Histoincompatibility-associated differences in the phenotypes of murine cardiac allograft infiltrating T cells.

Mechanisms of graft rejection may be governed in part by the kind and degree of histocompatibility differences between donor and recipient. Cardiac allograft rejection was studied in three murine models selected to provide disparity at different major histocompatibility complex (MHC), minor lymphocyte stimulating (Mls) and other minor histocompatibility loci. Graft survival for the A.TL to A.TH combination (M3) was significantly longer (median day of rejection 15.0 days) than both the B10.A to AKR (M2) or the C57BL/6 to C3H/HeN (M1) donor-recipient combinations (median days of rejection: 9.0 days and 9.0 days respectively; P < 0.001). The infiltration of grafts by T cells was examined by removal of grafts serially post-transplantation and culturing mechanically disrupted graft tissue with interleukin-2 (IL-2). Recovery of T cells by this method revealed highly reproducible characteristics (kinetic and phenotypic), unique to each donor-recipient combination. Cultures from M1 and M2 grafts had differing CD4/CD8 T-cell ratios at days 2 (1.8 versus 0.7, respectively) and 4 (1.6 versus 0.1, respectively) post-transplantation. The M3 model differed from M2 (at days 4, 8 and 10) and from M1 (at days 8 and 10). At these times, cultures of M3 grafts contained a significantly increased percentage of CD4 cells and significantly decreased percentage of CD8 cells (CD4/CD8 ratios 0.9-1.3) by comparison with M1 (CD4/CD8 ratios 0.02-0.04) and M2 (CD4/CD8 ratios 0.1-0.02). Long-surviving M3 grafts (greater than 30 days post-transplantation) were compared with grafts removed immediately upon cessation of graft function (days 14, 15 and 18 post-transplantation). There was a significant difference between these groups in the ratios of CD4/CD8 T-cell ratios (1.1 versus 0.4, respectively). This study suggests that the cellular rejection mechanism of a graft is a variable process driven by the individual histocompatibility antigen disparity between donor and recipient. These findings may have diagnostic and therapeutic applications in organ transplantation.

Phenotype and serine esterase production of human cardiac allograft-infiltrating lymphocytes.

Human cardiac allograft-infiltrating lymphocytes were studied by in vitro expansion in interleukin-2. Of 28 graft-infiltrating lymphocyte cultures from 17 recipients, 17 were comprised predominantly of CD4+ T cells and 10 predominantly CD8+ T cells; one culture had equal numbers of CD4+ and CD8+ cells. The mean percentages (+/- SE) of T-cell subsets for all cultures were as follows: CD4+, 49% +/- 29%; CD8+, 42% +/- 31%. No correlation was observed between the culture phenotype and histologic findings, length of time from transplantation, or number (or class) of mismatched HLA antigens. N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase (BLT-SE) is an enzyme associated with intracellular cytotoxic T-cell granules and with target-cell destruction. Sixteen cultures were tested for BLT-SE activity and had significantly increased enzyme activity as compared to untreated peripheral blood mononuclear cells from healthy control subjects (p < 0.002), or interleukin-2-treated control cells (p < 0.05). A low percentage (0.4% +/- 0.2%) of cells in the graft-infiltrating lymphocyte cultures expressed the phenotypic marker NKH-1, suggesting that the source of BLT-SE in these cultures was not natural killer or lymphokine-activated T cells. Elevated BLT-SE was observed in five of ten cultures containing predominantly CD4+ cells and five of six cultures containing predominantly CD8+ T cells. Mixed phenotype graft-infiltrating lymphocyte cultures depleted of either CD4+ or CD8+ T cells retained BLT-SE activity. Thus both CD4+ and CD8+ graft-derived T cells can produce this enzyme although much greater variability in enzyme production was seen for CD4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Accelerated rejection of murine cardiac allografts by murine cytomegalovirus-infected recipients. Lack of haplotype specificity.

Clinical studies have revealed a correlation between cytomegalovirus (CMV) infection and acute allograft rejection. Likewise, for a murine model we observed that C3H (H-2k) recipients infected with murine CMV (MCMV) rejected BALB/c (H-2d) cardiac allografts earlier than uninfected recipients (6.9 +/- 0.1 d compared with 8.1 +/- 0.6 d; P < 0.001). It has been hypothesized that MCMV epitopes crossreact with alloantigens and in this manner induce rejection. However, we also demonstrated that MCMV caused accelerated rejection in the reverse combination (C3H heart engrafted to BALB/c recipient; 7.2 +/- 0.3 and 9.4 +/- 0.4 d for infected and control animals, respectively; P < 0.001) and accelerated rejection of grafts of a third, unrelated haplotype (C57Bl/6; H-2b; 8.0 +/- 0.7 d compared with 10.1 +/- 0.6 for infected and control C3H recipients, respectively; P < 0.03). Ultraviolet (UV) inactivation of MCMV before administration to the graft recipient abrogated the ability to induce rapid rejection. Activated T lymphocytes were present in grafts from infected recipients 2 d before control recipients (P < 0.02) and, at the time of graft rejection, were almost exclusively CD8+ for both infected and control recipients. Thus, MCMV accelerated rejection appears not to result from crossreaction between MCMV epitopes and MHC products. The failure of UV-inactivated virus to accelerate rejection and the high proportion of CD8+ T cells recovered from all rejected grafts suggest that the class I pathway of antigen presentation may be important in the induction of early rejection.

