T-Lymphocyte Subsets in the Embryonic Spleen Undergoing a Graft-Versus-Host Reaction

Allogeneic immunocompetent T cells injected into chicken embryos induce a graft-versushost reaction (GVHR) whose most prominent manifestation is splenic hyperplasia. The highly inbred CC and CB strains of chickens used here are, respectively, homozygous for the B4 or B12 MHC haplotypes. By means of a panel of immunological reagents, including alloantisera and monoclonal antibodies against public domains of the T-cell receptor, CD4, CD8, and the inducible interleukin-2-receptor light chain (CD25), it is shown that the bulk of cells in the enlarged spleen are of host origin and do not express markers typical of mature T or B lymphocytes. Among recipient splenocytes, the quantitatively most important population consists of TCRαβ-TCRγδ-CD4-CD8+CD25+ (TCR0) lymphocytes. Donor cells encountered in the spleen prevalently exhibit a TCRαβ+CD4+CD8-CD25+ phenotype and proliferate in vivo. The data demonstrate that nonspecific host and potentially specific donorderived cellular elements contribute to splenomegaly.


INTRODUCTION
The development of graft-versus-host reactions (GVHR) depends on a special set of requirements each of which has the status of conditio sine qua non (for a review, see Bril and Benner, 1985;Parkman, 1989). First, graft and host have to differ in major or minor histocompatibility loci. Second, the allograft must contain immunologically competent alloreactive T cells. Third, the recipient needs to be immunodeficient, thus being incapable of rapidly eliminating the grafted cells by an immunological counterattack (host-versus-graft reaction). GVHR is a major complication of therapeutic bone marrow transplantation in patients and may become manifest in an acute or chronic fashion, then mimicking certain aspects of autoimmune diseases. Much of the pioneering work on GVHR has been performed in the chicken embryo, where it is induced either by placing immunocompetent allogeneic T cells onto *Corresponding author. the chorioallantoic membrane or by injecting them intravenously (Simonsen, 1957(Simonsen, , 1962Seto and Albright, 1965;D6sveaux-Chabrol and Dieterlen-Li/vre, 1987;D(sveaux-Chabrol et al., 1989). The latter procedure entails a systemic response whose most spectacular manifestation is splenomegaly, that is, an easily quantifiable increase of splenic mass.
Extensive studies have been carried out on the T-cell subsets required to trigger the reaction in the mammalian adult GVHR model (Gleichman et al., 1984;Korngold and Sprent, 1985;Sprent et al., 1990). In this model, the GVHR usually develops in an irradiated animal, whose own responding cells .have been largely eradicated. On the other hand, in the avian embryonic model,, host ceils proliferate actively especially in the early phase of the reaction, as shown earlier using sex chromosomes as markers (Owen et al., 1965;Seto and Albright, 1965;McBride, 1966;Nisbet and 'Simonsen, 1967). Precocious immunological maturation of the host immune system in serial transfer experiments was hypothesized to occur under the influence of allogeneic cells Lafferty et al., 1972;Simonsen, 1985). 164 -BRUNER et al. In these studies however, subpopulations of host and donor cells were not investigated for the want of specific markers, so that the, enlarged spleen remained largely a black box. We recently showed by means of a pan T-cell monoclonal antibody (mAb) (Yassine et al., 1989) that the T-cell population became amplified by a factor of 10 or more in the spleens of embryos undergoing a GVHR . For the present study, we have used two inbred chicken strains, carrying, respectively, the B4 and B12 MHC haplotypes, and have performed double staining of spleen cells, to combine detection of MHC haplotype and one of several functional markers.

FEDECKA
Panels of different monoclonal antibodies (mAbs) specific for chicken cluster of differentiation (CD) antigens have become available, allowing the study of the expression of clonotypic (TCR1, equivalent of mammalian y/TCR, Sowder et al., 1988; TCR2 a/ /J TCR, Chen et al., 1988) or invariant (CD3, Chen et al., 1986) components of the CD3/TCR complex, as well as the distribution of CD4, CD8 , and IL-2-receptor (IL-2R) light chains (CD25, Mr=50 kD, Hala et al., 1986;Schauenstein et al., 1988). Using these mAbs in conjunction with alloantisera, we evaluated the relative contribution of donor and recipient in an attempt to determine to which extent specific and nonspecific phenomena are involved in GVHR-induced splenomegaly.

