Expression of T-cell differentiation antigens on activated Thymocytes

The expression of Thy 1, TL, Lyt1, and Lyt2 antigens on resting and proliferating thymocytes activated by concanavalin A in the presence of interleukin 2 has been studied by conventional complement-dependent cytotoxicity assay. The predominant population of resting thymocytes has a TL⁺Lytl⁺Lyt2⁺ phenotype while the predominant population of proliferating thymocytes has a TL — Lytl⁺Lyt2⁺ phenotype. Using several separation procedures such as agglutination by peanut lectin, BSA density gradient centrifugation, and pretreatment with high dilutions of anti-H-2 serum it was impossible to obtain a 100% pure population of TL⁺Lytl⁺Lyt2⁺ cells, suggesting that the population of resting immature thymocytes contains small subpopulations of phenotypically differentiated cells. The population of proliferating thymocytes is also phenotypically heterogenous and contains cells bearing all phenotypes that were described for different stages of T-cell differentiation, including TL⁺Lytl⁺Lyt2^? and TL⁺Lytl^?Lyt2⁺ with the following approximate frequency: TL⁺Lytl⁺Lyt2⁺—27%, TL⁺Lytl⁺Lyt2^?—8%, TL⁺Lyt1^?Lyt2⁺—4%, TL^?Lytl⁺Lyt2⁺—45%, TL^?Lyt1⁺Lyt2^?—13%, TL^?Lyt1^?Lyt2⁺—3%.     

A new serologically defined locus,Qa-1, in theTla-region of the mouse

A new cell-surface antigen, specified by a gene betweenH-2D andTla is described. The provisional notationQa-1 is suggested for the locus determining this newly recognized cell surface component. Qa-1 is distinguished from known TL antigens by the following two criteria. Its expression is not confined to thymocytes — it occurs on lymph node cells (LNC) also; and the phenotypes of the new congenic recombinant strains B6.K1 and B6.K2, derived fromH-2D/Tla crossovers, are Qa-1⁺ Qa-2^–TL^– and Qa-l⁺Qa-2⁺TL^–. Qa-1 antigen is defined by reaction of the standard TL typing serum, (B6 × A -Tla ^b)F₁ anti-A strain leukemia ASL1, with lymph node cells (LNC) in the cytotoxicity assay. Qa-1 antigen evidently is expressed, at least, on a subpopulation of T cells as well as on thymocytes. The gene order isH-2D, Qa-1, Qa-2, Tla.Abbreviations used in this paper LNC lymph node cells pooled from inguinal, axillary, brachial, and mesentric nodes - BA⁺ (C57BL/6-Tla^axA)F1 - BA^– (C57BL/6 × A -Tla ^b)F₁ - PBS phosphate buffered saline pH 7.2 - Thy thymocytes - RMIg Rabbit anti-mouse immunoglobulin Please address proofs and communications concerning this paper to Dr. Thomas Stanton, Sloan Kettering Institute, 1275 York Ave., New York, N.Y. 10021     

Extension of the H-2 TLb molecular map

A region of the TL ^b locus encompassing T11 to T13 contains retroviral sequences TLev1 and TLev2. As part of a study to determine whether the retroviral elements are involved in the expression of TL genes, the genomic organization of this region was reexamined in greater detail. A result of these investigations is the extension of the H-2 TL^b molecular map. Two additional TL genes have been isolated from C57BL/6 mice, T14 and T15. The genomic organization of T9 through T15 is presented. The nucleotide sequence has been determined for exons 4, 5, and 6 of T13. As a result of a C to T conversion, a termination codon is introduced into exon 4, indicating that T13 either encodes a secreted protein or is a pseudogene. T13 was found to be more homologous to the H-2 genes outside the TL region. T14 has been physically disrupted by the integration of TLevl , and the H-2 sequences appear to have diverged greatly. The relationship of the TL regions of the b and c haplotypes has been investigated using numerous low copy probes. The genome of BALB/c (TL^c) is shown to lack a counterpart of the T13–T15 ^b region. Homologous regions exist in the two haplotypes; yet considerable polymorphism is observed. TL^b mice do not express TLa on the cell surface of normal thymocytes while TL^c mice do; TLa expression is activated in many TO leukemias. The diversity seen in the T13–T15 region may provide insights into the phenotypic expression or regulatory mechanisms of TL expression in these two haplotypes.     

