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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.

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.

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.

IL-10 is a cytokine secreted by a wide variety of cells type that has pleiotropic stimulatory and suppressive activities on both lymphoid and myeloid cells in vitro. To analyze the consequences of high IL-10 secretion by APCs in immune responses, we produced transgenic mice expressing human IL-10 directed by the MHC class II Ea promoter. Despite alterations in the development of T and B cells, no gross abnormalities were detected in peripheral lymphocyte populations or serum Ig levels. However, when immunized using conditions that give either a Th2-type or a Th1-type response, IL-10 transgenic mice failed to mount a significant T or B cell immune response to OVA. IL-10 transgenic mice were also highly susceptible to infection with intracellular pathogens like Listeria monocytogenes or Leishmania major, in contrast to IL-10 transgenic mice, where the transgene was express in T cells. Finally, the recently described stimulatory effect of IL-10 on CD8+ T cells was confirmed by the ability of IL-10 transgenic mice to limit the growth of immunogenic tumors by a CTL-mediated mechanism. These results demonstrate, that, depending on the type of immune response, IL-10 can mediate immunosuppressive or immunostimulatory activities in vivo.

The effects of the granulocyte (G) and macrophage (M) colony-stimulating factors (CSFs) on the growth of purified subpopulations of human fetal liver progenitors were investigated. In contradiction to the characterization of these cytokines as CSFs acting late in the course of hematopoiesis, both G-CSF and M-CSF were most potent in promoting the growth of fetal liver colony-forming cells (CFCs) that express high levels of CD34 and CD38 (CD34(++)CD38(+)) and are depleted of cells expressing a panel of lineage markers (Lin(-)). Cultures of these cells in serum-deprived conditions generated a mean of 11.2 and 39.1 low-proliferative potential (LPP)-CFCs per 1.0 x 10(3) CD34(++)CD38(+)Lin(-) cells grown in G-CSF and M-CSF, respectively. Cultures of more mature progenitors, isolated based on a lower level of CD34 expression (CD34(+) Lin(-)), generated few LPP-CFCs and 6.3 and 4.7 clusters per 1.0 x 10(3) CD34(+)Lin(-) cells in response to G-CSFs and M-CSF, respectively. G-CSF was also found to synergistically enhance colony growth by either kit-ligand (KL) or flt-3/flk-2 ligand (FL) in cultures of CD34(++)CD38(+)Lin(-) cells as well as the more primitive compartment of CD34(++)CD38(-)Lin(-) cells. Synergism between G-CSF and KL or FL was also observed in liquid cultures of CD34(++)CD38(-)Lin(-) cells. The effects of G-CSF on CD34(++)CD38(-)Lin(-) cells were further demonstrated by the ability of G-CSF to support the short-term survival of these cells in clonal cultures. In contrast, M-CSF did not affect the growth or survival of CD34(++)CD38(-)Lin(-) cells, a finding that was also supported by the observation that the receptor for M-CSF (CD115 or fms) was only expressed on CD34(++)CD38(+)Lin(-) cells. G-CSF receptor expression and flt-3/flk-2 expression were detected by flow cytometry on both the CD38(-) and CD38(+) subpopulations of CD34(++)Lin(-) cells, but these receptors were not detected on CD34(+) cells. Receptors for KL (CD117) and interleukin-3 (CD123), for which the ligands are active on a broad range of fetal liver progenitors, were detected on cells expressing both high and low levels of CD34. These data help to define the potential roles of cytokines in human fetal hematopoiesis.

Human allograft rejection was studied in SCID mice transplanted with human fetal liver and thymus tissue (SCID-hu mice). These SCID-hu mice have functional, mature T cells with a polyclonal TCR repertoire. Within 12 to 36 wk after construction, SCID-hu mice were transplanted with an HLA-mismatched human fetal pancreas. In contrast to control SCID mice transplanted with pancreas alone, cellular infiltration, induction of HLA-DR on pancreatic epithelial cells, and tissue destruction of the allogenic pancreata were observed in SCID-hu mice. In addition, human insulin was not detected in the serum of SCID-hu mice in which pancreas rejection occurred. The infiltrating cells were mainly human CD3+ T lymphocytes of thymic origin, expressing the CD45RO isoform. T cell lines and CD4+ T cell clones obtained from the rejected tissues proliferated vigorously when stimulated with EBV-transformed B cell lines of pancreas donor origin. Furthermore, the majority of these CD4+ T cell clones displayed strong allospecific cytotoxicity. In addition, CD8+ T cell clones cytotoxic for EBV-transformed B cell lines of pancreas donors were isolated. Blocking experiments with anti-HLA mAbs and panel studies with HLA-matched cell lines showed that these CD4+ and CD8+ T cell clones were specific for the HLA class II and class I molecules, respectively, expressed by the pancreas donor. These data indicate that human T lymphocytes developing in SCID-hu mice are able to mount in vivo responses against allogenic organs, resulting in tissue infiltration and rejection. In addition, they show that both CD4(+)- and CD8(+)-allospecific CTL can be isolated from rejected allogenic pancreata.

In the present study, the biological properties of cord blood cells were investigated. Cord blood mononuclear cells and T cells responded normally to activation by alloantigens in primary mixed leukocyte reactions (MLRs), indicating that cord blood T cells can be normally activated via their TcR and have normal proliferative capacities. In addition, they expressed normal levels of accessory molecules such as CD28 and LFA-1, which contribute to amplify their responses. In contrast, cord blood mononuclear cells, but not cord blood monocytes, had a reduced capacity to stimulate allogeneic cells in primary MLRs. In addition, cord blood monocytes express lower levels of HLA-DR and ICAM-1 compared to adult peripheral blood monocytes. Cord blood mononuclear cells were also impaired in their capacity to generate allogeneic cytotoxic activity in primary mixed leukocyte cultures (MLCs). In contrast, cord blood B cells were similar to adult B cells in their capacity to switch to immunoglobulin E producing cells when incubated with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody. We also demonstrated that IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) production by activated cord blood mononuclear cells was comparable to that observed with peripheral blood mononuclear cells isolated from normal adult donors. In contrast, interferon-gamma (IFN-gamma) was significantly decreased, whereas IL-4 and IL-5 were absent. Granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were in general higher in the supernatants of cord blood cells. Thus, cord blood immune responses differ from those of peripheral blood at several levels. Whether these differences account for a reduced capacity of transplanted cord blood cells to modulate graft vs. host disease remains to be determined.