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.

scholarly journals The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.

1987 ◽  
Vol 165 (6) ◽  
pp. 1675-1687 ◽  
Author(s):  
A G Rolink ◽  
T Radaszkiewicz ◽  
F Melchers
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
The Other ◽  
Cell Populations ◽  
Foreign Antigen ◽  
Alloreactive T Cells ◽  
Polyclonal Activator ◽  
Activated B Cells

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

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.

Multidimensional Flow Cytometric (MFC) Analysis of the Immune System of Multiple Myeloma (MM) Patients Achieving Long Term Disease Control

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 810-810
Author(s):  
Roberto J. Pessoa Magalhaes ◽  
María-Belén Vidriales ◽  
Bruno Paiva ◽  
Maria-Victoria Mateos ◽  
Norma C. Gutierrez ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Disease Control ◽  
B Cell ◽  
Nk Cells ◽  
Cell Populations ◽  
High Dose ◽  
Newly Diagnosed

Abstract Abstract 810FN2 Increasing evidence shows that a small fraction of MM patients (pts) treated with high-dose therapy followed by autologous stem cell transplantation achieve long-term remission. Interestingly, this is not restricted to pts in complete response (CR), since those that revert to a monoclonal gammopathy of undetermined significance (MGUS) profile may also achieve long-term remission, despite the persistence of residual myeloma plasma cells (PCs). These results suggest that in addition to the anti-myeloma therapy, other factors may play a role in the control of the disease. Herein, we used 8-color MFC for detailed characterization of the structural components of the immune system and hematopoietic precursor cells (HPC) in paired bone marrow (BM) and peripheral blood (PB) samples from 26 MM patients in long-term disease control (LTDC): 9 in continuous CR and 17 who reverted to an MGUS profile and that subsequently showed stable disease without treatment for ≥5 years (median of 9 years; range, 5–19). As controls, paired BM and PB samples from 23 newly-diagnosed MGUS and 16 MM pts, together with 10 healthy adults (HA), were studied in parallel. In all BM and PB samples the distribution of the major T- (CD4, CD8, Tregs and γδ), NK- (CD56dim and CD56bright) and B-cell subsets (Pro-B, Pre-B, naïve and memory), in addition to normal PCs, dendritic cell (DC) subsets (plasmacytoid, myeloid and monocytic), monocytes, and CD34+ HPC (myeloid and lymphoid), were studied. The percentage and absolute count of each cell population was analysed in the BM and PB, respectively. Comparison of the two groups of MM pts with LTDC (9 CR vs. 17 MGUS-like) showed similar (p>.05) cellular profiles in PB and BM, except for an increased number of BM and PB normal PCs in CR patients (P≤.04). Consequently, for all subsequent analyses, LTDC myeloma pts were pooled together. When compared to HA, patients with LTDC had increased numbers of CD8 T-cells and CD56dim NK-cells in both the BM and PB (p≤.03 and p≤.01, respectively). Despite this, the distribution of BM and PB CD4, CD8 and γδ T-cells among LTDC patients was similar (p>.05) to that of both newly-diagnosed MM and MGUS cases; in contrast, BM and PB Tregs were significantly decreased vs newly-diagnosed MM (P=.03) and MGUS (P=.04). Regarding B-cells and normal PCs, LTDC patients showed increased numbers of BM B-cell precursors (both Pro-B and Pre-B cells) and normal PCs vs. newly diagnosed MM (P≤.05), but not MGUS, together with increased numbers of naïve B-cells vs. both MM and MGUS pts (P≤.01); all such cell populations returned to levels similar (p>.05) to those of HA. As expected, this also included the number of CD34+ B-cell HPC which was increased among patients who achieved LTDC vs MM (p=.02), at levels similar (p>.05) to those of MGUS and HA. Regarding DC, LTDC patients showed normal DC numbers in PB (but with higher PB myeloid-DC numbers vs. MM; p=.02), in association with decreased numbers of plasmacytoid DC and increased monocytic-DC in the BM vs. HA (p≤.04). No differences were found for the numbers of BM and PB monocytes. In summary, here we investigated for the first time the immune cell profile of MM patients who achieve long-term disease control. Our results show that, as newly-diagnosed MM, patients that achieve long-term disease control also show increased numbers of cytotoxic T-cells and CD56dim NK-cells; however, in contrast to newly-diagnosed MM, among LTDC patients such increase is associated with lower numbers of T-regs and an almost complete recovery of the normal PC, B-cell precursor and naïve B-cell compartments both in BM and PB. Further investigations on the activation and functional status of these cell populations are warranted.MO (%)/SP (cels./μl)HA N= 10MGUS N= 23MM N= 16LTDC-MM N= 26T cells9.588110.6117313113711926    CD4+4.85004.6624^6*5085463    CD8+3.7∼216∼4.63865.32645.3431    TCR γδ.2426.3230.2428.3421    Treg.4137.4141^.54*38.3432NK cells.7∼87∼1.51982.11721.6212    CD56 dim.65∼79∼1.41922.21681.6202B cells2.81471.8104.97*68*1.9160    Pro B.11—.06—.02*—.07—    Pre B.6—.4—.08—.23—    Naive SP—80—57^—36*—118    Normal-PCS.18.9.11.7.008.72*.11.84DCs.3449.3653.6848.558    Monocytes2.22472.42853.43023.1315    m-DC SP—11—14—8*—12    MO-DC.11∼29.2036.434.2837    p-DC.2∼4.1.145.112.8.123.8CD34+.9∼1.46.61.1.261.4.431.4    Mie-HPC.8∼—.53—.26—.36—    Linfo-HPC.1—.07—.03*—.05—*p≤.05 LTDC vs MM: ^ p≤.05 LTDC vs MGUS; ∼ p≤.05 LTDC vs HA Disclosures: Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Martinez:Janssen: Honoraria; Celgene: Honoraria. Maiolino:Centocor Ortho Biotech Research & Development: Research Funding.

