Site-specific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes

Site-specific deletions in the tal-1 gene are reported to occur in 12- 26% of T cell acute lymphoblastic leukemias (T-ALL). So far two main types of tal-1 deletions have been described. Upon analysis of 134 T- ALL we have found two new types of tal-1 deletions. These four types of deletions juxtapose the 5' part of the tal-1 gene to the sil gene promoter, thereby deleting all coding sil exons but leaving the coding tal-1 exons undamaged. The recombination signal sequences (RSS) and fusion regions of the tal-1 deletion breakpoints strongly resemble the RSS and junctional regions of immunoglobulin/T cell receptor (TCR) gene rearrangements, which implies that they are probably caused by the same V(D)J recombinase complex. Analysis of the 134 T-ALL suggested that the occurrence of tal-1 deletions is associated with the CD3 phenotype, because no tal-1 deletions were found in 25 TCR-gamma/delta + T-ALL, whereas 8 of the 69 CD3- T-ALL and 11 of the 40 TCR-alpha/beta + T-ALL contained such a deletion. Careful examination of all TCR genes revealed that tal-1 deletions exclusively occurred in CD3- or CD3+ T- ALL of the alpha/beta lineage with a frequency of 18% in T-ALL with one deleted TCR-delta allele, and a frequency of 34% in T-ALL with TCR- delta gene deletions on both alleles. Therefore, we conclude that alpha/beta lineage commitment of the T-ALL and especially the extent of TCR-delta gene deletions determines the chance of a tal-1 deletion. This suggests that tal-1 deletions are mediated via the same deletion mechanism as TCR-delta gene deletions.     

Evidence that the T cell antigen receptor may not be involved in cytotoxicity mediated by gamma/delta and alpha/beta thymic cell lines

After culture in IL-2, thymocytes expressing either TCR-alpha/beta or - gamma/delta acquired the ability to lyse hematopoietic and solid tumor cell targets without deliberate immunization or apparent restriction by the MHC. Moreover, TCR-alpha/beta- and TCR-gamma/delta-bearing thymic cell lines demonstrated an essentially identical spectrum of cytolysis against several tumor cell targets. Cytotoxicity was not inhibited by antibodies against CD3 or CD2 and modulation of the CD3/TCR complex also failed to affect cytotoxicity. Thus, non-MHC-restricted cytotoxicity can be mediated by thymocytes with either TCR-alpha/beta or TCR-gamma/delta, but the TCR may not be responsible for target recognition.     

Developmental arrest of NK1.1+ T cell antigen receptor (TCR)- alpha/beta+ T cells and expansion of NK1.1+ TCR-gamma/delta+ T cell development in CD3 zeta-deficient mice

The relationship between the structure of the T cell antigen receptor (TCR)-CD3 complex and development of NK1.1+ T cells was investigated. The TCR complex of freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes contained CD3 zeta homodimers and CD zeta-FcR gamma heterodimers, whereas that of the majority of NK1.1- T cells did not contain FcR gamma. The function of CD3 zeta and FcR gamma in the development of NK1.1+ T cells was determined by analyzing CD3 zeta- and FcR gamma- deficient mice. The NK1.1+ T cells from wild-type and CD3 zeta- deficient mice had equal levels of CD3 expression. However, the development of NK1.1+ TCR-alpha/beta+ T cells was almost completely disrupted in thymus and spleen in CD3 zeta-deficient mice, whereas no alteration was observed in FcR gamma-deficient mice. In contrast, the number of novel NK1.1+ TCR-gamma/delta+ thymocytes expressing a surface phenotype similar to NK1.1+ TCR-alpha/beta+ thymocytes increased approximately six times in CD3 zeta-deficient mice. These findings establish the distinct roles of the CD3 zeta chain in the development of the following different thymic T cell compartments: NK1.1- TCR+, NK1.1+ TCR-alpha/beta+, and NK1.1+ TCR-gamma/delta+ thymocytes, which cannot be replaced by CD3 eta or FcR gamma chains.     