Analysis of hybrids between an H-2+, TL− lymphoma and an H-2+, TL+ lymphoma and its H-2−, TL− variant subline

Hybrids between an H-2⁺, TL⁺ lymphoma and an H-2⁺, TL^– lymphoma were studied for their expression of H-2 and TL antigens. The H-2 antigens of both parents were expressed, the TL specificity of the TL⁺ parent was retained, and the TL specificity characteristic of the mouse strain from which the TL^– lymphoma was derived was not expressed. There was no evidence that the genome of either parent altered the expression of the TL antigens coded for by the genome of the opposite parent. Hybrids between the H-2⁺, TL^– lymphoma and an H-2^–, TL^– variant line derived from the H-2⁺, TL⁺ cell line expressed both theK- andD-regioncoded H-2 antigens and the TL specificity characteristic of the parental cell line from which the variant cell was derived. This result is consistent with the defect in the variant cell's being the result of a mutation affecting a gene coding for a positive element necessary for expression of both TL and the serologically detectable H-2 antigens on the cell surface.     

The ability of H-2+ and H-2− cell lines to induce or be lysed by Cytotoxic T cells

We have studied the T cell-mediated lysis of two C58 lymphoma lines: R1(TL⁺), which bears serologically detectable H-2^k and TL1,2,3 antigens, and R1(TL^–), an immunoselected variant which lacks these antigens. Unlike R1(TL⁺) cells, the variant cells are not sensitive to specific lysis by T cells directed against either H-2^k or minor H antigens of C58 mice. An injection of R1(TL⁺) cells into allogeneic H-2-identical or H-2-different mice primes for an excellent secondary cytotoxic response in vitro to R1(TL⁺); but not to R1(TL^–). Immunization in vivo and in vitro with R1(TL)^–) leads to little or no priming or generation of cytotoxic T cells. Both cell lines, however, are sensitive to nonspecific lysis by cytotoxic cells in the presence of PHA or Con A, although even under these conditions, R1(TL⁺) is killed more effectively than is R1(TL^–). We conclude that R1(TL^–) does not express any form of H-2 antigen which can be detected by immunization or by sensitivity to cytotoxic T cells.     

Identification of a novel isoform of ZAP-70, truncated ZAP kinase

We identified a novel cDNA encoding truncated ZAP-70, which lacked the SH2 domain and a part of interdomain B, and named it truncated ZAP kinase (TZK). TZK was expressed in the thymus, spleen, and lymph nodes with ZAP-70. TZK was expressed in CD44⁺CD25⁻ thymocytes up to mature T cells, but ZAP-70 was not expressed in CD44⁺CD25⁻ or CD44⁺CD25⁺ thymocytes. ZAP-70 or TZK was transfected into P116 cells derived from a Jurkat T-cell line deficient in ZAP-70. The P116 cells with ZAP-70 induced the T-cell receptor-mediated signal transduction, but the cells expressing TZK did not. While ZAP-70 was accumulated at the immune synapse, TZK was not. Meanwhile, impaired phosphorylation of SLP-76, one of the substrates of ZAP-70, in P116 cells upon pervanadate stimulation was rescued in the cells expressing TZK. These findings show that TZK is a novel isoform of ZAP-70, which is expressed in pre-T-cell receptor-minus thymocytes and functions as a kinase not associated with T-cell receptor.     

Biochemical genetics of TL antigens

TL antigens are class I glycoproteins which are expressed on thymocytes and which are coded by the Tla region of the major histocompatibility complex of the mouse. Biochemical analysis of TL molecules from different strains of mice revealed structural variation determined by the Tla region which is detectable by peptide mapping, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gels, and by differential reactivity of allelic forms of TL molecules with a panel of anti TL reagents. The quantity of TL expressed on thymocytes is also influenced by the Tla region; three quantitative phenotypes were identified: high (Tla ^a, Tla ^d, Tla ^c), intermediate (Tla ^c, Tla ^f), and low (Tla ^b). (Relative amounts: 1000 : 100 : 1.) Some thymic leukemias arising in (Tla ^b, Tla ^c, Tla ^c) mice with genetically determined reduced levels of thymic TL were found to express TL molecules which were structurally indistinguishable from TL isolated from thymocytes but were present in larger amounts. This suggests that TL structural genes are intrinsically capable of full expression in all mice but that the Tla region of mice expressing an intermediate or low quantity of TL is marked by some feature which causes the thymocyte to express less than the full amount of TL possible.     

