Selected Contribution: Effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions

2003 ◽  
Vol 94 (5) ◽  
pp. 2085-2094 ◽  
Author(s):  
Michael J. Pecaut ◽  
Gregory A. Nelson ◽  
Luanne L. Peters ◽  
Paul J. Kostenuik ◽  
Ted A. Bateman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Psychological Stress ◽  
Space Shuttle ◽  
Splenic Lymphocytes ◽  
Immune Parameters ◽  
Population Distributions ◽  
First Time ◽  
T Helpers

There are several aspects of the spaceflight environment that may lead to changes in immunity: mission-related psychological stress, radiation, and changes in gravity. On December 5, 2001, the space shuttle Endeavor launched for a 12-day mission to examine these effects on C57BL/6 mice for the first time. On their return, assays were performed on the spleen, blood, and bone marrow. In response to flight, there were no significant differences in the general circulating leukocyte proportions. In contrast, there was an increase in splenic lymphocyte percentages, with a corresponding decrease in granulocytes. There was an overall shift in splenic lymphocytes away from T cells toward B cells, and a decrease in the CD4-to-CD8 ratios due to a decrease in T helpers. In contrast, there were proportional increases in bone marrow T cells, with decreases in B cells. Although the blast percentage and count were decreased in flight mice, the CD34+ population was increased. The data were more consistent with a shift in bone marrow populations rather than a response to changes in the periphery. Many of the results are similar to those using other models. Clearly, spaceflight can influence immune parameters ranging from hematopoiesis to mature leukocyte mechanisms.

scholarly journals INDUCTION OF A HEMOLYSIN RESPONSE IN VITRO

1971 ◽  
Vol 133 (6) ◽  
pp. 1325-1333 ◽  
Author(s):  
Klaus-Ulrich Hartmann
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Close Contact ◽  
Precursor Cells ◽  
Spleen Cells ◽  
High Concentrations ◽  
Thymus Cells ◽  
Bone Marrow Chimeras

Spleen cells of bone marrow chimeras (B cells) and of irradiated mice injected with thymus cells and heterologous erythrocytes (educated T cells) were mixed and cultured together (17). The number of PFC developing in these cultures was dependent both on the concentration of the B cells and of the educated T cells. In excess of T cells the number of developing PFC is linearly dependent on the number of B cells. At high concentrations of T cells more PFC developed; the increase in the number of PFC was greatest between the 3rd and 4th day of culture. Increased numbers of educated T cells also assisted the development of PFC directed against the erythrocytes. It is concluded that the T cells not only play a role during the triggering of the precursor cells but also during the time of proliferation of the B cells; close contact between B and T cells seems to be needed to allow the positive activity of the T cells.

scholarly journals Hematologic engraftment and immune reconstitution posttransplantation with anti-B1 purged autologous bone marrow

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 597-604
Author(s):  
KC Anderson ◽  
J Ritz ◽  
T Takvorian ◽  
F Coral ◽  
H Daley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Immune Reconstitution ◽  
Autologous Bone Marrow ◽  
Normal Numbers ◽  
Autologous Bone ◽  
Total Body ◽  
One Year

Hematologic engraftment and immune reconstitution were examined in patients who received cyclophosphamide and total body irradiation therapy followed by infusion of autologous bone marrow purged with anti- B1 monoclonal antibody (MoAb) and complement as therapy for non- Hodgkin's lymphoma. Hematologic engraftment was prompt with return of greater than or equal to 0.5 X 10(3)/microL granulocytes and greater than or equal to 2 X 10(4)/microL platelets at a median of 26 and 29 days posttransplant, respectively. Immunologic reconstitution, in contrast, was prolonged. Normal numbers of circulating B cells were consistently noted by five months posttransplant, whereas return of normal immunoglobulin levels in some patients did not occur for one year. Normal numbers of T cells were evident within the first month posttransplant, but a reversed T4:T8 ratio persisted in some patients up to three years. In vitro responses of either B cells to triggers of activation or of T cells to mitogens and antigens were not normal for at least three months posttransplant. Natural killer (NK) cells predominated early after transplant and may demonstrate cytotoxicity against tumor cells. Our studies demonstrate that transplantation with anti-B1 purged autologous bone marrow results in complete hematologic and delayed immunologic engraftment. No significant acute or chronic clinical toxicities have been observed.