Rearrangement of variable region T cell receptor gamma genes in acute lymphoblastic leukemia. V gamma gene usage differs in mature and immature T cells.

Using probes recognizing variable regions (V gamma) and joining regions (J gamma) of the T cell receptor (TCR) gamma gene, we have analyzed the usage of V gamma genes in 24 patients with T cell acute lymphoblastic leukemia (ALL) and 36 patients with B-precursor ALL. In CD3- T-ALL derived from immature T cells, V gamma genes more proximal to J gamma were frequently rearranged; V gamma 8, V gamma 9, V gamma 10, and V gamma 11 were used in 19 of 24 rearrangements. In contrast, CD3+ T-ALL derived from a more mature stage of T cell ontogeny, showed a high frequency of rearrangements involving V gamma genes distal to J gamma; V gamma 2, V gamma 3, V gamma 4, and V gamma 5 were used in 17 of 25 rearrangements. In B-precursor ALL, no notable bias of V gamma gene usage was observed. This probably reflects the possibility that TCR genes may not rearrange according to a T cell hierarchy when under control of a B cell gene program. Furthermore, deletions of those V gamma genes located 3' to rearranged V gamma genes were observed in all patients analyzed. This supports the theory that loop deletion is a major mechanism for TCR-gamma gene rearrangement.     

T cell receptor gamma and delta rearrangements in hematologic malignancies. Relationship to lymphoid differentiation.

We have studied recombinatorial events of the T cell receptor delta and gamma chain genes in hematopoietic malignancies and related these to normal stages of lymphoid differentiation. T cell receptor delta gene recombinatorial events were found in 91% of acute T cell lymphoblastic leukemia, 68% of non-T, non-B lymphoid precursor acute lymphoblastic leukemia (ALL) and 80% of mixed lineage acute leukemias. Mature B-lineage leukemias and acute nonlymphocytic leukemias retained the T-cell receptor delta gene in the germline configuration. The incidence of T cell receptor gamma and delta was particularly high in CD10+CD19+ non-T, non-B lymphoid precursor ALL. In lymphoid precursor ALL, T cell receptor delta was frequently rearranged while T cell receptor gamma was in the germline configuration. This suggests that TCR delta rearrangements may precede TCR gamma rearrangements in lymphoid ontogeny. In T-ALL, only concordant T cell receptor delta and gamma rearrangements were observed. Several distinct rearrangements were defined using a panel of restriction enzymes. Most of the rearrangements observed in T-ALL represented joining events of J delta 1 to upstream regions. In contrast, the majority of rearrangements in lymphoid precursor ALL most likely represented D-D or V-D rearrangements, which have been found to be early recombinatorial events of the TCR delta locus. We next analyzed TCR delta rearrangements in five CD3+TCR gamma/delta+ ALL and cell lines. One T-ALL, which demonstrated a different staining pattern with monoclonal antibodies against the products of the TCR gamma/delta genes than the PEER cell line, rearranges J delta 1 to a currently unidentified variable region.     

Transient rearrangements of the T cell antigen receptor alpha locus in early thymocytes

The dull Ly-1 double-negative (Ly-1dull, Lyt-2-, L3T4-) subpopulation appears to be the major precursor group of T lymphocytes in the thymus. In examining the status of the alpha, beta, and gamma chain genes for T cell receptors (TCR) in this population of cells and hybridomas made from them, we find that all of these loci appear to begin DNA rearrangements in a nearly simultaneous fashion. In the case of the gamma genes, these involve V gamma----J gamma C gamma gene rearrangements; with the beta chain genes, both D beta----J beta C beta rearrangement and V beta----D beta J beta C beta rearrangements are evident; and in the case of the alpha locus, assayed in part by pulsed-field gel electrophoresis, they take the form of a novel series of rearrangements occurring 80 kb or more 5' to the C alpha gene. These alpha locus rearrangements are well away from any of the J alpha gene segments found in cDNA clones to date and are deleted in most mature thymocytes and functional T cell lines. Therefore they appear to represent a distinct class of rearrangement that occurs before V alpha----J alpha joining. These distinctions between the character of the TCR gene rearrangements in these cells represent useful markers in further distinguishing different stages of T cell differentiation within this compartment of early T cells. In addition, the unexpected discovery of clonal rearrangements so far away from any of the expressed J alpha gene segments, and at a stage where there is little or no stable C alpha RNA present, has interesting implications for the hierarchy of TCR gene expression.     