RT1.P, rat class Ib genes related to mouse TL: evidence that CD1 molecules but not authentic TL antigens are expressed by rat thymus

 CD1 and TL were once thought to be genetic homologues because of their thymus-specific expression. We investigated their equivalents in the rat to clarify whether their structure and pattern of expression are conserved in rodents. Two rat class Ib genes, containing 3′ sequences very similar to mouse TL, were identified and designated RT1.P. Neither of them, however, can encode ordinary class I molecules due to the accumulation of harmful mutations in the 5′ regions that are unique to RT1.P, while the 3′TL-like regions still retain protein-coding capacity. Comparison of the structural organization of three types of TL family genes, which include mouse T3/T18-encoding TL antigens, mouse T1/T16, and rat RT1.P1/P2 pseudogenes, revealed the presence of a clear demarcation between the type-specific and TL-specific sequences at intron 3. This finding suggests that recombination plays an important role in creating the TL family genes in rodents. Characteristic features of TL, such as a low level of polymorphism and linkage to the major histocompatibility complex, were also observed in the rat. On the other hand, rat CD1 molecules were expressed at a high level on the surface of thymocytes. Absence of authentic TL antigens and thymic expression of CD1d molecules in the rat suggest the plasticity and conservation of class Ib genes in rodent evolution. Functions of TL may be substituted with CD1 or other class Ib molecules expressed by rat thymus. Received: 16 December 1996 / Revised: 11 March 1997     

Developmental regulation of αβ T cell antigen receptor assembly in immature CD4+CD8+ thymocytes

Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic αβ chains associated with invariant CD3-γδε components and ζ chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4⁺CD8⁺ thymocytes expressing only 10% of the number of αβ TCR complexes on their surfaces expressed by mature CD4⁺ and CD8⁺ T cells. Recent evidence demonstrates that low surface TCR density on CD4⁺CD8⁺ thymocytes results from the limited survival of a single TCR component within the ER, the TCRα chain, which has a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCRα proteins in immature CD4⁺CD8⁺ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of αβ TCR complexes in CD4⁺CD8⁺ thymocytes and comment on the functional significance of TCRα instability during T cell development.     

Fidelity of a BAC-EGFP transgene in reporting dynamic expression of IL-7Rα in T cells

Interleukin-7 receptor α chain (IL-7Rα)-derived signals are critical for normal T cell development, mature T cell homeostasis, and longevity of memory T cells. IL-7Rα expression in T cells is dynamically regulated at different developmental and antigen-responding stages. However, the molecular mechanism underlying the dynamic regulation is not completely understood. Here we describe generation of a bacterial artificial chromosome (BAC)-based reporter transgenic mouse strain, which contains 210 kb DNA sequence flanking the Il7r locus. We used in vitro validated EGFP reporter and insulator sequences to facilitate the reporter transgene expression. Consistent with endogenous IL-7Rα expression, the BAC transgene was expressed in mature T cells, a portion of natural killer cells but not in mature B cells. In the thymus, the EGFP reporter and endogenous IL-7Rα showed synchronized silencing in CD4⁺CD8⁺ double positive stage, were both upregulated in CD4⁺ or CD8⁺ single positive thymocytes, and both continued to be co-expressed in na?ve T cells in the periphery. Upon encountering antigen, the antigen-specific effector CD8⁺ T cells downregulated both endogenous IL-7Rα and the EGFP reporter, which were upregulated in synchrony in antigen-specific memory CD8 T cells. These results indicate that the BAC-EGFP transgene reports endogenous IL-7Rα regulation with high fidelity, and further suggest that the 210 kb sequence flanking the Il7r locus contains sufficient genetic information to regulate its expression changes in T lineage cells. Our approach thus represents a critical initial step towards systematic dissection of the cis regulatory elements controlling dynamic IL-7Rα regulation during T cell development and cellular immune responses.     

Antigenic relationship between medullary thymocytes and a subpopulation of peripheral T cells in the rat: description of a masked antigen.

