scholarly journals A subset of CD4+ memory T cells contains preformed CD40 ligand that is rapidly but transiently expressed on their surface after activation through the T cell receptor complex.

1995 ◽  
Vol 181 (4) ◽  
pp. 1293-1301 ◽  
Author(s):  
M Casamayor-Palleja ◽  
M Khan ◽  
I C MacLennan
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Phorbol Myristate Acetate ◽  
Cd4 T Cells ◽  
Cd40 Ligand ◽  
Surface Expression ◽  
Germinal Centers ◽  
Myristate Acetate ◽  
Receptor Complexes

Signaling through surface CD40 is essential for selecting B cells that have mutated their immunoglobulin variable region genes in germinal centers and is an important signal in the early stages of antibody responses to T cell-dependent antigens. It is shown that a subset of CD45RO+, CD4+ T cells isolated from human tonsil contains preformed 30-35-kD ligand for CD40. This is expressed on their surfaces within 5 min of their antigen-receptor complexes interacting with CD3 epsilon antibodies bound to ox erythrocytes. This surface expression does not require de novo protein synthesis and lasts for only 1-2 h. Preformed CD40 ligand (CD40L) was not detected in any CD4+ CD45RA+ T cells, but > 90% of all CD4+ T cells from the tonsil can be induced to express large amounts of CD40L on culture with phorbol myristate acetate and the calcium ionophore ionomycin. This expression of CD40L starts between 1 and 2 h, peaks at 6 h, and remains at a high level for > 20 h. It is totally prevented by adding a concentration of cycloheximide that inhibits CD25 synthesis by these activated cells. While CD3 epsilon antibody bound to ox red cells is a good inducer of surface expression of CD40L, it is a much less potent inducer of CD40L synthesis than phorbol myristate acetate with ionomycin. Immunohistological analysis of tonsil sections shows that cells containing CD40L are located mainly in the outer zone of germinal centers and the margins of the T zones that are rich in dendritic cells (interdigitating cells). The distribution of these cells is consistent with: (a) their interaction in T zones with B cells that have taken up and processed antigen and (b) their involvement in B cell selection in germinal centers.

In Vitro Expansion of Autologous Follicular Lymphoma-Specific T Cells for Adoptive Transfer.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1482-1482
Author(s):  
Seung-Tae Lee ◽  
Yun Fang Jiang ◽  
Soung-Chul Cha ◽  
Hong Qin ◽  
Larry W. Kwak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Cd40 Ligand ◽  
Autologous Tumor ◽  
T Cell Lines

Abstract Advanced stage follicular lymphoma remains an incurable disease with a median survival of 8 to 10 years that has not significantly changed over the last four decades. Therefore, novel treatment options are necessary to improve the clinical outcome in these patients. The observation of spontaneous regressions in a small percentage of patients suggested that augmenting the host immune response could potentially control this malignancy. Strategies using active specific immunotherapy with idiotype vaccines led to induction of clinical and molecular responses in a few patients but have met with only limited success possibly due to the low frequency of antigen-specific T cells induced in the patients. In contrast to active immunization, T cells of a given specificity and function may be selected and expanded in vitro to the desired number for adoptive cell transfer. Towards this goal, we stimulated tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from five follicular lymphoma patients with CD40 ligand-activated autologous tumor cells at approximately ten-day intervals in the presence of IL-2 and IL-15. After four rounds of stimulations, T cell lines generated from 3/5 patients recognized autologous unmodified tumor cells by producing significant amounts of TNF-α, GM-CSF and/or IFN-γ. By phenotypic analysis, the T cell lines were predominantly CD4+ T cells (> 70%), and intracellular cytokine assay showed that up to 40% of the CD4+ T cells were tumor-reactive. The inhibition of cytokine production by anti-HLA class II but not class I blocking antibodies confirmed that the CD4+ T cells were tumor-reactive. Further characterization revealed that the T cells from one patient recognized autologous tumor but not autologous normal B cells suggesting that they were tumor-specific. While in a second patient CD4+ T cell clones generated from the T cell line by limiting dilution recognized autologous tumor and autologous normal B cells but not autologous monocytes suggesting that they were B cell lineage-specific. We conclude that follicular lymphoma-specific T cells exist and can be efficiently expanded in vitro from both TILs and PBMCs using CD40 ligand-activated autologous tumor cells for adoptive T cell therapy. Additionally, identification of antigens recognized by these T cells could lead to development of novel immunotherapeutic strategies for lymphomas.

