Thymus Leukemia Antigen Research Articles

Nonclassical behavior of the thymus leukemia antigen: peptide transporter-independent expression of a nonclassical class I molecule.

The thymus leukemia (TL) antigen is a major histocompatibility complex-encoded nonclassical class I molecule. Here we present data demonstrating that expression of the TL antigen, unlike other class I molecules, is completely independent of the function of the transporter associated with antigen processing (TAP). The TL antigen is expressed by transfected TAP-2-deficient RMA-S cells when these cells are grown at 37 degrees C. In transfected RMA cells, the kinetics of arrival of TL antigen on the cell surface are similar to those of a classical class I molecule. The kinetics are not altered in TAP-deficient RMA-S cells, demonstrating that surface TL expression in TAP-deficient cells is not due to the stable expression of a few molecules that leak out by a TAP-independent pathway. Soluble TL molecules produced by Drosophila melanogaster cells are highly resistant to thermal denaturation, unlike peptide-free classical class I molecules synthesized by these insect cells. In addition, these soluble TL molecules are devoid of detectable bound peptides. The results demonstrate that the TL antigen is capable of reaching the surface without bound peptide, although acquisition of peptide or some other ligand through a TAP-independent pathway cannot be formally excluded. We speculate that the ability of the TL antigen to reach the cell surface, under conditions in which other class I molecules do not, may be related to a specialized function of the TL molecule in the mucosal immune system, and possibly in the stimulation of intestinal gamma delta T cells.

Surface expression of beta 2-microglobulin-associated thymus-leukemia antigen is independent of TAP2.

Mouse thymus-leukemia antigen (TL), like other major histocompatibility complex (MHC) class I-b antigens, displays signs of a specialized function. It is normally expressed at high levels on immature thymocytes and at moderate levels on gut epithelium and activated mature T cells. A promoter/enhancer region unique among class I genes accounts for this narrow range of tissue distribution. Like most other class I molecules, TL is dependent upon endogenous beta 2-microglobulin (beta 2m) for transport to the surface. However, here we show that unlike most other MHC class I molecules, TL is expressed efficiently in the absence of functional transporter associated with antigen processing subunit 2 (TAP2). A putative fourth TLa gene cloned from A.SL1 cells was expressed in RMA and RMA-S cells. In bulk transformants, TL expression is higher in TAP2-RMA-S cells than in wild-type RMA cells, and is not elevated by incubation at reduced temperatures or exposure to exogenous beta 2m. Analysis of immunoprecipitated molecules by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that TL is processed normally in RMA-S cells and is associated with beta 2m both intracellularly and at the cell surface. However, TL heavy chains expressed on the cell surface in the absence of TAP2 are cleaved to a predominant 38 kDa fragment, presumably the result of an altered conformation that renders TL more susceptible to proteolysis. These results suggest that while TL may normally acquire TAP2-dependent peptides, this class I-b molecule does not require them for efficient export to, and stable expression at the cell surface.

Ten polymorphic DNA loci, including five in the rat MHC (RT1) region, form a single linkage group on rat chromosome 20.

We have described ten markers for polymorphic loci on rat chromosome 20, including five in the rat MHC (RT1) region. These markers formed a single linkage group spanning a recombination distance of 0.40. The markers identified five expressed gene loci - RT1.N1 (thymus leukemia antigen 1), Tnfa (tumor necrosis factor {alpha}), Hspa1 (heat shock protein 70), Ggt1 ({gamma} glutamyl-transferase 1), and Prkacn2 (protein kinase C catalytic subunit binding inhibitor 2), two loci with sequences that are related to expressed genes - RT1.Aw2 (sequence related to a non-RT1A class I {alpha} chain) and Mt21 (sequence related to metallothionein 2), and three anonymous loci - D20Arb548, D20Arb234, and D20Arb249. These polymorphic markers should facilitate mapping studies and genetic monitoring of inbred rat strains. 18 refs., 2 figs., 3 tabs.