T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production

The antigen-specific receptors of T and B lymphocytes are generated by somatic recombination between noncontiguous gene segments encoding the variable portions of these molecules. The semirandom nature of this process, while desirable for the generation of diversity, has been thought to exact a high price in terms of sterile (out-of-frame) products. Historically, the majority of T lymphocytes generated in mammals were thought to be useless, either because they generated such sterile rearrangements or because the receptors generated did not appropriately recognize self-molecules (i.e., positive and negative selection). In the studies described here, we characterize the onset of T cell receptor (TCR) alpha and beta chain gene rearrangements and quantitate their progression throughout T cell development. The results show that T cell production efficiency is enhanced through (a) rearrangement of TCR-beta chain genes early during T cell development, with selective expansion of those cells possessing in-frame rearrangements; (b) deletion of sterile rearrangements at the TCR-alpha chain locus through ordered (proximal to distal) sequential recombination; and (c) modification of nonselectable alpha/beta heterodimer specificities through generation and expression of new TCR- alpha chains. In addition, we demonstrate strict correlations between successful TCR-beta gene rearrangement, the onset of TCR-alpha gene rearrangement, rapid cell division, and programmed cell death, which together serve to maintain cell turnover and homeostasis during T cell development.     

Involvement of the interleukin 2 pathway in the rearrangement and expression of both alpha/beta and gamma/delta T cell receptor genes in human T cell precursors

In this report, we have undertaken the phenotypic, functional and molecular characterization of a minor (less than 5%) subpopulation of adult thymocytes regarded as the earliest intrathymic T-cell precursors. Pro-T cells were immunoselected and shown to express different hematopoietic cell markers (CD45, CD38, CD7, CD5) and some activation-related molecules (4F2, Tr, HLA class II), but lack conventional T cell antigens (CD2-1-3-4-8-). TCR-gamma RNA messages are already expressed at this early ontogenic stage, while alpha and beta chain TCR genes remain in germline configuration. In vitro analyses of the growth requirements of pro-T cells demonstrated the involvement of the IL-2 pathway in promoting their proliferation and differentiation into CD3+ CD4+ or CD8+ mature thymocytes. Moreover, during the IL-2-mediated maturation process rearrangements and expression of both alpha and beta chain TCR genes occurred, and resulted in the acquisition of alpha/beta as well as gamma/delta (either disulphide-linked or non-disulphide-linked) heterodimeric TCR among the pro-T cell progeny.     

Generation of diversity in T cell receptor repertoire specific for pigeon cytochrome c

17 T cell clones and 3 T cell lines, specific for pigeon cytochrome c, were analyzed for fine specificity and rearranged T cell receptor (TCR) gene elements. Clones of similar fine specificities were grouped into one of four phenotypes, and correlations between phenotype differences and gene usage could be made. All the lines and clones rearranged a member of the V alpha 2B4 gene family to a limited number of J alpha regions. The beta chain was made up of one of three non-cross-hybridizing V beta regions, each rearranging to only one or two J beta s. The use of alternate V beta regions could be correlated with phenotype differences, which were manifested either as MHC- or MHC and antigen-specificity changes. In addition, the presence of alloreactivity, which defined a phenotype difference, could be correlated solely with the use of an alternate J alpha region. These observations were substantiated by prospective analyses of pigeon cytochrome c-specific T cell lines that were selected for alternate MHC specificity or alloreactivity and were found to express the correlated alpha and beta chain rearrangements. Previously, the TCR DNA sequences from two clones, each representing a variant of one phenotype, showed sequence differences only in the N regions of their TCR genes. Since only these two variants, using identical V alpha-J alpha and V beta-J beta gene elements, were repeatedly observed in this study, we would predict that the junctional diversity differences are selectable. In this T cell response, all the gene elements involved in the generation of diversity appear to be selected, and may therefore be important in the determination of TCR specificity. This high degree of receptor gene selection represents a fundamental difference from the diversity seen in several extensively analyzed antibody responses.     