Using differentially absorbed rabbit antisera to rat thoracic duct cells, an antigen is described which normally is expressed on the surface of T cells in thoracic duct lymph and lymph node, but which exists in a masked form on medullary thymocytes and apparently not at all on cortical thymocytes. This antigen is termed the rat masked thymocyte antigen (RMTA). RMTA on medullary thymocytes can be unmasked mechanically by sectioning in a cryostat or enzymatically by treating with neuraminidase. Trypsin destroys or removes RMTA. Nearly all the T cells in thoracic duct lymph and lymph node are RMTA⁺, whereas only 58–66% of T cells in spleen are RMTA⁺. RMTA⁺ T cells, which are cortisone resistant, reside in the paracortex and periarteriolar sheath regions of lymph node and spleen. RMTA^? T cells, which are cortisone sensitive, appear to reside in the red pulp of spleen. The results suggest that (i) two antigenically distinct populations of T cells exist in the rat, RMTA⁺ and RMTA^? T cells, (ii) medullary thymocytes are the immediate precursors of RMTA⁺ T cells, and (iii) cortical thymocytes may be the immediate precursors of RMTA^? cells.     

A novel MHC class I-related molecule expressed on mouse thymic stroma cells and mature lymphocytes

A monoclonal antibody (mAb) TP-3 has been established by immunizing rats with the BALB/c mouse thymic epithelial cell line TEL-2. The TP-3 antigen is expressed on stroma cells of thymus, spleen, and lymph node in syngeneic BALB/c mice (H-2 ^d ). This antigen is also expressed at a low level on the cell surface of immature thymocytes, and at a high level on mature T and B cells. In allogeneic mice such as C57BL/6 (H-2 ^b ) or C3H (H-2 ^k ), no cells expressed the TP-3 antigen. Using H-2 congenic mice, reactivity with mAb TP-3 was found to map to a region of H-2D ^d L ^d or between D ^d and Qa, suggesting that TP-3 is a major histocompatibility complex (MHC) class I antigen. However, immunoprecipitation analysis indicated that this antigen is not identical to the classical mouse class I molecules in terms of molecular size, antigenicity, and tissue distribution.     

Exposure of a Distinct PDCA-1+ (CD317) B Cell Population to Agonistic Anti-4-1BB (CD137) Inhibits T and B Cell Responses Both In Vitro and In Vivo

4-1BB (CD137) is an important T cell activating molecule. Here we report that it also promotes development of a distinct B cell subpopulation co-expressing PDCA-1. 4-1BB is expressed constitutively, and its expression is increased when PDCA-1⁺ B cells are activated. We found that despite a high level of surface expression of 4-1BB on PDCA-1⁺ B cells, treatment of these cells with agonistic anti-4-1BB mAb stimulated the expression of only a few activation markers (B7-2, MHC II, PD-L2), cytokines (IL-12p40/p70), and chemokines (MCP-1, RANTES), as well as sTNFR1, and the immunosuppressive enzyme, IDO. Although the PDCA-1⁺ B cells stimulated by anti-4-1BB expressed MHC II at high levels and took up antigens efficiently, Ig class switching was inhibited when they were pulsed with T-independent (TI) or T-dependent (TD) Ags and adoptively transferred into syngeneic recipients. Furthermore, when anti-4-1BB-treated PDCA-1⁺ B cells were pulsed with OVA peptide and combined with Vα2⁺CD4⁺ T cells, Ag-specific cell division was inhibited both in vitro and in vivo. Our findings suggest that the 4-1BB signal transforms PDCA-1⁺ B cells into propagators of negative immune regulation, and establish an important role for 4-1BB in PDCA-1⁺ B cell development and function.     

Stage-specific induction of terminal deoxynucleotidyl transferase in a T-lymphoid line upon coculture with a thymic stromal line