Intraepithelial lymphocytes and their recognition of non-classical MHC molecules.

Recent studies of the TCR alpha and beta chains expressed by normal human IELs suggest that these intestinal lymphocytes are directed at a limited set of antigens, presumably on intestinal epithelial cells in view of their anatomic location. The direct sequence analysis of these cells has indicated that they are oligoclonal and cannot, therefore, be responding to the complex mixture of antigens which are present in the lumen. The abundant expression of the CD8 accessory molecule by the IELs, in addition, indicates that these putative intestinal epithelial cell antigens are presented by MHC class I or I-like molecules. The expression of CD8 also suggests that these cells function biologically in part as cytolytic T lymphocytes which is consistent with a variety of functional studies. Taken together with their expression of the CD45RO isoform, these phenotypic and functional observations suggest that iIELs are cytolytic, memory cells which are responsive to an extremely limited number of antigens bound to major histocompatibility complex (MHC) class I or class I-like molecules. Several non-polymorphic MHC class I-like molecules such as Qa, the thymus leukemia antigen (TL) and CD1 in the mouse and CD1 in human represent important candidate ligands for these oligoclonal iIELs. TL and CD1 are expressed specifically by murine intestinal epithelial cells. In humans, CD1d is constitutively expressed by intestinal epithelial cells. In addition, we have isolated iIEL T cell clones which specifically recognize members of the CD1 gene family when expressed on a transfected B cell line that lacks HLA-A and B and have shown that the proliferation of peripheral blood T cells to intestinal epithelial cells is CD1d dependent. Thus, the evidence to date strongly implicate the nonpolymorphic, class Ib molecules as novel restriction elements for unique populations of lymphocytes within the intestinal epithelium.

Expression of mouse Tla region class I genes in tissues enriched for ?? cells

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to γδ T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for γδ T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain γδ T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing γδ T cells further extends the list of potential candidates for antigen presentation to γδ T cells.

<b>GENERALIZED EXPANSION OF EXTRATHYMIC T CELLS IN VARIOUS IMMUNE ORGANS OF TL-TRANSGENIC </b><b>MICE </b>

Thymus-leukemia (TL) * antigens are monomorphic class I MHC antigens in mice that are expressed in restricted sites (e.g., in thymocytes and epithelium of the intestine). Recent studies using γδ T cell clones and TL-transgenic mice have suggested that TL antigens are preferentially recognized by γδ T cells. TL-transgenic mice eventually display an abnormal expansion of γδ T cells in the thymus that indicates impaired development. In this study, we further characterized the properties of T cells in various immune organs of these mice, including the sites for extrathymic T-cell differentiation (i.e., liver and intestine)

Differential induction of major histocompatibility complex molecules on mouse intestine by bacterial colonization

The aim of this study was to determine what factors induce major histocompatibility complex (MHC) molecules on the mouse small intestinal epithelium by using immunohistochemical methods. In germ-free mice, although MHC class I molecules such as H-2K and thymus leukemia antigen (TLa) were expressed on the small intestinal epithelium, class II molecules were absent. The introduction of micro-organisms into germ-free mice induced characteristic MHC molecules on the small intestinal epithelial cells. The I-A molecule was induced on the villus tip and crypt epithelial cells 7 days after conventionalization, and the I-E molecule was induced on the mid villus and crypt epithelial cells 14 days after conventionalization. The staining intensity of the H-2K molecules was increased 4 days after conventionalization. In contrast, TLa did not change during conventionalization of germ-free mice. These results suggest that the expression of MHC molecules, except for the TLa, is greatly dependent on the presence of intestinal microorganisms.

Peripheral engraftment of fetal intestine into athymic mice sponsors T cell development: direct evidence for thymopoietic function of murine small intestine.

