Lymph Node Derived CLL Cells Have a More Activated Phenotype and Better Antigen Presentation Capabilities Compared To Those From The Peripheral Blood

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4119-4119
Author(s):  
Andrea GS Buggins ◽  
Marta Pasikowska ◽  
Elisabeth J Walsby ◽  
Kirsty M Cuthill ◽  
Maria Longhi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
Thymidine Incorporation ◽  
Activated T Cells ◽  
Hla Dr ◽  
Cell Expression ◽  
Antigen Presenting ◽  
Activation Marker Cd69

Abstract It is now well established that CLL is a highly proliferative disease with replication restricted to lymphoid tissue microenvironments. There is good evidence that interactions involving activated CD4+ T cells influence proliferation of the neoplastic B-cell clone. In this study we investigated the paradox that, whilst chronically activated T cells are found at these sites, in-vitro studies have shown that peripheral blood (PB) CLL cells suppress T cell activation. We hypothesized that the explanation for these contradictory findings is that lymph node (LN) CLL cells have increased antigen-presenting capacity compared to those in the PB. Accordingly, we obtained paired PB and LN fine needle aspirate (FNA) samples from a series of CLL patients and compared the expression of molecules associated with activation, antigen presentation and the ability to stimulate a third party mixed lymphocyte reaction (MLR). Using multi color flow cytometry, CD5+/CD19+ CLL cells were gated for expression of CD80, CD86, HLA-DR, CD25 and CD69. LN CLL cells had higher expression of markers associated with co-stimulation: CD80 (p=0.002), CD86 (p=0.037, n=6), antigen presentation: HLA-DR (p=0.04) and activation: CD25 (p=0.005) and CD69 (p=0.0018). We next used an MLR to determine the functional significance of these findings. Irradiated CLL cells from PB and LN were mixed at co-culture ratio’s of 1:1 and 1:10 and the proliferation/activation of CD4+ T cells measured using the expression of Ki67, CD69, and HLA-DR, and thymidine incorporation. T cell expression of the proliferation marker ki67 and activation marker CD69 was higher when co-cultured with LN CLL cells compared to PB derived cells. 1:1 co-culture ratio, mean Ki67 expression induced by LN CLL cells was 16.1% , SD ± 8.0 compared to 11.5%, SD± 7.7 induced by PB CLL cells (p=0.04) and at 1:10 ratio 8.4% , SD ± 3.6 compared to 3.9%, SD± 3.8 (p=0.02). Similarly, T cell expression of CD69 induced by LN CLL cells at 1:1 ratio was 37.0% , SD ± 15.5 compared to 26.6%, SD± 13.8 induced by PB-CLL cells (p=0.025) and at a 1:10 ratio 14.7% , SD ± 7.7 compared to 11.0%, SD± 7.4 (p=0.0027). LN-derived CLL cells also induced higher levels of T cell HLA-DR expression: 1:1 ratio 30.0%, SD ± 14.7 compared to 21.1%, SD± 9.1 (p=0.02) and at a 1:10 ratio 12.3%, SD ± 5.0 compared to 8.4%, SD± 3.7 (p=0.03). In accordance with these findings, T cell proliferation, measured by thymidine incorporation, was significantly higher in LN-CLL co-cultures compared to PB: 1:1 ratio, LN 2252, SD ± 1549 compared to PB 1615, SD± 1302 (p=0.008) and at 1:10 ratio, LN 910, SD ± 746 compared to, PB 416, SD± 366 (p=0.052). These data support our hypothesis that LN CLL cells have an increased capacity to activate T cells compared to those from the PB. We next investigated whether migration through the endothelium might play a role in promoting the antigen presenting function of CLL cells and hypothesized that this increased antigen presentation capacity of LN-CLL cells would be associated with markers of adhesion and migration. In keeping with this concept, we established that LN-CLL cells had higher expression of CD49d (p=0.008) and CD38 (p=0.018) and reduced CXCR4 (p=0.02) when compared to PB-CLL. To demonstrate this functionally we used a novel in vitro circulation system to compare the phenotype of cells that migrate through the endothelium with those that remain in circulation. CLL cells that migrated into the extravascular compartment in this system had a similar phenotype to LN CLL cells with elevated levels of CD49d and CD38 (P = 0.02 and P = 0.009 respectively), increased levels of CD80 (p=0.0014) and CD86 (p=0.015) and a trend towards increased expression of the activation marker CD69 (p=0.08). CLL cells that remained in the circulating compartment had a phenotype comparable to PB CLL cells. These results provide an explanation for the fact that CLL lymph nodes contain significant numbers of activated T cells, despite the known inhibitory effects of the tumor. Since activated T cells are in turn thought to promote tumor proliferation, our findings suggest that reciprocal interactions between CLL cells and LN T cells might play an important role in disease progression. In addition, our results show that transendothelial migration appears to activate CLL cells. Whether this, or other interactions such as stimulation of the B cell receptor, are responsible for the enhanced antigen presenting capacity of LN CLL cells remains to be determined. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Circulating CD3+HLA-DR+Extracellular Vesicles as a Marker for Th1/Tc1-Type Immune Responses

