Development of a Novel Method for in Vitro Analysis of CD8 Thymocyte Selection and Maturation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3235-3235
Author(s):  
Moutih Rafei ◽  
Alexandre Rouette ◽  
Juan Vanegas Ruiz ◽  
Claude Perreault
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Stromal Cells ◽  
T Cell Development ◽  
Cd8 T Cell ◽  
Cell Development ◽  
Expression Intensity

Abstract Abstract 3235 T cell development relies on the interaction between the T-cell receptor (TCR) on thymocytes and the self-major histocompatibility complex (MHC) expressed on thymic epithelial cells in the thymus. This process, called positive selection, rescues developing thymocytes from cell death while leading to their differentiation into mature T cells. Since it is believed that the proper development of CD8 T cells requires an intact thymus, several groups studied their development using fetal or reaggregation thymus organ cultures in vitro. Unfortunately, these models were shown to be cumbersome requiring a complicated set-up while generating limited cellular yield. Thus, we sought of developing a novel in vitro system using bone marrow-derived stromal cells to support CD8 T cell development and maturation in vitro. We selected the OTI system as a working model due to the availability of previously identified positively selecting peptides. Non-selected T-cell-committed double-positive (DP) OTI thymocytes (CD4+CD8+CD69−) were first fractionated based on the surface expression intensity of both TCR and CD5. These 3 subsets designated as TCRloCD5lo (DP1), TCRintCD5hi (DP2), and TCRhiCD5int (DP3) express different levels of ZAP70. Following fractionation, the DP subsets were co-cultured with bone marrow-derived stromal cells presenting OTI-selecting peptides. In the absence of cytokines, no CD8+ OTI cell development occurred in vitro. When repeated in the presence of γc-cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) only rIL4 and rIL7 were able to induce CD8 T cell development. Supplementing the co-culture system with rIL4 led to the generation of 50–60% single-positive (SP) CD8 T cells only from the DP3 fraction whereas rIL7 induced the development of a minor fraction of CD8 T cells from DP2s (3–4%) and a major population from DP3 (50–76%). Furthermore, we found that rIL4 treatment triggers the development of 2 distinct populations of SP OTI cells (based on their CD8 expression intensity) which we termed CD8int and CD8hi. When analyzed by flow-cytometry, ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44. In contrast, SP CD8 T cells developed in the presence of rIL7 did not upregulate these markers. Since IL7 promotes survival and proliferation of TCR-triggered DPs while IL4 affects their differentiation, we admixed both cytokines during the co-culture and found a dominant rIL4 effect: the phenotype of SP CD8 T cells was similar to that induced by rIL4 alone. Taken together, our findings demonstrate that some DP thymocytes are efficiently selected in our system by OTI-specific positively selecting peptides. Notably, the addition of rIL7 leads to the development and maturation of classic CD8 T cells whereas rIL4 induces both classic and innate CD8 T cells. This work was supported by grant a from CIHR. Disclosures: No relevant conflicts of interest to declare.

Kras Is Critical for CD8 T Cell Antiviral Function

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 284-284
Author(s):  
Yuhong Chen ◽  
Yongwei Zheng ◽  
Xiaona You ◽  
Gang Xin ◽  
Mei Yu ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Development ◽  
Cd8 T Cell ◽  
Cell Immune Response ◽  
Deficient Mice ◽  
Lcmv Infection