Adult athymic, lethally irradiated, F1-->parent bone marrow-reconstituted (AT x BM) mice were engrafted bilaterally with day 16-18 fetal intestine or fetal thymus into the kidney capsule and were studied for evidence of peripheral T cell repopulation of 1-12 wk postengraftment. Throughout that time period, both types of grafts were macroscopically and histologically characteristic of differentiated thymus or intestine tissues, respectively. Beginning at week 2 postengraftment, clusters of lymphocytes were present within intestine grafts, particularly in subepithelial regions and in areas below villus crypts. As determined by immunofluorescence staining and flow cytometric analyses, lymphocytes from spleen and lymph nodes of sham-engrafted mice (AT x BM-SHAM) were essentially void of T cells, whereas in AT x BM thymus-engrafted (AT x BM-THG) mice, which served as a positive control for T cell repopulation, normal levels of T cells were present in spleen and lymph nodes by week 3 postengraftment, and at times thereafter. Most striking, however, was the finding that T cell repopulation of the spleen and lymph nodes occurred in AT x BM fetal intestine-engrafted (AT x BM-FIG) mice beginning 3 wk postengraftment. Based on H-2 expression, peripheral T cells in AT x BM-FIG mice were of donor bone marrow origin, and consisted of CD3+, T cell receptor (TCR)-alpha/beta+ T cells with both CD4+8- and CD4-8+ subsets. Peripheral T cells in AT x BM-FIG mice were functionally mature, as demonstrated by their capacity to proliferate after stimulation of CD3 epsilon. Moreover, alloreactive cytotoxic T lymphocytes were generated in primary in vitro cultures of spleen cells from AT x BM-FIG and AT x BM-THG mice, though not in spleen cell cultures from AT x BM-SHAM mice. Histologic studies of engrafted tissues 3-4 wk postengraftment demonstrated that thymus leukemia (Tl) antigens were expressed on epithelial surfaces of intestine grafts, and that both TCR-alpha/beta+ and TCR-gamma/delta+ lymphocytes were present in intestine grafts. Collectively, these findings indicate that the murine small intestine has the capacity to initiate and regulate T cell development from bone marrow precursors, thus providing a mechanism by which extrathymic development of intestine lymphocytes occur.

Expression of mouse Tla region class I genes in tissues enriched for gamma delta cells.

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to gamma delta T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for gamma delta T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent and transcripts from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain gamma delta T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing gamma delta T cells further extends the list of potential candidates for antigen presentation to gamma delta T cells.

TL (thymus leukemia) antigen expressed on skin mediates graft rejection and CTL response

TL (thymus leukemia) antigen expressed on skin mediates graft rejection and CTL response

The thymus leukemia antigen binds human and mouse CD8.

The thymus leukemia antigen (TLA) is a class Ib, or 'nonclassical' class I molecule, one of several encoded within the Tla locus of the mouse major histocompatibility complex (MHC). It structurally resembles the H-2K, D, and L class I transplantation antigens, which present processed peptides to cytotoxic T lymphocytes (CTLs). Although their function(s) are unknown, there has been recent speculation concerning the possibility that class Ib molecules may present antigens to T cells that express gamma delta T cell antigen receptors (TCRs). In this report, using both a cell-cell adhesion assay and adhesion of T lymphocyte clones to purified plate-bound TLA, we provide evidence that TLA can bind to both human and mouse CD8. We also show that a chimeric class I molecule containing the peptide antigen binding site of Ld and the alpha 3 domain, transmembrane, and cytoplasmic segments of TLA, can support a CD8-dependent immune response by CTLs. These results demonstrate for the first time binding of a class Ib molecule to CD8 with a functional outcome, as is observed for the class I transplantation antigens. The capacity to interact with CD8 has been conserved despite the extensive sequence divergence of TLA in the peptide antigen binding site, suggesting this interaction is highly significant. TLA is expressed by epithelial cells in the mouse small intestine. As these epithelial cells are in close contact with intestinal intraepithelial lymphocytes that are nearly all CD8+, and many of which express the gamma delta TCR, the data are consistent with the hypothesis that TLA is involved in antigen presentation, perhaps to gamma delta-positive lymphocytes in this site.

Highly restricted expression of the thymus leukemia antigens on intestinal epithelial cells.