Murine CD4-CD8- thymocytes are stimulated by interleukin-2 to proliferate in vitro in chemically defined medium.

The ability of fetal and young adult CD4-CD8- thymocytes to proliferate in chemically defined (serum-free) medium in the presence and absence of IL-2 was examined. Dissociated thymocytes from day 15 and older fetal mice proliferated in vitro in the presence but not the absence of IL-2. The degree of proliferation was increased by including IL-1 with the IL-2. Inclusion of IL-1 in cultures of fetal thymocytes was associated with an increase in the number of IL-2 receptor positive cells, relative to cultures containing IL-2 alone. Although unfractionated thymocytes failed to proliferate in chemically defined medium, CD4-CD8- cells purified from thymic cell suspensions from young adult mice from several inbred strains proliferated to a limited extent in the absence of added cytokines. Proliferation was augmented 40-100 fold by inclusion of IL-2 in cultures. IL-1 stimulated some proliferation by young adult CD4-CD8- cells, but, unlike the effect of IL-1 and IL-2 on fetal thymocytes, combination of IL-1 with IL-2 did not have a notable additive effect on IL-2 induced proliferation. Proliferation stimulated by both IL-1 and IL-2 was completely abrogated by inclusion of anti-IL-2 receptor antibody in the cultures. Thymocytes from F1 progeny of inbred strains of mice proliferated to a greater extent in the absence of IL-2 than did thymocytes from either parent strain, although the response to IL-2 was not significantly different. The data demonstrate that both fetal and adult CD4-CD8- thymocytes area capable of proliferating in response to IL-2 in vitro, suggesting that, as is the case during antigen specific responses by mature T cells, IL-1 and IL-2 cooperate to stimulate T cell proliferation during development in vivo.

Growth of murine thymocytes in vitro in chemically defined medium.

Immature T cells proliferate, diversify their repertoire of antigen specificity, are selected for MHC-restricted function, are selected for non-self reactivity and undergo maturation in the thymus. The mechanisms underlying thymic development are poorly understood. One reason for this is that murine thymocytes generally die when cultured in vitro under conditions which normally support lymphocyte growth. We describe conditions under which CD4-CD8- thymocytes proliferate at a high rate and acquire maturation-associated markers in vitro in the absence of exogenous mitogenic stimuli. CD4+CD8- cells also multiplied in the absence of added lymphokines while CD4-CD8+, but not CD4+CD8+, cells proliferated in the presence of exogenous IL-2. Proliferation of CD4-CD8- cells was associated with production of both IL-1 and IL-2. Proliferation of unfractionated, CD4-CD8- and CD4+CD8- thymocytes was dependent upon interaction of IL-2 with its receptor. CD4-CD8- cells acquired CD4 and/or CD8 markers during culture, indicating that, in addition to the proliferation, some maturation occurred. Proliferation occurred in complexes containing one or more central stromal cells. The results are discussed in relation to their possible relevance to thymocyte development.

Flow cytometric analysis of lymphocyte subpopulations in infants with congenital heart disease.

Premortem diagnosis of the DiGeorge syndrome and its partial variants relies on the demonstration of a primary defect in cell-mediated immunity, generally in the setting of an infant with congenital heart disease, hypocalcemia, absence of a thymic shadow, and typical dysmorphic features. Although T-cell enumeration is considered a vital part of the diagnostic evaluation, no studies to date have addressed the issue of appropriate reference data in infants with congenital heart disease. We therefore undertook a prospective descriptive study of lymphocyte phenotype analysis in 27 nontransfused infants undergoing diagnostic cardiac catheterization. Striking differences were seen between patients and adult controls in means percentages and numbers of most lymphocyte subsets analyzed. Few differences were found in comparing the patient data to values for age-matched control infants without heart disease. The data are discussed with reference to published values for patients with partial DiGeorge syndrome. It is concluded that lymphocyte phenotype analysis in the diagnostic evaluation of patients with suspected DiGeorge syndrome must utilize appropriate reference values.

Lymphocyte phenotyping of infants with congenital heart disease: comparison of cell preparation techniques.

