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Utilizing a rat model of semiallogeneic intestinal transplantation, recipients were evaluated for accumulation of donor-specific cytotoxic T cells in spleen, mesenteric lymph nodes, Peyer's patches, lamina propria, and intraepithelial lymphocytes using limiting dilution analysis. Naive animals exhibited a low frequency of cytotoxic T cells in spleen and mesenteric lymph nodes with minimal activity detected in Peyer's patches and intraepithelial lymphocytes, but not detectable activity in lamina propria. Orthotopic intestinal transplantation resulted in significant increases in cytotoxic T-cell activity in recipient Peyer's patches as early as Day 6 and by Day 8 in spleen, mesenteric lymph nodes, lamina propria and intraepithelial lymphocyte populations. Graft lamina propria and intraepithelial lymphocytes exhibited significant cytotoxic T-cell activity as early as 4 days following transplant. The highest donor-specific cytotoxic T-cell activity was observed in graft intraepithelial lymphocytes on Day 8 posttransplant. These studies demonstrate rapid expansion of donor-specific cytotoxic T cells which migrate to the graft site within 4 days after semiallogeneic intestinal transplantation.

Primary murine thymus cell cultures consist of thymus epithelial cells, macrophages and interdigitating dendritic cells. Results of the present study indicated that the proportion of macrophage-like cells in cultures was directly related to the age of the thymus donors through 7 weeks of age. We standardized procedures to compare primary thymic cultures derived from mice, 1 day to 115 days of age. Cultures were grown 6 to 8 days and then tested for nonspecific esterase, acid phosphatase, phagocytosis of latex beads, Fc receptors, and MAC-1 staining. Less than 10% macrophage-like cells were identified in cultures from mice 1 to 3 days-old, but the proportion increased to 50% macrophage-like cells in cultures from 7 week-old mice. Thereafter the proportion plateaued although there was considerable variation among older mice. Further studies showed that the proportions of macrophage-like cells was not affected by the number of cells initially seeded into culture, nor by the amount of serum added to the culture medium. These data suggest that macrophage/interdigitating cell precursors immigrate into the thymus for 4 to 6 weeks after birth and support the hypothesis that these precursors may be influenced to differentiate into thymus macrophages or interdigitating cells by the thymic microenvironment.

Direct regulatory control of the immune system by the central nervous system has been postulated. In support of this view is a large body of literature describing immunoregulatory activities of neuropeptides isolated from the gastrointestinal tract. In this review we examine the evidence for expression of specific receptors for gut peptides on immune effector cells and further explore the regulatory effects of these peptides on immune function. Peptides to be discussed include substance P, somatostatin, vasoactive intestinal peptide (VIP), the opioid peptides leu and met enkephalin, calcitonin gene related peptide (CGRP), neuropeptide Y, and cholecystokinin (CCK).

Natural killer cells (NK) demonstrate cytotoxicity against a wide range of cultured cells without prior sensitization, and the sensitivity of many target cells to NK attack is altered by exposure to interferon. The structures on the target cells conferring sensitivity or resistance to NK are not known, but glycolipids are suggested to be related to NK susceptibility. To determine possible relationships between target cell glycolipids and NK cytolysis, the effects of human beta-interferon (IFN) on the neutral glycolipid composition and sensitivity to NK cytolysis of cultured cells from four human gliomas and two fetal brains were analyzed. Compared to MOLT-4 and Raji cells all six neural cell lines were quite resistant to NK, and IFN slightly increased this resistance. IFN also caused increases in the amounts of non-hydroxy fatty acid cerebroside, ceramide trihexoside, asialo-GM1, asialo-GM2 and globoside. Increased molar proportions of ceramide tri- and tetra-saccharides occurred in the two glioma lines which had the greatest increases in NK resistance following IFN exposure. It is concluded that neutral glycolipids may play a role in the mechanisms responsible for resistance of some glioma cells to NK cytolysis.

Ten human neural tumor lines and three established from normal human brain were analyzed for sensitivities to natural killer (NK) cytolysis. Compared to MOLT-4, fetal brain cells were sensitive, but those from adult brain and eight of ten neural tumor cell lines demonstrated marked NK resistance. The frequencies of target-binding cells (TBC) and single-cell lysis of glioma cells bound within tumor cell conjugates demonstrated that the resistance of two lines was explained either by a decrease in the frequencies of TBC or reduced ability of bound NK cells to lyse the tumor cell conjugates. A third resistant line demonstrated decreases in both TBC and tumor cell conjugate lysis. Two glioma lines with less NK resistance had greater frequencies of TBC or conjugate lysis than the resistant lines. Thus, NK resistance can result from decreased recognition of targets, diminished NK lysis of bound targets, or a combination of both.