The TL region of the major histocompatibility complex of the mouse contains dozens of tandemly arranged class I genes, including those encoding the thymus leukemia (TL) antigens. TL antigens have been thought to be expressed only on the surface of some T lineage cells, namely immature thymocytes of some mouse strains (TL+ strains), some leukemia cells, and activated T cells. While the function of TL antigens is unknown, recent studies have implicated the products of at least some TL region class I genes as molecules that present antigens to gamma/delta T cells. Since some gamma/delta T cells are known to be specifically associated with certain epithelial tissues, we have investigated the expression of some TL region class I genes in a variety of epithelium-containing tissues. Our results show that the TL antigen gene of C57BL/6 mice, T3b, and the TL antigen genes of BALB/c mice, T3d (previously T3c) and T18d (previously T13c), are highly expressed in the epithelium of the small intestine. In the case of T3b, we further show, using a T3 product-specific antibody, that its product is expressed on the surface of the columnar epithelial cells. In addition, we demonstrated that two other TL region class I genes of C57BL/6 origin, T9b and T21b, are also expressed nearly exclusively in intestinal epithelial cells. These results are consistent with the hypothesis that the products of these TL region class I genes are recognized by gamma/delta T cell receptors of intestinal intraepithelial lymphocytes, a subset of gamma/delta T cells that is localized in the intestinal epithelium and has a restricted V gamma repertoire. Finally, our study indicates that the relative levels of expression of the two homologous TL antigen genes, T3d and T18d, differ widely between the thymus and the intestine.

Cellular location of thymus-leukemia (TL) antigen as shown by immuno-cryoultramicrotomy

Thymus-leukemia (TL) antigen is a class I molecule of the major histocompatibility complex that is expressed on the surface of mouse cortical thymocytes. Though not expected, it has been reported that TL antigen can be found on isolated mitochondria of TL+ cells. We used immuno-cryoultramicrotomy to look for TL on mitochondria in situ, thereby avoiding the plasma membrane contamination that occurs when isolating organelles. Establishing optimal fixation conditions was crucial, as mitochondrial structure was not preserved by the low concentrations of fixative needed for detection of antibody labeling. The plasma membranes of tissue culture and thymus cells were labeled well with anti-TL antibody and protein A-gold conjugate, while mitochondria within the cells were not labeled. Isolation of mitochondria on a one-step Ficoll gradient resulted in a purer organelle preparation than did isolation of mitochondria by centrifugation alone. Generally, mitchondria within this purer preparation were not labeled. Our data show that under conditions where contamination by plasma membrane is not a major concern, TL antigen cannot be detected on mitochondria.

Recognition of MHC TL Gene Products by γδ T Cells

We have studied the ligand specificity of a gamma delta T-cell receptor (TCR) derived from a mouse T-cell hybridoma (KN6). KN6 cells reacted with syngeneic (C57BL/6) cells from various origins (splenocytes, thymocytes, peritoneal exudate cells, etc.) and cells from many different mouse strains. KN6 reactivity against cells from a panel of congenic and recombinant mouse strains demonstrated that the ligand recognized by KN6 is controlled by an MHC-linked gene that most probably maps in the TL region. We cloned this gene and formally proved that it does map in the TL region. This gene turned out to be a novel class I gene (designated T22b) belonging to a hitherto unidentified cluster of TL region genes in strain C57BL/6. This gene was expressed in many different tissues and cell types. We also examined the tissue expression of several other TL genes. One of these, the structural gene (T3b) encoding the thymus leukemia (TL) antigen from C57BL/6 mice, was specifically expressed in the epithelium of the small intestine. Since the intestinal epithelium of the mouse is known to be the homing site for a subset of gamma delta T cells (i-IEL) bearing diverse TCR with V7 rearranged gamma chains, we propose that the T3b gene product is part of the ligand recognized by some of the i-IEL. Our data support the idea that gamma delta T cells might be specific for non-classical class I or class I-like molecules and suggest that gamma delta TCR and non-classical MHC co-evolved for the recognition of a conserved set of endogenous or foreign peptides.

