CD4+CD25+FOXP3+ T regulatory cells reconstitute and accumulate in the bone marrow of patients with multiple myeloma following allogeneic stem cell transplantation

Abstract BACKGROUND: Immunosuppressive CD4+Foxp3+ T regulatory cells (Treg) play a vital role in immune regulation. Thus, Treg contribute to the prevention of autoimmune disease and graft-versus-host reactions following allogeneic stem cell transplantation (alloSCT) but also to the inhibition of effective anti-tumor T cell responses. It has previously been suggested that the frequency of Treg is increased in the peripheral blood of patients with multiple myeloma (MM). However, little is known about the presence of Treg in the bone marrow and it is unclear whether allogeneic stem cell transplantation might deplete Treg from this immune compartment. METHODS: In the present study, we analyzed percentages of CD4+Foxp3+ Treg as well as Treg expression of CD45RA and CCR7 in the bone marrow (BM) and in the peripheral blood of MM patients who had received alloSCT (N=42), in newly diagnosed MM patients (N=18), and in healthy controls (N=15) using flow cytometry. In addition, we performed inhibition assays in order to test the functional relevance of peripheral and BM-residing Treg. RESULTS: While newly diagnosed MM patients and healthy controls showed no significant difference in the proportions of CD4+Foxp3+ Treg in the bone marrow, percentages of BM-residing CD4+Foxp3+ T regulatory cells were markedly higher (p<0.001 and p<0.01) in patients post alloSCT (3.3±0.3%) than in normal BM (1.0±0.3%) or in BM of untreated MM patients (1.8±0.4%). In both groups of patients (p<0.05) as well as in the healthy controls (p<0.001) percentages of Treg were higher in the peripheral blood than in the bone marrow. While there were no differences regarding the percentages of peripheral Treg between the remaining groups, patients post alloSCT had higher percentages of peripheral Treg than newly diagnosed patients (5.6±0.8 vs. 3.2±0.7%, p<0.05). More than 90% of these donor-derived peripheral and BM-residing Treg expressed a memory T cell phenotype, being negative for CD45RA and CCR7. Importantly, peripheral as well as BM-residing Treg of patients post alloSCT were capable of inhibiting the proliferation of autologous non-Treg CD4+ T cells. CONCLUSION: Our study demonstrates for the first time an increased frequency of immunosuppressive Treg in the bone marrow of MM patients. Remarkably, in our patients these memory-type Treg were all donor-derived and led to an efficient replenishment of Treg in the periphery. These Treg might be necessary for the prevention of graft-versus-host disease in the transplanted MM patients, however, they might also contribute to the failure of an effective graft-versus-myeloma effect in the majority of the patients.

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Prognostic Significance of Molecular Remission Determined by Highly Specific PCR after Dose-Reduced Allogeneic Stem Cell Transplantation (SCT) in Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.4420.4420 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4420-4420

Author(s):

Maria V. Zagrivnaja ◽

Anita Badbaran ◽

Paolo Corradini ◽

Boris Fehse ◽

Axel R. Zander ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stem Cell ◽

Complete Remission ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Prognostic Significance ◽

High Rate ◽

Molecular Remission

Abstract Achieving molecular remission after standard allogeneic stem cell transplantation in multiple myeloma is associated with long term freedom of disease and possible cure. We recently reported a high rate of complete remission after an auto-allo tandem approach using fludarabine (150 mg/m²), melphalan (140 mg/m²) and anti-thymocyte globulin (ATG: 3 x 10–20mg/kg) three months after a cytoreductive autograft (melphalan 200 mg/m²). To determine the incidence of molecular remission we tried to develop allele-specific oligonucleotides (ASO) based upon the clonal rearrangement of immunoglobulin heavy chain genes and ASO - polymerase chain reaction (ASO-PCR) for each patient who achieved complete remission with negative immunofixation after SCT (n=27). The specificity of the ASO-PCR was tested using patient and control DNA. Patient DNA isolated from bone marrow (BM) samples obtained at diagnosis and after allografting at variable time points was used for further molecular analysis. For nine patients we were able to generate specific primers and MRD diagnostics have been performed for a median of two years after SCT. Molecular remission was strictly defined by the absence of any PCR-positivity. In 6 patients nested PCR remained negative after a median follow up of 22 months (range 10–24). Only one of these patients relapsed with extramedullary disease (14 months) still being PCR-negative in his bone marrow. In contrast, all those patients with complete remission but positive PCR (n=3) relapsed (at 11, 14 and 16 months after SCT, respectively) thus underlining the importance of achieving molecular remission after allo-SCT. We suppose that quantitative real-time PCR might be a useful tool to closely monitor MRD kinetics post allo-transplantation in order to verify efficiency of various treatment regimens, e.g. donor lymphocyte infusions.

