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S T Ildstad

Publications and source records attributed to S T Ildstad.

At least 73 records · Page 4Linked to original sources
PubMed1993

Coexpression of NKR-P1 and alpha beta-TCR on lymphoid cells in fully xenogeneic (rat-->mouse) chimeras and syngeneically reconstituted (A-->A) rats.

We have identified three populations of cells among rat splenocytes expressing NKR-P1, including cells of NKR-P1bright/alpha beta-TCR-, NKR-P1dim/alpha beta-TCR+, and NKR-P1dim/alpha beta-TCR- phenotypes. To study the phenotypic characteristics and development of these various cell populations, we have made use of transplanting untreated rat bone marrow into recipient rats (syngeneic) or mice (fully xenogeneic) conditioned with total body irradiation. Rat NK cells exhibit normal phenotypic markers (NKR-P1+, CD8+) and are normally functional by 28 days after reconstitution. We have found that the various populations of NKR-P1+ cells are enriched significantly in the spleen and follow a characteristic pattern of development in the first mo after reconstitution. After syngeneic bone marrow reconstitution (rat-->rat), NKR-P1dim and NKR-P1bright cells (3-15%) can be demonstrated among splenocytes as early as day 3 after bone marrow transplantation. By day 7, the NKR-P1+ cells reach peak levels and comprise as much as 80% of splenic lymphoid cells, with 35% being NKR-P1bright and 45% being NKR-P1dim. The percentage of NKR-P1+ cells decreases over the next several wk until they constitute "normal rat" levels with 8 to 20% being NKR-P1bright and only 1 to 5% being NKR-P1dim. These same populations are also present in fully xenogeneic chimeras. In both models, approximately 80 to 90% of the NKR-P1dim cells were found to coexpress alpha beta-TCR at all time points. These NKR-P1dim/alpha beta-TCR+ cells are not large granular lymphocytes and lack NK cell lytic activity against YAC-1 target cells. Additional analyses of cells derived from spleen, bone marrow, and thymus indicated that NKR-P1+ cells develop, for the most part, in a thymic-independent manner in our fully xenogeneic chimeras and syngeneically reconstituted rats. At present the developmental pathway of these NKR-P1dim cells remains speculative.

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PubMed1993

Characterization of the immunosuppressive effects of nitric oxide in graft vs host disease.

The generation of nitric oxide (.N = O) during in vitro assays involving lymphocyte-macrophage interaction can result in profound inhibition of lymphocyte proliferation. The present study examined whether .N = O synthesis plays a role in the suppression observed in immune function assays during graft vs host disease (GvHD). By using a parent to F1 model to induce GvHD (C57BL/6J to C57BL/6J x DBA 2J F1), a mild but transient increase in serum NO2- plus NO3- levels was observed on day 12 after inoculation. Resident peritoneal macrophages obtained from mice with GvHD demonstrated enhanced .N = O synthesis in response to LPS, compared with control F1 peritoneal macrophages. Similarly, when splenocytes from GvHD mice were cultured with Con A or LPS enhanced supernatant NO2- levels were observed, compared with control F1 mice. Addition of NG-monomethyl-L-arginine (NMA), a competitive inhibitor of .N = O synthesis, resulted in decreased NO2- levels and greatly enhanced proliferation in response to Con A. Addition of NMA to LPS-stimulated cultures did not enhance proliferation, perhaps as the result of the paucity of B cells in the GvHD population. LPS-induced .N = O synthesis by GvHD splenocytes was blocked by anti-IFN-gamma mAb, whereas Con A-induced .N = O synthesis was relatively unaffected by similar concentrations of anti-IFN-gamma mAb, suggesting different mechanisms of induction of .N = O synthesis. A proliferative response of splenocytes from mice with GvHD to third-party alloantigen was not detectable, even in the presence of NMA. The suppression observed when splenocytes from GvHD animals were added to control TNP-modified self cultures was partially reversed in the presence of NMA. These results demonstrate that .N = O synthesis in both splenocyte and peritoneal macrophage populations from GvHD mice is enhanced, revealing that in vivo priming of macrophages for .N = O synthesis occurs during GvHD. Some, but not all, in vitro tests of immune function by using GvHD splenocytes are suppressed by the generation of .N = O.

