Clonal predominance of CD8(+) T cells in patients with unexplained neutropenia

Abstract

Objective: T-cell-mediated autoimmunity may be involved in some cases of idiopathic neutropenia. We hypothesized that a precise T-cell receptor repertoire analysis may uncover cytotoxic T-cell (CTL) expansions that are less pronounced than those seen in T large granular lymphocyte leukemia (T-LGL), but are pathophysiologically analogous and thus can serve as markers of a T-cell-mediated process.

Materials and methods: Using rational algorithms for T-cell receptor analysis and in vivo tracking of CTL responses previously established in our laboratory, we studied patients with unexplained chronic neutropenia (n = 20), T-LGL (n = 15), and healthy controls (n = 12). We further investigated the involvement of soluble inhibitory factors by coculture assays. To determine the level of immune activation, we studied interferon-gamma expression in CD8(+)cells using Taqman polymerase chain reaction.

Results: Fifteen expanded (immunodominant) CTL clones were detected in 12 of 20 patients. In comparison to LGL leukemia, these clones were less immunodominant, but clearly discernible from subclinical lymphoproliferations in controls. As a surrogate of cytotoxic activity, we found markedly increased production of interferon-gamma in most of the neutropenia patients, irrespective of the presence of immunodominant CTL clones.

Conclusions: These results suggest that, while immunodominant CTL clones are detectable in a proportion of patients only, CTL-mediated pathophysiology may be a general mechanism operating in idiopathic neutropenia. Oligogoclonal CTL expansions in chronic neutropenia may indicate an ongoing autoimmune process, while highly polarized monoclonalities in a subset of neutropenic LGL patients may represent the "extreme" end of the clonal continuum.

Figure 1

Experimental strategy for the detection and molecular characterization of clonal immunodominant cytotoxic T-cell (CTL) populations. Organizational chart illustrates the rational diagnostic approach for the detection of cryptic clonal CTL expansions in neutropenia patients. For details see text.

Figure 2

Clonotypic analysis of patients with neutropenia. (A) Quantitative detection of disease-associated immunodominant clonotypes. Clonotypic size was measured using Taqman polymerase chain reaction (PCR) in five neutropenia patients, five large granular lymphocyte leukemia (LGL) patients and five healthy controls. Each patient-specific clonotypic assay was also performed on other patients and controls. For relative quantification the original patient sample harboring the expanded clonotype as initially identified by sequencing was used as a calibrator (*). Values are shown in log₁₀ scale. ND = not detectable. (B) Clonotypic T-cell receptor (TCR) CDR3 expansions in neutropenia, typical LGL leukemia and healthy controls. Expansions of 15 disease-associated immunodominant clonotypes in neutropenia patients (n = 12) are shown in relation to the 15 highly expanded monoclonal clonotypes in typical LGL leukemia (n = 15) and 25 redundant clonotypes in healthy controls (n = 12). (C) Levels of interferon-γ (IFN-γ) expression in patients with neutropenia. Expression of IFN-γ in sorted CD8⁺ cells of neutropenia patients and healthy controls was measured using Taqman PCR. Mean expression values of sorted CD8⁺ cells from 10 healthy controls were used as a baseline (calibrator). Gray bars represent patients without detectable immunodominant expansions (log₁₀-fold increase vs controls: 1.07 ± 0.69), black bars correspond to patients in whom expanded clones were identified (log₁₀ fold increase vs controls: 0.84 ± 1.42).