R. Lee Mosley

A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity

Because murine intestine intraepithelial lymphocytes (IEL) are dispersed throughout the intestine epithelium, it is important that IEL extraction procedures result in lymphocyte preparations of sufficient purity for use in in vitro and in vivo experimental systems. Here, we describe an improved technique for isolating murine IEL consisting of a single 30 min extraction followed by multiple nylon wool filtrations and centrifugation through Percoll. This procedure yields a preparation of IEL with high overall recovery and purity yet takes only 2-2.5 h. Evaluation of individual steps in the extraction process indicated that nylon wool filtration, in particular multiple filtrations, and Percoll fractionation both were important for achieving highly-enriched IEL populations by removal of enterocytes and cellular debris, and demonstrated that multiple nylon wool filtration improved the overall IEL recovery. This procedure has several advantages for studies of murine IEL in that the resultant IEL population is ideal for phenotypic, functional, or molecular analyses. Moreover, this technique is effective for isolating IEL on a single-animal basis, thereby permitting analyses of IEL from individual mice rather than as pooled IEL obtained from several animals.

Repopulation kinetics of intestinal intraepithelial lymphocytes in murine bone marrow radiation chimeras

The kinetics of lymphoid cell repopulation of murine intestinal intraepithelial lymphocytes (IEL) from BM stem cells were studied in F1----parent radiation chimeras and were compared with T cell repopulation of the thymus and the spleen. T cells arising from donor bone marrow were present in the gut epithelium as early as day 5 postreconstitution in radiation chimeras. By day 7 postreconstitution, and at times thereafter, the IEL consisted of 70-95% CD3+ donor bone marrow-derived T cells, most of which were CD8+ cells with variable Thy-1 expression. CD4+8- IEL also were detected between days 7 and 14 postreconstitution; however, the CD4+8+ IEL subset did not appear within the gut epithelium until several weeks later and was followed by a progressive increase in the intensity of CD8 expression on CD4+8+ IEL. Functionally mature T cell receptor gamma/delta + and alpha/beta + IEL were present throughout repopulation of the gut epithelium. In contrast to the IEL, T cells were not detected in the thymus or the spleen until days 14 and 21 postreconstitution, respectively, and evidence of T cell function in the spleen was not detected until day 21 postreconstitution. These findings have implications for human bone marrow transplantation in that they demonstrate that T cell repopulation of the gut epithelium begins prior to T cell repopulation of the thymus and the spleen, and indicate that even in the presence of a thymus some IEL proceed through an extrathymic developmental pathway.

Stimulation via the cd43 coreceptor augments T cell proliferation during the early phase of antigen-induced activation

R2/60 is a monoclonal antibody (mAb) that recognizes the murine CD43 molecule expressed on mature T cells, developing thymocytes, and a subset of bone marrow hematopoietic stem cells. Recent studies using R2/60 demonstrate that CD43 is a costimulatory receptor involved in the activation of murine T cells. In the present study we have examined the kinetics of CD43-mediated costimulation in murine T cell populations including purified CD4+ and CD8+ peripheral T cells, and T cells activated in antigen-induced mixed-lymphocyte cultures. In each population, CD43 stimulation significantly enhanced T cell proliferation compared to non-CD43 activated cultures. CD43 costimulation was greatest in conjunction with suboptimal CD3 stimulation, and was most efficient during the early phase of antigen-driven activation, suggesting that under normal biologic conditions the role of CD43 may be to augment T cell responses early in immune activation.

T cell receptor delta gene repertoire and diversity of intestinal intraepithelial lymphocytes in athymic mice

T cell receptor (TCR) delta gene rearrangements in intestinal intraepithelial lymphocytes (IEL) were studied in athymic radiation chimeras using polymerase chain reaction (PCR) and sequence analysis of DNAs spanning the variable (V), diversity (D), and junctional (J) genes. In both thymus-bearing and athymic mice, IEL delta gene rearrangements occurred for V delta 3, V delta 4, V delta 5 and V delta 6. V-D-J junctional-site sequence analyses of cloned DNAs from rearranged IEL delta genes in athymic mice revealed a predominance of in-frame rearrangements; junctional diversity consisting of nucleotide removal from V, D and/or J genes; N segment nucleotide insertions; and high overall gene diversity. Evaluation of PCR-amplified cDNAs made from IEL RNA indicated that all four rearranged V delta genes were expressed in IEL from athymic mice. The high diversity observed at the gene level also was present in amino acid sequences encoded by the V-D-J region of IEL delta genes in athymic mice. These data demonstrate that there is extensive diversity of rearranged delta genes in IEL which develop extrathymically, and suggest that the delta chain of IEL TCR-gamma delta+ T cells has the potential for interactions with polymorphic structures.