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A Characteristic Cytokine/Chemokine Milieu Is Present in the Bone Marrow Environment of Multiple Myeloma Patients Before and After Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.2795.2795 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2795-2795

Author(s):

Yanran Cao ◽

Tim Luetkens ◽

Sebastian Kobold ◽

York Hildebrandt ◽

Maja Gordic ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cell Lines ◽

Allogeneic Stem Cell Transplantation ◽

Myeloma Cell ◽

Luminex Technology ◽

Before And After

Abstract Abstract 2795 Poster Board II-771 Background: The interaction of Multiple Myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing, survival, and proliferation of the malignant plasma cells. In this study, we aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT) and might be involved in the pathogenesis of MM and/or Graft-versus-Host Disease (GvHD). Design and Methods: Applying Multiplex Bead-based Luminex technology and Quantikine ELISAs, we determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines as well as in the plasma derived from BM and peripheral blood (PB) samples of 10 newly diagnosed MM patients, 20 MM patients post alloSCT, and 20 healthy donors. Results: Besides factors known to be secreted by myeloma cell lines (IL-1RA, IL-8, MCP-1, MIP-1α, MIP1β, MIP-3α) we observed secretion of other cytokines/chemokines such as EGF, HGF, IL2R, IL-12p40/p70, IL-22, IP-10, MIG, and RANTES. In the BM plasma samples of MM patients, we detected elevated levels of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and MIG (p<0.01 or p<0.05). Most of these factors were significantly increased in the BM compared to PB. In addition to increased levels of the factors mentioned above, the BM plasma of MM patients post alloSCT showed selectively elevated concentrations of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin (p<0.01 or p<0.05). Eotaxin levels were particularly high in patients with chronic GvHD. Correlating the BM cytokine/chemokine levels with the clinical characteristics of MM patients, we observed that BM levels of IL-16 (r=0.829, p=0.003) and HGF (r=0.805, p=0.005) correlated positively with myeloma cell infiltration within the BM. In addition, IP-10 (r=0.770, p=0.009), HGF (r=0.645, p=0.044), and IL-6 (r=0.664, p=0.036) correlated significantly with the initial stage of disease. Conclusions: Our study demonstrates that myeloma cells themselves generate a significantly higher number of cytokines/chemokines than previously described. We show that characteristic cytokine/chemokine patterns exact in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin might not only be developed into diagnostic instruments and/or predictive biomarkers, but might also represent potential targets for future myeloma- or GvHD-specific therapies. Disclosures: No relevant conflicts of interest to declare.

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CTLA-4 Blockade following Relapse of Malignancy after Allogeneic Stem Cell Transplantation Is Associated with T Cell Activation But Not with Increased Levels of T Regulatory Cells

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2010.08.005 ◽

2011 ◽

Vol 17 (5) ◽

pp. 682-692 ◽

Cited By ~ 39

Author(s):

Jiehua Zhou ◽

Asad Bashey ◽

Ruikun Zhong ◽

Sue Corringham ◽

Karen Messer ◽

...

Keyword(s):

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

T Cell Activation ◽

Allogeneic Stem Cell Transplantation ◽

Cell Activation ◽

T Regulatory Cells ◽

Regulatory Cells

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The correlation between T regulatory cells and autologous peripheral blood stem cell transplantation in multiple myeloma

Turkish Journal of Hematology ◽

10.5152/tjh.2011.26 ◽

2011 ◽

Vol 28 (2) ◽

pp. 107-114

Author(s):

Ayse Pinar Ercetin ◽

Safiye Aktas ◽

Ozden Piskin ◽

Halil Ates ◽

Zeynep Filiz Zadeoglulari ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Peripheral Blood ◽

T Regulatory Cells ◽

Peripheral Blood Stem Cell ◽

Blood Stem Cell ◽

Regulatory Cells ◽

Blood Stem Cell Transplantation

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Cancer-testis antigens are commonly expressed in multiple myeloma and induce systemic immunity following allogeneic stem cell transplantation

Blood ◽

10.1182/blood-2006-04-014480 ◽

2006 ◽

Vol 109 (3) ◽

pp. 1103-1112 ◽

Cited By ~ 107

Author(s):

Djordje Atanackovic ◽

Julia Arfsten ◽

Yanran Cao ◽

Sacha Gnjatic ◽

Frank Schnieders ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stem Cell ◽

Immune Responses ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Antibody Responses ◽

Ct Antigens ◽

Cancer Testis

Abstract Immunotherapies using cancer-testis (CT) antigens as targets represent a potentially useful treatment in patients with multiple myeloma (MM) who commonly show recurrent disease following chemotherapy. We analyzed the expression of 11 CT antigens in bone marrow samples from patients with MM (n = 55) and healthy donors (n = 32) using reverse transcriptase–polymerase chain reaction (RT-PCR). CT antigens were frequently expressed in MM with 56% (MAGEC2), 55% (MAGEA3), 35% (SSX1), 20% (SSX4, SSX5), 16% (SSX2), 15% (BAGE), 7% (NY-ESO-1), and 6% (ADAM2, LIPI) expressing the given antigen. Importantly, CT antigens were not expressed in healthy bone marrow. Analyzing patients with MM (n = 66) for antibody responses against MAGEA3, SSX2, and NY-ESO-1, we found strong antibody responses against CT antigens preferentially in patients who had received allogeneic stem cell transplantation (alloSCT). Antibody responses against NY-ESO-1 correlated with NY-ESO-1–specific CD4+ and CD8+ T-cell responses against peptide NY-ESO-151-62 and CD4+ responses against NY-ESO-1121-140 in 1 of these patients. These allogeneic immune responses were not detectable in pretransplantation samples and in the patients' stem cell donors, indicating that CT antigens might indeed represent natural targets for graft-versus-myeloma effects. Immune responses induced by alloSCT could be boosted by active CT antigen–specific immunotherapy, which might help to achieve long-lasting remissions in patients with MM.

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