scholarly journals Ex Vivo Kinetics of Early and Long-Term Multifunctional Human Leukocyte Antigen E-Specific CD8+ Cells in Volunteers Immunized with the Ty21a Typhoid Vaccine

2010 ◽  
Vol 17 (9) ◽  
pp. 1305-1314 ◽  
Author(s):  
Rosângela Salerno-Goncalves ◽  
Rezwanul Wahid ◽  
Marcelo B. Sztein
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Typhoid Vaccine ◽  
Tnf Α ◽  
Ifn Γ ◽  
Kinetics Of

ABSTRACT T cells are likely to play an important role in the host defense against Salmonella enterica serovar Typhi, the causative agent of typhoid fever. We have shown that HLA-E can function as a restriction element for S. Typhi-specific CD8+ T cells. Because of the potential importance of HLA-E-restricted CD8+ responses in resistance to Salmonella infection, we characterized these responses and investigated their kinetics of appearance and persistence in volunteers immunized orally with the licensed attenuated Ty21a strain typhoid vaccine. Cells were obtained from volunteers before and at days 2, 4, 7, 10, 14, 28, 42, 56, 120, 180, 360, and 720 after immunization. An ex vivo multicolor staining panel including antibodies to CD107a and -b, interleukin-2, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) was used to functionally assess memory T-cell subsets by flow cytometry. Increases in cytokine-secreting CD8+ cells were observed in the T effector/memory (TEM) and CD45RA+ TEM (TEMRA) subsets as early as 4 days after immunization and persisted, particularly in the TEMRA subset, up to 2 years after immunization. The majority of HLA-E-restricted CD8+ cells 28 to 56 days after immunization coexpressed CD107, IFN-γ, and TNF-α, showing characteristic features of multifunctional T cells. In summary, the multifunctionality and longevity of the HLA-E-restricted CD8 responses observed in this study highlight their significance in adaptive immunity to S. Typhi. Finally, this is the first demonstration, in either animals or humans, of the presence of long-term multifunctional HLA-E-restricted CD8+ cells after immunization.

scholarly journals Differentiation of Antigen-Specific T Cells with Limited Functional Capacity during Mycobacterium tuberculosis Infection

2013 ◽  
Vol 82 (1) ◽  
pp. 132-139 ◽  
Author(s):  
Yun Hee Jeong ◽  
Bo-Young Jeon ◽  
Sun-Hwa Gu ◽  
Sang-Nae Cho ◽  
Sung Jae Shin ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Memory T Cells ◽  
Interleukin 2 ◽  
Effector Memory ◽  
Memory Cells ◽  
Content Type ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACTDespite the generation ofMycobacterium tuberculosis-specific T cell immune responses during the course of infection, only 5 to 10% of exposed individuals develop active disease, while others develop a latent infection. This phenomenon suggests defectiveM. tuberculosis-specific immunity, which necessitates more careful characterization ofM. tuberculosis-specific T cell responses. Here, we longitudinally analyzed the phenotypes and functions ofM. tuberculosis-specific T cells. In contrast to the functional exhaustion of T cells observed after chronic infection,M. tuberculosis-specific CD8+T cells differentiated into either effector (CD127loCD62Llo) or effector memory (CD127hiCD62Llo) cells, but not central memory cells (CD127hiCD62Lhi), with low programmed death 1 (PD-1) expression, even in the presence of high levels of bacteria. Additionally,M. tuberculosis-specific CD8+and CD4+T cells produced substantial levels of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), but not interleukin 2 (IL-2), uponin vitrorestimulation. AmongM. tuberculosis-specific CD8+T cells, CD127hieffector memory cells displayed slower ongoing turnover but greater survival potential. In addition, these cells produced more IFN-γ and TNF-α and displayed lytic activity upon antigen stimulation. However, the effector function ofM. tuberculosis-specific CD8+CD127hieffector memory T cells was inferior to that of canonical CD8+CD127himemory T cells generated after acute lymphocytic choriomeningitis virus infection. Collectively, our data demonstrate thatM. tuberculosis-specific T cells can differentiate into memory T cells during the course ofM. tuberculosisinfection independent of the bacterial burden but with limited functionality. These results provide a framework for further understanding the mechanisms ofM. tuberculosisinfection that can be used to develop more effective vaccines.