We previously reported an in vitro T-cell differentiation system in which the L4 lymphoid clone was cocultured with the St3 stromal line derived from the same murine thymic tumor, 15#4T. L4 cells in L4—St3 cocultures sequentially express Thy-1 and CD4 in a manner typical of normal thymocytes. In contrast, L4 cells grown in medium alone retain their Thy-1⁻CD4⁻ phenotype. We also isolated L4 subclones from the coculture with increasingly differentiated phenotypes with respect to Thy-1 and CD4. We now report induction of an additional thymocyte differentiation marker, terminal deoxynucleotidyl transferase (TdT) in 15#4T cells (and to a lesser extent subcloned L4 cells) upon coculture with St3 stroma. Coculture of 15#4T cells with St3 stroma resulted in expression of TdT as measured by ribonuclease protection for TdT RNA and Western immunoblotting for TdT protein. Cocultured L4 cells were induced for TdT expression to a lesser degree and for a shorter period of time. The magnitude of TdT RNA induction was maximal for cell lines with the least mature differentiation phenotype (15#4T and L4: Thy-1⁻CD4⁻) and decreased proportionally for subclones with increasingly mature phenotype, e.g., L4E cells (Thy-1⁺CD4⁺). TdT protein was undetectable by Western immunoblotting and immunofluorescent staining of the L4E subclone on or off stroma. Recombination-activating gene-1 (RAG-1), which is expressed in immature thymocytes during T-cell receptor rearrangement, but suppressed in mature thymocytes, was also examined using the ribonuclease protection assay. In contrast to TdT, RAG-1 expression was suppressed by coculture with St3 cells for 15#4T and also more mature subclones, indicating regulation by a mechanism independent from TdT. The ordered induction of TdT, Thy-1, and CD4, as well as regulation of RAG-1 in the 15#4T-St3 system, supports the conclusion that this in vitro system is a valuable model for characterizing regulation of these markers in normal thymocytes.     

Allele-specific expression of the mouse B-cell surface protein CD72 on T cells

 CD72 is a 45 000 M _r mouse B-cell surface glycoprotein involved in B-cell proliferation and differentiation. Expression of mouse CD72 is thought to be restricted to the B-cell lineage. We recently demonstrated that the monoclonal antibodies K10.6 and B9.689, previously defined as recognizing the mouse lymphocyte alloantigens Ly-19.2 and Ly-32.2, respectively, recognize specific alleles of CD72. Early studies using antibody-mediated cytotoxicity assays demonstrated that K10.6 and B9.689 react with B cells, several T-cell lines, and a subset of peripheral T cells. These findings led us to consider the possibility that CD72 might also be expressed on a subset of T cells. In this report we demonstrate that CD72 is constitutively expressed on a fraction of peripheral T cells isolated from strains of mice expressing the CD72 ^b allele, but not the CD72 ^a or CD72 ^c alleles. Three days after activating T cells with concanavalin A or plate-bound CD3-specific mAb, CD72 is expressed on a larger fraction of peripheral T cells as well as a fraction of thymocytes from mouse strains expressing the CD72 ^b allele. CD72 is expressed on both the CD4⁺ and CD8⁺ thymocyte and peripheral T-cell subsets. No CD72 expression is detected on activated thymocytes or peripheral T cells from mouse strains expressing the CD72 ^a or CD72 ^c alleles. Expression of CD72 ^b on peripheral T cells was confirmed by northern blot analysis demonstrating CD72 mRNA expression. These results demonstrate that CD72 expression is not restricted to B lineage cells in mouse strains expressing the CD72 ^b allele; instead, a population of T lineage cells in these mice also expresses CD72. Received: 18 June 1996 / Revised: 17 September 1996     

Role of T‐bet,the master regulator of Th1 cells,in the cytotoxicity of murine CD4+ T cells

Although CD4⁺ T cells are generally regarded as helper T cells, some activated CD4⁺ T cells have cytotoxic properties. Given that CD4⁺ cytotoxic T lymphocytes (CTLs) often secrete IFN‐γ, CTL activity among CD4⁺ T cells may be attributable to Th1 cells, where a T‐box family molecule, T‐bet serves as the “master regulator”. However, although the essential contribution of T‐bet to expression of IFN‐γ has been well‐documented, it remains unclear whether T‐bet is involved in CD4⁺ T cell‐mediated cytotoxicity. In this study, to investigate the ability of T‐bet to confer cytolytic activity on CD4⁺ T cells, the T‐bet gene (Tbx21) was introduced into non‐cytocidal CD4⁺ T cell lines and their cytolytic function analyzed. Up‐regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4⁺ T cells but not in untransfected parental cells. In one cell line, T‐bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas^? target cells. Although T‐bet was found to repress up‐regulation of CD40L (CD154), which controls FasL‐mediated cytolysis, the extent of CD40L up‐regulation on in vitro‐differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T‐bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.

     

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes.

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2^k and H-2^d antigens and a similar decrease in the expression of the TL antigen (in TL⁺ strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G_IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.     

