Ross D. Brown

The prognostic significance of T cell receptor β gene rearrangements and idiotype-reactive T cells in multiple myeloma

Clonal T cell populations with idiotype specificity are present in the peripheral blood of a proportion of patients with multiple myeloma. We have identified the presence of both T cell sub-populations with a specificity for autologous immunoglobin fragments and T cell receptor β gene rearrangements in peripheral blood samples of patients with myeloma. T cell receptor β gene rearrangements were detected in 38 of 119 patient samples (32%) and were more common in progressive disease (70%), than at diagnosis (25%) or in stable disease (23%). The 38 patients who had T cell receptor β gene rearrangements detected at any time had a better overall survival (median not yet achieved) than the patients who never had rearrangements detected (median 45 months, n = 49;χ 2 = 6.2, P < 0.01). All 12 patients with T cell receptor β gene rearrangements at diagnosis are still alive whereas the median survival for 28 patients with a germline configuration at diagnosis was 40 months (χ 2 = 5.8, P > 0.01). The presence of T cell receptor β gene rearrangements even conferred a survival advantage during progressive disease (median survival 44 months vs 19 months;χ2 = 8.7, P < 0.003). Two colour flow cytometry with biotinylated autologous immunoglobulin fragments demonstrated idiotype-reactive T cells in the peripheral blood of five out of 15 patients all of whom had T cell gene rearrangements. The remaining 10 patients had neither idiotype-reactive T cells nor a detectable T cell receptorβ gene rearrangement in concurrent samples. Thus in patients with myeloma there was a good correlation between the presence of T cell receptor β gene rearrangements and idiotype-reactive T cells. Patients with a rearranged T cell receptor β gene had a significantly better prognosis.

The labelling index of primitive plasma cells determines the clinical behaviour of patients with myelomatosis

For patients with multiple myeloma the most important laboratory correlate of prognosis and disease activity is the bromodeoxyuridine (BrdUrd) plasma cell labelling index (LI). However, the traditional immunofluorescent microscope LI technique, like other manual enumeration assays, can suffer from poor precision and accuracy. In this study the LI of different subpopulations of plasma cells (CD38++) as determined by flow cytometry was correlated with disease state. The mean LI of the total CD38++ population was significantly higher (2.7±0.4%) than the LI determined by the traditional slide technique (0.6±0.1%) for 65 samples tested. Primitive plasma cells (CD38++, CD45++) had a higher labelling index than mature plasma cells (CD38++, CD45−) (7.0±1.3% v 1.8%±0.3%) and in one patient the LI of the primitive plasma cells was 46%. In addition, the LI of the mature plasma cells was lower than the total plasma cell population. As expected, there was a significant difference between the LI of patients in plateau phase and progressive disease but this difference was greatest when the LI of the primitive plasma cells was studied (9.2±2.9% v 2.2±0.7%; z=19.9, P<0.001). This study has raised some concerns about the sensitivity and accuracy of the traditional labelling index and has shown that the increased LI associated with progressive disease is almost entirely attributable to an increase in the LI of the primitive plasma cell subpopulation and that the LI of primitive plasma cells provides a more clinically significant correlation with disease status than the traditional assay.

Long-term survival in multiple myeloma is associated with a distinct immunological profile, which includes proliferative cytotoxic T-cell clones and a favourable Treg/Th17 balance

Despite improved outcomes in multiple myeloma (MM), a cure remains elusive. However, even before the current therapeutic era, 5% of patients survived >10 years and we propose that immune factors contribute to this longer survival. We identified patients attending our clinic, who had survived >10 years (n=20) and analysed their blood for the presence of T-cell clones, T-regulatory cells (Tregs) and T helper 17 (Th17) cells. These results were compared with MM patients with shorter follow-up and age-matched healthy control donors. The frequency of cytotoxic T-cell clonal expansions in patients with <10 years follow-up (MM patients) was 54% (n=144), whereas it was 100% (n=19/19) in the long-survivors (LTS-MM). T-cell clones from MM patients proliferated poorly in vitro, whereas those from LTS-MM patients proliferated readily (median proliferations 6.1% and 61.5%, respectively (P<0.0001)). In addition, we found significantly higher Th17 cells and lower Tregs in the LTS-MM group when compared with the MM group. These results indicate that long-term survival in MM is associated with a distinct immunological profile, which is consistent with decreased immune suppression.