scholarly journals Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2520-2527 ◽  
Author(s):  
Yoshinobu Koguchi ◽  
Timothy J. Thauland ◽  
Mark K. Slifka ◽  
David C. Parker
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cd4 T Cells ◽  
De Novo ◽  
Cd40 Ligand ◽  
Surface Expression ◽  
Antigen Recognition ◽  
Memory Cd4 T Cells ◽  
Secretory Lysosomes

CD40 ligand (CD40L) is an essential effector cytokine for macrophage activation, dendritic cell licensing, and T-cell–dependent antibody responses. Although CD40L is known to be made de novo following antigen recognition, several reports have described surface mobilization of preformed, intracellular CD40L in certain CD4+ effector T cells. Here we show that rapid surface expression of preformed CD40L following antigen recognition is a general property of both effector and memory CD4+ T cells, including in vitro and in vivo activated T-cell–receptor transgenic T cells, memory phenotype CD4+ T cells from pathogen-free naive mice, and polyclonal virus–specific effector and memory T cells. Intracellular CD40L is stored in secretory lysosomes, and colocalizes more strongly with Fas ligand than with CTLA-4, two other molecules that are delivered to the cell surface following antigen recognition. Stimulated surface expression of preformed CD40L is found in memory CD4+ T cells from CD40-deficient mice, indicating that it does not depend on CD40-induced internalization for delivery to the secretory compartment. We suggest that delivery of preformed CD40L to antigen-presenting cells (APCs) could enable antigen-specific activation of APCs in transient interactions that are too brief to permit de novo synthesis of CD40L.

scholarly journals Mapping of the autophagosomal degradome identifies IL-7Rα as key cargo in proliferating CD4+ T-cells

2021 ◽  
Author(s):  
Dingxi Zhou ◽  
Mariana Borsa ◽  
Daniel J. Puleston ◽  
Susanne Zellner ◽  
Jesusa Capera ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Surface Expression ◽  
T Cell Proliferation ◽  
Gamma Chain ◽  
Direct Identification ◽  
The Common ◽  
First Time

CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naive and memory T cells, was reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy showed increased IL-7Rα surface expression, while no defect in internalisation was observed. Mechanistically, excessive surface IL-7Rα sequestrates the common gamma chain, impairing the IL-2R assembly and downstream signalling crucial for T cell proliferation. This study provides proof-of-principle that key autophagy substrates can be reliably identified with this model to help mechanistically unravel autophagy's contribution to healthy physiology and disease.

scholarly journals Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

2016 ◽  
Vol 213 (11) ◽  
pp. 2413-2435 ◽  
Author(s):  
Yi Wang ◽  
Cindy S. Ma ◽  
Yun Ling ◽  
Aziz Bousfiha ◽  
Yildiz Camcioglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Loss Of Function ◽  
Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection

2021 ◽  
Vol 118 (46) ◽  
pp. e2108157118
Author(s):  
Kerstin Narr ◽  
Yusuf I. Ertuna ◽  
Benedict Fallet ◽  
Karen Cornille ◽  
Mirela Dimitrova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Type I ◽  
Clonal Deletion ◽  
Cell Immunity ◽  
B Cell Immunity

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)–driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called “decimation,” of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I–driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell–intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell–mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell–based vaccination against persistent viral diseases.