Immunosuppressive effects of pentostatin.

1990 ◽  
Vol 8 (5) ◽  
pp. 848-855 ◽  
Author(s):  
E H Kraut ◽  
J C Neff ◽  
B A Bouroncle ◽  
D Gochnour ◽  
M R Grever
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Investigational Drug ◽  
Cd8 Cells ◽  
Immune Parameters ◽  
Before And After ◽  
Significant Depression ◽  
Normal Monocyte

The immune function of patients with hairy cell leukemia (HCL) and solid tumors was evaluated before and after treatment with the investigational drug 2'-deoxycoformycin (pentostatin; dCF). Thirteen HCL patients received doses of dCF of 2 to 4 mg/m2 intravenously at 2- to 6-week intervals for up to 15 courses. After completion of treatment, 12 of 13 patients had resolution of severe monocytopenia and five of nine had normal monocyte antibody dependent cellular cytotoxicity. There was statistically significant depression of total lymphocytes, T cells, and B cells. Evaluation of T subsets showed a decrease in CD4+ cells. Immunoglobulin G (IgG) in sera were decreased from baseline, while IgM and IgA were unaffected. There was no significant effect on skin-test reactivity or large granular lymphocyte numbers. Lymphoblastic transformation was variably affected. Natural-killer (NK) cell function was improved or unchanged after dCF treatment. Reevaluation of seven patients at 21 to 119 weeks after receiving dCF demonstrated that recovery to normal T- and B-cell numbers and subsets does occur. Five solid tumor patients were given dCF at 4 mg/m2 intravenously at 1- to 2-week intervals for up to five courses. There was significant reduction in T cells, B cells, CD4+, and CD8+ cells with no statistically significant effect on the other immune parameters. We conclude that low doses of dCF can cause persistent immunosuppression though recovery may occur after the drug is stopped. In patients followed after completion of dCF, there was no associated increase in second malignancies or unusual infections.

scholarly journals 5'-Nucleotidase in different populations of mouse lymphocytes.

1977 ◽  
Vol 25 (2) ◽  
pp. 97-103 ◽  
Author(s):  
R J Uusitalo ◽  
M J Karnovsky
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cell Populations ◽  
Consistent Difference ◽  
Splenic Lymphocytes ◽  
Splenic Cells ◽  
Ultrastructural Radioautography ◽  
Different Populations ◽  
Immature Cells ◽  
Mouse Lymphocytes

The activity of 5'-nucleotidase in different populations of intact lymphocytes was studied using biochemical, cytochemical and radioautographic methods. In some strains of mice the results showed a consistent difference in 5'-nucleotidase (AMPase) content between intact thymic and splenic lymphocytes. In the R III, C 57, BALB/c, CBA and Cd-1 strains AMPase activity in the isolated splenic cells was foru to 10 times the activity of intact thymocytes. In highly enriched populations of splenic T and B cells the average AMPase activity was about the same. From separate assays it was seen that the AMPase activity in highly enriched populations of lymphoctes was variable so that within one experiment the T cells seemed to have the higher AMPase activity while in other experiments B cells shown to be more active than T cells. Ultrastructural radioautography was done to count AMPase positive cells within T and B cell populations, the latter identified b binding of I125-labelled anti-immunoglobulin. It was seen that about 50% of B cells, but only about 10% of T cells, were positive for AMPase. It is suggested that there is a subpopulation within B and T cell populations with a high membrane AMPase activity and another subpopulation with less or no enzyme activity. It is also suggested that the activity and/or the proportion of these positive cells is changing within the splenic cell population. By using cortisone to deplete the immature cells from the thymus it was seen that the remaining mature cells have about the same AMPase activity as did the immaturecells, and thus mature T cells must gain their high acitivity after leaving the thymus. By incubating splenic lymphocytes with Concanavalin A it was also seen that the immature transformed cells had the same amount of enzyme as did untransformed cells.

