New Molecular Strategies for Detection and Quantitation of Clonal Cytotoxic T Cell Responses in Myelodysplasia and Aplastic Anemia.

Clinical similarities suggest that a T cell-mediated immune attack is involved not only in the pathophysiology of AA but also certain forms of MDS. Such a mechanism may result from a CTL attack directed against normal hematopoiesis or may reflect an immune surveillance reaction. Unlike in clonal T-LGL, immune responses in bone marrow failure states are polyclonal. We hypothesized that if immunodominant CTL clones are involved in the mechanisms of cytopenia in MDS and AA, the clonal size should correspond to the activity of disease. New molecular technologies based on TCR B-chain CDR3 region analysis allow for identification and monitoring of immunodominant T cell clones. These techniques, initially established in T-LGL (Wlodarski et al, Blood 2005), serve here as a basis for the systematic analysis of polyclonal CTL responses in bone marrow failure. We analyzed blood and paraffin-embedded marrow specimens from 30 AA, 21 PNH, 36 MDS and 60 T-LGL patients. By crude flow cytometric VB utilization analysis, we observed a continuum of VB skewing patterns from extreme monoclonal VB over-representation in T-LGL to multiple smaller expansions identified in AA and MDS. To determine whether the VB expansions are due to oligoclonal CTL proliferations, we have amplified and cloned the VB CDR3 regions of expanded VB families and sequenced large numbers of clonotypes. In both AA and MDS, most of the expanded VB families showed oligoclonality consistent with the presence of immunodominant clones. Pathogenic immunodominant clonotypes were not present in HLA-matched sibling donors or healthy identical twins discordant for the disease. A novel TCR VB multiplex PCR was applied for direct identification of immunodominant clones in the corresponding paraffin-embedded biopsies. Using this technique we retrospectively demonstrated the presence of skewed pattern of CTL clones in marrow biopsies of AA and some MDS patients; we also identified immunodominant clones in most of the biopsies studied.

Moreover, biopsy-derived clonotypes were detected in blood; in many instances these clonotypes were also immunodominant as precisely quantitated by a novel clonotypic Taqman PCR utilizing CB- and clonotype-specific primer and JB-specific Taqman probes. Using a modified clonotypic PCR, blood-derived clonotypes were also found in patients’ bone marrow biopsies, but not in controls. The frequency of dominant VB CDR3 clonotypes and the overall TCR repertoire were measured before and after administration of immunosuppressive therapy. Notably, the size of immunodominant clones decreased parallel to hematopoietic improvement, while the overall variability of TCR repertoire increased. Conversely, nonpathogenic clones remained unchanged. Globally, our analysis showed that immunodominant CTL clonotypes can be detected at comparable rates in AA and MDS irrespective of the morphologic subtype. Immunodominant clones derived from blood or even historical marrow specimens can be used as markers of the activity of the disease. Beyond the previously reported molecular TCR analysis in AA and MDS, we show that the CDR3 sequences of immunodominant clones can be exploited to design individualized quantitative assays that would facilitate recognition of clonotypic patterns in patients. In the future, the analysis of CDR3 amino acid sequence patterns may be used for structural comparisons of CTL clones in MDS and AA.