A Detailed Phenotypic Analysis Using Six Color Flow Cytometry of Lymphocyte Subsets Mobilized with AMD3100 Compared to G-CSF.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 408-408 ◽  
Author(s):  
Yoshiyuki Takahashi ◽  
S. Chakrabarti ◽  
R. Sriniivasan ◽  
A. Lundqvist ◽  
E.J. Read ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Lymphocyte Subsets ◽  
Phenotypic Analysis ◽  
Color Flow ◽  
Cd34 Cells

Abstract AMD3100 (AMD) is a bicyclam compound that rapidly mobilizes hematopoietic progenitor cells into circulation by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. Preliminary data in healthy donors and cancer patients show large numbers of CD34+ cells are mobilized following a single injection of AMD3100. To determine whether AMD3100 mobilized cells would be suitable for allografting, we performed a detailed phenotypic analysis using 6 color flow cytometry (CYAN Cytometer MLE) of lymphocyte subsets mobilized following the administration of AMD3100, given as a single 240mcg/kg injection either alone (n=4) or in combination with G-CSF (n=2: G-CSF 10 mcg/kg/day x 5: AMD3100 given on day 4). Baseline peripheral blood (PB) was obtained immediately prior to mobilization; in recipients who received both agents, blood was analyzed 4 days following G-CSF administration as well as 12 hours following administration of AMD3100 and a 5th dose of G-CSF. AMD3100 alone significantly increased from baseline the PB WBC count (2.8 fold), Absolute lymphocyte count (ALC: 2.5 fold), absolute monocyte count (AMC: 3.4 fold), and absolute neutrophil count (ANC: 2.8 fold). Subset analysis showed AMD3100 preferentially increased from baseline PB CD34+ progenitor counts (5.8 fold), followed by CD19+ B-cells (3.7 fold), CD14+ monocytes (3.4 fold), CD8+ T-cells (2.5 fold), CD4+ T-cells (1.8 fold), with a smaller increase in CD3−/CD16+ or CD56+ NK cell counts (1.6 fold). There was no change from baseline in the % of CD4+ or CD8+ T-cell expressing CD45RA, CD45RO, or CD56, CD57, CD27, CD71 or HLA-DR. In contrast, there was a decline compared to baseline in the mean percentage of CD3+/CD4+ T-cells expressing CD25 (5.5% vs 14.8%), CD62L (12.1% vs 41.1%), CCR7 (2.1% vs 10.5%) and CXCR4 (0.5% vs 40.9%) after AMD3100 administration; similar declines in expression of the same 4 surface markers were also observed in CD3+/CD8+ T-cells. A synergistic effect on the mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF (Figure). In those receiving both AMD3100 and G-CSF, a fall in the % of T-cells expressing CCR7 and CXCR4 occurred 12 hours after the administration of AMD3100 compared to PB collected after 4 days of G-CSF; no other differences in the expression of a variety activation and/or adhesion molecules on T-cell subsets were observed. Whether differences in lymphocyte subsets mobilized with AMD3100 alone or in combination with G-CSF will impact immune reconstitution or other either immune sequela (i.e. GVHD, graft-vs-tumor) associated with allogeneic HCT is currently being assessed in an animal model of allogeneic transplantation.

Cultured Human B Cell APCs Expanded Using Il-4 and CD40 Ligand Preferentially Promote a Type 2 T Cell Response to Alloimmune Stimulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2668-2668
Author(s):  
Abdul Tawab ◽  
Yoshiyuki Takahashi ◽  
Childs Richard ◽  
Kurlander J. Roger
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Cd40 Ligand ◽  
T Cell Response ◽  
Ifn Γ