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 Rosette formation between murine lymphocytes and erythrocytes. A new locus in the H-2 region.

1979 ◽  
Vol 149 (6) ◽  
pp. 1349-1359 ◽  
Author(s):  
D Primi ◽  
G K Lewis ◽  
R Triglia ◽  
J W Goodman
Keyword(s):  
T Cells ◽  
B Cells ◽  
Rosette Formation ◽  
Chromosome 17 ◽  
Splenic Lymphocytes ◽  
Recombinant Strains ◽  
Surface Immunoglobulin ◽  
Self Recognition

More than 5% of murine splenic lymphocytes form rosettes with syngeneic erythrocytes. This property was maximally expressed when the lymphocytes were cultured for 24 h before rosetting. About 70% of the rosetting lymphocytes were B cells and 30% were T cells on the basis of surface immunoglobulin and the Thy-1-antigen. Capping surface immunoglobulin had no effect on the capacity of lymphocytes to form rosettes, indicating that the receptor in question was not immunoglobulin. The capacity of lymphocytes to form rosettes with erythrocytes from other strains of mice was H-2 restricted. Extensive pairings of congenic and recombinant strains as donors of lymphocytes and erythrocytes showed that none of the known loci within the H-2 region-controlled rosetting. The involvement of regions on chromosome 17, telomeric or centromeric to H-2, was also excluded. The data were only compatible with the conclusion that this form of self-recognition is associated with a new locus (or loci) mapping between H-2G and H-2D.

scholarly journals Human allogeneic stem cell maintenance and differentiation in a long- term multilineage SCID-hu graft

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1680-1693 ◽  
Author(s):  
CC Fraser ◽  
H Kaneshima ◽  
G Hansteen ◽  
M Kilpatrick ◽  
R Hoffman ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
B Cells ◽  
Myeloid Cells ◽  
Cell Populations ◽  
Multilineage Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Populations

