In the present study we used an adoptive transfer model with athymic nude mice to characterize the T cells involved in initiating and mediating skin allograft rejection. It was found that skin allograft rejection in nude mice required the transfer of immunocompetent T cells and that such reconstitution did not itself stimulate the appearance of T cells derived from the nude host. Reconstitution with isolated populations of Lyt-2+/L3T4- T cells resulted in the rapid rejection of MHC class I-disparate skin allografts, whereas reconstitution with isolated populations of L3T4+/Lyt-2- T cells resulted in the rapid rejection of MHC class II-disparate and minor H-disparate skin allografts. By correlating these rejection responses with the functional capabilities of antigen-specific T cells contained within the reconstituting Lyt-2+ and L3T4+ T cell populations, it was noted that skin allografts were only rejected by mice that, as shown by in vitro assessment, contained both lymphokine-secreting Th cells and lymphokine-responsive Tk cells specific for the alloantigens of the graft. The ability of two such functionally distinct T cell subsets to interact in vivo to reject skin allografts was directly demonstrated in H-Y-specific rejection responses by taking advantage of the fact that H-Y-specific Th cells are L3T4+ while H-Y specific Tk cells are Lyt-2+. Finally, the importance of in vivo interactions between functionally distinct Th/T-inducer cells and T killer (Tk)/T-effector cells in skin allograft rejection was demonstrated by the observation that normal B6 mice retain Qala and Kbm6 skin allografts because of a selective deficiency in antigen-specific Th cells, even though they contain T-effector cells that, when activated, are able to reject such allografts. Thus, the ability to reject skin allografts is neither unique to a specialized subset of T cells with a given Lyt phenotype, nor unique to a specialized subset of helper-independent effector T cells with so-called dual function capability. Rather, skin allograft rejection can be mediated by in vivo collaborations between T-inducer cells and T-effector cells, and the two interacting T cell subsets can express different Lyt phenotypes as well as different antigen specificities.

https://rupress.org/jem/article-pdf/165/5/1296/1096976/1296.pdf

Phenotype, specificity, and function of T cell subsets and T cell interactions involved in skin allograft rejection.

The capacity for fluorescence excitation by beams of different wavelengths at separate points along the sample stream, and the capacity for computer analysis of multiparameter data thus obtained, are now available in flow cytometer/sorter systems from commercial producers. It is now readily apparent to most experienced users of flow cytometers that such multiparameter analysis offers the most convenient solution to the problem of characterizing subpopulations of cells within a mixed population. The use of multiple beams facilitates resolution of fluorescence signals from several probes within or upon a single cell and widens the range of analytical alternatives available to experimenters. This critical review discusses the history of the instrumentation, the parameters now measurable and the probes used for their measurement, and the methods for data analysis. Required sensitivity and precision are discussed, leading to the conclusion that many of the advantages of multistation, multiparameter flow cytometry can be made available in less complex and less costly instruments using less powerful sources and less elaborate computer hardware than are presently incorporated in commercial apparatus.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.990030402

Multistation multiparameter flow cytometry: A critical review and rationale

The capacity for fluorescence excitation by beams of different wavelengths at sepa- rate points along the sample stream, and the capacity for computer analysis of multipar- ameter data thus obtained, are now avail- able in flow cytometer/sorter systems from commercial producers. It is now readily ap- parent to most experienced users of flow cytometers that such multiparameter anal- ysis offers the most convenient solution to the problem of characterizing subpopula- tions of cells within a mixed population. The use of multiple beams facilitates resolution of fluorescence signals from several probes within

REVIEW ARTICLE Multistation Multiparameter Flow Cytometry: A Critical Review and Rationale'

