Circulating subpopulations of non-cytotoxic ILCs in diffuse large B-cell lymphoma.

Non-cytotoxic innate lymphoid cells (ILCs) have been added to the list of immune cells that may contribute to the tumor microenvironment. Elevated levels of total ILCs and their subgroups have been reported in peripheral blood and tissue samples from patients with solid tumors, but their frequency in non-Hodgkin lymphomas, particularly diffuse large B-cell lymphoma (DLBCL), has not been clearly established. This study examined frequency and subset distribution in newly diagnosed DLBCL patients (nodal and extra-nodal) and compared it with blood specimens from healthy donors. The percentage of total ILCs (Lin - CD127+) was assessed by flow cytometry, as well as the four ILC subsets, defined as ILC1 (Lin - CD127 + cKit - CRTH2-), ILC2 (Lin - CD127 + cKit+/- CRTH2+), ILCp NCR- (Lin - CD127 + cKit + CRTH2- NKp46-) and NCR + ILC3 (Lin - CD127 + cKit + NKp46+). In the studied group of patients (n = 54), significantly lower levels of circulating total ILCs, ILC1, and ILCp NCR- were observed compared to the control group (n = 43). Similarly, there was a statistically significant decrease in the median frequency of NKp46 + ILC3 cells in lymphoma patients. Analysis of the ILC2 subpopulation showed no significant differences. The correlation of the distribution of individual subpopulations of ILCs with the stage and location of the tumor was also demonstrated. Our results suggest that circulating ILCs are activated and differentiated and/or differentially recruited to the lymph nodes or tumor microenvironment where they may be involved in antitumor defense. However, our observations require confirmation in functional studies.

Increased Efficacy of Stem Cell Chemomobilization with Intermediate-Dose Cytarabine Plus Granulocyte Colony-Stimulating Factor (G-CSF) Compared with G-CSF Alone in Patients with Multiple Myeloma: Results of a Randomized Trial.

Mobilization of hematopoietic stem cells for patients with multiple myeloma (MM) may be done using either steady-state granulocyte colony-stimulating factor (G-CSF) or a combination of chemotherapy with G-CSF. The goal of this randomized, open-label, phase 3 trial was to compare the efficacy of chemomobilization using intermediate-dose cytarabine (ID-AraC) plus G-CSF with G-CSF alone in patients with MM referred for tandem autologous stem cell transplantation (autoSCT). The percentage of patients with stem cell yield of at least 5 × 106 CD34+ cells/kg was the primary endpoint. Ninety patients were enrolled, including 44 assigned to the ID-AraC arm and 46 in the G-CSF arm. The threshold number of CD34+ cells was reached in 43 patients (98%) in the ID-AraC arm and in 32 patients (70%) in the G-CSF arm (P = .0003). The median number of collected CD34+ cells was 20.2 × 106 cells/kg in the ID-AraC arm versus 5.9 × 106 cells/kg in the G-CSF arm (P < .000001). A single apheresis was sufficient to achieve the required number of harvested CD34+ cells in 37 patients (86%) in the ID-AraC arm and in 13 patients (41%) in the G-CSF arm (P = .00008). The times to both neutrophil and platelet recovery after autoSCT were significantly shorter in the patients mobilized with ID-AraC. This study provides the first evidence of the advantage of chemomobilization over G-CSF monotherapy in terms of efficacy. ID-AraC with G-CSF should be the preferred chemomobilization protocol for patients with MM scheduled to undergo tandem autoSCT.

Evaluation of proinflammatory and immunosuppressive cytokines in blood and bone marrow of healthy hematopoietic stem cell donors.