The ability to determine the functional capacity of putative human hematopoietic stem cell (HSC) populations requires in vivo assays in which long-term multilineage differentiation can be assessed. We hypothesized that if human fetal bone was transplanted adjacent to a fetal thymus fragment in severe combined immunodeficient (SCID) mice, a conjoint organ might form in which HSC in the human bone marrow (BM) would mimic human multilineage differentiation into progenitor cells, B cells, and myeloid cells; undergo self-renewal; and migrate to and differentiate into T cells within the thymic microenvironment. To test this possibility, SCID mice were transplanted subcutaneously with HLA class I mismatched fetal bone, thymus, and spleen fragments (SCID-hu BTS). We found that the BM of SCID-hu BTS grafts maintained B cells, myeloid cells, CD34+ cells for at least 36 weeks posttransplant. Assayable hematopoietic progenitors colony-forming units-granulocyte- macrophage were present in 100% (66/66) of grafts over a period of 28 weeks. Cells with a HSC phenotype (CD34+Thy-1+Lin-) were maintained for 20 weeks in SCID-hu BTS grafts. These CD34+Thy-1+Lin- cells had potent secondary multilineage reconstituting potential when isolated and injected into a secondary HLA mismatched SCID-hu bone assay and analyzed 8 weeks later. In addition, early progenitors within the BM of SCID-hu BTS grafts were capable of migrating to the human thymus and undergoing differentiation through immature CD4+CD8+ double-positive T cells and produce mature T cells with a CD4+CD8- or CD8+CD4- phenotype that could be detected for at least 36 weeks. Phenotypically defined human fetal liver (FL) and umbilical cord blood (UCB) hematopoietic stem cell populations were injected into irradiated SCID-hu BTS grafts to assess their multilineage repopulating capacity and to assess the ability of the BTS system to provide an environment where multiple lineages might differentiate from a common stem cell pool. Injection of irradiated grafts with FL HSC or UCB HSC cells resulted in donor- derived B cells, myeloid cells, immature and mature T cells, and CD34+ cells in individual grafts when analyzed 8 weeks postreconstitution, further showing the multipotential nature of these stem cell populations. In addition, a strong correlation was observed between maintenance of host graft-derived CD8+ cells and failure of donor stem cell engraftment.(ABSTRACT TRUNCATED AT 400 WORDS)

CD154 Blockade Prevents Allosensitization through Inhibiting Both T- and B-Cell Activation and Decreasing Expression of IFN-γ and IL-10 in T-Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1346-1346
Author(s):  
Hong Xu ◽  
Jun Yan ◽  
Yiming Huang ◽  
Paula M. Chilton ◽  
Michael K. Tanner ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Skin Grafting ◽  
Cell Populations ◽  
B Cell Activation ◽  
T And B Cells ◽  
Time Points ◽  
Ifn Γ

Abstract Recipient sensitization to MHC antigens from transfusion therapy and prior graft rejection is among the most critical of problems in clinical transplantation. Sensitized patients reject vascularized organ or bone marrow transplants within minutes to hours as a result of preformed anti-donor Abs. Preventing allosensitization at the time recipients are exposed to donor alloantigens would be of obvious clinical benefit. B cell activation and the generation of memory B cells depends upon T cell responses via signaling from the co-stimulatory molecule CD154 (on activated T cells) to CD40 (on B cells). We have demonstrated in an allogeneic mouse model [BALB/c (H2Kd) to B6 (H2Kb)] that blockade of T and B cell interactions with anti-CD154 induces B cell tolerance, as defined by complete abrogation of the generation of donor-specific Ab after skin grafting. Furthermore, anti-CD154 treatment promotes successful subsequent bone marrow transplantation in these recipients, confirming that sensitization was prevented. In this study, we evaluated the effect of anti-CD154 mAb on T- and B-cell populations, activation state, and cytokine expression by T cells. B6 recipients were treated with anti-CD154 (day 0 and +3) or isotype hamster IgG control around the time receiving BALB/c skin grafts (day 0), and the number of T-cells (CD4+ and CD8+), total B-cells (CD19+), immature B-cells (CD19+CD24highCD23low), and follicular B-cells (CD19+CD24lowCD23high) in the spleen was enumerated by 4 color flow cytometry at day 7, 15 and 25 after skin grafting. No significant difference in absolute number of T- and B-cell subpopulations was seen between anti-CD154 and control IgG treated groups at the time points tested. By measuring the percentage CD71+ cells in the CD8+ or CD4+ gate or CD69+ in the CD19+ gate, activated T and B cell populations were evaluated. In vivo blockade of CD154 resulted in a significantly reduced activation of alloreactive T- and B-cells: the percentage of CD8+/CD71+ T cells was significantly lower at day 7 and the percentage of CD4+/CD71+ T cells was significantly lower at all time points compared with control mice (P < 0.05). The percentage of CD19+/CD69+ B cells at day 7 and 25 was significantly lower compared with control IgG treated mice (P < 0.05). To determine the effect of anti-CD154 treatment on Th1 and Th2 cytokine production, intracellular IFN-γ and IL-10 expression was analyzed. The IFN-γ expression in both CD8 and CD4 T-cells was inhibited at day 7 and reached significance (P < 0.01) by day 15 compared with control IgG treated group. IL-10, a cytokine which promotes B-cell activation and differentiation expression, was similar at day 7 between the two groups, but significantly decreased in both CD8 and CD4 T-cells at day 15 in mice treated with anti-CD154. Therefore, these data suggest that blockade of CD154 during initial antigen exposure mechanistically interferes with activation of both allo antigen-specific T and B-cells and inhibits the generation of allogeneic Ab (allosensitization). These effects are associated with suppression of IFN-γ and IL-10 cytokine secretion. Figure Figure