Abstract In vitro stimulation of human peripheral blood B cells with recombinant IL-4 and CD40 ligand (CD40L) markedly increases their expression of MHC and costimulatory molecules, thus enhancing antigenic peptide presentation to T cells. Because these cells proliferate extensively in vitro (unlike monocytes or dendritic cells), they represent a promising and convenient reagent for the generation and maintenance of antigen-specific T cells for use in a variety of experimental or therapeutic settings. However, the impact of this type of B cell APC on cytokine production by responder T cells has hitherto not been examined. To address this issue, we stimulated normal human T cells with either allogeneic B cells (generated in vitro) or with MNCs obtained from the same donor. After 7 days, T cells were washed and re-challenged with the same APCs. The resulting alloreactive cytokine response was measured using quantitative ELISPOT methods and expressed as the frequencies of IFN-γ, IL-4, and IL-5 producing cells per thousand responder cells added. B cell- and MNC-primed cell lines both produced vigorous lymphokine responses, but B cell-stimulated T cells consistently produced more IL-5 spots (mean of 265 vs. 98/1000 responders, p<0.002) and fewer IFN-γ spots (163 vs 386/1000 cells, p<0.005) than MNC-stimulated cells. Further, the ratio of IFN-γ to IL-5 spots was almost ten-fold lower in B cell-stimulated cultures compared to MNC-induced cultures (0.67 vs. 5.2, p<0.001). ELISPOT studies assessing the ratio of IFN-γ to IL-4 spots and ELISA assays comparing IFN-γ and IL-5 levels from culture supernatants demonstrated the same pattern of marked type 2 skewing by B cells. This pattern was unaffected by the presence of anti-IL-4 antibody suggesting type 2 skewing was not mediated by IL-4. Cytokine skewing produced by B cells or MNC could be partially reversed by swapping MNC and B cells during re-stimulation on day 7, but this plasticity was markedly reduced after 3 (weekly) cycles of B cell or MNC re-stimulation in vitro. Type 2 skewing by B cells was enhanced when monocytes were removed from responder T cell populations by either depleting CD14+ positive cells or by positive selection of T cells prior to stimulation. In contrast, type 2 polarization could be prevented using recombinant IL-12. Not all cells of B-cell origin share the same propensity to type 2 skewing observed with IL-4/CD40L-stimulated B cells; under identical conditions, EBV-transformed B cells stimulated alloimmune T cells to produce a strong type 1 cytokine response comparable to that produced by MNCs. In summary, IL-4/CD40L-stimulated B cells strongly promote a type 2 T cell response during primary alloimmune challenge; this skewing can become fixed after repeated B cell stimulation. Investigators using these cells as APC should be aware of this potential phenomenon, particularly during primary T cell responses. It is also important to consider the factors described above that may exacerbate or ameliorate this effect.

Targeting STAT3 Signaling to Augment the Immunogenicity of B-Cell Lymphomas

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 708-708
Author(s):  
Hongwei Wang ◽  
F. Cheng ◽  
K. Wright ◽  
J. Tao ◽  
M. Smith ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Dna Binding ◽  
B Cell ◽  
Cd4 T Cells ◽  
Mutant Form ◽  
B Cell Lymphomas ◽  
Stat3 Signaling ◽  
Antigen Presenting