DATA on the T-cell antigens, Lyt 1, 2, 3 (refs 1, 2), relating to functional T-cell subsets, suggest that some peripheral T cells express restricted Lyt phenotypes, that is, Lyt 1+23− or Lyt 1−23+ (ref. 3). Evidence has been presented showing that two major subpopulations of lymphocytes, Lyt 1+ T cells and Lyt 23+ T cells, are committed to particular lines of functional differentiation in the immune response before encountering exogenous antigen in peripheral lymphoid tissue4,5. It has been suggested that the two major subpopulations representing T-cell help (Lyt 1+) and T-cell cytotoxicity or suppression (Lyt 23+) arise in the periphery from peripheral Lyt 123+ cells6. Recent studies have concluded that as stem cells mature in the thymus, the thymic epithelium is important in determining which H–2 specificities the maturing T cells are able to recognise as self7. Previous cytotoxicity data have been interpreted as indicating that Lyt 1, 2, 3 antigens are expressed on each of most if not all, mouse thymocytes; that is, that most thymocytes are Lyt 123+ (refs 1–4, 8–10). However, the data presented here suggest that the commitment to differentiation of the Lyt 1+ or the Lyt 123+ phenotype, and thus presumably the relevant functional differentiation occurs before cell migration from the thymus. Using flow microfluorometry (FMF) and anti-Lyt antisera, we demonstrate a normal thymocyte subpopulation which is not expressing Lyt 2 or Lyt 3 antigens (Lyt 1+23−). This subpopulation represents a significant constant proportion (10%) of normal thymocytes which is markedly increased following in vivo cortisone treatment. The data also show that Lyt 1 expression, rather than being decreased on cortisone-resistant thymocytes (CRT), as would have been predicted from previous cytotoxicity data2,8, is expressed in increased amounts on CRT.

An Lyt differentiated thymocyte subpopulation detected by flow microfluorometry.

A convenient, preparative scale, nonlytic separation of mouse T lymphocytes into Lyt2.2+ and Lyt2.2- populations is reported. Immunoglobulin-negative (Ig-) spleen cells, Ig- lymph node cells, and peanut lectin-unagglutinated (PNA-) thymocytes were incubated under sterile conditions at 0 degree C with monoclonal mouse antibody to the Lyt2.2 T cell differentiation antigen. The antibody-treated cells were washed and placed in polystyrene tissue culture dishes that had been precoated with antibody to mouse Ig. Nonadherent populations were depleted to Lyt2.2+ cells and were essentially devoid of cytotoxic T lymphocyte precursors (CTLp), but contained helper activity for in vivo T-dependent IgM, IgG and IgA antibody formation. Adherent cell populations were enriched for Lyt2.2+ cells and for CTLp. The graft-vs.-host activity of the separated, adherent (Lyt2.2+) and nonadherent (Lyt2.2-) cells in the Simonsen spleen assay in neonatal (C57BL/6 x BALB/c)F1 mice was less than of unfractionated cells, but the activity of remixed Lyt2.2+ plus Lyt2.2- cells was higher than the sum of the contributions of these cells tested separately, and equal to that of the unfractionated cells. PNA- thymocytes were also separated into Ly2.2+ and Lyt2.2- populations by fluorescence-activated cell sorting. Nonlytic separation allows the recovery of the Lyt1+2+ population, which is lost in cytotoxic elimination experiments. Under the conditions described for the plate separation, the purity of the separated cells and recovery of activity approaches that of cells separated by sorting. Therefore, the plate separation offers a convenient alternative to fluorescence-activated cell sorting when large numbers (i.e. up to 5 x 10(7) positively selected cells) are needed, as in studies of in vivo cell-mediated immune reactions.

Preparative nonlytic separation of Lyt2+ and Lyt2− T lymphocytes, functional analyses of the separated cells and demonstration of synergy in graft-vs.-host reaction of Lyt2+ and Lyt2− cells†

A computer system was developed at NIH, NCI, to add data acquisition, storage, and processing capability to two different flow microfluorometer/cell separators (FMF). Data can be collected from either or both cell separators while previously collected data are being simultaneously processed. Measured cell parameters can be accumulated in the computer memory as arrays of cell counts over a range of intensities or stored as raw data in a list mode on magnetic tape. The computer system hardware includes a refresh display, two disks, industry standard magnetic tape, 32K core, an incremental plotter, a link to the central facility, and links to the FMFs. For convenience in program development and to give direct access to additional data analysis resources, a direct 1200 baud link is available to a Central Facility PDP‐10. Programs have been developed to display the data in two dimensions and to create three‐dimensional projections which can be rotated. Two‐dimensional contour maps at user‐selected thresholds a...

A. R. Schultz

Papers

Rapid data collection, analysis, and graphics for flow microfluorometry instrumentation.