scholarly journals Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

2020 ◽  
Vol 28 (1) ◽  
pp. 75-89
Author(s):  
Ion Bogdan Manescu ◽  
Doina Ramona Manu ◽  
Georgiana Mihaela Serban ◽  
Minodora Dobreanu
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Healthy Subjects ◽  
Ex Vivo ◽  
Intracellular Cytokine Staining ◽  
Interleukin 2 ◽  
Cd8 Cells ◽  
Tnf Α ◽  
Ifn Γ ◽  
Synthetic Capacity

AbstractPeripheral blood lymphocytes (PBL) are able to synthesize various cytokines that play key roles in the immune response and intercellular signaling. Since alterations in cytokine production and/or activity occur in many pathological processes, the study of cytokine synthetic capacity of PBL is a valuable tool for assessing the immune profile. In this paper, we aimed to investigate the variability of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) synthetic capacity of CD4+/CD8+ T-cells stimulated ex-vivo in healthy subjects, by means of a commercial intracellular cytokine staining (ICS) protocol. Peripheral blood mononuclear cells were isolated from 16 healthy subjects by Ficoll gradient centrifugation and activated ex-vivo with PMA/Ionomycin/Brefeldin-A for 4 hours. Activated PBL were surface-stained for CD3/CD4/CD8, fixed and permeabilized. ICS was performed using anti-human IL-2/TNF-α/IFN-γ and samples were analyzed on a BD-FACSAria-III flow cytometer. We recorded high post-isolation and post-activation mean viabilities: 82.1% and 82.4% respectively, p=0.84. Both CD4+/CD8+ subpopulations were found to partially produce each of the three cytokines, but in different proportions. On average, a significantly greater percentage of CD4+ cells was shown to produce IL-2 and TNF-α, compared with CD8+ cells (61.5%+/-5.8 vs. 25%+/-5.6 and 26.9%+/-11 vs. 7.5%+/-3.3 respectively, p---lt---0.0001 for both). Contrarily, IFN-γ was produced by a higher proportion of CD8+ cells (8.4%+/-3.9 vs. 6.8%+/-3.2, p=0.01). These results show that the employed ICS protocol elicits a satisfactory and consistent cytokine response from PBL of healthy subjects. The collected data may be used to outline a preliminary reference range for future studies on both healthy/pathological subjects.

Human CMVpp65-Specific Stem Cell-like Memory T-Cells (TSCM) Are a Principal Source for Rapid Repopulation of Immunodominant Central Memory (TCM) and Effector Memory (TEM) in the Circulation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5728-5728
Author(s):  
Tzu-Yun Kuo ◽  
Aisha Hasan ◽  
Manuel Guerreiro ◽  
Richard J. O'Reilly
Keyword(s):  
T Cells ◽  
Memory T Cells ◽  
Amino Acid Sequences ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Tnf Α ◽  
Artificial Antigen ◽  
Ifn Γ ◽  
Antigen Presenting

Abstract Latent CMV infection is controlled by a limited repertoire of immunodominant T-cells specific for viral peptides, particularly CMVpp65 and CMV IE-1. The antigen-specific T-cell subsets responsible for maintaining memory T-cells in this repertoire and repopulating them in response to periodic viral reactivations remain unclear. In this study, we generated T-cells specific for CMVpp65 from naïve (TN), TSCM, TCM and TEM subsets isolated from the blood of HLA-A*0201 normal seropositive donors and then comparatively characterized NLV-HLA-A*0201 Tetramer+ T-cells from each of these subsets. Following in vitro sensitization with artificial antigen-presenting cells transduced to express HLA-A*0201 and CMVpp65 and expansion with IL-7, IL-15 and IL-2, Tet+ T-cells were regularly generated from CD62L+CD45RO-CD95- TN and from CD62L+CD45RO-CD95+ TSCM, as well as TCM and TEM. Isolated Tet+ TN, TSCM, TCM and TEM were each able to generate IFN-γ, TNF-α and granzyme B. Each Tet+ subset also expressed similar level of PD-1 and KLRG-1. However, Tet+ TN and TSCM expressed higher levels of CD27 and lower levels of CD57 than TCM or TEM. Tet+ TSCM were distinguished from Tet+ TN, TCM and TEM by a significantly greater level of proliferation and by their rapid and selective expansion of NLV-specific T-cells bearing TCRs identical by amino acid sequences to those expressed by TEM and TCM in the blood. Thus, Tet+ TSCM rather than Tet+ TN constitute the principal repertoire for repopulation of immunodominant memory T-cells sustained in the circulation. Disclosures Hasan: Atara: Patents & Royalties.

