The proportion of multiple myeloma patients in long-term complete response (LTCR-MM) for more than 6 years after autologous stem cell transplantation (ASCT) is small. To evaluate whether this LTCR is associated with a particular immune signature, peripheral blood samples from 13 LTCR-MM after ASCT and healthy blood donors (HBD) were analysed. Subpopulations of T-cells (naïve, effector, central memory and regulatory), B-cells (naïve, marginal zone-like, class-switched memory, transitional and plasmablasts) and NK-cells expressing inhibitory and activating receptors were quantified by multiparametric flow cytometry (MFC). Heavy/light chains (HLC) were quantified by nephelometry. The percentage of CD4+ T-cells was lower in patients, whereas an increment in the percentage of CD4+ and CD8+ effector memory T-cells was associated with the LTCR. Regulatory T-cells and NK-cells were similar in both groups but a particular redistribution of inhibitory and activating receptors in NK-cells were found in patients. Regarding B-cells, an increase in naïve cells and a corresponding reduction in marginal zone-like and class-switched memory B-cells was observed. The HLC values were normal. Our results suggest that LTCR-MM patients express a particular immune signature, which probably reflects a ‘high quality’ immune reconstitution that could exert a competent anti-tumor immunological surveillance along with a recovery of the humoral immunity.
Multiple myeloma (MM) is a biologically heterogeneous disease with great variability among patients in terms of response to therapy and overall survival (OS). In the last decade, several novel agents such as immunomodulators (thalidomide, lenalidomide and pomalidomide) and proteasome inhibitors (bortezomib and carfilzomib) have been approved for the treatment of MM.1, 2, 3, 4 With these new drugs and the introduction of high-dose therapy followed by autologous stem cell transplantation (HDT/ASCT) in eligible patients, the response rates and OS have significantly improved, changing life expectancy of MM patients.1, 5, 6, 7
It is well documented that achieving a complete response (CR) after HDT/ASCT is associated with a longer survival, thus making CR the most important prognostic factor in this setting.1, 8, 9, 10, 11 However, the spectrum of long-term outcomes in patients who achieve CR is heterogeneous, including patients who will lose their CR status during the first year and few patients who will remain in sustained CR for more than 10 years, who may be considered as potentially cured.12 The identification of these patients is crucial, but little is known about other characteristics apart from the persistence of a negative minimal residual disease (MRD) that could predict a sustained CR.13, 14, 15, 16, 17 Interestingly, several studies revealed that the absolute lymphocyte count recovery is associated with a longer OS in MM,18, 19 suggesting that immune reconstitution after HDT/ASCT determines the duration of the relapse free period. As a matter of fact, many qualitative and quantitative alterations of different immune parameters have been described in MM patients.20, 21 Among others, there is an expansion of cytotoxic lymphocytes that could partially control the progression of the disease,22, 23 but the tumoral cells will eventually escape their control in probable relationship to a marked immunosuppressive microenvironment, as supported by the reported increased in regulatory T-cells (Tregs) and the Treg/Th17 ratio in relapsed MM patients.24, 25
The ‘Heavy/Light Chain’ (HLC) nephelometric/turbidimetric assay accurately quantifies the specific pairs of each HLC of the involved and uninvolved immunoglobulin (Ig) in the serum (IgGκ, IgGλ, IgAκ, IgAλ), enabling the calculation of the monoclonal/polyclonal Ig ratios (HLC ratios). This test specifically allows the quantification of the tumoral Ig of the patient and, even more interesting, evaluates the immunoparesis in terms of suppression of the specific pair of the uninvolved Ig of the same isotype of the tumor.26, 27, 28
Even though the quantitative recovery of lymphocytes may play a critical role in the prognosis of MM patients, it is not yet known whether LTCR-MM patients after HDT/ASCT are characterized by a particular immune signature reflecting the recovery of an efficient anti-tumor surveillance.
Our aim was therefore to correlate different parameters of immune reconstitution, including the HLC and the quantification of different lymphoid subpopulations, with the achievement of LTCR following HDT/ASCT in MM patients.