The diagnostic evaluation of infants with suspected DiGeorge syndrome includes peripheral blood lymphocyte phenotype analysis by flow cytometry. Mononuclear cells are typically concentrated from infants' blood samples by Ficoll-Hypaque (FH) density gradient centrifugation prior to monoclonal antibody (Mab) staining. Having observed that lymphocyte percentages in samples prepared by this technique were low more often than anticipated based on the prevalence of the suspected diagnosis, we undertook a prospective study of 55 infants with congenital heart disease comparing FH with a whole-blood (WB) technique. Sixty healthy adult controls were similarly studied. We report the observation that FH-prepared mononuclear cells from blood samples of infants less than 4 months of age yield substantially different phenotypes than WB-prepared samples. The differences are related to red blood cell (RBC) contamination. No such difference was seen with adult samples. Unlike FH-prepared adult samples, contaminating RBCs are indistinguishable from lymphocytes through at least 4 months of age when assessed by the standard cytometric gating parameters of forward and 90 degrees light scatter. The use of a WB ammonium chloride lysis technique is recommended as most appropriate for the preparation of infants' blood samples.

DNA content of human squamous cell carcinoma cell lines. Analysis by flow cytometry and chromosome enumeration.

Fifteen squamous cell carcinoma cell lines derived from nine patients were examined for DNA content by flow cytometry and chromosome counts. Using human peripheral blood leukocytes and nucleated trout and chicken red blood cells as standards, the DNA indexes of the squamous cell carcinoma cell lines were found to range from 1.1 to 3.3. The DNA content was a stable characteristic of individual cell lines in multiple passages over a seven-month period. Although flow cytometry could detect abnormal DNA content even in diploid tumor lines, the chromosome number correlated well with the DNA content by flow cytometry. In cases in which more than one cell line was established from the same patient, the individual cell lines were found to differ in their DNA content. The cell lines established from metastatic or recurrent tumors usually had a lower DNA content and chromosome number and exhibited a more aggressive in vitro growth pattern than the primary tumor or earlier recurrence. We hypothesize that "streamlined" and aggressive cell populations may evolve in vivo from more slowly growing hyperploid precursor tumor cell populations when in the course of random loss of DNA or chromosomes those that confer no growth advantage are lost, while those that do confer growth advantage are retained.

A new Salmonella serotype isolated from Saudi Arabia.

The isolation and identification of a new sub-genus I Salmonella is described. The complete antigenic formula is 3,10:z29:enx; the serotype has been named Salmonella everleigh. The strain is atypical in that it ferments salicin, produces indole, and fails to produce acid in tartrate media.

In vitro characterization of human intestinal intraepithelial lymphocytes.

Enriched populations of human intestinal and colonic intraepithelial lymphocytes were isolated separate from lamina propria lymphocytes. These populations, and peripheral blood mononuclear cells, were characterized for membrane features, lymphocyte subsets, and proliferative potential to the nonspecific mitogenic lectins phytohemagglutinin, concanavalin A, and pokeweed mitogen. All three populations were predominantly T lymphocytes as measured by sheep red blood cell rosettes and monoclonal antibodies. Peripheral blood mononuclear cells had a T4:T8 ratio of 2.15 while intraepithelial lymphocytes were enriched for T8+ cells with a T4:T8 ratio of 0.06-0.14. Peripheral blood mononuclear cells and lamina propria lymphocytes registered vigorous proliferative responses to phytohemagglutinin, concanavalin A, and pokeweed mitogen. Intraepithelial lymphocytes, in contrast, failed to respond to the lectins. Control experiments with peripheral blood mononuclear cells indicated that the isolation reagents and procedures had no adverse effect on lectin-stimulated proliferation or on T-cell subset proportions. Viability of the intraepithelial lymphocytes after separation (88%) and after 3 days in culture with lectin (77%-85%) was confirmed by trypan blue dye exclusion, by light microscopy, by electron microscopy, and by ability to affect pokeweed mitogen-induced immunoglobulin synthesis when added to autologous and heterologous peripheral blood mononuclear cells. The failure of intraepithelial lymphocytes to respond to lectins was not due to failure of lectin binding, macrophage depletion, or absence of T-cell growth factor in culture. The possibility that the T-cell subset proportions (T4:T8 ratio) affect the proliferative response of isolated intraepithelial lymphocytes is discussed.

Evaluation of suppressor immune regulatory function in idiopathic congestive cardiomyopathy and rheumatic heart disease.

Several diseases with autoimmune features have recently been shown to be characterised by defects in suppressor cell immune regulation. Aberrant immune mechanisms of primary importance have been sought but not yet demonstrated for idiopathic congestive cardiomyopathy and rheumatic heart disease. We tested whether defective immunoregulatory function might explain certain features of these diseases. Peripheral blood mononuclear cells from patients with both diseases showed normal proliferative responses in the mixed leucocyte reaction. Concanavalin A induced similar suppressor activity, quantified in mixed leucocyte reaction as a suppression index, among control subjects, patients with rheumatic heart disease, and patients with idiopathic congestive cardiomyopathy. Similarly, patient serum supported induction of suppressor activity in normal leucocytes equal to that of control serum. A chronic immunoregulatory defect thus does not appear necessary for the development of idiopathic congestive cardiomyopathy or rheumatic heart disease.