T cell receptor alpha-chain gene rearrangements in B-precursor leukemia are in contrast to the findings in T cell acute lymphoblastic leukemia. Comparative study of T cell receptor gene rearrangement in childhood leukemia.

We have analyzed T cell receptor alpha-chain gene configuration using three genomic joining (J) region probes in 64 children with acute lymphoblastic leukemia (ALL). 11 out of 18 T-ALLs were T3 positive; alpha-chain gene rearrangements were demonstrated in only two of 18, indicating that the majority of T-ALLs would have rearrangements involving J alpha segments located upstream of these probes. In contrast, 15 out of 46 B-precursor ALLs showed rearrangements of the alpha-chain gene and J alpha segments located approximately 20-30 kb upstream of the constant region were involved in 13 of these patients. Nine of 15 B-precursor ALLs with rearranged alpha-chain genes had rearrangements of both gamma- and beta-chain genes, whereas the remaining six had no rearrangements of gamma- and beta-chain genes. These findings indicated that alpha-chain gene rearrangement is not specific for T lineage cells and gamma- and/or beta-chain gene rearrangement does not appear essential for alpha-chain gene rearrangement, at least in B-precursor leukemic cells.     

The presumptive CDR3 regions of both T cell receptor alpha and beta chains determine T cell specificity for myoglobin peptides.

The T cell receptor alpha/beta (TCR-alpha/beta) is encoded by variable (V), diversity (D), joining (J), and constant (C) segments assembled by recombination during thymocyte maturation to produce a heterodimer that imparts antigenic specificity to the T cell. Unlike immunoglobulins (Igs), which bind free antigen, the ligands of TCR-alpha/beta are cell surface complexes of intracellularly degraded antigens (i.e., peptides) bound to and presented by polymorphic products of the major histocompatibility complex (MHC). Therefore, antigen recognition by T cells is defined as MHC restricted. A model has been formulated based upon the similarity between TCR-alpha/beta V region and Ig Fab amino acid sequences, and the crystal structure of the MHC class I and Ig molecules. This model predicts that the complementarity determining regions (CDR) 1 and 2, composed of TCR V alpha and V beta segments, primarily contact residues of the MHC alpha helices, whereas V/J alpha and V/D/J beta junctional regions (the CDR3 equivalent) contact the peptide in the MHC binding groove. Because polymorphism in MHC proteins is limited relative to the enormous diversity of antigenic peptides, the TCR may have evolved to position the highly diverse junctional residues (CDR3), where they have maximal contact with antigen bound in the MHC peptide groove. Here, we demonstrate a definitive association between CDR3 sequences in both TCR alpha and beta chains, and differences in recognition of antigen fine specificity using a panel of I-Ed-restricted, myoglobin-reactive T cell clones. Acquisition of these data relied in part upon a modification of the polymerase chain reaction that uses a degenerate, consensus primer to amplify TCR alpha chains without foreknowledge of the V alpha segments they utilize.     