Abstract Small GTPases regulate multiple signaling pathways and individual Ras member can have distinct biological function. To overcome embryonic lethality of Kras-deficient mice, we generated and examined mice with hematopoietic- and T cell-specific deletion of Kras. In VavCreKrasfl/fl mice with hematopoietic deletion of Kras, thymic T-cell development was normal based on the presence of normal populations of total, CD4- CD8-, CD4+ CD8+, CD4+ and CD8+ thymocytes. The populations of splenic CD4+ and CD8+ T cells were also comparable between VavCreKrasfl/fl relative to control mice. In addition, no consistent defects in the 3 H-thymidine incorporation rate of Kras-deficient splenic CD4+ or CD8+ T cells in response to anti-CD3 or anti-CD3 plus IL-2 was detected. Nonetheless, we studied the effect of Kras deficiency on CD8 T-cell immune response to acute infection of the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). Sub-lethally irradiated Rag1-deficient mice transplanted with bone marrow (BM) cells from VavCreKrasfl/fl or control mice were subjected to LCMV infection. Infection-induced expansion of CD8 T cells and generation of LCMV epitope gp33-specific CD8 T cells were markedly reduced in the recipients that received the BM from VavCreKrasfl/fl relative to control mice. Following in vitro stimulation with the LCMV epitope gp33, the induction of IFNg-expressing CD8 T cells from LCMV-infected recipients that received the BM from VavCreKrasfl/fl mice was dramatically reduced. Further, BM chimeric mice with CD8 T cell-specific deficiency generated by transplantation of lethally irradiated CD8 T cell-depleted CD45.1 congenic mice with a mixture of BM cells from VavCreKrasfl/fl mice and BM cells from CD8 T cell-deficient mice exhibited an impaired CD8 T-cell immune response to LCMV infection. Lastly, we examined the role of Kras in TCR signaling. The level of total TCR-activated Ras (Ras-GTP) was markedly reduced in Kras-deficient relative to control CD8 T cells. Importantly, TCR-induced ERK1/2 activation was impaired in Kras-deficient relative to control CD8 T cells. Consistently, TCR-induced activation of Raf-1 and MEK1/2 was markedly reduced in mutant CD8 T cells. However, TCR-induced JNK and p38 activation as well as Ca2+ flux were normal in Kras-deficient CD8 T cells. Of note, TCR-induced activation of Ca2+ flux, JNK and p38 as well as ERK1/2, MEK1/2 and Raf1 was normal in Kras-deficient relative to control CD4 cells. Taken together, these data demonstrate that Kras is dispensable for T cell development or TCR-induced proliferation of CD4 or CD8 T cells in vitro, but regulates TCR-induced activation of the Raf-1/MEK/ERK pathway in CD8 but not CD4 T cells and intrinsically controls CD8 T-cell immune response to viral infection. Disclosures No relevant conflicts of interest to declare.

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 Adrenergic signaling controls early transcriptional programs during CD8+ T cell responses to viral infection

2021 ◽  
Author(s):  
Leonardo Estrada ◽  
Didem Agac Cobanoglu ◽  
Aaron Wise ◽  
Robert Maples ◽  
Murat Can Cobanoglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Receptor Activation ◽  
Cell Development ◽  
Primary Response ◽  
Cell Responses

Viral infections drive the expansion and differentiation of responding CD8+ T cells into variegated populations of cytolytic effector and memory cells. While pro-inflammatory cytokines and cell surface immune receptors play a key role in guiding T cell responses to infection, T cells are also markedly influenced by neurotransmitters. Norepinephrine is a key sympathetic neurotransmitter, which acts to suppress CD8 + T cell cytokine secretion and lytic activity by signaling through the beta2-adrenergic receptor (ADRB2). Although ADRB2 signaling is considered generally immunosuppressive, its role in regulating differentiation of effector T cells in response to infection has not been investigated. Using an adoptive transfer approach, we compared the expansion and differentiation of wild type (WT) to Adrb2-/- CD8 + T cells throughout the primary response to vesicular stomatitis virus (VSV) infection in vivo. We measured the dynamic changes in transcriptome profiles of antigen-specific CD8 + T cells as they responded to VSV. Within the first 7 days of infection, WT cells out-paced the expansion of Adrb2-/- cells, which correlated with reduced expression of IL-2 and the IL-2Ralpha; in the absence of ADRB2. RNASeq analysis identified over 300 differentially expressed genes that were both temporally regulated following infection and selectively regulated in WT vs Adrb2-/- cells. These genes contributed to major transcriptional pathways including cytokine receptor activation, signaling in cancer, immune deficiency, and neurotransmitter pathways. By parsing genes within groups that were either induced or repressed over time in response to infection, we identified three main branches of genes that were differentially regulated by the ADRB2. These gene sets were predicted to be regulated by specific transcription factors involved in effector T cell development, such as Tbx21 and Eomes. Collectively, these data demonstrate a significant role for ADRB2 signaling in regulating key transcriptional pathways during CD8 + T cells responses to infection that may dramatically impact their functional capabilities and downstream memory cell development.