scholarly journals Spontaneous Cytokine Production and Its Effect on Induced Production

2002 ◽  
Vol 9 (5) ◽  
pp. 1049-1056 ◽  
Author(s):  
Derrick Walker ◽  
Janine Jason ◽  
Kelly Wallace ◽  
Justin Slaughter ◽  
Virginia Whatley ◽  
...  
Keyword(s):  
T Cells ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Healthy Adults ◽  
Multiparameter Flow Cytometry ◽  
Parasitic Infections ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) by peripheral-blood B lymphocytes, T cells, CD8− T cells, CD8+ T cells, CD3− CD16/56+ lymphocytes (natural killer [NK] cells), CD3+ CD16/56+ lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-α production occurred most frequently, followed in descending order by IFN-γ, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-α; for 12 of these patients, TNF-α was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-α and IFN-γ, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.

scholarly journals Rapid quantification of naive alloreactive T cells by TNF-α production and correlation with allograft rejection in mice

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 819-826 ◽  
Author(s):  
Michael A. Brehm ◽  
Julie Mangada ◽  
Thomas G. Markees ◽  
Todd Pearson ◽  
Keith A. Daniels ◽  
...  
Keyword(s):  
T Cells ◽  
Allograft Rejection ◽  
Ex Vivo ◽  
Graft Function ◽  
Effector Memory ◽  
Skin Allograft ◽  
Alloreactive T Cells ◽  
Tnf Α ◽  
Ifn Γ

Abstract Allograft transplantation requires chronic immunosuppression, but there is no effective strategy to evaluate the long-term maintenance of immunosuppression other than assessment of graft function. The ability to monitor naive alloreactive T cells would provide an alternative guide for drug therapy at early, preclinical stages of graft rejection and for evaluating tolerance-inducing protocols. To detect and quantify naive alloreactive T cells directly ex vivo, we used the unique ability of naive T cells to rapidly produce TNF-α but not IFN-γ. Naive alloreactive T cells were identified by the production of TNF-α after a 5-hour in vitro stimulation with alloantigen and were distinguished from effector/memory alloreactive T cells by the inability to produce IFN-γ. Moreover, naive alloreactive T cells were not detected in mice tolerized against specific alloantigens. The frequency of TNF-α–producing cells was predictive for rejection in an in vivo cytotoxicity assay and correlated with skin allograft rejection. Naive alloreactive T cells were also detected in humans, suggesting clinical relevance. We conclude that rapid production of TNF-α can be used to quantify naive alloreactive T cells, that it is abrogated after the induction of tolerance, and that it is a potential tool to predict allograft rejection.

HTLV-II down-regulates HIV-1 replication in IL-2–stimulated primary PBMC of coinfected individuals through expression of MIP-1α

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2760-2769 ◽  
Author(s):  
Claudio Casoli ◽  
Elisa Vicenzi ◽  
Andrea Cimarelli ◽  
Giacomo Magnani ◽  
Paolo Ciancianaini ◽  
...  
Keyword(s):  
T Cells ◽  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Human T Cell ◽  
Cc Chemokines ◽  
Kinetics Of ◽  
Hiv 1