T cell receptor alpha-, beta-, and gamma-genes in T cell and pre-B cell acute lymphoblastic leukemia

We examined alpha-, beta-, and gamma-T cell receptor (TCR) gene activation within acute lymphoblastic leukemias (ALLs) that represent early stages of B and T cell development. We wished to determine if TCR rearrangement and expression was lineage restricted, showed any developmental hierarchy, or could identify new subsets of T cells. Rearrangement of gamma and beta TCR genes occurred early in development but in no set order, and most T-ALLs (22/26) were of sufficient maturity to have rearranged both genes. T-ALLs preferentially rearranged C gamma 2 versus the C gamma 1 complex; no preference within the beta locus was apparent. Once rearranged, the beta TCR continued to be expressed (11/13), whereas the gamma TCR was rarely expressed (3/14). The alpha TCR was expressed only in more mature T-ALLs (8/14) that usually displayed T3. The 3A-1 T cell associated antigen appeared earliest in development followed by T11 and T3. Within pre-B cell ALL a higher incidence of lineage spillover was noted for gamma TCR rearrangements (8/17) than for beta rearrangements (3/17). This also contrasts with the only occasional rearrangement of immunoglobulin (Ig) heavy chains (3/25) in T-ALL. However, in pre-B ALL the pattern of gamma TCR usage was distinct from that of T cells, with the C gamma 1 complex utilized more frequently. Almost all ALLs could be classified as pre-B or T cell in type by combining Ig and TCR genes with monoclonal antibodies recognizing surface antigens, although examples of lineage duality were noted. Unique subpopulations of cells were discovered including two genetically uncommitted ALLs that failed to rearrange either Ig or TCR loci. Moreover, two T lymphoblasts were identified that possessed the T3 molecule but failed to express alpha plus beta TCR genes. These T-ALLs may represent a fortuitous transformation of T cell subsets with alternative T3-Ti complexes.     

Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation.     

A novel subset of CD2-, CD3/T cell receptor alpha/beta+ human peripheral blood T cells. Phenotypic and functional characterization of interleukin 2-dependent CD2-CD3+ T cell clones

It is generally believed that CD2 (T11, sheep erythrocyte receptor) is expressed on all human T cells. In the present study we have identified and characterized a minor subset of CD2- CD3/TCR alpha/beta+ T cells in the peripheral blood of healthy individuals. CD2-CD3+ T cells were enriched in PBMC depleted of plastic-adherent macrophages, E-rosetting (i.e., CD2+) T cells and surface Ig+ B cells. CD2-CD3+ T cells accounted for 0.1-0.8% of PBMC in six individuals. IL-2-dependent long-term clones of CD2-CD3+ T cells neither reacted with a panel of anti-CD2 mAbs nor expressed detectable levels of CD2 mRNA by Northern blot analysis. These clones, however, expressed a full-length TCR C beta mRNA and reacted with mAbs against TCR-alpha/beta, CD3, and CD4, and thus were mature T cells. CD2-CD3/TCR+ T cell clones could be triggered into proliferation, IL-2 production, and cytotoxic effector activity by anti-CD3 and anti-TCR mAbs. We conclude that (a) a minor subset of CD2-, CD3/TCR-alpha/beta+ T cells is present in normal peripheral blood; and (b) expression of CD2 at the level of protein and/or mRNA is not required for T cell signaling via the CD3/TCR molecular complex.     