INTRODUCTION: Cytokine composition of bone marrow microenvironment in comparison to blood is poorly explored. The goal of this study was to investigate the levels of cytokines present in peripheral blood and bone marrow of healthy hematopoietic stem cells donors. The data obtained on this subject with addition to cytometric analysis can provide new insight into the hematopoietic stem cells microenvironment. METHODOLOGY: Study consisted of cytokine concentration analysis performed by ELISA tests of peripheral blood of healthy peripheral blood stem cells donors and bone marrow of healthy bone marrow donors. Additionally we have tested the expression of CD47 and CD274 proteins on the surface of hematopoietic stem cells by the flow cytometry analysis. RESULTS: The results has shown different composition of analyzed cytokines (IL-1 ß, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-ß1, IFN-γ and TNF-α) present in bone marrow and blood of stem cells donors. The hematopoietic stem cells in peripheral blood are subjected to higher levels of proinflammatory cytokines whilst the lower level of those cytokines in bone marrow with a very high level of TGF-ß1 which possibly creates a more immunosuppressive environment. The IL-10 level was significantly higher in peripheral blood of PBSC donors after the administration of mobilizing factor (G-CSF). The percentage of CD47+HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors.

Reduction of DMSO concentration in cryopreservation mixture from 10% to 7.5% and 5% has no impact on engraftment after autologous peripheral blood stem cell transplantation: results of a prospective, randomized study.

The procedure of autologous peripheral blood stem cell transplantation (autoPBSCT) requires cryopreservation of cells in a mixture containing dimethyl sulfoxide (DMSO). DMSO is necessary to secure cell viability, however, its infusion may be toxic to stem cell recipient. The aim of this study was to prospectively evaluate the impact of DMSO concentration on engraftment after autoPBSCT.One-hundred-fifty patients were randomly assigned to one of three study arms; their leukapheresis products were cryopreserved in 10%, 7.5% or 5% DMSO. The study groups did not differ with regard to the diagnosis (mainly lymphomas and multiple myeloma), age, conditioning regimen, and the number of transplanted hematopoietic stem cells. 143 patients were treated with autoPBSCT. The frequency of adverse effects during and shortly after infusion was the lowest in 5% DMSO arm (p = 0.02 compared to 10% DMSO). 4 patients died due to infection before the engraftment. The median time to leukocyte and neutrophil recovery was 10 days in all study groups (p = 0.36 and p = 0.2). As well, the median day of platelet recovery was the same for all DMSO concentrations and equaled 15 days (p = 0.61).In view of these results, 5% DMSO mixture may be considered a new standard in cryopreservation of hematopoietic stem cells.

Immunological properties of bone marrow microenvironment 1 year after allogeneic hematopoietic stem cell transplantation.

Regeneration of the bone marrow microenvironment after transplantation of allogeneic hematopoietic stem cells is poorly explored. The goal of our study was to investigate this process focusing on immunologic factors: concentrations of selected cytokines, expression of immunosuppressive proteins CD47 and CD274 on hematopoietic stem cells, and frequency of T regulatory lymphocytes (Tregs). Bone marrow samples were collected before transplantation, on the day of transplantation, and at the 1-year follow-up. As a control group, we used bone marrow from healthy donors. Prior to the conditioning, the percentage of Tregs and concentration of interleukin-10 were higher in the bone marrow of patients than in healthy donors. The conditioning regimen resulted in increased concentrations of interferon-γ and expression of CD274 on hematopoietic stem cells. Twenty-eight days after transplantation, level of Tregs, expression of CD47, and concentration of interleukin-10 and latency-associated peptide 1 were increased compared with the period before conditioning. Starting from day 100 after transplantation, the microenvironment tended to normalize; the level of Tregs and concentrations of most cytokines were similar to values in the bone marrow of healthy donors.

Thymic Activity and T Cell Repertoire Recovery after Autologous Hematopoietic Stem Cell Transplantation Preceded by Myeloablative Radiotherapy or Chemotherapy.

