkinson et al. 1985, Lo et al. 1986, von Boehmer & Schubiger 1984, Marrack et al. 1988). Besides clonal deletion, two other possible mechanisms for T-cell tolerance have been proposed: clonal anergy involving specific inactivation of self-reactive T cells (Nossal & Pike 1983, Rammensee et al. 1990), and suppression of self-reactive T cells by other T cells (Gershon & Kondo 1971). Elegant studies using transgenic mice

Tolerance to Alloantigens and Recognition for “Allo + X” Induced in Humans by Fetal Stem Cell Transplantation

Transplantation of normal hemopoietic stem cells from the fetal liver can cure a number of diseases in experimental animals as well as in humans. Because of immune immaturity of the human fetus during the first trimester of gestation, fetal liver cells of 8–12 weeks post-fertilization are devoid of any T lymphocyte ; therefore they do not induce graft-versus-host disease (GvHD) after transplantation into an allogeneic host, despite full mismatch [1]. Prevention of rejection of these transplanted stem cells can be ensured by immunodeficiency disease of the host, by immunosuppressive treatment or by immune immaturity of the host when the transplant is performed into a fetal patient [2]. The stem cells from the fetal donor progressively differentiate into T lymphocytes within the environment of host antigens, and give rise to mature T cells exerting their functional activities and restoring immune defenses to the patient [3]. In most cases, the fetal donor and the patient host are fully mismatched at the class I and the class II loci of the major histocompatibility complex (MHC). T lymphocytes deriving from the donor stem cells are therefore confronted with HLA-different host cells (monocytes-macrophages, B lymphocytes, NK cells, target cells of various kinds, etc) after they have matured in this HLA-different host environment. This situation provides us with a unique model to study recognition of “self” and “allo” by helper and cytotoxic T cells, as well as the acquisition of tolerance during T cell ontogeny. Positive and negative selection processes are further demonstrated to be separate phenomena, likely to be induced by distinct cell categories.

Structural and functional aspects of the T-cell differentiation antigens T3, T6, and T8

The progressive diversification of T-lymphocytes begins within the thymus gland. Since cell-cell interactions may play a major role in this process, the study of the expression of thymic surface markers would aid our understanding of thymic differentiation. On murine thymocytes, selective expression of genes coding for cell surface markers has been studied with alloantisera [l]. More recently, monoclonal antibodies have made possible the study of selective expression of cell surface glycoproteins on human thymocytes. Thus, as in the mouse, early and late events in the thymic differentiation have been recognized [2]. Although the precise function of these thymic differentiation antigens remains to be determined, it seems plausible that they may govern associative recognition among cooperative cells sets. One could therefore surmise that inappropriate expression of some of the thymic differentiation antigens may playa role in leukemogensis.

Down-Regulatory Role of CD8 Molecules in CD2 MAb and CD3 MAb Induced Nonspecific Cytotoxicity of Cytotoxic T Lymphocyte Clones

The CD8 (T8) antigen is thought to function as an additional stabilizer of the interaction of the cytotoxic T lymphocyte (CTL) with the target cell by binding to monomorphic determinants on the HLA class I molecules. Recent evidence, however, indicates that the CD8 molecules do not merely serve as passive adhesion structures, but also exert regulating effects on T-cell activity (1). Therefore, we decided to reinvestigate the role of the CD8 antigen in CD8+ HLA class I and class II allospecific human CTL clones.