We have examined the frequency and alloantigen specificity of CTL that accumulate in sponge allografts (sponges seeded with allogeneic splenocytes) in sponge isografts (sponges seeded with syngeneic splenocytes), and in splenocyte-free sponge implants. Using limiting dilution analysis (LDA), we observed that sponge isografts and splenocyte-free sponge implants from C57BL/6 (H-2b) mice usually acquire small numbers of CTL (less than 250 cells per graft) with DBA/2 (H-2d)-reactivity or C3H/HeJ (H-2k)-reactivity. These alloreactive CTL are not detectable in conventional 51Cr-release assays, presumably because they are too infrequent and/or because they are inactive CTL precursors. When we examined the accumulation of alloreactive CTL in sponge allografts, we observed that DBA/2 sponge allografts from C57BL/6 recipients accumulate 10 to 100 times more DBA/2-reactive CTL than alloantigen-free sponge grafts. Nonetheless, these donor-reactive CTL rarely constitute more than 0.5% of the T cells recovered from sponge allografts, even at the peak of the rejection response. This raises questions concerning the remaining 99.5% of the allograft-infiltrating T cells. We were unable to detect by LDA any host-reactive CTL in sponge allografts, thus excluding the possibility that some of the remaining T cells were host-reactive CTL of donor origin which diluted graft-reactive T cells. However, using LDA we did detect a significant number of third-party (C3H/HeJ)-reactive CTL in sponge allografts, suggesting that the intense immune response at a graft site might facilitate indiscriminate recruitment of T lymphocytes. Alternatively, this enhanced third-party alloreactivity might reflect the proliferation of donor-reactive CTL with incidental crossreactivity for C3H/HeJ alloantigens. While testing these two alternatives, we observed that LDA cultures designed to detect third-party-reactive CTL could also support the growth of the in vivo-activated, donor-reactive CTL from sponge allografts; This compromised enumeration by LDA of the less frequent, third-party-reactive CTL by LDA. Although LDA is the only method that detects the growing population of third-party-reactive CTL in sponge allografts, technical restraints exclude LDA as a method of determining whether donor-reactive CTL and third-party-reactive CTL are separate or overlapping CTL subpopulations. Hence, it remains unclear if third-party-reactive CTL are a significant or insignificant proportion of the CTL that infiltrate sponge allografts.(ABSTRACT TRUNCATED AT 400 WORDS)

The in vivo generation of donor-specific alloantibody was examined using a sponge matrix allograft model. This experimental model has not been previously used to study the humoral immune response during allograft rejection. We chose this model on the basis of its ability to permit recovery of antibody directly from the graft site without the use of elution and dialysis protocols. C57BL/6 (H-2b) mice were implanted with polyurethane sponges injected with DBA/2 (H-2d) splenocytes, C57BL/6 splenocytes, or phosphate-buffered saline (PBS). Thirteen days later, only those sponges injected with allogenic (DBA/2) splenocytes contained high levels of complement-dependent donor-reactive cytolytic antibody. Donor-reactive cytotoxic antibody was detected by its ability to lyse donor-derived target cells but not host-derived target cells. In contrast, cell-free control sponges injected with PBS or syngeneic sponges injected with C57BL/6 splenocytes contained no detectable H-2d-reactive cytolytic activity. Donor specificity of sponge fluid antibody was determined by removal of H-2d cytotoxic activity following absorbtion with DBA/2 thymocytes. Control absorptions with C57BL/6 or third-party C3H/HeJ thymocytes were ineffective in removing this activity. To determine the rate at which antibody would accumulate in sponge implants we examined the sponges for antibody activity at varying times following implantation. Detectable levels of donor-specific antibody were observed as early as 7 days postimplantation. Antibody activity in the sponges subsequently increased to maximal levels by day 10, and was maintained through day 17, which was the last day examined. An accelerated rate of antibody accumulation was observed in alloimmune recipients, which had previously rejected DBA/2 skin grafts. To determine whether antibody activity observed in the sponge fluid reflected similar activity present in the circulation, we compared donor-reactive antibody activity in sera and sponge fluids from the same recipients on various days following sponge implantation. In this experiment donor-specific cytotoxic antibody was first detected in both the sponge fluids and sera on day 10, although the level of activity was higher in the sera than in the sponge fluids. Levels of cytotoxic antibody in sponge fluids remained constant through day 14, and cytotoxic antibody activity in the sera decreased by day 14 to levels observed in the sponge fluids. The cytotoxic antibody activity in sponge allograft fluids paralleled the accumulation of donor-reactive IgM, rather than the accumulation of total donor-reactive antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

We have studied the influence of Cyclosporine on accumulation of donor-reactive CTL, donor-reactive cytotoxic alloantibody and as an immunosuppressive drug in individual murine sponge matrix allografts. During these studies, we observed that: (1) levels of CsA in sponge matrix allografts rapidly reach levels that are immunosuppressive in vitro, i.e. greater than 100 ng/ml, following daily i.m. or subsponge CsA injections of 30 mg/kg; (2) donor-reactive CTL continue to accumulate and develop cytolytic activity in sponge allografts from mice treated i.m., but not subsponge with CsA; (3) delay or interruption of subsponge CsA therapy decreases the ability of CsA to block CTL accumulation in sponge matrix allografts; (4) relatively high local concentrations of CsA appeared to be necessary to influence CTL behavior in sponge matrix allografts; (5) CsA therapy had a different effect on the development of donor-reactive cytotoxic alloantibody. Intramuscular CsA therapy potentiated alloantibody production whereas subsponge CsA therapy temporarily delayed alloantibody production. These studies demonstrate that CsA can differentially impair the accumulation of donor-reactive CTL, but not donor-reactive alloantibody in sponge matrix allografts. These results have several theoretical and clinical implications. They also illustrate the utility of sponge matrix allografts from concomitant in vivo immunologic and pharmacologic studies at the site of a localized immune response.