Application of the Molecular Analysis of the T-Cell Receptor Repertoire in the Study of Immune-Mediated Hematologic Diseases

Abstract

The basis for the vast recognition spectrum of the T-cell receptor (TCR) can be determined by the rearrangement and recombination of the variable, diversity and joining regions of the variable portions of beta (B) and alpha (A) chains as well as their recombination and modification. Analysis of the TCR rearrangement has been routinely used to detect clonality for the diagnosis of lymphoid malignancies. However, molecular analysis of the TCR repertoire can be a powerful tool in the study of T-cell responses to pathogens and in autoimmune diseases. The concept of the oligoclonality in the context of cellular immune responses is based on the presence of immunodominant T-cell clones within distinct T-cell subpopulations used for analysis. Under normal circumstances, a limited number of clones undergo periodic expansions in reaction to foreign antigens. Under pathologic conditions, though, the derailment of immune regulation allows expansions of specific and potentially pathogenic T-cell clones. For example, large granular lymphocyte (LGL) leukemia illustrates an extreme expansion of a single T-cell clone associated with a distinct autoimmune pathology, which suggests an exaggerated clonal response to a specific antigenic target. In immune-mediated bone marrow failure syndromes, clonal rearrangement of the TCR cannot be detected in unseparated blood or marrow. Nevertheless, individual T-cell clones can significantly expand and may allow for demonstration of oligoclonality in selected T-cell populations. These subpopulations are defined, for example, by a specific beta (B)-chain usage or other phenotypic markers. Given the diversity of the TCR recognition spectrum, the task of identifying immunodominant clonotypes derived from unique complementarity determining region-3 (CDR3) sequences is very complex. However, expanded T-cell clones likely represent immunodominant responses which can be detected on the molecular level using analysis of the individual TCR VB-chain representation, CDR3 size fragment skewing, and determination of the frequency of individual clonotypic sequences. In the future, TCR VB clonotypes may be applied as a diagnostic tool, analogous to serologic markers. As an investigative tool in hematology, molecular analysis of the TCR utilization pattern and the detection of immunodominant clonotypes represents a novel approach in the study of immune-mediated hematologic diseases, such as aplastic anemia (AA), some forms of myelodysplasia (MDS), anti-leukemic immune surveillance, graft-versus-leukemia effects and graft-versus-host disease (GvHD).

Prevalence and clinical association of clonal T-cell expansions in Myelodysplastic Syndrome

“…17,[23][24][25]30,45 Expansion of these pathologic T-cell populations may reflect an underlying autoimmune, antigen-driven process. 25,45 We studied a cohort of 52 patients for the presence of clonal T-cell expansion using combined molecular and flow cytometric methods.…”

“…21 An increased proportion of T cells with a restricted CDR-3 TCR-Vb nucleotide length, which is representative of clonal T-cell expansion, is known as 'TCR-Vb-repertoire skewing'. [22][23][24][25] In normal T-cell homeostasis, a limited number of clones undergo periodic expansion in response to foreign antigens. However, exaggerated clonal T-cell expansion is associated with viral infections, autoimmune diseases and clonal T-cell malignancies.…”

Molecular Analysis of TCR Clonotypes in LGL: A Clonal Model for Polyclonal Responses

“…Somatic rearrangements between the V, D, and J regions give the TCR both its heterogeneity and its fine specificity for Ags. The complementarity-determining region 3 (CDR3) of the ␤-chain (VB) of the TCR is the structure recognizing antigenic peptides in the context of HLA, and the sequence of this unique Ag-specific region can act as a molecular marker, or clonotype, of the disease-specific T cells (32). During rearrangement, transferases add or remove nucleotides at the V␤-D␤ and D␤-J␤ junctions, resulting in CDR3 regions that can vary in length by several amino acids.…”

Large granular lymphocyte (LGL)-like clonal expansions in paroxysmal nocturnal hemoglobinuria (PNH) patients

“…6,7 The TCR repertoire is physiologically extremely diverse, as a result of somatic V-(D)-J rearrangement and the contribution of nongermline N nucleotides; 8 dominance of a few TCRs over the heterogeneous repertoire is typical of oligoclonal T-cell immune response. 9 TCR clonality may be assessed by flow cytometric analysis of Vb usage 10 and by molecular studies, 11,12 including size-distribution analysis and sequencing of the highly variable complementarity determining region 3 (CDR3; the site of peptide binding), giving a unique signature of a T-cell clone or clonotype. Our group has systematically studied cellular immune responses in different hematologic diseases characterized by bone marrow failure.…”