RESULTS
Relative Contribution of Donor and Host Ceils to Splenomegaly As depicted in Fig. 1, the numeric contribution of lymphoid cells increases in an overproportional fashion during GVHR-induced splenomegaly (Figs. 1A and 1B). Among these lymphoid elements, a significant portion exhibits allogeneic markers of the donor MHC haplotype (B4) and appears to vigorously proliferate in vivo, increasing by a factor of 5.1 from E18 to 20 of embryonic age and reaching up to 6.5 xl0 cells (Fig. 1C). Given that probably only a fraction of the injected PBL (10 cells) homes to the recipient's spleen, the increase of donor cells probably is more important than suggested by these numbers. Nonetheless, the majority of cells (approximately 90% at E18 and 80% at E20) present in the hyperplastic spleen (about 10 times control weight at E18 to E20) is of host. origin. Thus, in situ proliferation of recipient cells in the enlarging spleen and/or recruitment of cells from other organs quantitatively outnumber donor cells.
Characterization,of T-Lymphocyte Subpopulations Splenomegaly produced in the (B4) donor-(B12) recipient combination is accompanied by an increase of CD8 + cells (from about 2 xl0 in controls to 5 xl0" 160" Lymphoid cell numbers and donor-cell contribution in GVHR-induced splenomegaly. One million B or B 12 lymphocytes (3to 4-week-old donors) were injected i.v. into E13 old B 12 embryos. At E18, E19, or F.20 of embryonic age, splenic weight (A), total leukocyte numbers (B), and the number of cells reacting with a B4)-specific alloantiserum (C) were determined. 86 + 2% of cells isolated from spleens undergoing GVHR on E18 to E20 were stained by a B12-specific alloantiserum. Only E20 untreated (0) or syngeneic controls (syng.) are shown; these control values do not significantly change from E18 to E20. Note the overproportional increase in lymphoid cells and cells of the donor phenotype. Values were determined by microscope counting for (B) and by microscope counting and cellsorter analysis for (C).
T SUBSETS IN GVHR SPLEEN 165 on E20 in embryos undergoing GVHR), as well as by the appearance of cells that are practically absent in control spleens, namely, lymphoid cells exhibiting CD4, TCR1, TCR2, IL-2R, or a B-cell marker (Fig. 2). From E18 to 20, the percentage of the CD8 / cells among splenocytes increases significantly, whereas the percentages of other cell populations remain relatively constant (although their absolute number increases, Fig. 1). Nonetheless, the majority of splenocytes isolated from chicken embryos undergoing GVHR lack surface antigens normally found on lymphocytes. In untreated or syngeneic controls, CD25 is faintly expressed only on a small (< 2%) subpopulation of embryonic spleen cells. In contrast, up to 15% of cells strongly express CD25 in spleens of chicks undergoing GVHR. The majority of these CD25 / cells bear the Class MHC recipient phenotype (Table 1). Most CD8 / cells (84%) and a large portion of CD4 / cells (65%) express CD25 on E20, that is, 7 days after injection of allogeneic adult PBL (Table 1). These findings strongly suggest that IL-2-mediated proliferation and/or differentiation are implicated in the expansion of these cell subsets.