T‐cell expansions in patients with multiple myeloma have a phenotype of cytotoxic T cells

The presence of T‐cell clones in peripheral blood has been previously shown to be associated with a survival advantage in patients with multiple myeloma and suggests that the expanded T‐cell populations may be involved in an anti‐tumour response. We studied the T‐cell receptor (TCR) repertoire of 38 patients with myeloma to identify and characterize the expanded T‐cell populations by flow cytometry. T‐cell expansions were found in 79% of the patients. The expansions occurred randomly among the 21 variable regions of the TCR β chain (Vβ) studied, representing 62% of the V‐β repertoire, and were stable during an 18‐month follow‐up. The phenotype of the expanded V‐β populations was predominantly CD8+, CD57+, CD28− and perforin+, which differed significantly from the other non‐expanded Vβ populations. The expression of the apoptosis markers Fas (CD95) and bcl‐2 were similar between the expanded and non‐expanded Vβ populations. In conclusion, expanded T‐cell populations were frequent in patients with myeloma, they remained unchanged during follow‐up and had phenotypic characteristics of cytotoxic T cells. These data add further support to the concept that the T‐cell expansions may have an immunoregulatory role in myeloma.

The Expression of T Cell Related Costimulatory Molecules in Multiple Myeloma

Presentation of tumour antigen by malignant cells not expressing costimulatory molecules is considered to be a major cause of the failure of the hosts immune response against tumours. This study has determined the expression of the B7 family of costimulatory molecules on malignant plasma cells and the expression of the counter receptor molecules, CD28 and CD152 (CTLA-4), on T cells of patients with multiple myeloma. CD28 expression was present on most CD4 cells but was lower on CD8 cells especially from those patients who also showed evidence of expanded T cell clones (median 40%. z=2.4; p<0.02). CD152 expression was increased in 50% (9/18) of patients with myeloma. CD80 (B7-1) expression was present on the plasma cells of only 1 of 27 samples but CD86 (B7-2) expression within the normal range was present on the plasma cells of 14 of 27 samples. Primitive plasma cells (CD38++ CD45++) had a higher expression of CD86 (median 78%) than mature plasma cells (CD38++ CD45-) (median 19%, z=3.7; p<0.01). Thus patients with expanded T cell clones have a downregulated T cell CD28 expression and lack B7-1 expression on their malignant plasma cells. These results are consistent with the concept that engagement of the T cell receptor by tumour antigen on B7-1 deficient malignant plasma cells would result in T cell anergy rather than productive immunity.

CD86+ or HLA-G+ can be transferred via trogocytosis from myeloma cells to T cells and are associated with poor prognosis

The transfer of membrane proteins between cells during contact, known as trogocytosis, can create novel cells with a unique phenotype and altered function. We demonstrate that trogocytosis is more common in multiple myeloma (MM) than chronic lymphocytic leukemia and Waldenstrom macroglobulinaemia; that T cells are more probable to be recipients than B or natural killer cells; that trogocytosis occurs independently of either the T-cell receptor or HLA compatibility; and that after trogocytosis, T cells with acquired antigens can become novel regulators of T-cell proliferation. We screened 168 patients with MM and found that CD86 and human leukocyte antigen G (HLA-G) were antigens commonly acquired by T cells from malignant plasma cells. CD3+ CD86acq+ and CD3+ HLA-Gacq+ cells were more prevalent in bone marrow than peripheral blood samples. The presence of either CD86 or HLA-G on malignant plasma cells was associated with a poor prognosis. CD38++ side population cells expressed HLA-G, suggesting that these putative myeloma stem cells could generate immune tolerance. HLA-G+ T cells had a regulatory potency similar to natural Tregs, thus providing another novel mechanism for MM to avoid effective immune surveillance.

Either interleukin-12 or interferon-γ can correct the dendritic cell defect induced by transforming growth factor β1 in patients with myeloma

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44+) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up‐regulate the expression of the B7 co‐stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor β1 (TGFβ1) and interleukin (IL)‐10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti‐TGFβ1. As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up‐regulation was reduced in both stage I and stage III. It was demonstrated that both IL‐12 and interferon‐γ neutralized the failure to stimulate CD80 up‐regulation by huCD40LT in vitro. IL‐12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123−) suggesting that there would be a predominantly T‐helper cell type response. The addition of IL‐12 or interferon‐γ to future immunotherapy trials involving these patients should be considered.