Functional heterogeneity of murine intestinal intraepithelial lymphocytes: studies using TCR-αβ+ IEL lines and fresh iel isolates reveal multiple cytotoxic subsets differentiated by CD5, CD8α/α, and CD8α/β expression

Three T-cell lines, isolated from murine small intestine epithelia, have been studied with respect to phenotypic properties and cytotoxic activity. All lines were TCR-alpha beta+, Thy-1+, CD3+, CD4-, CD8+ but differed in that one line was CD8 alpha/alpha+, CD5-; one line was CD8 alpha/alpha+, CD5+; and one line was CD8 alpha/beta+, CD5+. Both the CD8 alpha/alpha+, CD5-, and the CD8 alpha/beta+, CD5+ lines lysed antigen-bearing target cells; however, the latter line also spontaneously lysed natural killer (NK)-sensitive target cells. The CD8 alpha/alpha+, CD5+ IEL line was nonlytic for antigen-bearing target cells, for NK-sensitive target cells, and in assays that detect lytic activity regardless of specificity. Three-color flow cytometric analyses of intestinal intraepithelial lymphocytes (IEL) in freshly-extracted preparations indicated that cells with the phenotypes of the three IEL lines are normally present in the murine intestine epithelium, and revealed considerable variability in the distribution and density of CD5 expression on murine IEL. In freshly extracted IEL depleted of CD5 or CD8 beta by cell sorting, cytotoxicity was found to reside both within the CD5+ and the CD5- IEL subsets, as well as in the CD8 beta-depleted (i.e., CD8 alpha/alpha+) subset. These findings demonstrate: that cytotoxicity of murine IEL resides among multiple phenotypic subsets; that the distribution and density of CD5 on IEL is more complex than previously described; and that T-cell lines of IEL origin are valuable for dissecting functional properties of specific IEL subsets, particularly those that constitute a small proportion of the total IEL.

Immune recognition by cytotoxic T lymphocytes of minor histocompatibility antigens expressed on a murine colon carcinoma line

We have characterized in vivo and in vitro responses of mice to the BALB/c-derived carcinoma, C26. BALB/c mice were highly susceptible, in a dose-dependent fashion, to local tumor development following subcutaneous injection of C26. Other strains of mice, including allogeneic strains and major histocompatibility complex compatible strains of different minor histocompatibility (H) backgrounds, were resistant to C26-induced tumors. The basis for resistance of mice to C26 was studied using an in vitro-derived C26 line as target cells in microcytotoxicity assays, and as a source of antigen for in vivo priming. An H-2d-specific alloreactive cytotoxic T lymphocyte (CTL) line was isolated from C57BL/6 mice primed with C26, demonstrating the expression, and immune recognition, of MHC class I antigens on C26. C26 also expressed minor H antigens of BALB background as demonstrated by the ability of CTL specific for BALB minor H antigens to selectively lyse C26. Conversely, minor H antigens on C26 were immunogenic across a minor H barrier as demonstrated by the ability to raise anti-minor H CTL to C26 from minor H disparate strains. Collectively, those experiments indicate that C26 may be useful for immunologic and biochemical studies of murine minor H antigens, and for in vivo and in vitro studies of local immunity.

Expression of the Asialo GM1 Determinant on Murine Intestinal Epithelia

Abstract Murine intestinal epithelial cells were studied by flow cytometric analyses for the expression of lymphocyte-associated membrane antigens. Three lymphocyte antigens were found to be expressed at high density on most nonhematopoietic intestinal epithelial cells. These included major histocompatibility complex class II antigens, the T cell-associated CT carbohydrate determinant, and the asialo GM1 (aGM1) neutral glycolipid. Examination of aGM1 determinant density on epithelial cells, estimated by fluorescence intensity, indicated that aGM1 was expressed at levels equal to those present on lymphoid cells known to be aGM1 +. The potential role for lymphocyte antigenic determinants on nonhematopoietic cells of murine epithelia with respect to local regulation of intestinal lymphocytes is discussed.