Multiple myeloma causes clonal T-cell immunosenescence: identification of potential novel targets for promoting tumour immunity and implications for checkpoint blockade.

Tumour-induced dysfunction of cytotoxic T cells in patients with multiple myeloma (MM) may contribute to immune escape and be responsible for the lack of therapeutic efficacy of immune checkpoint blockade. We therefore investigated dysfunctional clonal T cells in MM and demonstrated immunosenescence but not exhaustion as a predominant feature. T-cell clones were detected in 75% of MM patients and their prognostic significance was revalidated in a new post-immunomodulatory drug cohort. The cells exhibited a senescent secretory effector phenotype: KLRG-1+/CD57+/CD160+/CD28-. Normal-for-age telomere lengths indicate that senescence is telomere independent and potentially reversible. p38-mitogen-activated protein kinase, p16 and p21 signalling pathways known to induce senescence were not elevated. Telomerase activity was found to be elevated and this may explain how normal telomere lengths are maintained in senescent cells. T-cell receptor signalling checkpoints were normal but elevated SMAD levels associated with T-cell inactivation were detected and may provide a potential target for the reversal of clonal T-cell dysfunction in MM. Low programmed death 1 and cytotoxic T-lymphocyte-associated antigen 4 expression detected on T-cell clones infers that these cells are not exhausted but suggests that there would be a suboptimal response to immune checkpoint blockade in MM. Our data suggest that other immunostimulatory strategies are required in MM.

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.

Characterisation and relevance of CD138-negative plasma cells in plasma cell myeloma.

INTRODUCTION: The use of CD138 to isolate CD138(+) plasma cells (PCs) from plasma cell myeloma (PCM) patients' bone marrow samples has been used extensively in myeloma research. We sought to highlight the problem with this selection process, by demonstrating that a subpopulation of CD138⁻ plasma cells exists which is not included in these analyses. METHODS: Retrospective analysis of a patient database was carried out on all PCM patient bone marrow biopsies taken between 4/9/07 and 18/2/09 (n = 218). CD138(+) and CD138⁻ cell populations were separated using flow cytometry cell sorter then analyzed for percentage of cells in S phase using plasma cell labeling index as an indicator of proliferation. RESULTS: Database results indicated a CD138⁻ PC population in all PCM patient samples which also had a significantly increased (r = 0.53; P < 0.0001) CD45 expression, an indicator or immaturity. Flow cytometric analysis demonstrated the presence of a more immature, higher proliferating CD138⁻ PC population through a significantly (t = 3.26; P < 0.02) higher number of CD138⁻ PCs in S phase compared with the CD138(+) cells. CONCLUSION: We have characterised the CD138⁻ PCs as more immature and with a significantly higher proliferative potential. The current trend to ignore this more immature and proliferative subpopulation of malignant PCs may have serious implications when determining gene expression, classifications and drug sensitivity of the malignancy.

Influence of fibrinogen and haematocrit on erythrocyte sedimentation kinetics.

This study investigates the influence of haematocrit, fibrinogen concentration and fibrinogen availability (amount of fibrinogen per red blood cell) on erythrocyte sedimentation. The Westergren technique was applied to blood samples from 36 subjects and to their blood manipulated to haematocrits of 10, 20, 30 and 40%. Readings were taken every 10 minutes for 300 minutes. Previous studies indicate that erythrocyte sedimentation occurs in three phases. In this study, we show that haematocrit has little influence on either the rate of fall of particles in the first phase (m1) or the duration of the first phase. This is also true for fibrinogen availability and for fibrinogen concentration at low haematocrits. At high haematocrits m1 increases with fibrinogen concentration. The rate of fall of rouleaux during phase 2 (m2) and ESR60 both decrease exponentially with haematocrit and increase linearly with fibrinogen concentration. While m2 is more closely correlated to fibrinogen availability than to fibrinogen concentration or to haematocrit, this is not the case for ESR60. Thus haematocrit, fibrinogen concentration and fibrinogen availability are more important to the velocity of sedimentation in the second phase than to the sedimenting velocity during phase 1 or to the duration of phase 1.

Erythrocyte sedimentation in columns and the significance of ESR.

Erythrocyte Sedimentation Rate (ESR) is a simple, non-specific clinical test. Most models of erythrocyte sedimentation (ES) are formulated as a sigmoid function but consider the ES process to consist of three distinct phases: single-cell fall; fall of rouleaux and aggregates; cell packing. Recently, a piecewise (three-phase) continuous model has been developed. Our study applies ES data from 29 haematologically normal subjects to this model and re-evaluates the mechanism of ES using the derived model parameters. Using the Westergren technique, ES readings were taken every 10 minutes for 300 minutes. Three subjects remained in the first phase, while 26 displayed three discrete phases. For the 26 subjects, the average rate of fall of the sedimenting particles in the first phase 87 microns/min, while that of the second phase was 176 microns/min. The ratio of these two values suggests an alternative nature of sedimenting particles in the first phase. Further, the average duration of the first phase was 62 minutes, suggesting that, in 50% of subjects, aggregate formation is incomplete when ESR is measured at 60 minutes.

Human recombinant interleukin 4 induces Fc epsilon R2/CD23 on normal human monocytes.

rIL-4 (B cell stimulatory factor 1) induces the expression of Fc epsilon R2/CD23 on normal human monocytes (Mo). Fc epsilon R2/CD23 induction was detectable both by flow cytometry using anti-CD23 mAbs as well as soluble IgE, and by the immunoprecipitation with CD23-specific mAb or IgE of a 45-kD band from 125I-lactoperoxidase-labeled Mo. Fc epsilon R2/CD23 was fully expressed after a 24-h incubation with rIL-4, and was still detectable after 72 h from the addition of IL-4. This effect was specific, because none of the other rILs tested (IL-1, IL-2, IL-3, IL-5, B cell stimulatory factor 2, granulocyte-macrophage colony stimulating factor, and IFN-gamma) could induce FC epsilon R2/CD23, either alone or in various combinations. No synergism was observed between IL-4 and other ILs. IFN-gamma was not able to inhibit the IL-4-induced expression of Fc epsilon R2/CD23 on Mo, neither when added to the culture together with IL-4, nor when added 36 h earlier.