Materials and methods
Study patients and samples
The study included a total of 13 LTCR-MM patients (7 males and 6 females; median age: 61) after HDT/ASCT and 15 voluntary and altruist age-matched healthy blood donors (HBD) as control arm. The patients were in LTCR for more than 6 years and less than 19 years after ASCT. Samples were collected randomly at different times, but minimum 6 years after ASCT. The specific time point when samples were acquired for analysis is indicated in Table 1. In order to confirm their specific immune signature, the analysis was repeated in the same LTCR-MM patients 1 year after the first analysis was done. All patients were treated since diagnosis at the Haematology Department of the University Hospital La Princesa in Madrid (Spain) during the years 1995–2009. Patients and HBDs signed an informed consent form according to the Declaration of Helsinki. The study was approved by the local ethical committee.
Analysis of the immunophenotype and the minimal residual disease by multiparametric flow cytometry
EDTA-anticoagulated fresh peripheral blood (PB) and a serum tube were obtained from each subject. Serum was immediately separated by centrifugation (20 min at 2000 g), aliquoted and stored at −80 °C. Erythrocyte-lysed whole PB samples were immunophenotyped with an immunofluorescence stain-and-then-lyse technique, using a comprehensive 8-color flow cytometry panel within the 24 h after the samples were obtained. The monoclonal antibody (mAb) combinations for T-, B-, Treg and NK-cells subsets in PB are shown in Table 2. Following the consensus guidelines, the presence of minimal residual disease (MRD) was studied in the bone marrow (BM) of patients by 8 color MFC29 with the Ab combination showed in Table 2.
The data acquisition was performed in a FACSCanto II flow cytometer (Becton Dickinson Biosciences (BD), San Jose, CA, USA) using the FACSDiva software (version 6.1). For the PB phenotyping, 100 000 events were acquired from tubes T, B and NK, and 1 000 000 from Treg tube. To study MRD in the BM, 5000 plasmatic cells (PCs) were analysed from each tube.
The following maturation-associated CD19+CD20+ B-cell subsets were identified: naïve B-cells (CD27−IgM+IgD+), class-switched memory B-cells (CD27+IgM−IgD−), marginal zone-like B-cells (CD27+IgM+IgD+), transitional (CD27−CD10+CD38+) and plasmablasts (CD20−CD27++CD38++). In relation to CD4+ and CD8+ T-cells, the following subsets were identified: naïve T-cells (CD27+CCR7+CD45RA+), central memory T-cells (CD27+CCR7+CD45RA−) and effector memory T-cells (CD27−CCR7−CD45RA+/−). NK and Treg cells were defined as CD3−CD56+ and CD3+CD4+CD25highCD127−, respectively.
The comprehensive flow cytometry strategy to identify the different lymphoid subsets is shown in the Supplementary Figure 1.
Heavy/light chain (HLC) assay and serum parameters
Each serum sample was tested for total Ig concentrations by immunonephelometry (Immage800, Beckman Coulter, CA, USA) and, depending on the isotype, the corresponding HLC concentration (that is, IgGк, IgGλ, IgAк, IgAλ or IgMк, IgMλ) was quantified by immunonephelometry using Hevylite reagents in a Binding Site SPAPLUS Analyser provided by the manufacturer (The Binding Site, Birmingham, UK). HLC ratios were determined for each isotype (IgGк/IgGλ, IgAк/IgAλ, IgMк/IgMλ).
Immunoparesis was defined as a reduction below the lower normal limit in the levels of 1 or 2 of the uninvolved Igs30 (for example, in a MM IgG patient, a reduction of >25% of the lower normal reference range of IgA and/or IgM concentrations). Conventional parameters such as beta-2-microglobulin, FLC ratios, creatinine, calcium, total protein, albumin and haemoglobin were also assessed by standard techniques. Immunoelectrophoresis and immunofixation were run on Sebia Hidrasys (Sebia, Evry, France).
Quantitative variables are expressed as measures of central tendency (mean) and dispersion (s.d.) and were analyzed using the Kruskal–Wallis test for heteroscedasticity variances to evaluate differences among the studied groups. A posteriori test was carried out to compare the control group with the two patient’s group (patients and patients +1 year), independently of each other. A Wilcoxon-matched test was used to compare a patient under group ‘patients’ with the status of the same patient in the second group ‘patients +1 year’. Significance was set at a value of P⩽0.05 (*P<0.05; **P<0.01). Statistical analysis was performed using GraphPad Prism software (version 5.0; GraphPad, La Jolla, CA, USA).
