Human γδ T Cells Express a Higher TCR/CD3 Complex Density Than αβ T Cells

The aim of our study was to compare CD3 expression on γδ T cells and αβ T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human γδ T cells constitutively express approximately twofold more of the TCR/CD3 complex than αβ T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of γδ T cells compared to αβ T cells. These clinical laboratory results confirm the fundamental data described elsewhere. γδ T cells deserve further clinical investigations to understand their precise role in human immunity.     

Recombination pattern of the TCR γ locus in human peripheral T-cell lymphomas

The recombination events of the γ and β T-cell receptor (TCR) loci were analysed in a series of 39 peripheral T-cell lymphomas (PTCLs) in association with the expression of TCR chains. In TCR αβ PTCLs, 22/23 cases showed a γ-gene rearrangement while only 18/23 showed a concomitant β-gene rearrangement. The germline configuration of the β locus was found in angioimmunoblastic lymphadenopathy and lymphoepithelioid lymphomas. Three γδ PTCLs rearranged both γ and β genes. TCR silent PTCLs showed three different patterns of γ- and β-gene rearrangements. Three cases were in germline configuration for both loci; five cases had a rearranged γ and a germline β locus; and five cases had the two loci rearranged. Regarding the variable genes in the γ-rearranged alleles, members of the VγI subgroup were the most frequently presented (39/50), followed by VγII, VγIII, and VγIV (9/50, 1/50, and 1/50, respectively). Joining segment usage was as follows: J1 or J2 (32/50), JP1 or JP2 (17/50), and JP (1/50). Taken together, these data demonstrate that the γ locus is more frequently rearranged whatever the TCR expression. The γ-locus analysis provides a better diagnostic yield than the β locus in the study of PTCL clonality.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Evidence for recombinatorial hot spots at the T cell receptor Jα locus

The complex genomic organization of the murine T cell receptor (TcR) δ-α region has hindered detailed studies of α gene rearrangement and Jα gene usage in individual differentiating T cell precursors. We have isolated a novel set of Jα probes which, in combination with a few restriction enzyme digests, enable a reliable, simple and nearly complete analysis and location of any rearrangement at the Jα locus by conventional Southern blotting. The probes were used to analyze TcR α gene rearrangements in T cell hybridomas derived from an in vitro culture system that supports T cell differentiation of bone marrow cells. Our results indicate that Jα genes are unequally accessible for rearrangement and two hot spots for rearrangement could be demonstrated. In addition, only a restricted set of Jα genes was rearranged in each culture indicating that the slightly variable composition of factors can influence the recombinatorial accessibility of Jα genes. The hot spots for rearrangement were not only limited to T cells differentiating in vitro but could also be demonstrated among functional T cell clones based on the published sequence information from isolated TcR α gene rearrangements. The demonstration and the location of the hot spots for rearrangement in the T cell differentiation culture system opens up the possibility to study factors and mechanisms that regulate recombinatorial accessibility of TcR α genes.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β-/- mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre-TCR complex.     

Molecular characterization and clinical presentation of HKαα and anti‐HKαα alleles in southern Chinese subjects

The HKαα allele is a rearrangement occurring in the α‐globin gene cluster containing both the ‐α^3.7 and ααα^anti4.2 unequal crossover junctions. The anti‐HKαα allele is the reciprocal product containing both the ‐α^4.2 and ααα^anti3.7 unequal crossover junctions, which had been predicted but had not been detected previously. The phenotypic feature and population frequency of these two unusual alleles were not described. We report the identification of nine individuals carrying the HKαα allele and two individuals carrying the anti‐HKαα allele in southern China and describe their phenotype and haplotype data. The molecular structures of HKαα allele and anti‐HKαα allele were confirmed by two‐round nested polymerase chain reaction assay. The mechanism of origin of both alleles is related to probably simultaneous double crossover. Heterozygotes of HKαα or anti‐HKαα allele show a normal hematological phenotype. Finally, we report the carrier rates of these both alleles in the Guangxi Zhuang Autonomous Region of southern China, namely, ∼0.07% for the HKαα allele and ∼0.02% for the anti‐HKαα allele.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

