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

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.

Phenotypic differences between healthy effector CTL and leukemic LGL cells support the notion of antigen-triggered clonal transformation in T-LGL leukemia.

T cell large granular lymphocyte leukemia (T-LGL) is a chronic clonal lymphoproliferation of CTL. In many ways, T-LGL clones resemble terminal effector CTL, including down-modulation of CD28 and overexpression of perforin, granzymes, and CD57. We studied the transcriptome of T-LGL clones and compared it with healthy CD8+CD57+ effector cells as well as CD8+CD57- populations. T-LGL clones were sorted based on their TCR variable beta-chain restriction, and controls were obtained by pooling cell populations from 14 donors. Here, we focus our analysis on immunological networks, as immune mechanisms play a prominent role in the etiology of bone marrow failure in T-LGL. Informative genes identified by expression arrays were studied further in an independent cohort of patients using Taqman PCR, ELISA assays, and FACS analysis. Despite a strikingly similar gene expression profile between T-LGL clones and their healthy counterparts, important phenotypic differences were identified, including up-modulation of TNFRS9, myeloid cell leukemia sequence 1, IFN-gamma, and IFN-gamma-related genes, and several integrins/adhesion molecules. In addition, T-LGL clones were characterized by an overexpression of chemokines and chemokine receptors that are typically associated with viral infections (CXCL2, Hepatitis A virus cellular receptor 1, IL-18, CCR2). Our studies suggest that immunodominant LGL clones, although phenotypically similar to effector CTL, show significantly altered expression of a number of genes, including those associated with an ongoing viral infection or chronic, antigen-driven immune response.

Hemochromatosis-associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts.

We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional iron-overload. Hence, we hypothesized RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this myelodysplastic syndromes (MDS) subset. We studied a cohort of 140 MDS patients: 42 with RARS, 10 with increased ringed sideroblasts, and 96 with other forms of MDS (43 RA, 27 RAEB, 17 RAEB-T, 8 MDS/MPD, 1 CMML). Patients were genotyped using restriction fragment length polymorphism, designed to detect C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygosity for C282Y mutation in RARS patients compared with a large control population of matched race individuals (21 vs. 9.8% in controls, P = 0.03); H63D genotype was not significantly increased. Frequency of HFE variation in other MDS subtypes failed to differ significantly from controls. Within this group, we included patients with a rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt). 10/14 RARSt patients were carriers of either C282Y or H63D allele significantly increased compared with the combined prevalence in a healthy population (71 vs. 33%, P < 0.01). We found expected distribution of mutant HFE alleles in patients with other forms of MDS (9.1 vs. 9.8%, P = 0.82). Increased prevalence of HFE gene mutations is not a generalized feature of MDS, but some subgroups of MDS, especially those characterized by excessive accumulation of ringed sideroblasts, exhibit C282Y mutations at a higher frequency than in other forms of MDS and healthy controls.

Immunogenetic factors determining the evolution of T-cell large granular lymphocyte leukaemia and associated cytopenias.

T-cell large granular lymphocyte leukaemia (T-LGL) is a chronic clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGL presents with cytopenias, often accompanied by autoimmune diseases, suggesting clonal transformation arising from an initially polyclonal immune response. Various immunogenetic predisposition factors, previously described for both immune-mediated bone marrow failure and autoimmune conditions, may promote T-LGL evolution and/or development of cytopenias. The association of T-LGL was analysed with a number of immunogenetic factors in 66 patients, including human leucocyte antigen (HLA) and killer-cell immunoglobulin-like receptor (KIR) genotype, KIR/KIR-L mismatch, CTLA-4 (+49 A/G),CD16-158V/F, CD45 polymorphisms, cytokine single nucleotide polymorphisms including: TNF-alpha (-308G/A), TGF-beta1 (codons 10 C/T, 25 G/C), IL-10 (-1082 G/A), IL-6 (-174 C/G), and IFN-gamma(+874 T/A). A statistically significant increase in A/A genotype for TNF-alpha-308, IL-10-1082, andCTLA-4 +49 was observed in T-LGL patients compared with control, suggesting that the G allele serves a protective role in each case. No association was found between specific KIR/HLA profile and disease. KIR/KIR-L analysis revealed significant mismatches between KIR3DL2 and KIR2DS1 and their ligands HLA-A3/11 and HLA-C group 2 (P = 0.03 and 0.01 respectively); the biological relevance of this finding is questionable. The significance of additional genetic polymorphisms and their clinical correlation to evolution of T-LGL requires future analysis.

Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome.

Immune mechanisms are involved in the pathophysiology of aplastic anemia (AA) and myelodysplastic syndrome (MDS). Immune inhibition can result from cytotoxic T cell (CTL) attack against normal hematopoiesis or reflect immune surveillance. We used clonally unique T-cell receptor (TCR) variable beta-chain (VB) CDR3 regions as markers of pathogenic CTL responses and show that while marrow failure syndromes are characterized by polyclonal expansions, overexpanded clones exist in these diseases and can serve as investigative tools. To test the applicability of clonotypic assays, we developed rational molecular methods for the detection of immunodominant clonotypes in blood and in historic marrow biopsies of 35 AA, 37 MDS, and 21 paroxysmal nocturnal hemoglobinuria (PNH) patients, in whom specific CDR3 sequences and clonal sizes were determined. CTL expansions were detected in 81% and 97% of AA and MDS patients, respectively. In total, 81 immunodominant signature clonotypes were identified. Based on the sequence of immunodominant CDR3 clonotypes, we designed quantitative assays for monitoring corresponding clones, including clonotypic Taqman polymerase chain reaction (PCR) and clonotype-specific sequencing. No correlation was found between clonality and disease severity but in patients treated with immunosuppression, truly pathogenic clones were identified based on the decline that paralleled hematologic response. We conclude that immunodominant clonotypes associated with marrow failure may be used to monitor immunosuppressive therapy.