Patient characteristics and therapy details are summarized in Table 1. All patients included in this study were in stringent complete response (sCR) for at least 6 years according to the International Myeloma Working Group (IMWG) criteria31 and had no evidence of autoimmune disease. Furthermore, the percentage and phenotype of PC in the BM was studied by MFC. Supporting a sCR, all of the patients but two whose BM were not available, had less than 1.5% of PC and were MRD-negative at a sensitivity level of 10−4 up to 10−5 (Table 3).
The median follow-up in sCR in this study was 8 years (range 6–19). The response before ASCT was also evaluated according to the IMWG criteria.31, 32 Induction treatment was performed with vincristine-doxorubicin-dexamethasone (VAD) and alternate VBCMP/VBAD (vincristine-carmustine-melphalan-cyclophosphamide-prednisone/vincristine-carmustine-doxorubicin-dexamethasone) chemotherapy. As conditioning therapy for ASCT, high-dose melphalan was used in seven patients and busulphan plus melphalan in six patients. Two patients received a new drug: low-dose thalidomide as maintenance during 2 years post-ASCT. Apart from these two cases and a short period of monthly low-dose dexamethasone in other four patients during less than 2 years, no other maintenance treatment was administered to this population.
LTCR-MM patients have decreased proportion of naïve T-cells, and a corresponding increased percentage of effector T-cells in the peripheral blood
The distribution of T-cell subsets in the PB of LTCR-MM patients and HBD was compared. The patients had a lower percentage of total CD4+ T-cells (P=0.0004) together with a decrease in the naïve CD4+ T-cells (CD27+CCR7+CD45RA+) population (P=0.0004) and an increment of the effector memory CD4+ T-cells (CD27−CCR7−) (P=0.0028), which include both CD27−CCR7−CD45RA− and CD27−CCR7−CD45RA+ cells (Figures 1a–c).
Similar results were found within the CD8+ T-cells. Although no differences were observed in the proportion of total CD8+ T-cells (P=0.1236; Figure 1d), an increment in the percentage of effector memory CD8+ T-cells (P=0.0084; Figure 1f), both CD27−CCR7−CD45RA− and CD27−CCR7−CD45RA+). Conversely, naïve CD8+ T-cells (CD27+CCR7+CD45RA+) were clearly decreased in patients (P=0.0007; Figure 1e).
When the analysis was repeated in the same LTCR-MM patients 1 year after the first analysis, no changes were detected neither when analysed as a group (see columns ‘patients+1 year’ in Figure 1) nor when analysed individually (data not shown).
No differences were observed in the Tregs defined as CD4+CD25highCD127− (data not shown).
LTCR-MM patients have increased percentage of NK-cells expressing KIR2DL1+ but a decrease of those expressing NKp46+ and a decreased number of CD8+ T-cells expressing NKG2D+
The proportion of total NK-cells was similar in patients and HBD (Figure 2a). However, patients showed higher expression of the inhibitory receptor KIR2DL1+ (P=0.0326), a higher expression of NKG2A+ in the NK-cell population (P=0.0206) and lower expression of the activating receptor NKp46+ (P=0.0146) (Figures 2b–d). A reduction in the expression of NKG2D+ in the CD8+ T-cell population was also observed in patients (P=0.0019) (Figure 2e). Similar to T-cells, this NK-cell profile was maintained after 1 year in the same patients when analysed individually (data not shown).
Distribution of the B-cell subpopulations in the peripheral blood of LTCR-MM patients and healthy blood donors
The mean of the percentage of total B-cells (CD19+CD20+) in the patients was within the normal range and no significant differences were found when compared to HBD (Figure 3a). Even though the range of percentage values in patients was quite wide, the specific percentage of B-cells was maintained in the same patient 1 year after (data not shown). However, naïve B-cells (CD27−IgD+IgM+) proportion was higher in patients (Figure 3b; P=0.0308), and a corresponding reduction of marginal zone-like B-cells (CD27+IgD+IgM+, P=0.0047) and class-switched memory B-cells (CD27+IgD−IgM−, P=0.0043) was observed (Figures 3c and d). The immune signature of the B-cells was unchanged when patients were evaluated one year later either as a whole (see columns ‘patients+1 year’ in Figure 3) or individually (data not shown).