Long-Term Chimerism and B-Cell Function After Bone Marrow Transplantation in Patients With Severe Combined Immunodeficiency With B Cells: A Single-Center Study of 22 Patients

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2923-2930 ◽  
Author(s):  
Elie Haddad ◽  
Françoise Le Deist ◽  
Pierre Aucouturier ◽  
Marina Cavazzana-Calvo ◽  
Stephane Blanche ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Severe Combined Immunodeficiency ◽  
Combined Immunodeficiency ◽  
B Cell Function ◽  
Host Origin

We retrospectively analyzed the B-cell function and leukocyte chimerism of 22 patients with severe combined immunodeficiency with B cells (B+ SCID) who survived more than 2 years after bone marrow transplantation (BMT) to determine the possible consequences of BMT procedures, leukocyte chimerism, and SCID molecular deficit on B-cell function outcome. Circulating T cells were of donor origin in all patients. In recipients of HLA-identical BMT (n = 5), monocytes were of host origin in 5 and B cells were of host origin in 4 and of mixed origin in 1. In recipients of HLA haploidentical T-cell–depleted BMT (n = 17), B cells and monocytes were of host origin in 14 and of donor origin in 3. Engraftment of B cells was found to be associated with normal B-cell function. In contrast, 10 of 18 patients with host B cells still require Ig substitution. Conditioning regimen (ie, 8 mg/kg busulfan and 200 mg/kg cyclophosphamide) was shown neither to promote B-cell and monocyte engraftment nor to affect B-cell function. Eight patients with B cells of host origin had normal B-cell function. Evidence for functional host B cells was further provided in 3 informative cases by Ig allotype determination and by the detection, in 5 studied cases, of host CD27+ memory B cells as in age-matched controls. These results strongly suggest that, in some transplanted patients, host B cells can cooperate with donor T cells to fully mature in Ig-producing cells.

Mezagitamab Induces Immunomodulatory Effect in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Michael Abadier ◽  
Jose Estevam ◽  
Deborah Berg ◽  
Eric Robert Fedyk
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Cell Populations ◽  
Landscape Changes ◽  
Ki 67 ◽  
Immune Cell Subsets

Background Mezagitamab is a fully human immunoglobulin (Ig) G1 monoclonal antibody with high affinity to CD38 that depletes tumor cells expressing CD38 by antibody- and complement-dependent cytotoxicity. CD38 is a cell surface molecule that is highly expressed on myeloma cells, plasma cells, plasmablasts, and natural killer (NK) cells, and is induced on activated T cells and other suppressor cells including regulatory T (Tregs) and B (Bregs) cells. Data suggest that immune landscape changes in cancer patients and this may correlate with disease stage and clinical outcome. Monitoring specific immune cell subsets could predict treatment responses since certain cell populations either enhance or attenuate the anti-tumor immune response. Method To monitor the immune landscape changes in RRMM patients we developed a mass cytometry panel that measures 39-biomarkers to identify multiple immune cell subsets, including T cells (naïve, memory, effector, regulatory), B cells (naïve, memory, precursors, plasmablasts, regulatory), NK cells, NKT cells, gamma delta T cells, monocytes (classical, non-classical and intermediate), dendritic cells (mDC; myeloid and pDC; plasmacytoid) and basophils. After a robust analytical method validation, we tested cryopreserved peripheral blood and bone marrow mononuclear cells from 19 RRMM patients who received ≥ 3 prior lines of therapy. Patients were administered 300 or 600 mg SC mezagitamab on a QWx8, Q2Wx8 and then Q4Wx until disease progression schedule (NCT03439280). We compared the percent change in immune cell subsets at baseline versus week 4 and week 16. Results CD38 is expressed at different levels on immune cells and sensitivity to depletion by mezagitamab generally correlates positively with the density of expression. CD38 is expressed at high densities on plasmablasts, Bregs, NK-cells, pDC and basophils at baseline and this was associated with reductions in peripheral blood and bone marrow (plasmablasts, 95%, Bregs, 90%, NK-cells, 50%, pDC, 55% and basophils, 40%) at week 4 post treatment. In contrast, no changes occurred in the level of total T-cells and B-cells, which is consistent with low expression of CD38 on most cells of these large populations. Among the insensitive cell types, remaining NK-cells acquired an activated, proliferative and effector phenotype. We observed 60-150% increase in activation (CD69, HLA-DR), 110-200% increase in proliferation (Ki-67), and 40-375% increase in effector (IFN-γ) markers in peripheral blood and bone marrow. Importantly, NK-cells which did not express detectable CD38, also exhibited a similar phenotype possibly by a mechanism independent of CD38. Consistent with these data, the remaining CD4 and CD8 T-cell populations exhibited an activated effector phenotype as observed by 40-200% increase in activation, 60-200% increase in proliferation and 40-90% increase in effector markers in peripheral blood. A potential explanation for this acquisition of activated effector phenotypes could be a reduction in suppressive regulatory lymphocytes. Next, we measured levels of Tregs and Bregs, and observed that Bregs which are CD24hiCD38hi were reduced to 60-90% in peripheral blood and bone marrow. In contrast, total Tregs were reduced by only 5-25% because CD38 expression in Tregs appears as a spectrum where only ~10-20% are CD38+, and thus CD38+ Tregs were reduced more significantly (45-75%), reflecting the selectively of mezagitamab to cells expressing high levels of CD38. CD38+ Tregs are induced in RRMM patients, thus we looked at the phenotype of CD38-, CD38mid, and CD38high -expressing Tregs. We observed higher level of markers that correlate with highly suppressive Tregs such as Granzyme B, Ki-67, CTLA-4 and PD-1 in CD38high Tregs. Accordingly, the total Treg population exhibited a less active phenotype after exposure to mezagitamab, which selectively depleted the highly suppressive CD38+ Tregs. Conclusions Chronic treatment with mezagitamab is immunomodulatory in patients with RRMM, which is associated with reductions in tumor burden, subpopulations of B and T regulatory cells, and characterized by conventional NK and T cells exhibiting an activated, proliferative and effector phenotype. The immune landscape changes observed is consistent with the immunologic concept of converting the tumor microenvironment from cold-to-hot and highlights a key mechanistic effect of mezagitamab. Disclosures Berg: Takeda Pharmaceuticals Inc: Current Employment.

