Limited heterogeneity of T cell receptor BV usage in aplastic anemia

Abstract

Immune mediation of aplastic anemia (AA) has been inferred from clinical responsiveness to immunosuppressive therapies and a large body of circumstantial laboratory evidence. However, neither the immune response nor the nature of the antigens recognized has been well characterized. We established a large number of CD4 and CD8 T cell clones from a patient with AA and analyzed their T cell receptor (TCR) usage. Most CD4 clones displayed BV5, whereas most CD8 clones displayed BV13. We found sequence identity for complementarity determining region 3 (CDR3) among a majority of CD4 clones; the same sequence was present in marrow lymphocytes from four other patients with AA but was not detected in controls. The dominant CD4 clone showed a Th1 secretion pattern, lysed autologous CD34 cells, and inhibited their hematopoietic colony formation. In three of four patients, successful immunosuppressive treatment led to marked decrease in clones bearing the dominant CDR3 BV5 sequence. These results suggest surprisingly limited heterogeneity of the T cell repertoire in an individual patient and similarity at the molecular level of the likely pathological lymphocyte response among multiple patients with AA, consistent with recognition of limited numbers of antigens shared by individuals with the same HLA type in this disease.

Figure 1

Analysis of TCR BV CDR3 region size profiles. CDNA of 22 BV families from P1, P2, P3, and P4 were amplified using BV primer and a fluorescent BC primer, and the PCR products were analyzed for CDR3 size. x-axis, CDR3 size; y-axis, relative fluorescence units.

Figure 2

Strategy to establish T cell clones. Activated T cells (bearing CD2 and CD69 antigens) were obtained from BMMCs by flow cytometric sorting. Cells fractions were resuspended in complete medium, cultured with IL-2 and CD28, and then immortalized with SHV-2, after limiting dilution, 112 CD4⁺ T cell clones were obtained. TCR BV usage was analyzed by PCR: BV5 was used in 26 of 32 T cell clones, and BV5 sequence analysis showed 20 of 26 have an identical CDR3 region.

Figure 3

Characterization of the JZ1.1 T cell clone. (a) Immunophenotype by flow cytometry. JZ1.1 was stained with mAb’s directed to CD3, CD4, CD8, CD45RA, CD45RO,CD28, HLA-DR, TCRa/β, CD95, CD86, CD11a (dashed lines) as described in Methods; subclass-matched IgG reagents served as controls (solid lines). (b) Cytokine secretion analysis by flow cytometry. JZ1.1cells were subjected to intracellular staining using fluorescent conjugated-mAb’s to IL-2, IL-4, IL-10, or IFN-γ. Data are representative of three independent experiments.

Figure 4

Functional analysis of JZ1.1. (a) Cytotoxicity for autologous CD34 cells, normal CD34⁺ cells (N1, HLA-matched for DR2; N2, DR-mismatched). CD34 cells were labeled with calcein-AM and incubated with differing numbers of JZ1.1 cells in a 4-hour cytotoxicity assay. (b) Blocking of cytotoxicity. Autologous CD34⁺ cells were incubated with JZ1.1 cells in medium alone or with addition of 10 μg/ml of the indicated mAb’s. Mean percentage of specific lysis was determined from triplicate cultures after 4 hours of incubation. (c) Effect of JZ1.1 on autologous hematopoietic progenitor cell growth. Ten thousand freshly isolated CD34 cells were incubated in medium alone or with 1 × 10⁵ JZ1.1 cells for 16 hours and then mixed with methylcellulose medium containing hematopoietic growth factors; colonies were enumerated after 2–3 weeks of culture. Data are expressed as mean ± SD of replicate plates.

Figure 5

TCR BV5 usage in AA patients in Southern blot analysis. BV5 PCR products from P2, P3, P4, P5 and normal individuals were electrophoresed on a 1.5% agarose gel (lower panel) and subjected to Southern hybridization using a biotinylated specific probe for the JZ1.1 N-D-N region sequence (upper panel). The numbers represent the calculated ratio between the signal intensities obtained with the specific BV5 /JZ1.1 compared with a universal BV5-probe. Note that the decrease in the BV5 JZ1.1 / BV5 ratio corresponds to the increase in the contribution of the specific clone to the total BV5 spectrum detected in a particular patient.

Figure 6

BV5 CDR3 size pattern and response to therapy. BV5 cDNA from P1, P2, P3, and P5 were amplified using a specific primer and a fluorescent BC primer; and the size of the PCR products was compared before and after treatment. x-axis, CDR3 size; y-axis, relative fluorescence units.