The influence of human T-cell leukemia/lymphoma virus type II (HTLV-II) in individuals also infected with HIV-1 is poorly understood. To evaluate the reciprocal influence of HTLV-II and HIV-1 infection, primary peripheral blood mononuclear cell (PBMC) cultures from coinfected individuals were established in the presence of interleukin 2 (IL-2). In these cultures, the kinetics of HTLV-II replication always preceded those of HIV-1. Noteworthy, the kinetics of HIV-1 production were inversely correlated to the HTLV-II proviral load in vivo and its replication ex vivo. These observations suggested a potential interaction between the 2 retroviruses. In this regard, the levels of IL-2, IL-6, and tumor necrosis factor- (TNF-) were measured in the same coinfected PBMC cultures. Endogenous IL-2 was not produced, whereas IL-6 and TNF- were secreted at levels compatible with their known ability to up-regulate HIV-1 expression. The HIV-suppressive CC-chemokines RANTES, macrophage inflammatory protein-1 (MIP-1), and MIP-1β were also determined in IL-2–stimulated PBMC cultures. Of interest, their kinetics and concentrations were inversely related to those of HIV-1 replication. Experiments were performed in which CD8+ T cells or PBMCs from HTLV-II monoinfected individuals were cocultivated with CD4+ T cells from HIV-1 monoinfected individuals separated by a semipermeable membrane in the presence or absence of antichemokine neutralizing antibodies. The results indicate that HTLV-II can interfere with the replicative potential of HIV-1 by up-regulating viral suppressive CC-chemokines and, in particular, MIP-1. This study is the first report indicating that HTLV-II can influence HIV replication, at least in vitro, via up-regulation of HIV-suppressive chemokines.

scholarly journals Complementary Dendritic Cell–activating Function of CD8+ and CD4+ T Cells

2002 ◽  
Vol 195 (4) ◽  
pp. 473-483 ◽  
Author(s):  
Robbie B. Mailliard ◽  
Shinichi Egawa ◽  
Quan Cai ◽  
Anna Kalinska ◽  
Svetlana N. Bykovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Class I ◽  
Chronic Infections ◽  
Cd40 Ligation ◽  
Activation Level ◽  
Tnf Α ◽  
Ifn Γ

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of “T helper (Th)” signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood– or peripheral blood–isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-γ at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I–presented epitopes by antigen-specific CD8+ T cells results in the TNF-α– and IFN-γ–dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I–restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I–presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.

Activation-Dependent Expression of an Interleukin-2 Transgene Mediated by Transduction with a Lentiviral Vector Enhances T Cell Functions.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5273-5273
Author(s):  
Qi Sun ◽  
Karen Chorney ◽  
Carol Stine ◽  
Kenneth G. Lucas
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Immune Functions ◽  
Cell Functions ◽  
Genetically Engineer ◽  
Ifn Γ

Abstract Adoptive T cell immunotherapy (ATCI) is an evolving strategy that explores antigen-specific T cells manipulated ex vivo as therapeutic agents. Although the concept of ATCI has been tested clinically, with success in the treatment of post-transplant EBV induced lymphoproliferative disease, one of the major obstacles hindering its application to other malignancies is the procurement of tumor-specific T cells that possess potent anti-tumor functions even in the inhibitory environment at the tumor sites. This study aims to genetically engineer enriched viral specific T cells for improved immune functions. A self-inactivating lentiviral vector (SIN) CD69p-IL2 was constructed to encode the transgene interleukin-2 (IL2) under the control of a human CD69 promoter (CD69p), and this vector was tested in ex vivo cultivated EBV-specific T cells. SIN vector allows a high degree of autonomy for the internal promoter, and CD69 expression in the T cells is closely associated with T cell activation. Experiments showed that the SIN vectors efficiently transduced EBV-specific T cells, both CD4 and CD8. Furthermore, the newly cloned CD69p exhibited a higher degree of responsiveness to physiological antigen stimulation than the early promoter from the cytomegalovirus (CMVp). In response to stimulation by EBV-infected B cells, the percentage of IL2 expressing cells was 2 fold higher for the activated CD69p-IL2 transduced T cells than the non-transduced, or the CMVp-IL2 transduced, counterparts. In correlation with the stronger IL2 expression, 3 fold more T cells expressed the anti-viral cytokine interferon-γ (IFN-γ) in the CD69p-IL2 transduced T cells than the CMVp-IL2 transduced, and the IFN-γ expression at the single cell level was 2 fold higher in the former, indicating an enhanced functionality. While the culture supernatant from the CMVp-IL2 transduced T cells contained IL2 at a concentration 2000 fold higher than the non-transduced T cells, the IL2 level in the media from the CD69p-IL2 transduced T cells was comparable to that in the control, suggesting the IL2 expression mediated by the CD69p more relevant to T cell functions than the CMVp. These results may serve as a foundation for the further development and clinical application of specific T cells engineered for enhanced immune functions.

Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1071-1071
Author(s):  
Melody M. Smith ◽  
Cynthia R. Giver ◽  
Edmund K. Waller ◽  
Christopher R. Flowers
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect. Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml. Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset. Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. Figure Figure

In Vitro Co-Culture of CLL-B Cells Reveals Long-Term Survival, Proliferation, and Maintenance of Telomere Length

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 350-350
Author(s):  
Ceri H Jones ◽  
Thet Thet Lin ◽  
Elisabeth Jane Walsby ◽  
Guy E Pratt ◽  
Christopher Fegan ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Telomere Length ◽  
Erosion Rate ◽  
Ex Vivo ◽  
Effector Memory ◽  
Long Term Culture

Abstract Telomere length is a prognostic factor in Chronic Lymphocytic Leukemia (CLL) with short telomere length a powerful predictor of early time to first treatment and reduced overall survival. However, little is known about telomere dynamics through the course of an individual patient's disease. Our recent longitudinal analysis of CLL B-cell telomere length revealed very little dynamic change within individual patients with a mean erosion rate of -52bp/year (p=0.05). In marked contrast, T-cells derived from the same patients showed a significantly higher mean erosion rate of -119bp/year (p=0.02) with a median follow up time of 69 months. Here we present data derived from long-term in-vitro co-culture of peripheral blood from CLL patients coupled with temporal analysis of their telomere length dynamics. We utilized a multi-cellular co-culture system, comprised of autologous T-cells and CD40L-expressing mouse fibroblasts, to maintain CLL cells in long-term culture. Patient-derived peripheral blood mononuclear cells (n=16) were maintained for a median of 70 days (range 54-154); samples were analyzed every two weeks for tumor cell telomere length and evidence of proliferation. We used fluorescence-activated cell sorting (FACS) to sort populations of CD19+CD5+ CLL B-cells and CD3+ T-cells from each of the cultures. We then performed high-resolution single telomere length analysis (STELA) on these sorted subsets of cells and analyzed their telomere dynamics over this extended time course. Analysis of CLL B-cells from these cultures revealed significantly increased Ki-67+ at day 14 when compared to day 0 (p<0.001) and this was evident for the duration of the cultures. Despite sustained tumor cell proliferation, we observed no significant difference in the CLL B-cell telomere length with a mean TL at the start of 4.5kb vs 4.3kb at the end (p=0.14). The presence of T-cells was shown to be critical for the maintenance of the long-term cultures in two ways. Firstly, cultures that were treated with 4μM fludarabine showed a catastrophic reduction in T-cells (p=0.01), which was associated with a significantly shorter duration of survival of CLL B-cells when compared to untreated controls (median 17.5 days (range 7-70); p<0.001). Secondly, it proved impossible to maintain T-cell depleted, purified CLL B-cells, in long-term culture. T-cells isolated from the long-term cultures showed evidence of proliferation with Ki-67+ again being increased at day 14 in comparison to baseline (p=0.003). Furthermore, T-cells derived from these cultures showed a significant alteration in subset composition over time with a decrease in the numbers of naive CD4+ (p=0.05) and CD8+ (p=0.02) T-cells and a corresponding increase in effector memory (p=0.2) and terminally differentiated effector memory (EMRA) subsets (p=0.07). In conclusion, this study demonstrates that we have developed a robust, long-term culture method for the maintenance of CLL cells. Despite evidence of sustained CLL proliferation, CLL B-cells showed little telomere length erosion during long-term co-culture and this is compatible with our recent ex-vivo analysis, which showed that the telomere length of CLL B-cells are remarkably stable with a mean erosion rate of only -52bp/year. In both ex-vivo and in-vitro analysis, telomere erosion correlated with starting telomere length (r2=0.14, p=0.04 and r2=0.3 p=0.03 respectively). Taken together, our in-vitro and ex-vivo data imply that the radically short telomeres observed in some CLL patients are not the result of increased proliferation of the malignant B-cell, but rather the mutagenic event occurs in a B-cell which already has short telomeres. Furthermore, our novel long-term culture model has reinforced the vital role of T-cells in sustaining CLL B-cells viability and proliferation in-vitro. Given the consistent skewing of the T-cell pool towards a memory phenotype it seems unlikely that this is driven in-vitro by cognate TCR antigen recognition but rather a cytokine-mediated response. Disclosures Fegan: Gilead Sciences: Honoraria; Roche: Honoraria; AbbVie: Honoraria.

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