No differences were observed in the percentage of transitional B-cells (CD27−CD10+CD38+) or plasmablasts (CD27++ CD38++) in the PB of the two groups (data not shown).
HLC ratios values of LTCR-MM patients
To accurately evaluate the CR status and the immune reconstitution in our patients, we quantified all the Igs as well as the individual isotypes of the patient’s affected Ig (Table 4). With the only exception of patient 1 who showed a moderate hypogammaglobulinemia, no immunosuppression and/or altered HLC ratios were observed in LTCR-MM patients, agreeing with the CR stage of the patients and the complete reconstitution of their humoral immunity (Table 4).
Recent studies have reported a survival rate of 15% approximately after 10–15 years of the HDT/ASCT5, 6 when the relapse rate is very low. These patients could have achieved an ‘operational cure’ associated to a particular immune reconstitution profile, which has not been defined yet.12 Therefore, we aimed to analyse the distribution of T-, NK- and B-cell subpopulations and the immunoparesis in LTCR-MM patients after ASCT. An important increment of the percentage of both CD4+ and CD8+ effector memory T-cells was found in parallel to an increase of naïve B-cells proportions and a redistribution of activating and inhibitory NK-cell receptors. Furthermore, this immune profile was confirmed 1 year later in the same LTCR-MM patients, suggesting a specific immune signature after the ASCT that remains stable while being in CR.
In particular, we evidence an increment of naïve B-cells and a reduction of marginal and class-switched B-cells, which would restore the repertoire of the humoral immune response and the recovery of normal plasma cells. The absence of immunoparesis in these patients is in accordance to this recovery. These findings on B-cells are similar to those obtained by Pessoa de Magalhães et al., although the differences between their LTCR-MM and controls did not reach statistical significance, in probable relationship to different immunophenotypic approaches to identify the B-cell subsets.33
In LTCR-MM patients, we also find a reduction of total CD4+ T-cells and a decrease of naïve CD4+ and CD8+ T-cells compared to HBD. On the contrary, there is an increase of the effector CD4+ and CD8+ T-cell populations and normal proportions of Tregs, suggesting an efficient mechanism to control tumor growth, achieve a CR and maintain an efficacious immune surveillance. Altogether, these results reinforce the idea of the relevant role of T-cells, including conventional T-cells, Tregs and exhausted T-cells, in myelomagenesis and in the clinical outcome of MM patients, as has been thoroughly reviewed by Dosani et al.21 The knowledge of the role of the immune system in the achievement of a long-term remission is of significance, not only for the development of immunotherapies, but also for the identification of predictive immune biomarkers. In fact, the T-cell exhaustion/senescence ratio has been recently proposed as an interesting immune biomarker, since relapsing patients have higher numbers of exhausted T-cells at +3 months after the transplant but before detection of clinical disease, thus identifying MM patients who could benefit from an early immunotherapy after the ASCT.34, 35
The NK-cell population, functionally controlled by the balance of activating and inhibitory signals,36 also plays an important role in controlling the progression of MM.37 Different activating receptors such as the natural cytotoxic receptor NKp46, DNAM-1 and NKG2D have all been implicated in tumor recognition and killing, including MM.38, 39 In our study, we observed a decreased number of activating receptors like NKp46 together with a trend of increased expression of the inhibitory molecules NKG2A and KIR2DL1 in LTCR-MM patients compared to controls. Therefore, some inhibitory signals seem to dominate in the NK-cells of LTCR-MM patients. It is possible that, in early stages after ASCT or even before, a continuous cytotoxic activity against myeloma cells is exerted by the activating signals of the NK-cells to achieve a CR, which would lead to a downregulation of these activating signals and to an increased expression of inhibitory receptors (NKG2A and KIR2DL1), conforming the characteristic phenotype of terminally differentiated NK-cells.40
NKG2D has been described as a costimulatory receptor for human naïve CD8+ T-cells.41 This specific population (CD8+NKG2D+ T-cells) not only plays a critical role in identifying and killing autologous myeloma cells but also seems to improve the survival rate after transplantation.42 Interestingly, we find a reduction of NKG2D+ in CD8+ T-cells from LTCR-MM patients compared to controls, which could reflect the expansion of terminally differentiated CD8+ T-cells that might have downregulated this receptor.
