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.

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.

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.

We reassessed the influence of ABO blood group, sex, and age on plasma levels of von Willebrand factor (vWF) antigen, vWF:ristocetin cofactor, vWF:collagen binding assay, and factor VIII coagulant (FVIII:C). Data show that levels of vWF and FVIII:C increase with increasing age (P < .001 for all parameters) and that the ABO blood group influences plasma levels such that O group levels are significantly less than non-O group levels. There was no significant association with sex and Rh status. The selection of normal ranges based on ABO blood groups may influence the clinical diagnosis of von Willebrand disease (vWD) but might not be clinically relevant or help identify people at increased risk of bleeding. Differences in ABO-related ranges were more extensive at the high end of the ranges. This is of particular interest because high levels of vWF and FVIII are associated with thrombosis risk, and an ABO relationship also has been described. O group individuals may or may not be at greater risk for bleeding (they have lower levels of vWF and FVIII:C) and are more likely to be diagnosed with vWD. It also is possible that O group status may be protective for thrombosis.

Discrepancies in the literature between regulatory T cell (T reg ) and pro-infl ammatory T helper 17 (T h17 ) numbers in multiple myeloma (MM) can be largely explained by technical diff erences in methodology and patient selection. In this study, T reg cells were defi ned as CD3 ؉ CD4 ؉ CD25 ؉ ؉ CD127 lo cells. Patients with MM ( n ‫؍‬ 20) had a signifi cant imbalance in T reg /T h17 ratio when compared with either aged-matched controls ( n ‫؍‬ 28) or other monoclonal gammopathies, and this was associated with a signifi cantly worse survival. The percent T reg in bone marrow of patients with MM was higher than that in matched peripheral blood samples ( p ‫؍‬ 0.02), although FOXP3 expression within bone marrow T cells was lower ( p ‫؍‬ 0.02). We observed increased T reg function, both in vivo and in vitro , due at least partially to an increase in CTLA-4 expression by concurrent treatment with dexamethasone and immune modulatory compounds (iMiDs). We suggest that immunoregulatory balance is important during active chemotherapy and that conclusions related to the immunostimulatory eff ect of iMiDs based on in vitro testing must be considered with caution.

CD8+CD57+ terminal effector T (TTE) cells are a component of marrow-infiltrating lymphocytes and may contribute to the altered immune responses in multiple myeloma (MM) patients. We analyzed TTE cells in the bone marrow (BM) and peripheral blood (PB) of age-matched controls and patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and newly diagnosed (ND) MM using flow cytometry, mass cytometry, and FlowSOM clustering. TTE cells are heterogeneous in all subjects, with BM containing both CD69− and CD69+ subsets, while only CD69− cells are found in PB. Within the BM-TTE compartment, CD69− and CD69+ cells are found in comparable proportions in controls, while CD69− cells are dominant in MGUS and SMM and predominantly either CD69− or CD69+ cells in NDMM. A positive relationship between CD69+TTE and CD69−TTE cells is observed in the BM of controls, lost in MGUS, and converted to an inverse relationship in NDMM. CD69−TTE cells include multiple oligoclonal expansions of T-cell receptor/Vβ families shared between BM and PB of NDMM. Oligoclonal expanded CD69−TTE cells from the PB include myeloma-reactive cells capable of killing autologous CD38hi plasma cells in vitro, involving degranulation and high expression of perforin and granzyme. In contrast to CD69−TTE cells, oligoclonal expansions are not evident within CD69+TTE cells, which possess low perforin and granzyme expression and high inhibitory checkpoint expression and resemble T resident memory cells. Both CD69−TTE and CD69+TTE cells from the BM of NDMM produce large amounts of the inflammatory cytokines interferon-γ and tumor necrosis factor α. The balance between CD69− and CD69+ cells within the BM-TTE compartment may regulate immune responses in NDMM and contribute to the clinical heterogeneity of the disease.

Introduction von Willebrand disease (VWD) is the most common inherited bleeding disorder and caused by an absence, deficiency or defect in von Willebrand factor (VWF). VWD is currently classified into six different types: 1, 2A, 2B, 2N, 2M, 3. Notably, 2M VWD is more often misdiagnosed as 2A or type 1 VWD than properly identified as 2M VWD. Aim To describe an algorithmic approach to better ensure appropriate identification of 2M VWD, and reduce its misdiagnosis, as supported by sequential laboratory testing. Methods Comparative assessment of types 1, 2A, 2B and 2M VWD using various laboratory tests, including VWF antigen and several VWF activity assays, plus DDAVP challenge data, ristocetin‐induced platelet agglutination (RIPA) data, multimer analysis and genetic testing. Results Types 1, 2A, 2B and 2M VWD give characteristic test patterns that can provisionally classify patients into particular VWD types. Notably, type 1 VWD shows low levels of VWF, but VWF functional concordance (VWF activity/Ag ratios >0.6), with both baseline assessment and post‐DDAVP. Types 2A, 2B and 2M VWD show VWF functional discordance (low VWF activity/Ag ratio(s)) dependent on the defect, but type 2M separates from 2A/2B VWD based on specific test patterns, especially with collagen binding vs glycoprotein Ib binding assays. RIPA identifies 2B VWD. Multimers separate 2M from 2A/2B. Conclusion We provide strategies to improve correct diagnosis of VWD, especially focussed on 2M VWD, and which can be used by most diagnostic haemostasis laboratories, reserving genetic analysis (if required) for confirmation.

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