Expression of the thymus leukemia antigen in mouse intestinal epithelium.

The Qa and Tla regions of the mouse major histocompatibility complex contain a series of genes encoding proteins with structural similarity to the class I transplantation antigens of the same complex. In contrast to the genes encoding the transplantation antigens, the Qa and Tla genes show very little polymorphism. Function(s) of the proteins encoded by the Qa and Tla loci remain an enigma. Recently, the protein products of the Qa and Tla loci, often referred to as class Ib major histocompatibility complex molecules, have been proposed to present antigen to gamma delta T cells. In mice, gamma delta T cells have been found concentrated in several epithelial barriers and in the skin; yet, expression of serologically detectable Tla antigens is believed restricted to thymocytes, activated T lymphocytes, and some T-cell leukemias. Here we report that luminal epithelial cells of the mouse small intestine express the thymus leukemia antigen (TLA). We also find that, unlike T cells in Peyer's patches, a significant fraction of intestinal epithelial lymphocytes also express TLA. RNA prepared from intestinal cells contains transcripts of the T18d gene, which encodes TLA. These data extend the known expression profile of TLA molecules to mature lymphocytes and to nonhematopoietic cells. These data also demonstrate the specific expression of TLA on antigen-presenting cells in a site enriched for T cells that express gamma delta T-cell antigen receptor.

Regulation of expression of TL genes of the mouse Mhc

The expression of thymus leukemia (TL) antigens and genes in thymocytes and activated T cells was examined by immunoprecipitation, flow cytometric, northern, and nuclear run-off transcription analyses. Cell surface forms of TL were detectable by immunoprecipitation on activated peripheral T cells from Tla haplotypes except Tla(b), in agreement with expression observed on thymocytes. Approximately 40%-50% of concanavalin A (Con A) or anti-CD3-activated T cells were TL+, with expression detected on both the CD4 and CD8 subsets by dual-color analysis. Activated T cells expressed detectable levels of TL mRNA 48 h after stimulation, but no TL transcripts were detectable in unstimulated splenocytes. However, TL mRNA expression in mature activated T cells did not precisely mimic thymocyte expression: the level of expression was considerably lower in activated T cells, and in most haplotypes the transcripts produced in activated T cells appeared to represent a subset of the transcripts produced in thymocytes. By run-off transcription assays in isolated nuclei, TL gene expression was detected in activated but not resting T cells indicating that lack of expression of TL in resting T cells is not due to message instability. These data demonstrate that TL genes are inducible and transcriptionally regulated.

Genomic organization of the mouse Tla locus: study of an endogenous retroviruslike locus reveals polymorphisms related to different Tla haplotypes.

A retrovirus element (TLev1) is located within the Thymus leukemia antigen (Tla) locus of the C57BL/10 mouse major histocompatibility complex. Low-copy probes have been isolated from sequences flanking the TLev1 integration site to examine the distribution of TLev1 among inbred mouse strains having genotypically determined variations in TL-antigen expression. It was found that the low-copy probes cross-hybridize to regions within the Tla locus in a genotype-specific manner. Although a strong association was found between TL mouse strains and TLev1, the presence or absence of the TLev1 locus did not exclusively correlate with expression or nonexpression of TL antigens. Analysis of different Mus subspecies indicates that TLev1 integrated into a common ancestor of the species Mus musculus. It is suggested that the loss of the TLev1 locus from certain mouse genomes reflects evolutionary rearrangements in the TL region; the resulting diversity may relate to the differential expression of TL antigens among mouse strains. The probes described here provide a useful tool for examining the genomic expansions and contractions which have occurred during the evolution of the Tla locus.

Influence of 5' flanking sequences on TL and H-2 expression in transfected L cells.