Junctional sequences of fetal T cell receptor beta chains have few N regions

T cell receptors (TCRs) and immunoglobulins (Igs) derive a large fraction of their repertoire from diversity generated at the junctions of the V, D, and J coding segments. This diversity is derived both from the random deletion of nucleotides from the ends of coding regions and from the subsequent addition of nontemplated N region nucleotides. While the vast majority of TCRs and Igs from adult mice have N regions, less than 5% of both TCR-gamma/delta and Ig from fetal and neonatal mice have N regions. This study analyzed the ontogeny of junctional diversity of TCR-alpha/beta. Genomic DNA or C beta-primed cDNA was prepared from thymocytes of mice at varying stages in ontogeny, and the rearranged V beta 8 or V beta 5 sequences were amplified by polymerase chain reactions. Sequencing of the V beta-D beta-J beta junctions showed few N regions early in ontogeny, although the fraction of sequences with N regions exceeded that previously reported for Ig and for TCR-gamma/delta. N regions were found in 13% of V beta junctional sequences from day 18-19 fetal thymocytes, 33% of sequences from newborn thymocytes, 76% of sequences from day 4 postnatal thymocytes, and 88% of sequences from 5-wk-old thymocytes. In addition, nonrandom usage of the D beta and J beta segments was observed in both fetal and adult TCR sequences. While the usage of each of the six J beta 2 segments was different, the same pattern of usage was seen regardless of whether D beta 1 or D beta 2 was used, suggesting that a factor controlling the rate of usage of each J segment is intrinsic to the J gene itself. Since TCRs derive so much of their diversity from N regions, the relative paucity of N regions in fetal alpha/beta T cells would create a fetal TCR-alpha/beta repertoire that would be quite different from, and smaller than, the adult repertoire. The lack of N regions might be predicted to limit the range of affinities of TCR-MHC + peptide interactions, which may have important consequences for positive and negative selection of fetal and newborn T cells.     

Development of CD8alpha/alpha and CD8alpha/beta T cells in major histocompatibility complex class I-deficient mice.

Peripheral CD8(+) T cells mainly use CD8alpha/beta, and their development is mainly dependent on the major histocompatibility complex (MHC) class I proteins K(b) and D(b) in H-2(b) mice. In this report, we have shown that the development of CD8alpha/beta TCR-alpha/beta cells in lymphoid organs as well as in intestinal intraepithelial lymphocytes (iIELs) is dependent on the MHC class I K(b) and D(b) proteins. In contrast, TCR-alpha/beta CD8alpha/alpha cells are found mainly in iIELs, and their numbers are unaffected in K(b)D(b) double knockout mice. Most of the TCR-gamma/delta cells in the iIELs also bear CD8alpha/alpha, and they are also unaffected in K(b)D(b) -/- mice. In beta2-microglobulin (beta2m)-deficient mice, all of the TCR-alpha/beta CD8alpha/alpha and CD8alpha/beta T cells disappear, but TCR-gamma/delta cells are unaffected by the absence of beta2m.     

Human lymphocytes bearing T cell receptor gamma/delta are phenotypically diverse and evenly distributed throughout the lymphoid system

A direct quantitative and phenotypic cytofluorographic analysis of TCR-gamma/delta+ lymphocytes as well as an immunohistologic study of their tissue distribution and microanatomy was made possible by the availability of two mAbs (anti-TCR-delta 1 and anti-C gamma M1) specific for framework determinants on human TCR gamma and delta chains, respectively. TCR-gamma/delta+ lymphocytes, ranging between greater than 0.5 and 16% of CD3+ cells, were found in fetal and postnatal thymus, fetal and adult peripheral lymphoid organs, and adult peripheral blood. While TCR-gamma/delta+ lymphocytes comprised a small subpopulation of T cells (mean, approximately 4%) occasionally greater than 10-16% of CD3+ cells expressed TCR-gamma/delta. Virtually all TCR-gamma/delta+ thymocytes/lymphocytes expressed CD7, CD2, and CD5 but were heterogeneous with respect to their expression of CD1, CD4, CD8, CD28, CD11b, CD16, and Leu-7. Human TCR-gamma/delta+ cells populate both organized lymphoid tissues (thymus, tonsil, lymphnode, and spleen) as well as the gut- and skin-associated lymphoid systems at similar frequencies without obvious tropism for epithelial microenvironments. TCR-gamma/delta+ lymphocytes tend to be located within a given organ wherever TCR-alpha/beta+ lymphocytes are found. This study shows that TCR-gamma/delta+ lymphocytes constitute a small but numerically important, phenotypically diverse T cell population distributed throughout the body. These results support the concept that TCR-gamma/delta+ cells comprise a distinct, functionally heterogeneous, mature T cell sublineage that may substantially broaden the T cell repertoire at all immunologically relevant sites.