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Ryutaro Oba ◽  
Motomichi Isomura ◽  
Akira Igarashi ◽  
Kinya Nagata
Keyword(s):  
T Cells ◽  
T Cell ◽  
Extracellular Vesicles ◽  
Inflammatory Diseases ◽  
Cell Subset ◽  
Activated T Cells ◽  
Ebv Infection ◽  
Hla Dr

Extracellular vesicles (EVs) are known to contain unique proteins that reflect the cells of origins. Activated T cells are reported to secrete various EVs. To establish T cell subset-specific biomarkers, we performed proteomic analysis with Th1- and Th2-derived EVs and identified HLA-DR as a Th1-dominated EV membrane protein. We designed a measurement system for CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+EVs to specifically determine EV subpopulations derived from CD4+, CD8+, and Th1-type T cells, respectively.In vitroanalysis showed that CD3+CD4+EVs and CD3+CD8+EVs were selectively secreted from activated CD4+and CD8+T cells, respectively, and that CD3+HLA-DR+EVs were actively secreted from not only Th1 but also activated CD8+T (probably mostly Tc1) cells. To evaluate the clinical usefulness of these EVs, we measured the serum levels in patients with inflammatory diseases, including Epstein-Barr virus (EBV,n=13) infection, atopic dermatitis (AD,n=10), rheumatoid arthritis (RA,n=20), and osteoarthritis (OA,n=20) and compared the levels with those of healthy adults (n=20). CD3+CD4+EVs were significantly higher in all of EBV infection, AD, RA, and OA while CD3+CD8+EVs were higher in EBV infection, lower in RA, and not different in AD and OA relative to the control. The levels of CD3+HLA-DR+EVs were markedly higher in EBV infection and significantly lower in AD. The results suggest that these EV subpopulations reflectin vivoactivation status of total CD4+, total CD8+, and Th1/Tc1-type T cells, respectively, and may be helpful in T cell-related clinical settings, such as cancer immunotherapy and treatment of chronic infection, autoimmune diseases, and graft-versus-host disease.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

scholarly journals Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.

1992 ◽  
Vol 176 (5) ◽  
pp. 1431-1437 ◽  
Author(s):  
M Croft ◽  
D D Duncan ◽  
S L Swain
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 Cells ◽  
Peptide Antigen ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Antigen Presenting

Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.

scholarly journals Development of Stable Chimeric IL-15 for Trans-Presentation by the Antigen Presenting Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Manoj Patidar ◽  
Naveen Yadav ◽  
Sarat K. Dalai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Half Life ◽  
Therapeutic Potential ◽  
Chimeric Protein ◽  
Antigen Presenting Cells ◽  
T Cell Response ◽  
Antigen Presenting

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rβ. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8+ T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8+ T cell responses.

scholarly journals Venetoclax enhances T cell-mediated anti-leukemic activity by increasing ROS production

Blood ◽  
2021 ◽  
Author(s):  
JongBok Lee ◽  
Dilshad H. Khan ◽  
Rose Hurren ◽  
Mingjing Xu ◽  
Yoosu Na ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mechanism Of Action ◽  
Adoptive Cell Therapy ◽  
Complete Response ◽  
Ros Generation ◽  
Activated T Cells ◽  
Treatment Naïve