Myeloid derived suppressor cells are numerically, functionally and phenotypically different in patients with multiple myeloma

Abstract Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells that have been implicated as inhibitors of lymphopoiesis in patients with malignancies. They have a consensus phenotype of CD33+/CD11b+/HLA-DRlo/− and can be further divided into CD15 + granulocytic (G-MDSC) and CD14 + monocytic (M-MDSC) subsets. We characterized MDSCs in patients with multiple myeloma (MM) and found a significant increase in G-MDSCs in the blood of patients with progressive MM. Flow-sorted MDSCs from patients with MM induced the generation of regulatory T cells (Treg). MDSCs from both patients with MM and aged-matched controls demonstrated a dose-dependent inhibition of lymphocyte proliferation in carboxyfluorescein succinimidyl ester (CFSE)-tracking experiments. Granulocyte colony stimulating factor (G-CSF) administered to induce stem cell mobilization caused an increase in the number of MDSCs in the peripheral blood of patients with MM and a concentration of these immune-suppressive cells in peripheral blood stem cell collections. MDSCs are likely to cause immune dysfunction in patients with MM.

CD10-(CALLA)-Positive Lymphocytes in Myeloma: Evidence that they are a Malignant Precursor Population and are of Germinal Centre Origin

CD10 antigen has been repeatedly detected on putative lymphoid precursor populations in both the bone marrow and circulation of multiple myeloma patients, as well as on the plasma cells in some cases of myeloma. The presence of these CD10-positive cells has raised questions regarding the ontogeny of the proliferating precursor cell in myeloma. The majority opinion has implicated a CD10-positive haemopoietic progenitor cell. However, the CD10 antigen has been detected on some mature B cells, i.e. germinal centre B cells. In this paper we postulate that the proliferating precursor cell in myeloma arises from the germinal centre. The germinal centre is the site of affinity maturation of antibody responses via somatic mutation and of isotype switching. Thus the siting of the clonogenic cell in myeloma in the germinal centre explains the overwhelming predominance of IgG and IgA myelomas, the phenomenon of point mutation which occurs in myeloma proteins in the presence of stable immunoglobulin gene rearrangements and the impaired primary immune response in myeloma. It is also consistent with the requirement for antigenic exposure in the development of myelomatosis.

Trough serum testosterone predicts the development of polycythemia in hypogonadal men treated for up to 21 years with subcutaneous testosterone pellets

OBJECTIVES Testosterone formulations that have more steady-state pharmacokinetics, such as subcutaneously implanted testosterone pellets, may cause less erythrocytosis than i.m. injections of shorter acting androgen esters. We, therefore, sought to define the prevalence, predictors, and proximate basis (role of erythropoietin) for polycythemia (hematocrit >0.50) in hypogonadal men receiving testosterone implants long term. DESIGN A cross-sectional study was conducted in an academic andrology center with a longitudinal subgroup analysis. PATIENTS A total of 158 hypogonadal men aged 14-84 years (mean age 46.7 years) treated on average for 8 years (range 0-21 years). MEASUREMENTS Trough blood testosterone and hematocrit. Serial serum erythropoietin concentrations were measured in 16 volunteers. RESULTS Positive univariate associations between polycythemia (hematocrit >0.50) and log(testosterone) (odds ratio (OR) 24.7, 95% confidence interval (CI): 4.3, 141.2, P<0.01) and age (OR 1.1, 95% CI: 1.0, 1.1, P=0.03) and a borderline relationship with current smoking (OR 4.2, 95% CI: 0.9, 20.0, P=0.08) were unveiled. A sensitivity analysis using alternate definitions of polycythemia was performed to capture all potential covariants. Multivariate regression analysis incorporating all potential covariants disclosed the independent OR of developing polycythemia (after adjusting for smoking and age) for log(testosterone) to be 15.0 (95% CI: 2.5, 90.8). Duration of testosterone therapy did not alter the risk of polycythemia. A direct relationship between testosterone and erythropoietin was observed (P=0.05). CONCLUSIONS Higher trough serum testosterone concentrations but not duration of treatment predict the development of polycythemia in men receiving long-acting depot testosterone treatment.