CDR3 of the BV chain of the TCR is a nongermline–encoded hypervariable region directly related to T cell recognition of specific peptides in the appropriate HLA context. CDR3 thus defines a unique clonotype. Somatic rearrangement leads to six to eight amino acid differences among CDR3 within each BV chain, detectable by a variety of molecular methods and apparent as deviation from the normal Gaussian size distribution. Murine studies have shown that the cellular immune response to even complex protein antigens is relatively restricted, to one or a few CDR3 size peaks, from which a few dominant specific T cell clones have been inferred (31
). Both the normal immune response and autoimmune disease show CDR3 restriction, consistent with either epitope-driven or BV region–restricted responses, and both public and private TCR specificities have been identified in animal models.
Parallel investigations of patients with immune-mediated disease have been more difficult to interpret. First, peripheral blood cells may not reflect local and pathological T cell infiltration. Second, in vitro growth may artificially skew representation of BV. Third, many studies compare patients with diverse clinical manifestations and/or different histocompatability backgrounds. Fourth, the human immune response is both intrinsically more complex than the murine and evolves in an individual over many decades. Fifth, both young and old humans can show surprising degrees of BV skewing and nonpathological expansion of T cell clones (24
). Nevertheless, investigations of CDR3 in humans have generally validated animal observations, as applied to the immune response to viral infections (35
), immunity against tumors and during graft-versus-host disease, as part of graft-versus-tumor effects (37
), and during recovery from stem cell transplant (24
). TCR usage has been extensively applied to human autoimmune diseases, and although the confidence of identification of driving epitopes in these conditions varies, this analysis has often served as a surrogate for antigen specificity studies when the autoantigen is unknown (7
). In many human autoimmune diseases, restricted CDR3 usage has been inferred from skewed size patterns, which may be particularly marked if locally infiltrating T cells are examined; the T cell response, as in animals, has been limited, and both private and shared public specificities have been described in detail when the antigens, such as myelin basic protein, were known. Indeed, these studies have been the basis for T cell vaccination strategies, in which the TCR itself is the antigen and the anti-idiotype response is therapeutically beneficial.
To maximize the likelihood of establishing this pattern in acquired AA, a disease in which there is still considerable debate as to underlying pathophysiology, the mechanism of action of effective therapies, and no suitable animal model, we chose first to examine the TCR only in lymphocytes that were activated and derived from the target organ, the marrow. In addition, our patients were similar in crucial but also common clinical characteristics, in showing evidence of T cell activation in blood, a good response to immunosuppressive therapy, the presence of an expanded PNH clone, and HLA-DR2 phenotype. To perform extensive functional and phenotypic characterization, we also immortalized the selected T cells; although artificial biasing of T cell representation as a result of viral infection is theoretically possible, SHV-2 has not been reported to produce TCR skewing in other published studies (25
). Spectra typing did show the presence of multiple skewed BV families in patient bone marrow, whereas a single dominant clone emerged after cell culture and viral transformation; nevertheless, this dominant clone, and not other BV clones obtained from the same bone marrow, showed a cytokine profile suggestive of activation, suggesting that viral transformation does not result in obligatory alteration in T cell function. We obtained functionally active T cell clones that were specifically cytotoxic for autologous marrow CD34 cell targets from bone marrow obtained at disease presentation. That such a large number of both CD4 and CD8 clones in our first patient were identical suggests oligoclonal expansion in this individual. That the same TCR (for CD4) was detected in large proportions of activated lymphocyte populations from other patients indicated that public specificities were being detected (and also argued against artifact due to the viral infection protocol used to obtain the clones); as the CDR3 region from the AA cases was not detectable in normal individuals, their association with disease seems likely. The proportion of T cells bearing the abnormal TCR fell with effective treatment in three of four cases, consistent with a pathophysiological role. Our data thus provide strong direct evidence of an autoimmune pathophysiology of AA and describe similarity of the molecular genetic level between this form of bone marrow failure and other types of organ-specific, immune-mediated tissue damage and destruction.
In the current work, we present data derived from studies of CD4 cell clones, mainly because of the readier availability of other class II–matched patients and controls. Although CD4 cytotoxic lymphocytes are well described, our CD4 cell clones were particularly potent in this assay, perhaps reflecting their clonal origin from activated cells. Similar high activity has been described for other CD4 cell clones derived from AA patients (43
). In our study, both the CD4 and CD8 clones appeared to be Th1/Tc1 cells, but the immortalized CD8 cell clones await more intensive study utilizing class I matched target cells.
For AA, our results should be compared to a few previous studies of limited numbers of patients. When Swiss investigators performed spectratyping of unfractionated blood and marrow lymphocytes of severely affected patients, the expression pattern was broadly normal (45
). Individual patients showed size skewing, indicative of selective CDR3 usage, but the BV subfamilies differed for each case, and there was no correlation of CDR3 size distribution and enhanced BV usage (both BV5 and BV13 were among a total of seven abnormal CDR3 patterns). Nakao and coworkers studied patients whose immunological disease could be inferred from the dependence of their blood counts on CsA administration; these patients showed a skewed CDR3 size pattern, in contrast to results with refractory or remitted cases (46
). Furthermore, two patients showed homology in the amino acid sequence of their expanded CDR3 domains. Individual helper (43
) and cytotoxic (44
) T cell clones have been isolated and functionally characterized as responsive to or cytotoxic for target hematopoietic cells. CDR3 skewing was found in multiple BV families, and BV15 CDR3 sequencing showed dominant clones in these five patients. An abnormal T cell repertoire has also been found in PNH (47
), which is often diagnosed concurrently with AA.
In our study, the CDR3 genotype shared among all five patients is probably the signature of a pathogenic T cell clone. If verified on examination of larger numbers of patients of similar clinical characteristics, CDR3 analysis may be a highly sensitive clinical assay for diagnosis and to evaluate the effectiveness of treatment. For example, we noted that our current therapeutic regimen reduced but did not eradicate evidence of this clone, consistent with the notoriously high rate of relapse after immunotherapy. CDR3 fine analysis may serve to distinguish subsets of marrow failure, such as post-hepatitis AA and immune-mediated pancytopenia in the myelodysplastic syndromes. Ultimately, the generation of immortalized T cell clones derived from cells clearly involved in the pathophysiology of the marrow disease offers a method for the identification of the elusive antigen(s) in AA, through screening of cell lines, subcellular fractions, peptide libraries, and randomly generated tetramers. Even at a comparatively early stage of investigation, however, we observed that T cell cytotoxicity was relatively specific for putative target antigens present only at disease presentation. Although the precise nature of the antigen must be determined, these data suggest that persistence of low levels of pathogenic T cell clones may be innocuous until antigen reappears to drive their expansion.