Venetoclax, a Bcl-2 inhibitor, in combination with the hypomethylating agent, Azacytidine, achieves complete response with or without count recovery in approximately 70% of treatment-naïve elderly patients unfit for conventional intensive chemotherapy. However, the mechanism of action of this drug combination is not fully understood. We discovered that Venetoclax directly activated T cells to increase their cytotoxicity against AML in vitro and in vivo. Venetoclax enhanced T cell effector function by increasing ROS generation through inhibition of respiratory chain supercomplexes formation. In addition, Azacytidine induced a viral-mimicry response in AML cells by activating the STING/cGAS pathway, thereby rendering the AML cells more susceptible to T-cell mediated cytotoxicity. Similar findings were seen in patients treated with Venetoclax as this treatment increased ROS generation and activated T cells. Collectively, this study demonstrates a new immune mediated mechanism of action for Venetoclax and Azacytidine in the treatment of AML and highlights a potential combination of Venetoclax and adoptive cell therapy for patients with AML.

scholarly journals The Activated Type 1–Polarized Cd8+ T Cell Population Isolated from an Effector Site Contains Cells with Flexible Cytokine Profiles

1999 ◽  
Vol 190 (8) ◽  
pp. 1081-1092 ◽  
Author(s):  
Anthony G. Doyle ◽  
Kathy Buttigieg ◽  
Penny Groves ◽  
Barbara J. Johnson ◽  
Anne Kelso
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Expression Profiles ◽  
Lung Parenchyma ◽  
Activated T Cells ◽  
Cytokine Genes ◽  
Effector Site

The capacity of activated T cells to alter their cytokine expression profiles after migration into an effector site has not previously been defined. We addressed this issue by paired daughter analysis of a type 1–polarized CD8+ effector T cell population freshly isolated from lung parenchyma of influenza virus–infected mice. Single T cells were activated to divide in vitro; individual daughter cells were then micromanipulated into secondary cultures with and without added IL-4 to assess their potential to express type 2 cytokine genes. The resultant subclones were analyzed for type 1 and 2 cytokine mRNAs at day 6–7. When the most activated (CD44highCD11ahigh) CD8+ subpopulation from infected lung was compared with naive or resting (CD44lowCD11alow) CD8+ cells from infected lung and from normal lymph nodes (LNs), both clonogenicity and plasticity of the cytokine response were highest in the LN population and lowest in the activated lung population, correlating inversely with effector function. Multipotential cells were nevertheless detected among clonogenic CD44highCD11ahigh lung cells at 30–50% of the frequency in normal LNs. The data indicate that activated CD8+ T cells can retain the ability to proliferate and express new cytokine genes in response to local stimuli after recruitment to an effector site.

IκB kinase 2 but not NF-κB–inducing kinase is essential for effective DC antigen presentation in the allogeneic mixed lymphocyte reaction

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 983-991 ◽  
Author(s):  
Evangelos Andreakos ◽  
Clive Smith ◽  
Claudia Monaco ◽  
Fionula M. Brennan ◽  
Brian M. Foxwell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mixed Lymphocyte Reaction ◽  
Dominant Negative ◽  
Allogeneic Mixed Lymphocyte Reaction ◽  
Iκb Kinase ◽  
Antigen Presenting

AbstractAlthough dendritic cells (DCs) are the most potent antigen-presenting cells involved in numerous physiologic and pathologic processes, little is known about the signaling pathways that regulate DC activation and antigen-presenting function. Recently, we demonstrated that nuclear factor (NF)-κB activation is central to that process, as overexpression of IκBα blocks the allogeneic mixed lymphocyte reaction (MLR), an in vitro model of T-cell activation. In this study, we investigated the role of 2 putative NF-κB–inducing components, NF-κB–inducing kinase (NIK), and IκB kinase 2 (IKK2). Using an adenoviral gene transfer method to efficiently express dominant-negative (dn) forms of these molecules in monocyte-derived DCs, we found that IKK2dn but not NIKdn inhibited the allogeneic MLR. When DCs were fixed, this inhibitory effect of IKK2dn was lost, suggesting that IKK2 is involved in T-cell–derived signals that enhance DC antigen presentation during the allogeneic MLR period and does not have an effect on viability or differentiation state of DCs prior to coculture with T cells. One such signal is likely to be CD40 ligand (CD40L), as IKK2dn blocked CD40L but not lipopolysaccharide (LPS)–induced NF-κB activation, cytokine production, and up-regulation of costimulatory molecules and HLA-DR in DCs. In summary, our results demonstrate that IKK2 is essential for DC activation induced by CD40L or contact with allogeneic T cells, but not by LPS, whereas NIK is not required for any of these signals. In addition, our results support IKK2 as a potential therapeutic target for the down-regulation of unwanted immune responses that may occur during transplantation or autoimmunity.