The HLC assay is a promising tool since it can evaluate the immune reconstitution due to its capacity of measuring the suppression of the uninvolved HLC-pair (for example, measurement of IgAκ in a patient with IgAλ).26, 28 None of our LTCR-MM patients had altered HLC ratio and therefore, they had no HLC-pair suppression, in accordance with the recovery of a normal humoral immunity.43
Immune dysfunction is related to the development and progression of MM.20, 21 In particular, lymphoid cells are significantly altered in myelomagenesis, and their dysfunction closely mirrors the course of the disease.21 They are therefore appealing candidates for immunotherapy approaches aimed at exploiting, increasing or restoring the myeloma immunosurveillance and disease control. In particular, both T- and NK-cells are already known to have integral roles in the mechanisms of action of currently explored immunotherapies, particularly those of immunomodulatory drugs and mAbs—including elotuzumab, daratumumab and immune checkpoints modulators.44 It is not in vain that the most efficacious drugs in the maintenance therapy after ASCT are lenalidomide and bortezomib, which rely on the functional activity of the different cytotoxic lymphocytes (cytotoxic T-cells, NK-cells and NKT cells) and the inhibition of Tregs to exert some of their anti-MM effects. Moreover, both lenalidomide and bortezomib have demonstrated to enhance the anti-MM activity of elotuzumab and daratumumab.45, 46 Our study provides knowledge of the immune profile that might be worth aiming for through modern immunomodulatory maintenance therapies, which consists on an increment of effector T-cells and terminally differentiated NK-cells that probably exert a competent immune surveillance. Conversely, the increase of naïve B-cells may guarantee the humoral response homeostasis, including the recovery of normal plasma cells that might compete with myelomatous cells for normal bone marrow niches. This replenishment of B-cells may contribute to the absence of immunoparesis, what can be better currently evaluated by the HLC-pair suppression. Possibly, in the near future, the HLC assay could be used not only as a marker of MRD but also as a potential marker of robust immune recovery.
The precise role of these refined immune studies in the monitoring and therapeutic decisions in MM patients, and also in the duration of sCR, should be defined in the future. As a first step in this effort, Paiva et al. has recently published that immune profiling in BM might allow the identification of patients with different outcomes in transplant-ineligible MM patients.47 Of importance, prospective similar studies with the different therapies currently used are required to determine whether the immune recovery pattern of LTCR-MM patients differs from that of patients who achieve sCR and that will eventually relapse. In other words, it is important to determine whether these findings can be used as a prognostic predictive testing and if they are reproducible in the current therapeutic scenario which includes new proteasome inhibitors, IMIDs, mAbs and other immune strategies to evaluate the role of immunomodulatory drugs and maintenance strategies.
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We thank all patients and clinical staff who made this study possible. We are very grateful to Ana Ramírez and Víctor López for their valuable help with flow cytometry, to Lorena Vega for statistics advice and to Lawrence Baron for his help with the writing. The grants include Grant PI12/00494P and PI15/02085 from the Fondo de Investigaciones Sanitarias to CMC supported this work. CMC was co-financed by FEDER funds. AAL is supported by a research grant from the JL Castaño Foundation since September 2015. BA and MGP were granted for the Spanish Leukemia and Lyphoma Foundation.
AA is a member of Advisory Boards of Janssen-Cilag, Celgene and Amgen. The remaining authors declare no conflict of interest.
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Arteche-López, A., Kreutzman, A., Alegre, A. et al. Multiple myeloma patients in long-term complete response after autologous stem cell transplantation express a particular immune signature with potential prognostic implication. Bone Marrow Transplant 52, 832–838 (2017) doi:10.1038/bmt.2017.29
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