Alloreactive T-cell clones. Ly phenotypes predict both function and specificity for major histocompatibility complex products

We have studied the association of Ly phenotype with function and specificity for major histocompatibility complex (MHC) products by examining the properties of 21 T-cell clones derived from B10 anti-B 10.D2 and B10.A anti-B10.D2 mixed lymphocyte cultures (MLC). T cells were selected after MLC solely on the basis of Ly phenotype, cloned by limiting dilution, and tested for stability of Ly phenotype, function and specificity for class I or class II MHC products. Sixteen Ly-1⁺2^– and five Ly-1^–2⁺ T-cell clones were tested. The clones selected for the Ly-1 ⁺2^– phenotype maintained this phenotype, expressed helper but not lytic function, and recognized class II MHC products (I-A^d or I-Ed). All Ly-1^–2^– clones maintained this phenotype, possessed cytolytic but not helper activity, and recognized class I MHC products (D^d and L^d). Our data therefore confirm at the clonal level the original observations of a remarkably consistent correlation between Ly markers, MHC specificity, and. function. They suggest that the expression of Ly antigens on T-cell clones forms part of a genetic program for each of these specialized cells that also determines their function and MHC specificity.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - TCGF T cell growth factor (Interleukin-2) - Con A Concanavalin A - DME Dulbecco's modified Eagle's medium - PHA phytohaemagglutinin - LPS lipopolysaccharide - TRF-C T cell replacing factor required for induction of cytolytic cells from thymocytes - PBS phosphate-buffered saline (pH 7.4)     

Expression of Ly-6A/E alloantigens in thymocyte and T-lymphocyte subsets: variability related to the Ly-6 a and Ly-6 b haplotypes

We have studied the cellular basis for differential expression of the Ly-6A/E alloantigen on T cells obtained from mice of the Ly-6 ^a (10–20% Ly-6A/E ⁺) and Ly-6 ^b (50–60% Ly-6A/E ⁺) haplotypes. During T-cell ontogeny only a small fraction (< 12 %) of thymocytes expressed Ly-6A/E. By 4 weeks of age adult levels of Ly-6A/E bearing lymphocytes were seen in peripheral lymphoid tissue. Immunohistochemical studies of the thymus revealed that Ly-6A/E⁺ cells were located predominantly in the medulla with small clusters of Ly-6A/E⁺ cells throughout the cortex. Consistent with this result, phenotypic studies showed that in the adult thymus the majority of Ly-6A/E expression was on mature CD4⁺ CD8^– and CD4^– CD8⁺ cortisone-resistant and precursor CD4^– CD8^– thymocytes. However, a much higher percentage of CD4⁺ CD8^– and CD4^– CD8^– thymocytes as well as CD4⁺ CD8^– peripheral T cells expressed Ly-6A/E from Ly-6 ^b mice. Furthermore, although gamma interferon induced increased Ly-6A/E expression in certain thymocyte and T-cell subsets, this induction functioned preferentially for cells obtained from Ly-6 ^b mice. Studies using F₁ hybrid mice (Ly-6 ^a × Ly-6 ^b) indicated that the basal level of Ly-6A/E expression on these subsets appeared to be under codominant genetic control, whereas gamma interferon-induced regulation of Ly-6A/E expression appeared to be under dominant genetic control. Collectively, these results suggest that the expression of Ly-6A/E on a particular T-cell subset is established in the thymus and is a stable characteristic of each haplotype. In addition, the low levels of Ly-6A/E expression for the Ly-6 ^a haplotype appear to be partially due to the inability of the majority of resting CD4⁺ T cells to express Ly-6A/E and to the relatively poor induction of this protein by gamma interferon.     

Enzymatic relationship between human Tγ+ lymphocytes and thymocytes

The lactate dehydrogenase isoenzyme pattern has been determined in human thymocytes and in peripheral blood T lymphocytes, Tγ⁺, and Tμ⁺ lymphocytes. Thymocytes have a highly significant lower activity in LDH-1 and LDH-2 together with a highly significant higher activity in LDH-3 and LDH-4 than peripheral T lymphocytes. The same differences are seen when Tγ⁺ and Tμ⁺ cells are compared. On the contrary there are almost no differences in pattern between the peripheral T lymphocytes and Tμ⁺ cells on one hand and between thymocytes and Tγ⁺ cells on the other hand. These findings suggest that both thymocytes and Tγ⁺ cells exhibit a very immature LDH pattern with regard to the Tμ⁺ cell population.