Longitudinal Analysis Reveals Telomere Length Maintenance In CLL B-Cells But Marked Erosion In CLL Patient T-Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1617-1617
Author(s):  
Ceri Jones ◽  
Thet Lin ◽  
Guy Pratt ◽  
Chris Fegan ◽  
Duncan Baird ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Telomere Length ◽  
Erosion Rate ◽  
B Lymphocyte ◽  
Cell Populations ◽  
Aggressive Disease ◽  
Telomere Dynamics

Abstract Telomere length (TL) is a prognostic factor in Chronic Lymphocytic Leukemia (CLL) with short TL being a predictor of time to first treatment, progression-free survival and overall survival. However, little is known about telomere dynamics through the course of the disease. Most studies conducted on CLL patients have measured TL in unselected peripheral blood mononuclear cell populations, often at a single time point. In this context, longitudinal analysis of TL is problematic as patients who undergo disease progression and/or treatment may have a significantly altered proportion of CLL cells in their peripheral blood compared to T-cells. In order to ensure that we specifically analyzed the TL of the tumor cells, we used fluorescence-activated cell sorting (FACS) to sort populations of CD19+CD5+ CLL B-cells and CD3+T-cells from samples taken from individual patients at different time points throughout their disease (n=30). We then performed high-resolution single telomere length analysis (STELA) on these sorted subsets of cells and analyzed their telomere dynamics over time (median follow up 69 months). We found a signifcant difference in the CLL B-cell TL (p=0.05) with a mean erosion rate of -52base pairs/year. TL change in the 18/30 patients who remained untreated at all time points was -51bp/year. In the 6/30 patients who received treatment following their initial TL measurements the mean TL change was -40bp/year. Finally, for 11/30 patients samples were only available in the post treatment setting, for these patients the TL change was lower at -29bp/year. The difference in TL erosion between these different groups was not statistically significant. These data shows that the TL erosion in CLL B-cells is modest and similar to that of an age-matched population (Steenstrup et al 2013). Furthermore, CLL B-cells showed no reduction in TL standard deviation signifying the maintenance of intraclonal diversity (p=0.78). In contrast to the modest changes in TL observed in the CLL B-cells, TL change in the T-cell populations were much more pronounced with mean change of -119bp/year (p=0.02). Furthermore, there was a trend towards increased erosion in the treated patient group when compared with the untreated group (-230bp/year vs -85bp/year, p=0.14) suggesting that therapy may have an impact on the composition of T-cell populations in treated CLL patients. In keeping with this notion, the T-cells derived from CLL patients showed a significant reduction in TL standard deviation (P=0.02), implying that the T-cell repertoire is significantly altered during the course of the disease. In conclusion, this study of TL in ex vivo CLL B-cell samples shows TL erosion during long-term follow-up that is comparable to that seen in non-leukemic leukocyte and lymphocyte samples (Lansdorp et al 1999). In keeping with a recent study, we showed that the erosion rate correlated with starting TL, with the longest telomeres showing the largest erosion and the shortest telomeres showing elongation (Rosenquist et al 2013). This implies that the radical telomere shortening observed in some CLL patients is an early disease event which is in keeping with our previous data demonstrating that a proportion of stage A patients possess very short dysfunctional telomeres (Lin et al 2010). Given that short TL is associated with an inferior clinical outcome, our data indicates that part of the explanation for the clinical heterogeneity seen in CLL may be telomere dependent whereby if the mutagenic event occurs in a B lymphocyte which already has shorter TL then a more aggressive disease occurs whereas if it occurs in a B lymphocyte with longer TL then the outcome is less aggressive disease. Finally, T-cells in CLL patients show markedly more TL erosion corroborating previous studies suggesting there is extensive and abnormal T-cell proliferation in CLL. Whether the CLL cells themselves are driving T-cell TL erosion is at present unknown. Disclosures: No relevant conflicts of interest to declare.

