Oligoclonal TRBV gene usage among CD8+ T cells in monoclonal B lymphocytosis and CLL
Year: 2009
Type: article
Abstract: Whereas most malignancies are associated with decreased numbers of circulating T cells, in chronic lymphocytic leukaemia (CLL) T cell numbers are elevated 2–4 times normal. Although T cell numbers are increased, the T cell repertoire is significantly contracted in CLL, with oligoclonal and monoclonal subsets (Goolsby et al, 2000; Rezvany et al, 1999). Rather than promoting an anti-tumour response, these qualitative and quantitative T cell abnormalities may contribute to a tumour microenvironment that promotes progression of the malignant clone (Ghia & Caligaris-Cappio, 2000). Chronic lymphocytic leukaemia patients with mutated immunoglobin heavy chain variable region (IGHV) genes follow a more indolent disease course than those with unmutated IGHV. It has been proposed that IGHV unmutated CLL arise from B cells that mature through a T cell independent pathway, because CD4+ T cell help is required for somatic mutation and isotype switching in the germinal centre reaction. Therefore, IGHV mutated and unmutated CLL may have differing capacities for T cell interaction and immune modulation. A recent report (Palmer et al, 2008) showed that increased numbers of T and ‘natural killer’ (NK) cells relative to the number of CLL lymphocytes in patients with newly diagnosed CLL was associated with both a mutated IGHV and improved prognosis, suggesting that T and NK cell responses precede clinical CLL. Sensitive flow cytometry has been used to determine that small populations of cells with a typical CLL immunophenotype can be found in older adults (Ghia et al, 2004; Rawstron et al, 2002). This condition is termed monoclonal B lymphocytosis (MBL). Because MBL may progress to CLL (Rawstron et al, 2008; Landgren et al, 2009), we sought to clarify if either the number or T cell receptor (TCR) repertoire of T cells differed between IGHV mutated CLL, and IGHV unmutated CLL, and MBL. Twenty untreated CLL patients were identified from a longitudinal CLL patient cohort at Duke University or the Durham VA Medical Centres between October 2006 and August 2007. The clinical and molecular characteristics of this longitudinal cohort of patients have been previously described (Weinberg et al, 2007). A priori, an equal number of IGHV mutated and unmutated patients were selected for this study. No other clinical or molecular features were used to identify or stratify patients. A diagnosis of CLL was based on standard criteria (Weinberg et al, 2007). MBL subjects were identified by flow cytometric screening of unaffected relatives of patients with familial CLL at Duke University or the Durham VA Medical Centre. Eight subjects from five different families with MBL provided blood for T cell analysis. This study was approved by the Institutional Review Boards at Duke University and the Durham VA Medical Centres, and all subjects provided written informed consent. Chronic lymphocytic leukaemia and MBL subject characteristics are summarized in Table I. Patients with IGHV unmutated and mutated CLL were similar in regards to white blood cell (WBC) count, age, lymphocyte doubling time and duration since diagnosis. Similar to prior reports, unmutated IGHV correlated with ZAP70 expression (Fishers exact test, P < 0·01) and surface expression of CD38 (Fishers exact test, P < 0·05). Patients with IGHV unmutated CLL were significantly more likely to be intermediate or high Rai stage (χ2df = 2, P < 0·01). The mean age of CLL patients was 62 years, and the mean MBL age was 71 years. This difference was not statistically significant (NS; two-sided t-test, P = 0·1). Lymphocyte subset enumeration was performed using multi-channel flow cytometry (Table II and Appendix SI). The absolute B cell count was not significantly different in IGHV unmutated compared to mutated CLL patients. Among MBL subjects, the mean absolute B cell count was 0·6 × 109 cells/l and the MBL clone size was small (< 0·15 × 109 cells/l) in seven of the eight subjects. The CD4+ count was similar in IGHV unmutated CLL, IGHV mutated CLL and MBL subjects. The CD8+ count was higher in IGHV unmutated compared to IGHV mutated CLL patients (0·89 vs. 0·59 × 109 cells/l, NS), and was significantly higher in IGHV unmutated CLL than MBL subjects (0·89 vs. 0·27 × 109 cells/l, P < 0·05). Similarly, the ratio of CD4+:CD8+ T cells was significantly lower in IGHV unmutated CLL compared to IGHV mutated CLL or MBL (1·6 vs. 2·6, P < 0·05 and 1·6 vs. 4·5, P < 0·01, respectively). NK T cell numbers were not significantly different among IGHV unmutated CLL compared to IGHV mutated