Plasma and Intracellular Concentration of TNF and Its Receptors in Peripheral Blood and Bone Marrow in Different Stages of B-CLL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4781-4781
Author(s):  
Jacek Rolinski ◽  
Agnieszka Bojarska-Junak ◽  
Iwona Hus ◽  
Anna Dmoszynska
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Biological Effects ◽  
Leukemic Cells ◽  
Control Group ◽  
Stage Of Disease ◽  
Positive Correlation

Abstract TNF has been proposed to play a role in the regulation of growth and death of leukemic B-CLL cells. However, the biological effects of TNF on leukemic cells, as well as its role as a prognostic factor need to be further investigated. The aim of the study was to eevaluate the correlation of TNF and its receptors in peripheral blood (PB) and bone marrow (BM) with the stage of B-CLL and some other clinical parameters. PB and BM were taken from 44 newly diagnosed, untreated B-CLL. patients. The control group consisted of 20 healthy subjects. We used flow cytometry technique to assess the capability of T and B lymphocytes to produce TNF and ELISA method to measure plasma levels of TNF and their soluble receptors. We found, that PB and BM plasma TNF concentration in the patients was significantly higher than in the healthy control (2.61 pg/ml. vs 0.62 pg/ml; and 2.91 pg/ml vs 0.75 pg/ml, respectively p<0.001). TNF concentration in PB and BM was significantly higher in Rai stage III–IV than in early stages (p<0.01). There was a correlation between the PB and BM TNF level and lymphocytosis (p<0.005) and the total tumor mass (TTM) (p<0.0001). The PB and BM TNF concentration positively correlated with the percentage of T CD3+ lymphocytes producing intracellular TNF (p<0.01). The percentage of T cells from PB an BM expressing cytoplasmic TNF was significantly higher in patients (PB:39.11±16.97%; BM:40.73±18.19%) than in normal controls (PB:15.74±7.95%; BM:18.80±12.93%) (p< 0.00001; p<0.005, respectively). In PB and BM from B-CLL patients the percentage of CD3+ cells expressing intracellular TNF was significantly higher than the percentage of CD19+/TNF+ cells (p<0.0001). Besides, it was found that the percentage of T cells expressing cytoplasmic TNF positively correlated with the stage of disease (p<0.01). In PB positive correlation were found between the number of T CD3+/TNF+ cells and lymphocytosis (p<0.05) and TTM (p<0.001). The percentage of leukaemic B cells positive for TNF did not correlate with the stage of disease. There was increased expression of TNF-RI and TNF-RII in leukaemic B cells in comparison to normal B-cells was observed (p<0.0001). We found positive correlation between the number of CD5+ B lymphocytes and the levels of soluble TNF-RII (sTNF-RII) (p< 0.05). The sTNF-RII levels in PB and BM significantly correlated with the stage of disease acc. Rai (p<0.0001). Furthermore, the sTNF-RII concentration positively correlated with lymphocytosis and TTM (p<0.0001). These results strongly support the key role TNF in B-CLL pathogenesis. Our results suggest that TNF may function as growth factor for B-CLL cells. CD3+T cells may be the important source of this cytokine in advanced B-CLL. It seems that changes in T cells capability to produce cytoplasmic TNF are associated with disease progression. However, further studies are required to confirm the key role of TNF in B-CLL pathogenesis.