CONCLUDING REMARKS
The data shown in this paper suggest the following scenario of GVHR-induced splenomegaly. After intravenous injection, a portion of donor lympho-  Fig. 1) were subjected to immunofluorescence analysis using a panel of monoclonal antibodies. No significant difference was found-in lymphoid cell number and lymphocyte subset distribution between E18 and E20 syngeneic controls (E20 values shown). The  lymphocytes in the spleen undergoing GVHR probably also stimulate the nonspecific proliferation of hematolymphopoietic elements. Thus, the proliferation of donor lymphocytes is outnumbered by the expansion and/o r recruitment of host cells either of lymphoid (TCR0), prelymphoid or nonlymphoid type. Experiments about local GVHR also detected a predominance of host cells over cells expressing the donor MHC haplotype in chlorioallantoic pocks (Penninger et al., 1989(Penninger et al., , 1990. To summarize, the data reported here point to a significant donor as well as host contribution to qF double staining followed by microscope counting and cell-sorter GVHR in the chicken embryo and reveal the existanalysis were applied to splenocytes from E20 embryos undergoing GVHR. ence of specific and nonspecific components in bND=ntdetermined" splenomegaly development. It should indeed be emphasized that about 80% of white blood cells activated and start proliferating in response to present in the enlarged spleens do not seem to allogeneic antigens, mainly MHC determinants, once belong to the lymphoid population. The embryo they become expressed during ontogeny. Because challenged with an IL-2 stimulation appears to the expression of Class II MHC antigen precedes respond by amplifying the cell types present at that that of Class I, the latter being detectable only time, that is, CD8+ cells and cells that are probably shortly before hatching (Kroemer et al., 1990), hemopoietic precursors. It remains to be investipredominantly CD4 / (i.e., Class II-restricted) T cells gated whether proliferating cells display the same are activated. These CD4 / ("helper")lymphocytes of subpopulation profile, when GVHR occurs in neodonor origin express the high affinity IL-2R and natal rather than embryonic hosts or in adult organprobably produce their own IL-2 and proliferate in isms made immunodeficient by artificial means. an autocrine fashion via the IL-2/IL-2R pathway, as do mammalian CD4 / cells of the TH0 or TH1 phenotype (Smith, 1988). At the same period, CD8/TCR0 MATERIALS AND METHODS cells of the recipient become activated either by the antigenic makeup of the donor-cell population or Induction and Quantitation of GVHR more likely by the presence of T helper-derived lymphokines. CD8 / cells have been described by Highly inbred chickens homozygous for either the Max Cooper's group in the embryonic and adult B (CC line) or B 12 (CB line) MHC haplotypes chicken (Bucy et al., 1989(Bucy et al., , 1990. No TCR has been (obtained from the Czechoslovakian Academy of found on these cells, which have been interpreted as Sciences, Prague, or from the INRA station in Le candidate natural killer cells in this species. Practi-Magneraud, France) were used in all experiments. cally all CD8/TCR0 cells in the GVHR spleen dis-106 peripheral blood lymphocytes (PBL) were isoplay the IL-2R, thus being capable of proliferating in lated from heparinized blood from 3to 4-week-old response to endogenous or exogenous IL-2. In sup-donors by differential centrifugation, washed in port of a central role of IL-2 in the pathogenesis of phosphate-buffered saline (PBS, pH 7.2), and splenomegaly, local as well as systemic GVHR of injected into a chorioallantoic vein of 13-day-old mice or humans are suppressed by monoclonal (E13) syngeneic or allogeneic embryos. Necropsy antibodies directed against the IL-2R (Ferrara et al., was performed after 5, 6, or 7 more days of incuba-1986; Volk et al., 1986;Cavazzana-Calvo et al., tion (E18 to E20). 1990). Similarly, local GVHR in the chicken embryo can be prevented by functional IL-2R blockade with Cytofluorochemical Analysis   (Penninger et al., 1989), that is, a murine mAb that neither binds chicken complement After mechanical dissociation of the embryonic nor exerts direct toxic effects (Schauenstein et  heat-inactivated bovine serum, and subjected to immunofluorescence analysis. 1 x106 cells/ml were incubated in a first step, either with biotinylated B or Bla-specific alloantisera (gift from Dr. D. Bouret) or with the monoclonal antibodies (mAbs) CT4 (specific for CD4, Chan et al., 1988), CT8 (CD8, Chan et al., 1988), TCR1 (y/ TCR, Sowder et al., 1988; kindly, provided by Dr. Max Cooper), TCR2 (c//J TCR, Chen et al., 1988; gift from Dr. Cihak),  presented (mean+ standard error of the mean, SEM) three to eight experimental series (each comprising two to five animals) were performed. Group differences were calculated using the Mann-Whitney test.