It was previously postulated that pretransplant myeloablative treatment may impair thymopoiesis, contributing in this way to delayed reconstitution of T cells after hematopoietic stem cell transplantation (HSCT). On the other hand, de novo generation of T cells after HSCT requires a competent thymus. Various myeloablative conditioning regimens (total body irradiation [TBI] or high-dose chemotherapy) routinely used in clinical practice may have potentially different impacts on the thymus. However, no comparative study on thymic output and T cell repertoire in autologous (auto)HSCT model has been presented so far. Here we evaluated thymic output and TCR diversity in 45 lymphoma patients submitted to autoHSCT differing in respect to conditioning regimen: high-dose chemotherapy as monotherapy (BEAM, n = 22) or combination of total body irradiation with cyclophosphamide chemotherapy: Cy/TBI (n = 23). Thymic output was assessed before and on days +100, +180, and +365 after autoHSCT by flow cytometric counts of recent thymic emigrant (RTE) cells (CD31(+) CD62L(+) CD45RA(+) CD4(+)) and quantification of signal joint TCR receptor excision circles (sjTRECs) by quantitative PCR. T cell repertoire diversity was analyzed on day +365 after autoHSCT by spectra-typing of the CDR3 region in the TCRVß chain. The BEAM group, in contrast to the Cy/TBI group, manifested significantly higher proportions of RTE cells and sjTREC copy numbers on days +100 and +180. Analysis of TCRVß spectra-types on day +365 revealed more restricted (monoclonal or oligoclonal) T cell repertoires in the Cy/TBI versus BEAM group (48.8% versus 18.2%, P = .0002). In conclusion, the conditioning scheme based on BEAM chemotherapy may be performed with lower risk of thymic destruction and T cell repertoire distortion than Cy/TBI scheme. This finding may help to potentially improve conditioning schemes to efficiently perform myeloablation and maintain active thymopoiesis.

A faster reconstitution of hematopoiesis after autologous transplantation of hematopoietic cells cryopreserved in 7.5% dimethyl sulfoxide if compared to 10% dimethyl sulfoxide containing medium.

Our previous in vitro studies proved a higher clonogenic potential of peripheral blood progenitor cells cryopreserved in 7.5% dimethyl sulfoxide (Me2SO) than in 10% Me2SO containing medium. Based on this findings 7.5% Me2SO cryopreservation medium was introduced to our protocol and both the hematopoietic recovery and infusion-related toxicity were compared with that obtained with standard 10% Me2SO containing solution. Two cohorts of consecutive patients treated with autologous hematopoietic stem cell transplantation were included in the analysis: 56 patients with PBPCs cryopreserved in 7.5% Me2SO solution and 52 patients who obtained cells cryopreserved in 10% Me2SO. Both study groups did not differ significantly with regard to age, diagnosis, and the number of transplanted CD34(+) cells. The time to leukocyte recovery was shorter for patients in the 7.5% Me2SO treated group than in the 10% one. Reconstitution of platelets and the frequency of adverse events did not differ in both groups. Reduction of Me2SO concentration from 10% to 7.5% in cryoprotective mixture has a beneficial impact on leukocyte recovery. These findings require verification in a prospective, randomized trial.

Association of circulating regulatory T cell number with the incidence and prognosis of diffuse large B-cell lymphoma.

Regulatory T (Treg) cells are essential for maintaining immune tolerance. High Treg frequencies have been reported in peripheral blood and tissue samples of patients with solid tumors while their role in lymphomas, including diffuse large B-cell lymphoma (DLBCL) has not been clearly established. In this study, we analyzed the circulating Treg numbers in 27 patients with newly diagnosed DLBCL and 17 healthy individuals. Tregs were detected by flow cytometry based on CD4(+) CD25(high) FoxP3(+) phenotype. In addition, the expression of CD45RA, HLA-DR, CD62L, CD39, and CTLA4 was analyzed. The number of circulating Treg cells was lower in patients with DLBCL than in healthy controls: median 23 (range, 4-107)/µL vs. 41 (19-104)/µL (P = 0.04). In particular, the number of Tregs expressing CD45RA (naïve Tregs), HLA-DR (marker of activation), and CD62L (L-selectin) was decreased in the DLBCL group. Lower (below median) number of circulating Tregs was associated with reduced chance of achieving complete remission (29% vs. 69%, P = 0.05) and reduced probability of even-free survival (24% vs. 84% at 1 yr, P = 0.0004), independently on the International Prognostic Index. We conclude that low number of circulating Tregs may be associated with poor prognosis in patients with DLBCL. However, our observations require confirmation in larger patient population.