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PubMed1993

Intracellular labeling of T-cells with superparamagnetic contrast agents.

Isolated rat T-cells have been labeled intracellularly, using endocytosis uptake of two superparamagnetic contrast agents, AquaMag100 and BMS180549, which are both iron-oxide particles coated with dextran. No deterioration of cell proliferation response to mitogen stimulation was observed after labeling with either superparamagnetic contrast agent. AquaMag100 particles show aggregation and co-precipitation in culture media for T-cells. BMS180549 particles not only produce no observable aggregation or co-precipitation, but also have a higher efficiency for labeling T-cells than AquaMag100. The efficiency of cell labeling was determined by measuring the decrease in the spin-spin relaxation time of the water proton in cell samples containing 1 x 10(7) labeled T-cells/milliliter of 2% w/w gelatin. After optimization of the labeling procedures, a shortening of the spin-spin relaxation time by a factor of approximately 7 to 10 has been demonstrated. Under the present experimental conditions, the up-regulation of low density lipoprotein receptor does not increase the labeling efficiency by endocytosis. Our results suggest that intracellular labeling of specific cell types can be achieved with good efficiency and the labeled cells can be detected by magnetic resonance imaging in rat testicles in vivo.

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PubMed1993

Characterization of maturation and function of natural killer cells in xenogeneic (rat-->mouse) bone marrow chimeras. Evidence that rat NK cells are present and functional in a xenogeneic environment.

Reconstitution of B10 recipient mice, conditioned with total body irradiation (950 rads), with 40 x 10(6) untreated F344 or WF rat bone marrow cells results in stable rat stem-cell engraftment with multilineage lymphohematopoietic chimerism. We have now characterized NK cell generation, maturation, and function in fully xenogeneic chimeras (WF rat-->B10 mouse; F344-->B10 mouse). Early during xenogeneic reconstitution, rat-derived NK cells predominated in splenic lymphoid tissue, composing 14-18% of total cells at week 1 and increasing to 35.6-59.9% of total cells at week 2. By week 6, levels of rat NK cells had decreased and stabilized to that expected for normal rat (9-14.2%). The NK chimerism was reliably stable for up to 7 months following reconstitution. Most importantly, rat-derived NK cells were functional in both YAC tumor cytolysis and ADCC assays, suggesting that the xenogeneic mouse host environment was sufficient to support the generation, maturation, and function of rat-derived NK cells.

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PubMed1993

CD4+ T cells, but not CD8+ T cells, mediate the breaking of tolerance in mixed allogeneic chimeras (B10 + B10.BR-->B10).

Reconstitution of mouse recipients with a mixture of syngeneic plus allogeneic bone marrow (A+B-->A) results in stable mixed lymphohematopoietic chimerism and donor-specific transplantation tolerance. Previously, it was reported that administration of large numbers of unmanipulated host-type splenocytes to neonatal or adult radiation bone marrow chimeras resulted in a loss of chimerism and donor-specific transplantation tolerance. To characterize the phenotype(s) of cells that were responsible for this loss of chimerism, we performed depletion of various subsets of unmanipulated B10 splenocytes prior to infusion into mixed allogeneic chimeras (B10 + B10.BR-->B10). Recipients were followed serially to identify changes in the level of donor chimerism and by in vitro functional assays of tolerance. We report here that CD4+ T cells, but not CD8+ T cells, were sufficient to mediate the loss of donor chimerism. In all recipients in which allogeneic chimerism became undetectable, there was a simultaneous loss of donor-specific transplantation tolerance.

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PubMed1993

Evidence for early Th 2 T cell predominance in xenoreactivity.