scholarly journals SHP-1 Acts as a Key Regulator of Alloresponses by Modulating LFA-1-Mediated Adhesion in Primary Murine T Cells

2016 ◽  
Vol 36 (24) ◽  
pp. 3113-3127 ◽  
Author(s):  
Martin G. Sauer ◽  
Jessica Herbst ◽  
Ulf Diekmann ◽  
Christopher E. Rudd ◽  
Christian Kardinal
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphatase ◽  
Activated T Cells ◽  
Sirna Knockdown ◽  
Antigen Presenting ◽  
Pharmaceutical Agents ◽  
Interfering Rna ◽  
Clinical Potential

The clinical potential of transplantation is often reduced by T cell-mediated alloresponses that cause graft rejection or graft-versus-host disease. Integrin-mediated adhesion between alloreactive T cells and antigen-presenting cells is essential for allorejection. The identity of the signaling events needed for the activation of integrins such as LFA-1 is poorly understood. Here, we identified a novel role of the protein tyrosine phosphatase SHP-1 in the regulation of murine LFA-1-mediated adhesion in an allograft setting. Upon alloactivation, SHP-1 activity is reduced, resulting in an increase in LFA-1 adhesion compared to that for syngeneically activated T cells. The importance of these differential activation properties was further indicated by small interfering RNA (siRNA) knockdown of SHP-1 in syngeneically and allogeneically stimulated T cells. Mechanistically, SHP-1 modulated the binding of SLP-76 to ADAP by dephosphorylation of the YDGI tyrosine motif of ADAP, a known docking site for the Src family kinase Fyn. This novel key role of SHP-1 in the regulation of LFA-1-mediated adhesion may provide a new insight into T cell-mediated alloresponses and may pave the way to the development of new immunosuppressive pharmaceutical agents.

scholarly journals Fyn-Binding Protein (Fyb)/Slp-76–Associated Protein (Slap), Ena/Vasodilator-Stimulated Phosphoprotein (Vasp) Proteins and the Arp2/3 Complex Link T Cell Receptor (Tcr) Signaling to the Actin Cytoskeleton

2000 ◽  
Vol 149 (1) ◽  
pp. 181-194 ◽  
Author(s):  
Matthias Krause ◽  
Antonio S. Sechi ◽  
Marlies Konradt ◽  
David Monner ◽  
Frank B. Gertler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Actin Cytoskeleton ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Signaling ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Antigen Presenting ◽  
Syndrome Protein

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76–associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3–coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.

scholarly journals Abortive Proliferation of Rare T Cells Induced by Direct or Indirect Antigen Presentation by Rare B Cells In Vivo

1998 ◽  
Vol 187 (10) ◽  
pp. 1611-1621 ◽  
Author(s):  
Sarah E. Townsend ◽  
Christopher C. Goodnow
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Antigen Presentation ◽  
Cell Fate ◽  
T Cell Activation ◽  
Cell Activation ◽  
Antigen Presenting Cells ◽  
Antigen Presenting

Antigen-specific B cells are implicated as antigen-presenting cells in memory and tolerance responses because they capture antigens efficiently and localize to T cell zones after antigen capture. It has not been possible, however, to visualize the effect of specific B cells on specific CD4+ helper T cells under physiological conditions. We demonstrate here that rare T cells are activated in vivo by minute quantities of antigen captured by antigen-specific B cells. Antigen-activated B cells are helped under these conditions, whereas antigen-tolerant B cells are killed. The T cells proliferate and then disappear regardless of whether the B cells are activated or tolerant. We show genetically that T cell activation, proliferation, and disappearance can be mediated either by transfer of antigen from antigen-specific B cells to endogenous antigen-presenting cells or by direct B–T cell interactions. These results identify a novel antigen presentation route, and demonstrate that B cell presentation of antigen has profound effects on T cell fate that could not be predicted from in vitro studies.

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