In vivo application of mAb directed against the γδ TCR does not deplete but generates “invisible” γδ T cells

mAb targeting the γδ TCR have been used for γδ T‐cell depletion with varying success. Although the depletion‐capacity of the anti‐γδ TCR mAb clone GL3 has been disputed repeatedly, many groups continue to use γδ T‐cell depletion protocols involving the mAb clone UC7‐13D5 and find significant biological effects. We show here that treatment with both GL3 and UC7‐13D5 antibodies does not deplete γδ T cells in vivo, but rather leads to TCR internalization and thereby generates “invisible” γδ T cells. We addressed this issue using anti‐γδ TCR mAb injections into WT mice as well as into reporter TCR delta locus‐histone 2B enhanced GFP knock‐in mice, in which γδ T cells can be detected based on an intrinsic green fluorescence. Importantly, the use of TCR delta locus‐histone 2B enhanced GFP mice provided here for the first time direct evidence that the “depleted” γδ T cells were actually still present. Our results show further that GL3 and UC7‐13D5 mAb are in part cross‐competing for the same epitope. Assessed by activation markers, we observed in vitro and in vivo activation of γδ T cells through mAb. We conclude that γδ T‐cell depletion experiments must be evaluated with caution and discuss the implications for future studies on the physiological functions of γδ T cells.     

Transendothelial chemotaxis of human αβ and γδ T lymphocytes to chemokines

Two subpopulations of human T lymphocytes expressing different antigen receptors, α / β and γ / δ, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of α / β and γ / δ T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β stimulated similar, dose-dependent chemotaxis of purified γ / δ T cells, whereas MCP-1, RANTES, and MIP-1α pro duced greater chemotaxis of purified α / β T cells than MIP-1β. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-γ inducible protein-10 (IP-10) did not promote chemotaxis of either α / β or γ / δ T cells. Three γ / δ T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mono nuclear cells confirmed the results with purified γ / δ T cells. Our data demonstrate that human peripheral blood α / β and γ / δ T cells can transmigrate to MCP-1, RANTES, MIP-1α, and MIP-1β, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.     

α-thalassaemia due to a single codon deletion in the α-1-globin gene. Computational structural analysis of the new α-chain variant

A new unstable α-globin chain associated with α-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the α-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the α-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 or DK61 results in a structurally abnormal α-globin chain, not previously described, named Hb Clinic, which leads to the α-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this α-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer. Hum Mutat 11:412, 1998. © 1998 Wiley-Liss, Inc.     

Expression of HNF-1α and HNF-1β in various histological differentiations of hepatocellular carcinoma

Hepatic nuclear factor 1 (HNF-1) regulates genes in a hepatocyte-specific manner. It has been previously reported that the ratio of HNF-1α and HNF-1β mRNA is related to histological differentiation hepatocellular carcinoma (HCC). In this study, the expression levels of the HNF-1α and HNF-1β proteins were analysed relatively and quantitatively in various histologically differentiated HCC and surrounding non-cancerous tissues, and HNF-1α binding activity for the AT element of the B domain of the human α-fetoprotein enhancer was examined. Western blot analysis demonstrated that HNF-1α protein was expressed at a higher level in well-differentiated HCC tissues than in the surrounding non-HCC tissues; on the other hand, the HNF-1α protein was expressed at lower levels in moderately and poorly differentiated HCCs than in the surrounding non-HCC tissues. The levels of HNF-1β expression in well-differentiated and poorly differentiated HCCs were similar to and higher than those found in the respective surrounding non-cancerous portions. In binding assays, HNF-1 binding activity was high in well-differentiated HCC and lower in moderately and poorly differentiated HCCs. Most well-differentiated HCC cases showed immunohistochemical expression of HNF-1α. These findings show that poor histological differentiation of HCC correlates with decreases in the level and activity of HNF-1α proteins. © 1998 John Wiley & Sons, Ltd.