CLL or MBL. To estimate the CD4+ and CD8+ T cell repertoire diversity, we coupled polymerase chain reaction (PCR) amplification of the CDR3 length polymorphism of 23 TRBV families (‘spectratyping’) with a novel, quantitative method for analysis of TCR repertoires by using SpA (Kepler et al, 2005) (Appendix SI, Fig S1). CD4+ and CD8+ T cells were positively selected using anti-CD4 and anti-CD8 monoclonal antibodies conjugated to magnetic beads according to the manufacturer’s directions (Dynabeads®; Invitrogen, Carlsbad, CA, USA). Table II shows the calculated mean divergence coefficients (a calculated unitless value to quantify the degree of ‘skewness’ from a pseudo-Gaussian distribution) for the T cell spectratypes. Combined analysis of all CLL patients showed the mean CD8+ TCR spectratype divergence was 0·32, significantly higher than the mean CD4+ divergence (0·07, two-sided t-test, P < 10−6). Oligoclonal and monoclonal TRBV families were frequently observed among CD8+ cells, but rarely observed among CD4+ cells. We did not observe restriction of any specific TRBV families across patients. Given our hypothesis that IGHV unmutated CLL and mutated CLL may have different capacities for T cell interaction, we compared the TCR repertoire between these patient groups. The mean CD8+ TCR spectratype divergence was greater in IGHV unmutated CLL than in mutated CLL among CD8+ cells, but this difference was not statistically significant (0·36 vs. 0·29, two-sided t-test, P = 0·08, NS). Next, we evaluated whether MBL have a restricted TCR repertoire. The mean CD8+ and CD4+ divergence among MBL subjects was similar to that observed in CLL patients (mean CD8+ divergence: 0·35 for MBL vs. 0·32 for all CLL; mean CD4+ divergence: 0·06 for MBL vs. 0·07 for all CLL, Table II). We performed a multivariate correlation to identify variables associated with restriction of the CD8+ repertoire in MBL. Patient characteristics, the absolute number of different lymphocyte subsets and ‘MBL characteristics’ (ZAP70 status, CD38 status and the ‘MBL percentage’, defined as the number of MBL cells divided by the total number of CD19+ B cells) were considered. A correlation between the absolute number of CD4+ cells and the diversity of the CD8+ repertoire was observed: higher numbers of CD4+ cells correlated with more diverse CD8+ repertoire (ρ = −0·82, P < 0·05). Loss of specific CD4+ subsets may cause contraction of the CD8+ repertoire; alternatively some CLL and MBL may have the capacity to modulate both the CD4+ and CD8+ subsets as independent effects. Of the remaining variables, a correlation was observed between the MBL percentage and mean CD8+ divergence (ρ = 0·52, NS). There was no correlation of mean CD8+ divergence with MBL subject age (ρ = 0·20, NS). Because abnormalities in the T cell compartment are detectable at time of CLL diagnosis (Palmer et al, 2008), we speculate that MBL lymphocytes may have a capacity for T cell immune modulation similar to CLL lymphocytes. The absolute number of MBL lymphocytes was quite small in this cohort of MBL subjects, suggesting that acquisition of an immunomodulatory phenotype is an early event in CLL leukaemogenesis. Further investigation is required to test this hypothesis. Furthermore, MBL is relatively common among the elderly (Ghia et al, 2004), and the great majority of MBL probably do not progress to CLL (Rawstron et al, 2008). We identified qualitative abnormalities in the T cell repertoire in this otherwise healthy group of elderly individuals, suggesting that MBL may contribute to accelerated immune senescence. MC Lanasa is a Fellow of the Leukaemia and Lymphoma Society of America. This research is supported by the Leukaemia and Lymphoma Society of America, the Bernstein Fund for Leukaemia Research, the VA Research Service, and a grant from the Institute for Medical Research, Inc. We thank the study subjects for their willingness to participate in this study, and the haematology-oncology nurses and physician assistants for their special help. The authors thank Drs Nelson Chao, Congxiao Liu, Thomas Kepler and Min He for their helpful discussions. Flow cytometry was performed in the Duke Human Vaccine Institute Flow Cytometry Core Facility that is supported by the National Institutes of Health award AI-51445. Figure S1. Example spectratype histograms. Appendix SI. Lymphocyte enumeration and CDR3 size spectratyping methods. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Source: British Journal of Haematology
Language: English
Subfield: Genetics
Field: Medicine
Domain: Health Sciences
Open Access status: green
Awards: G2755446375, G5064229337
OpenAlex ID: W2166979726