Abstract STAT3 signaling has emerged as a negative regulator of inflammatory responses in immune cells. In bone-marrow derived antigen-presenting cells (APCs), genetic or pharmacologic disruption of STAT3 led to inflammatory cells that effectively prime antigen-specific T-cell responses and restore the responsiveness of tolerized T-cells. In contrast, enhanced Stat3 activity in APCs resulted in increased production of the immunosuppressive cytokine IL-10 and induction of T-cell tolerance1. B-cell lymphomas being tumors derived from B-lymphocytes display intrinsic antigen-presenting capabilities. Augmentation of this APC function has been shown to result in effective anti-lymphoma immunity2. In this study we determined whether targeting Stat3 signaling might influence the intrinsic APC function of malignant B-cells and the responsiveness –or not- of antigen-specific CD4+ T-cells. First, we specifically block STAT3 signaling in A20 lymphoma B-cells by using a dominant negative variant of STAT3, Stat3b. Inhibition of STAT3 resulted in tumor cells capable not only of fully priming naïve antigen-specific CD4+T-cells but also able of restoring the responsiveness of tolerant T-cells from lymphoma bearing mice. Conversely, transfection of A20 B-cells with Stat3c, a constitutively activated mutant form of STAT3, led to T-cell unresponsiveness. Of note, manipulation of STAT3 in B cell tumors was associated with changes in the mRNA expression and protein levels of IL-10. Second, we evaluated the effects of two novel Stat3 inhibitors, CPA-7 (a platinum-containing compound that disrupts STAT3 DNA binding activity) and S3I-201 (inhibitor of Stat3:Stat3 complex formation and Stat3 DNA binding and transcriptional activities) in a murine model of Mantle Cell Lymphoma (MCL). In vitro treatment of FC-muMCL1 cells - derived from a tumor elicited in Em-Cyclin D1 transgenic mice- with increasing concentrations of either CPA-7 or S3I-201 resulted in an enhanced presentation of OVA-peptide to naïve CD4+ T-cells specific for a MHC class II restricted epitope of ovalbumin (OT-II cells). Indeed, these T-cells produce higher levels of IL-2 and IFN-gamma compared to anti-OVA T cells that encountered cognate antigen in untreated FC-muMCL1 cells. More importantly, MCL cells treated with CPA-7 restored the responsiveness of tolerized anti-OVA CD4+ T-cells. Finally, in vivo treatment of MCL-bearing mice with CPA-7 (5 mg/kg/iv given on days +21, +24 and +27 after tumor challenge) resulted in significant inhibition of p-Stat3 in malignant B-cells and augmentation of their APC function. Taken together, STAT3 signaling is involved in the regulation of the antigen-presenting capabilities of B-cell lymphomas and as such represents a novel molecular target to augment the immunogenicity of these tumors.

Donor B Cells in Transplants Augment Clonal Expansion and Persistence of Pathogenic Donor CD4+ T Cells in Autoimmune-Like Chronic Graft-Versus-Host Disease,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4021-4021
Author(s):  
James Sundblom Young ◽  
Tao Wu ◽  
Yuhong Chen ◽  
Dongchang Zhao ◽  
Heather F Johnston ◽  
...  
Keyword(s):  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Graft Versus Host Disease ◽  
Tissue Damage ◽  
Cd4 T Cells ◽  
Clonal Expansion ◽  
Graft Versus Host

Abstract Abstract 4021 Chronic graft-versus-host disease (cGVHD) manifests with autoimmune symptoms (i.e. increased serum levels of autoantibodies, donor T cell infiltration in skin and salivary gland tissues, and collagen deposition in skin tissues). Donor B cells have been indicated to play an important role in the pathogenesis of cGVHD in mouse models as well as in patients, but the mechanisms remain unclear. In the current studies, using a cGVHD mouse model of DBA/2 donor to MHC-matched BALB/c host, we have observed that donor B cells are activated by donor CD4+ T cells in transplants to upregulate MHC II and co-stimulatory molecules and produce IgG autoantibodies; in turn, donor B cells mediated clonal expansion of autoreactive donor-type CD4+ T cells, as judged by TCR spectratyping and in vitro T cell proliferation in response to donor- and host-type APCs. Kinetic studies showed that the presence of donor B cells in transplants was associated with persistence of GVHD target tissue damage (i.e. sclerodermatous skin) and persistence of donor CD4+ T infiltration in the tissues in which there is an expansion of Th1 and Th2 but not Th17. The presence of donor B cells in transplants also markedly augmented tissue damage in prototypical cGVHD targets such as the salivary gland. Sorted donor CD4+ T cells from primary recipients given donor B cell-containing transplants but not from the primary recipients given B cell-depleted transplants caused cGVHD-like tissue damage in the skin and salivary gland of adoptive recipients. These results indicate that donor B cells in bone marrow transplants play an important role in the generation and expansion of pathogenic CD4+ T cells that mediate chronic GVHD tissue damage. Disclosures: No relevant conflicts of interest to declare.