TL (thymus leukemia) antigens are encoded by genes in the major histocompatibility complex (MHC) of the mouse. Although similar in overall structure to other class I MHC antigens (H-2, Qa), TL expression is regulated in a highly distinctive fashion. In contrast to the broad distribution of H-2 and the intermediate distribution of Qa, TL expression is restricted to cells of T-cell derivation during development in the thymus and is lost when T cells migrate to the periphery. Some mouse strains do not express TL antigens on thymocytes (TL- strains), but leukemias occurring in these mice can have a TL+ phenotype, indicating activation of normally silent TL genes. In transfection studies with H-2 or TL genes in L cells (mouse fibroblasts), H-2 is expressed at high levels, whereas TL is poorly expressed. To identify genetic elements that regulate expression in transfected L cells, chimeric genes were constructed by transposing the 5' and 3' regions of TL and H-2 genes. Antigen expression was not influenced by transposing the cytoplasmic domain and 3' untranslated region. In contrast, interchanging the 5' flanking sequences and exon 1 had a marked influence on antigen expression, with 5' sequences from the H-2 gene increasing TL expression 10- to 50-fold, and 5' sequences from the TL gene markedly decreasing H-2 expression. With both the parental TL gene (p20-TL) and the highly expressed chimeric TL gene (construct 3), levels of TL mRNA and TL antigen correlated with the number of transfected gene copies. However, in cells transfected with equal copy numbers, much higher levels of TL mRNA and TL antigen were found in construct-3 transfectants than in p20-TL transfectants. In addition, there was marked heterogeneity in TL mRNA size in L cells transfected with p20-TL, in contrast to a more homogeneous transcript size in construct-3 transfectants. These results point to regulatory sequences in the 5' flanking region of class I genes that control proper initiation and processing of TL transcripts.

M241 (CD1) expression on B lymphocytes.

The human thymus leukemia-like antigens (CD1a-c) consist of three similar glycoproteins found on subpopulations of normal thymocytes, T cell acute leukemias, and cutaneous dendritic cells. The CD1c antigen recognized by the M241 monoclonal antibody was detected on the circulating mononuclear cells of three children with severe combined immunodeficiency disease (SCID). Two-color immunofluorescence analysis demonstrated that M241 expression (43 to 95%) was limited to cells expressing the B cell-restricted antigens B4 (CD19), B1 (CD20), and surface immunoglobulin. To confirm M241 expression on normal cells of the B lineage rather than aberrant expression limited to SCID B cells, its expression was demonstrated serologically and biochemically on purified B cells from spleen, tonsil, and peripheral blood. Parallel analyses with monoclonal antibodies NA1/34 and 4A76 demonstrated that the CD1a and CD1b molecules were negative on all B cells that were studied. It has been hypothesized that the CD1 molecules represent the human counterpart of the murine thymus leukemia antigens due to their similar size, limited tissue distribution, and association with beta 2-microglobulin. This study suggests that a subset of CD1 antigens detected by M241 (CD1c) may represent a human analog of a murine Qa antigen due to its extended distribution on normal peripheral B cells.

T-cell markers in the thymus during methylnitrosourea-induced leukemogenesis

T-cell leukemias were induced in adult BDF1 mice by a single i.v. injection of 50 mg/kg of methylnitrosourea (MNU). During the latency period and in leukemic mice the expression of peanut agglutinin (PNA) receptors, terminal deoxynucleotidyl transferase (TdT) and thymus leukemia antigen (TLm4) in the thymus was studied. First, TLm4-positive thymocytes were found 6 wk after MNU. Further, there was a continuous reduction of the percentage of PNA-positive thymocytes. The expression of TdT remained unaltered late in the latency period and in leukemic mice. Four phenotype patterns could be defined for the expression of TdT and PNA in thymocytes: PNA + TdT +, PNA + TdT −, PNA − TdT + and PNA − TdT −. Thymuses negative for TdT were rare, the majority was PNA + TdT +. However, thymuses late after MNU treatment and leukemic thymus were PNA − TdT +. The discrepancy in the expression of PNA and TdT as cellular markers in leukemic mice as compared to normal cell populations is discussed.