Two distinct subsets of CD4+ Th lymphocytes have been characterized by their cytokine profiles: Th 1 (TH1) and Th 2 (TH2). While TH1 cells predominate in cell-mediated responses, TH2 cells support the humoral response. We have examined the mRNA cytokine profile of normal mouse lymphocytes in response to alloantigen versus xenoantigen (rat) in MLC, and present evidence to suggest that early in proliferative responses, alloreactivity is dominated primarily by TH1-type lymphocytes, while xenoreactivity is predominantly TH2. Normal mouse lymphocyte-responding cells were cultured in a one-way MLR with either allo or xeno antigen and examined for production of mRNA for cytokines characteristically produced by TH1 (IL-2, IFN-gamma) or TH2 (IL-4, IL-10) cells. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mouse IL-2, IL-4, IL-10, and IFN-gamma mRNA. In the mouse anti-rat xeno response, mRNA for TH2 gene products were upregulated, with greater levels of IL-4 and IL-10 at 24 and 48 hr when compared with controls. In contrast, upregulation of mRNA for TH1 gene products occurred in the mouse anti-mouse allo response, with higher levels of IL-2 and IFN-gamma at 24 and 48 hr. In the anti-xeno response, upregulation of all 4 cytokines occurred by day 4 and peak levels of mRNA for all cytokines examined were 2-3 times that seen for the peak anti-allogeneic response. These data suggest that early xenorecognition may differ from allorecognition by differential activation of the TH2 subset. A better understanding of the balance between Th subset function and cytokine profile in allo and xeno reactivity may allow a more targeted and specific approach to control the early events in xenograft rejection.

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PubMed1993

Cross-species graft-versus-host-disease is accompanied by a donor-derived cellular immune response.

Acute graft-versus-host-disease is classically described as reactivity of donor lymphocytes to recipient alloantigens. To date there is debate as to whether GVHD can be induced across a species barrier. We recently reported the induction of stable xenogeneic chimerism (rat-->mouse) and donor-specific transplantation tolerance using the transplantation of untreated rat bone marrow cells into B10 mouse recipients. Survival of chimeras was excellent, and there was no evidence of GVHD. We now describe the induction of xenogeneic GVHD by transplanting large numbers of donor rat spleen cells with the bone marrow inoculum. All chimeras that received bone marrow and untreated spleen cells developed an external appearance compatible with GVHD and had a median survival time of 14 days. Mice that received equivalent numbers of untreated rat bone marrow alone appeared healthy, had no evidence for GVHD, and survived > 90 days. The usual epithelial target tissues for allogeneic GVHD in those mice that received xenogeneic bone marrow and spleen cells showed the presence of tissue injury and histologic features compatible with GVHD. Donor rat MHC class I and class II positive cells were prominent cell types present in the tongues of mice that developed features of GVHD, and this was accompanied by a significant inflammatory tissue response with loss of the dermal-epidermal architecture. In contrast, fully xenogeneic chimeras without GVHD had no evidence for tissue injury or pathologic cellular infiltrates when examined by immunohistochemical analysis. These data suggest that although fully xenogeneic chimeras resist GVHD, GVHD can be induced across a species barrier if sufficient numbers of donor rat spleen cells are added to the bone marrow inoculum. Further comparisons of these models may provide an approach to study the mechanisms responsible for xenoreactivity in vivo.

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PubMed1992

Cross-species transplantation tolerance: rat bone marrow-derived cells can contribute to the ligand for negative selection of mouse T cell receptor V beta in chimeras tolerant to xenogeneic antigens (mouse + rat----mouse).

Mixed xenogeneic bone marrow reconstitution (mouse + rat----mouse) results in stable mixed lymphopoietic chimerism (1-48% rat), long-term survival, and the induction of stable functional donor-specific transplantation tolerance to xenoantigens in vivo. To examine the role of negative selection of potentially xenoreactive T lymphocytes during tolerance induction across a species barrier, mixed xenogeneic chimeras (mouse + rat----mouse) were prepared and analyzed using a mixture of mouse and rat bone marrow cells for relative T cell receptor (TCR)-V beta expression on mouse T cells. In mixed xenogeneic chimeras (B10 mouse + rat----B10 mouse), T cell maturation proceeded normally in the presence of rat bone marrow-derived elements, and functional donor-specific tolerance to rat xenoantigens was present when assessed by mixed lymphocyte reactivity in vitro. V beta 5, which is expressed at high (undeleted) levels in normal B10 mice, was consistently deleted in B10 recipients of Wistar Furth (WF), but not F344 rat bone marrow, whereas the coadministration of either F344 rat or WF rat bone marrow with B10 mouse bone marrow cells resulted in a significant decrease in expression of TCR-V beta 11. Taken together, these data demonstrate for the first time that rat bone marrow-derived cells can contribute in a strain-specific manner to the ligand for negative selection of specific mouse TCR-V beta during tolerance induction across a species barrier.