Targeting The T Cell Component Of The Tumour Microenvironment In Chronic Lymphocytic Leukaemia; A Potential Therapeutic Strategy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4147-4147
Author(s):  
Kirsty M Cuthill ◽  
Andrea Gail Sherman Buggins ◽  
Pj Chana ◽  
Stephen Devereux
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Lymphocytic Leukaemia ◽  
Nuclear Translocation ◽  
Tumour Microenvironment

Abstract It has recently become clear that B cell receptor (BCR) activation plays an important role in the pathogenesis of chronic lymphocytic leukaemia (CLL); a fact that is underlined by the marked efficacy of drugs that inhibit components of this pathway. Although the underlying mechanisms remain unclear, CLL BCRs have been shown to recognize a variety of autoantigens and there is evidence of ongoing activation of a number of downstream signaling molecules including Syk, Erk, Akt and the NFkB and NFAT family of transcription factors. In addition to BCR activation, it is thought that signals from other cells in the tumour microenvironment such as T cells, the vascular endothelium and other stromal cells may also play a role in promoting the growth of the disease. In the present study we chose to revisit the effects of ciclosporin (CsA), a calcineurin antagonist with effects on antigen receptor signaling, in CLL. When this agent is used to treat the autoimmune complications of CLL, concurrent responses in the underlying disease have been noted in about 20% of patients, although the underlying mechanism has not been thoroughly investigated. Since CsA primarily inhibits T cell activation we hypothesized that its effects in CLL might be due to a reduction in T cell mediated co-stimulation in the lymph nodes. We therefore investigated the effect of CsA on the activation of CLL B and T cells using conventional and multispectral imaging flow cytometry to measure the expression of activation markers and the nuclear translocation of NFAT and NFKB family transcription factors. Cells were collected from eight unselected patients with a confirmed diagnosis of CLL for each study. T and B cells were purified by negative immunomagnetic selection and activated by incubation with phorbol ester and ionomycin (PMA/I) or CD40L transfected fibroblasts in the presence of absence of CsA. The activation of CD4+ T cells and CD19+ CLL cells was assessed by staining for CD69/interferon gamma (IFNΥ) and CD69/CD25 respectively. Nuclear translocation of NFATc2 and NFKB p65 was measured by image flow cytometry (Amnis Imagestream). Leukaemia and Lymphoma Research provided the funding for this study. NFkB(p65) translocation at 30 minutes was inhibited by a mean of 22.5% (p=0.0003) in activated CLL CD4+ T cells treated with CsA compared to those treated with vehicle control (VC). Similarly, in the presence of CsA, NFAT-c2 translocation was inhibited by a mean of 24.3% (p=0.008) at 10 minutes in CLL CD4+ T cells compared to those treated with VC. NFkB(p65) translocation was not inhibited (mean of differences=0.63%, p=0.645) and NFAT-c2 translocation was minimally inhibited (mean of differences = -4%, p = 0.007) in activated CLL B Cells treated with CsA. The proportion of activated CLL CD4+ T cells expressing both CD69 and IFNΥ was reduced by 13.2% (p=0.003) in the presence of CsA whereas there was no inhibition of CD25(-1.5, p=0.16) and CD69(-1.4, p=0.5) expression in activated CLL B cells treated with CsA. In summary, CsA had a profound effect on CD4+ T cell activation in patients with CLL, as demonstrated by the reduction in NFkB (p65), NFAT-c2 nuclear translocation and CD69/IFNΥ expressing cells. In contrast, there was a minimal effect on NFAT-c2 translocation in activated CLL B cells and no impact on NFkB (p65) translocation or the expression of CD25 and CD69. These findings suggest that the previously documented activity of CsA in CLL is not due to a direct effect on the tumour but is instead indirect and mediated through inhibition of other microenvironment derived signals such as those provided by activated CD4+ T cells. Since it is likely that these co-stimulatory effects act in concert other signals, such as those induced by BCR activation, reexamination of CsA and similar agents in CLL would thus seem warranted. Disclosures: No relevant conflicts of interest to declare.

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