Citations

Abstract We have studied the molecular diversity of the allogeneic TCR repertoire specific for the HLA-DR4 variant, DRB1*0404, by analyzing the V beta gene segment usage of CD4+ T cell clones from nine different donors and comparing the V beta repertoire in allospecific and unselected T cell populations. To investigate the forces shaping the repertoire of the alloreactive T cell response we compared the diversity of TCR specificities utilized in very similar and in disparate responder/stimulator combinations. Six of the responders shared the HLA-DRB1*0401 allele, which differs from the HLA-DRB1*0404 allele by only two amino acid substitutions at positions 71 and 86 of the HLA-DR beta 1 chain; three responders expressed HLA-DRB1 alleles with extensive polymorphisms compared with the stimulator HLA-DRB1 allele. In all nine responders, TCR specificities recruited to recognize HLA-DRB1*0404 were strongly biased toward the dominant usage of one to three V beta elements. Despite some degree of heterogeneity between different responders, the overall pattern was very similar with a hierarchy of TCR V beta elements expressed by HLA-DRB1*0404 specific T cells. A core group of V beta elements (V beta 6, 5, 2, 13.2, 18, and 7) was preferentially used, whereas other V beta elements including V beta 3, 4, and 8 were infrequently used or not used at all. Sequencing of the VDJ beta region from selected T cell clones showed no junctional bias associated with the preferential V beta gene segment usage. Surprisingly, the T cell diversity in focused and in complex alloreactive responses were equally biased. The finding that HLA-DR4+ and DR4- donors exhibited a similar bias supports the interpretation that the alpha-helical HLA-DR structure of the stimulator cell has a dominant contribution in shaping the alloreactive repertoire.

Abstract Glucocorticoid (GC) is an effective anti-myeloma agent by directly inducing apoptosis and growth inhibition in myeloma cells. The anti-myeloma action of GC is mediated by its intracellular receptor, the glucocorticoid receptor (GR). The response to GC is variable among myeloma patients and resistance to glucocorticoid treatment develops almost invariably. The mechanism regulating glucocorticoid-sensitivity in not well understood, although it has been hypothesized that the regulation might occur at both the receptor (GR) and post-receptor level. In MM.1 myeloma cell lines, we observed significant down-regulation of GR expression as the cells develop GC-resistance, suggesting that GR expression level is an important determinant of GC-response. Here we present evidence that GR expression level directly correlates with the clinical outcome in myeloma patients who were treated with GC-containing regimens. First, using a quantitative real-time PCR analysis, we performed a retrospective analysis of GR gene expression on bone marrow plasma cell samples from 35 patients with newly diagnosed myeloma who were then treated on the ECOG study E1A00. This is a phase 3 trial which randomized patients to dexamethasone (dex) alone vs. dex in combination with thalidomide. Pre-treatment expression levels of GR-alpha, GR-beta and GR-P splicing isoforms were measured for 35 patients, of whom, 10 had post-treatment samples available. Interestingly, a large variation of the GR expression levels was observed across the patient samples, with the GR-alpha being the predominant isoform. However, a clear correlation between GR expression and treatment response was not seen, likely due to the small sample size. To access a larger patient pool, we retrospectively analyzed the GR expression levels using the cDNA microarray data from the Arkansas group on 351 newly diagnosed myeloma patients treated with Total Therapy 2, of which dexamethasone was an essential component throughout the treatment course. Of the 351 patients, both event-free (EFS) and overall survivals (OS) were compared between 49 patients (14%) with low levels of GR expression and the rest (86%) with high levels of GR expression. Strikingly, the cases with low levels of GR expression were associated with statistically significantly worse EFS and OS compared to those with high levels of GR expression, with the 5-year EFS of 20% vs. 52% and OS of 34% vs. 68%, P < 0.01. In summary, we have shown for the first time that the baseline level of glucocorticoid receptor expression in newly diagnosed myeloma patients predicts their clinical response to GC-containing regimen and the overall clinical outcome.