Search

Lymphoproliferative disease of granular lymphocytes (LDGL) is characterized by more than 0.5 x 109/L of proliferating granular lymphocytes in the peripheral blood. Because of its rarity, the characteristics of LDGL with T-cell receptor (TCR) gammadelta phenotype (gammadeltaT-LDGL) have not yet been identified. This report describes the clinical, hematological, and immunological findings of four patients with this disease. In two cases, the clinical course was indolent and the other two patients required various therapies. The cells had a common immunophenotype: CD3+, CD4-, CD16+, CD56-, CD57-, CD122-, TCR-gammadelta+, and three were CD8-positive. The immunopurified TCR-gammadelta cells from the patients expressed only Vgamma9 and Vdelta1. Spectratyping and sequencing showed mono- or oligoclonality for TCRgamma and TCRdelta subunit genes. Soluble Fas ligand in sera was significantly elevated in all patients. These findings suggest that gammadeltaT-LDGL qualifies as a distinct disease entity.

To study the relevance of gamma delta T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) gamma delta repertoire and the antigen reactivity of gamma delta clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype. Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.

While V gamma 9/V delta 2 cells dominate among peripheral blood gamma delta T cells in healthy adults, the majority of gamma delta T cells in most HIV-1-infected individuals express V delta 1. We asked whether these elevated levels of V delta 1 T cells were due to clonal expansion. Three-color flow cytometry with monoclonal antibodies against V gamma 2/V gamma 3/V gamma 4, V gamma 4 and V gamma 9 was used to investigate V gamma usage in 27 patients with elevated numbers of V delta 1 T cells. While the relative proportion of V gamma 9 cells among gamma delta T cells was significantly reduced in HIV-1+ individuals (10 +/- 11% vs. 80 +/- 17%, p < 0.001), the fraction of gamma delta T cells using V gamma 5 or V gamma 8 was significantly increased (54 +/- 15% vs. 7 +/- 11%, p < 0.001). In 1 patient, 76% of the V delta 1 cells expressed V gamma 2 or V gamma 3, suggesting clonality of the V delta 1 population. In line with this assumption, analysis of the V delta 1-J delta junctional regions by reverse transcription-polymerase chain reaction (RT-PCR) resulted in products of only one junctional length, as demonstrated by electrophoresis on denaturing gels, and 12 out of 16 (75%) in-frame junctional sequences were identical in this patient. In other HIV-1+ patients, RT-PCR resulted in products of several distinct sizes, also indicating a highly restricted repertoire. After sequencing the V delta 1-J delta junctional regions of 3 additional patients, we found repeated but patient-specific in-frame junctions accounting for 10-30% of the sequenced clones. However, limited V delta 1-J delta junctional diversity was also seen in healthy donors. RT-PCR products from 10 healthy individuals resulted in distinct bands on denaturing gels. In 1 of them exhibiting a single prominent band, 10 out of 17 (58%) sequenced junctions were identical. Two other healthy donors displayed 2/14 and 5/18 identical junctional sequences, respectively. Taken together, our results reveal significant alterations of V gamma usage in HIV-1+ patients, while the V delta 1 junctional repertoire is similarly restricted in HIV-1+ and HIV-1- individuals. Therefore, these data argue against an obligatory clonal expansion of V delta 1-expressing cells during HIV-1 infection.

The V gamma 5 chain of T cell receptor gamma delta is preferentially expressed by murine fetal thymocytes. The fetal V gamma 5 gene is known to be homogeneous and lacks N nucleotide addition. We previously reported that V gamma 5+ cells were detected among donor-derived thymocytes from irradiated C3H/He mice soon after reconstitution with AKR/J fetal liver (FL) cells, but that only a few V gamma 5+ were detected among donor-derived thymocytes from irradiated C3H/He mice reconstituted with adult bone marrow (ABM) cells. The results suggest that preferential use of V gamma 5 is determined at the level of T cell precursor generation. In the present report, we analyzed whether the junctional region of FL-derived V gamma 5 genes in the FL chimeras is of fetal type. Sequencing analysis showed that V gamma 5 genes from FL chimeras contained extensive N nucleotide addition and were diverse both in nucleotide sequences and deduced amino acid sequences. V gamma 5 genes from donor-derived thymocytes of ABM chimeras, which by polymerase chain reaction were revealed to be less frequent than those of FL chimeras, also showed extensive N addition. Furthermore, the terminal deoxynucleotidyl transferase (TdT) gene, which encodes an enzyme that adds N nucleotides into the junctional region, was expressed at high level in the donor-derived thymocytes of FL chimeras and normal 8-week-old AKR/J thymocytes but at low level in day 17 fetal AKR/J thymocytes. Our results suggest that the preferential rearrangement of the V gamma 5 gene is determined at the level of T cell precursor generation but the homogeneous V gamma 5 sequence of fetal type is generated only in the fetal thymic microenvironment where TdT gene expression is low or absent. The nomenclature of murine TcR gamma chains is according to Reilly et al. (Nature 1986. 321: 878). The relationship between the different nomenclature systems is summarized in Takagaki et al. (J. Immunol. 1989. 141: 2112).