Cucurbitacins, a Family of Natural Compounds That Effectively Disrupt Stat3 Signaling in Antigen-Presenting Cells (APCs) Are Promising Agents To Overcome Tumor-Induced CD4+ T-Cell Tolerance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1485-1485
Author(s):  
Fengdong Cheng ◽  
Hongwei Wang ◽  
Alfonso Suarez ◽  
Pedro Horna ◽  
Said Sebti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Cd4 T Cells ◽  
Natural Compounds ◽  
Dual Inhibitor ◽  
Ifn Gamma ◽  
Stat3 Signaling ◽  
Cognate Antigen ◽  
Antigen Presenting

Abstract Signal transducer and activator of transcription 3 (Stat3) is a key mediator of several cytokines and growth factors signaling pathways. On myeloid cells, activation of Stat3 to its phosphorylated form (pStat3) has been shown to negatively regulate inflammatory responses. Recently, we have unambiguously demonstrated that Stat3 signaling in APCs also play a central role in the decision leading to immune activation versus immune tolerance of antigen-specific T-cells1. In spite of these advances, there is however a paucity of therapeutic strategies targeting this signaling pathway in immune cells. Using a high throughput cytoblot screening for phospho-Stat3 inhibition, we have recently identified a family of natural compounds known as Cucurbitacins that effectively disrupt Stat3 signaling at different levels2. Three compounds have been identified, Cucurbitacin A (CuA) that inhibits phospho-JAK-2, Cucurbitacin I (CuI) a dual inhibitor of p-JAK2 and p-Stat3 and Cucurbitacin Q (CuQ) a selective inhibitor of p-Stat3. In vitro treatment of peritoneal elicited macrophages (PEM) and bone marrow-derived dendritic cells (DCs) with increasing concentrations of CuA or CuI resulted in inhibition of p-Stat3 and enhanced antigen presentation to naive CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). Indeed, these clonotypic T cells displayed increased antigen-specific proliferation and IL-2 production as compared to clonotypic T cells encountering cognate antigen on untreated APCs. Furthermore, unlike untreated PEM or DCs, which are unable to trigger IFN-gamma production by CD4+ T-cells, Cucurbitacin-treated APCs efficiently trigger the production of this cytokine by naïve CD4+ T-cells in response to cognate antigen. Given the above results, we explored next whether inhibition of Stat3 signaling in B-cell lymphomas by Cucurbitacins might increase the intrinsic antigen-presenting capabilities of these malignant B-cells. Reminiscent of our findings with bone marrow derived APCs, Cucurbitacin-treated A20 lymphoma cells also display enhanced antigen-presenting cell function leading to increased proliferation, IL-2 and IFN-gamma by naive antigen-specific CD4+ T-cells. More importantly, tolerant CD4+ T-cells (isolated from lymphoma bearing mice) exposed to Cucurbitacin-treated A20 B-cells regained their ability to proliferate and produce significant amounts of IL-2 and IFN-gamma in response to cognate antigen stimulation. Taken together, the ability of Cucurbitacins to inhibit p-Stat3 in normal APCs as well as in malignant B-cells make these natural compounds a promising agents to overcome the remarkable barrier that tolerance to tumor antigens has imposed to cancer immunotherapeutic strategies.