Animals↗
PubMed1992

Cross-species transplantation: NK cell number and function are normal in fully xenogeneic chimeras (rat----mouse).

When untreated F344 rat bone marrow is transplanted into B10 mouse recipients conditioned with total body irradiation, stable fully xenogeneic chimerism (rat----mouse) results. Chimeras are specifically tolerant to the donor strain of rat, survival is excellent (greater than 80% at 8 months), and all stem-cell-derived lineages are produced by the rat stem cell. We have previously demonstrated normal function of T-lymphocytes in these chimeras, but have not examined the immune function of natural killer (NK) cells present. Because NK cells play a critical role in immune surveillance, absence of function could result in a serious immunodeficiency state. We present data here to suggest that rat NK cells that have developed in a mouse stromal environment are normal in function as well as number. In all fully xenogeneic chimeras tested from 8 weeks to 8 months following bone marrow transplantation, NK cells were present at a normal level (10% to 16%). NK cells function in these chimeras, as tested by spontaneous lysis of YAC tumor cell targets, was normal or superior to normal F344 rat and B10 mouse NK cells.

Animals↗
PubMed1992

Long-term survival of donor-specific pancreatic islet xenografts in fully xenogeneic chimeras (WF rat----B10 mouse).

We recently reported that reconstitution of lethally irradiated B10 mouse recipients with 40 x 10(6) untreated WF rat bone marrow cells resulted in stable fully xenogeneic chimerism (WF rat----B10 mouse). In these animals, the tolerance induced for skin xenografts was highly MHC specific in that donor-specific WF rat skin grafts were significantly prolonged while MHC-disparate third-party xenografts were rapidly rejected (median survival time [MST] = 9 days). We have now examined whether islet cell xenografts placed under the renal capsule of chimeras rendered diabetic with streptozotocin would be accepted and remain functional to maintain euglycemia. Animals were prepared, typed for chimerism at 6 weeks, and diabetes induced with streptozotocin. Donor-specific WF (Rt1Au) islet cell xenografts were significantly prolonged (MST greater than 180 days) in WF----B10 chimeras, while MHC-disparate third-party F344 rat (Rt1A1) grafts were rejected with a time course similar to unmanipulated B10 mice (MST = 8 days). The transplanted donor-specific islet cells were functional to maintain euglycemia, since removal of the grafts at from 100 to 180 days in selected individual chimeras uniformly resulted in return of the diabetic state. These data suggest that donor-specific islet cell xenografts are accepted and remain functional in mice rendered tolerant to rat xenoantigens following bone marrow transplantation.

Animals↗
PubMed1992

Evidence that nitric oxide production by in vivo allosensitized cells inhibits the development of allospecific CTL.

The oxidative metabolism of L-arginine to its bioactive product, nitric oxide (.N = O) has been shown to inhibit rat splenocyte mixed lymphocyte reactions. To determine if alloantigen-induced .N = O production might be operative in vivo, cells that had infiltrated a rat sponge matrix allograft were tested for de novo .N = O production as well as .N = O production upon restimulation with the sensitizing alloantigen. When graft-infiltrating cells were placed in culture, a peak in de novo .N = O production was observed by day 6 graft-infiltrating cells, the time when donor-specific CTL activity by the graft-infiltrating cells was first observed. Upon restimulation with alloantigen, allograft-infiltrating cells produced greatly increased levels of .N = O, and this production was associated with inhibition of lymphocyte cytolytic function. The addition of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of oxidative L-arginine metabolism, inhibited .N = O production and promoted allospecific CTL development. Both observed effects of NMA were reversed by addition of excess L-arginine. Cytokine(s) able to induce proliferation of the IL-2-dependent T cell line CTLL-2 could be detected in alloantigen-stimulated cultures in both the presence and absence of NMA. However, proliferation of the graft-infiltrating cells in response to these cytokines was observed only in the presence of NMA. The immunosuppressive macrolide FK506 was a potent inhibitor of .N = O production in these cultures, presumably acting by inhibiting the production of those cytokines that induce the oxidative L-arginine pathway.

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