Simultaneous Monitoring of Drug Responses on Distinct Hematopoietic Cell Populations Allow Assessment of Direct and Indirect Cytotoxic Effects of Targeted Therapies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3515-3515
Author(s):  
Muntasir M Majumder ◽  
Aino Maija Leppä ◽  
Caroline A Heckman
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Drug Responses ◽  
Nk T Cells

Abstract Introduction Off-target cytotoxicity resulting in severe side effects and compromising patient survival often hampers the development of new cancer therapeutics. Understanding the complete drug response landscape of different cell populations is crucial to identify drugs that selectively eradicate the malignant cell population, but spare healthy cells. Here, we developed a high content, no wash, multi-parametric flow cytometry based assay that enables testing of blood cancer patient samples and simultaneously monitors the effects of several drugs on 11 hematopoietic cell types. The assay can be used to i) dissect malignant from healthy cell responses and predict off-target effects; ii) assess drug effects on immune cell subsets; iii) identify drugs that can potentially be repositioned to new blood cancer indications. Methods Mononuclear cells were prepared from bone marrow aspirates of 7 multiple myeloma (MM) and 3 acute myeloid leukemia (AML) patients plus the peripheral blood from a healthy donor, which were collected following informed consent and in compliance with the Declaration of Helsinki. Optimal cell density, antibody dilutions, incubation time, and wash versus no wash assay conditions for the selected antibody panels were determined. Cells were incubated at a density of 2 million cells/ml in either 96- or 384-well plates for 3 days. The antibodies were tested in two panels to study the effects of 6 drugs in 5 dilutions (1-10000 nM) (clofarabine, bortezomib, dexamethasone, navitoclax, venetoclax and omipalisib) on 11 cell populations, namely hematopoietic stem cells (HSCs) (CD34+CD38-), common progenitor cells (CPCs) (CD34+CD38+), monocytes (CD14+), B cells (CD45+CD19+), cytotoxic T cells (CD45+CD3+CD8+), T helper cells (CD45+CD3+CD4+), NK-T cells (CD45+CD3+CD56+), NK cells (CD45+CD56+CD3-), clonal plasma cells (CD138+CD38+), other plasma cells (CD138+CD38-) and granulocytes (CD45+, SSC++). Annexin-V and 7AAD were used to distinguish live cell populations from apoptotic and dead cells. After 1 h incubation with antibodies, the plates were read with the iQue Screener PLUS instrument (Intellicyt). Counts for each population were used to generate four parameter nonlinear regression fitted dose response curves with GraphPad Prism 7. Three samples were tested in duplicate to assess reproducibility. Results To decrease the complexity of the assay, we tested all antibodies under wash and no wash conditions, and found that results from both conditions were comparable. To minimize the amount of sample needed as well as maximize the number of drugs tested and cell populations that can be detected, we set up the assay in both 96- and 384-well plates. The assay was highly reproducible when samples were tested in replicate and was scalable to a 384-well format without compromising sensitivity to detect rare populations such as plasma cells. Due to the differentiation of immature cells to specialized cell types, the drug responses of specific populations tended to drift. HSCs (CD34+CD38-) were shown to be refractory to the tested drugs compared to CPCs characterized as (CD34+CD38+) and other cell types. Interestingly, the proteasome inhibitor bortezomib was cytotoxic to all cell populations except for CD138+CD38- plasma cells. Clofarabine, a nucleoside analog used to treat ALL, effectively targeted CPC, NK and B cells, while HSCs and plasma cells were resistant. The glucocorticoid and immunosuppressive drug dexamethasone specifically targeted B and NK cells compared to T cell populations (CD8+, CD4+), while NK-T cells were modestly sensitive. The cell population response patterns were similar in samples derived from MM, AML and healthy individuals, highlighting that the drug responses are highly cell type specific. Summary Using a high content, multi-parametric assay, we could rapidly assess the effect of several drugs on specific cell populations in individual patient samples. Our results demonstrate that many drugs preferentially affect different hematological cell lineages. Although heterogeneity was observed between individual patients, the pattern of cytotoxic response exhibited by specific cell types was consistent among samples derived from MM, AML and healthy donors. The assay will be useful to identify drugs with maximal on-target and minimal off-target specificity, and can potentially be used to guide treatment decision and predict patient response Disclosures Heckman: Celgene: Research Funding; Pfizer: Research Funding.

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