Flow Cytometry Primary 10 Colours Panel for Chronic Lympho Proliferative Diseases Diagnosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5190-5190
Author(s):  
Jonathan Brauner ◽  
Ingrid Beukinga ◽  
Zoulikha Amraoui ◽  
Zaina Kassengera ◽  
Michel Toungouz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Lymphocytes ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
Cd10 Expression ◽  
Complete Characterisation

Abstract Abstract 5190 Objectives: Definition of the primary antibodies panel for 10 colours flow cytometry able to describe normal and clonal T, B lymphocytes and plamocytes in blood and bone marrow. Once clonalities are detected, the complete characterisation of Chronic Lymphoproliferative Diseases (CLPD) is supported by secondary panels chosen based on the results of CD5/CD10 expression for clonal B lymphocytes, CD27/CD38 for plasmatocytes and CD3/CD27 for clonal T cells. Materials and Methods: Blood and bone marrow of patients (N=50) with CLPD (mainly B-CLL). Samples are enumerated by haematology analyzer DxH 800 then 106 cells are washed three times, stained with the antibodies combination and red blood cells lysed with Versalyse (TM. Beckman Coulter). The samples were analysed on a 10 colours Navios flow cytometer (Beckman Coulter Fullerton, CA). The staining panel consists of 14 antibodies (CD45, CD8, CD4, CD5, CD3, CD19, CD38, λ, κ, CD23, CD5, CD10, CD14, CD27) conjugated with 10 different fluorochromes. The fixed gating strategy allows linking Navios analysis software to the middleware Remisol which drives the choice of the secondary panel. In some cases a third tube is performed for Ki67 or Zap-70 intra-cytoplasmic staining. Results: Monocytes are removed on the basis of their CD14/CD4 expression. B lymphocytes are CD19 positive. Normal naïve/memory B cells, hematogones and plasma cells are defined by their CD27, CD10 and CD38 expression. Eventual monoclonality is sought by analysis of the distribution of Kappa and Lambda light chains. A first classification of B cell lymphoma is achieved with the CD5 and CD10 expression of the clone (CD5+/CD10−: B-CLL MCL and few MZL, CD5−/CD10−: MZL and related, CD5−/CD10+ DLBCL and FL). Analysis of CD27, CD20 and CD23 expression allows discriminating between CD5+/CD10- lymphomas. All the 50 samples were correctly detected as CLPD and the automated Remisol choice of the second panel fit to the final diagnosis of all the cases of this small series. T lymphocytes are defined by their CD3 and CD5 expression. The analysis of CD4/CD8 balance and CD27/CD5 distribution are first line test when T cell clonality is suspected. There is a special gating to detect CD3-CD4+ T cell lymphoma and double negativity of CD4 and CD8 is a surrogate marker for gamma/delta T cells. NK cells are mentioned as not-T not-B lymphocytes, without specific staining. Conclusion/Discussion:This 10 colours 14 antibodies panel allows describing in one tube normal T and B cells, hematogones, memory and naives B cells plasma cells and detects T and B clonalities. This panel follows a similar logic than the Euroflow LST tube but with 10 colours and with Beckman Coulter's technology and antibodies. Moreover, this combination helps discriminating rapidly the CD5+/CD10- lymphomas while the complete characterisation of CD5 negative lymphomas only require less than 6 antibodies second tube. This is a paperless (all the process is driven and controlled by Remisol), fast and inexpensive diagnostic approach (always less than 20 antibodies required). Disclosures: Pradier: Beckman Coulter: Consultancy, Membership on an entity's Board of Directors or advisory committees.

IL-21 and IL-6 Mediate Interactions Between T Cells and Malignant B Cells in the Bone Marrow Microenvironment in Waldenstrom's Macroglobulinemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1554-1554
Author(s):  
Lucy S. Hodge ◽  
Steve Ziesmer ◽  
Frank J Secreto ◽  
Zhi-Zhang Yang ◽  
Anne Novak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Bone Marrow Microenvironment ◽  
Waldenström’S Macroglobulinemia ◽  
Waldenström's Macroglobulinemia

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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