scholarly journals A new in vitro model of polymyositis reveals CD8+ T cell invasion into muscle cells and its cytotoxic role

Rheumatology ◽  
2019 ◽  
Vol 59 (1) ◽  
pp. 224-232
Author(s):  
Mari Kamiya ◽  
Fumitaka Mizoguchi ◽  
Akito Takamura ◽  
Naoki Kimura ◽  
Kimito Kawahata ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Muscle Injury ◽  
In Vitro Model ◽  
Muscle Cells ◽  
Cd8 T Cell ◽  
Class I ◽  
Vitro Model

Abstract Objectives The hallmark histopathology of PM is the presence of CD8+ T cells in the non-necrotic muscle cells. The aim of this study was to clarify the pathological significance of CD8+ T cells in muscle cells. Methods C2C12 cells were transduced retrovirally with the genes encoding MHC class I (H2Kb) and SIINFEKL peptide derived from ovalbumin (OVA), and then differentiated to myotubes (H2KbOVA-myotubes). H2KbOVA-myotubes were co-cultured with OT-I CD8+ T cells derived from OVA-specific class I restricted T cell receptor transgenic mice as an in vitro model of PM to examine whether the CD8+ T cells invade into the myotubes and if the myotubes with the invasion are more prone to die than those without. Muscle biopsy samples from patients with PM were examined for the presence of CD8+ T cells in muscle cells. The clinical profiles were compared between the patients with and without CD8+ T cells in muscle cells. Results Analysis of the in vitro model of PM with confocal microscopy demonstrated the invasion of OT-I CD8+ T cells into H2KbOVA-myotubes. Transmission electron microscopic analysis revealed an electron-lucent area between the invaded CD8+ T cell and the cytoplasm of H2KbOVA-myotubes. The myotubes invaded with OT-I CD8+ T cells died earlier than the uninvaded myotubes. The level of serum creatinine kinase was higher in patients with CD8+ T cells in muscle cells than those without these cells. Conclusion CD8+ T cells invade into muscle cells and contribute to muscle injury in PM. Our in vitro model of PM is useful to examine the mechanisms underlying muscle injury induced by CD8+ T cells.

BK Polyomavirus–Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination

2020 ◽  
Author(s):  
Maud Wilhelm ◽  
Amandeep Kaur ◽  
Marion Wernli ◽  
Hans H Hirsch
Keyword(s):  
T Cells ◽  
T Cell ◽  
Kidney Transplant ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Bk Polyomavirus

Abstract Background BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. Methods Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. Results BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1–) and cytotoxic. Conclusions Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

scholarly journals IL4I1 Accelerates the Expansion of Effector CD8+ T Cells at the Expense of Memory Precursors by Increasing the Threshold of T-Cell Activation

2020 ◽  
Vol 11 ◽  
Author(s):  
Marie-Line Puiffe ◽  
Aurélie Dupont ◽  
Nouhoum Sako ◽  
Jérôme Gatineau ◽  
José L. Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Lymphocytic Choriomeningitis ◽  
T Cell Clone

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

scholarly journals Phenotypic and Functional Separation of Memory and Effector Human CD8+ T Cells

1997 ◽  
Vol 186 (9) ◽  
pp. 1407-1418 ◽  
Author(s):  
Dörte Hamann ◽  
Paul A. Baars ◽  
Martin H.G. Rep ◽  
Berend Hooibrink ◽  
Susana R. Kerkhof-Garde ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Fas Ligand ◽  
Cytotoxic T Lymphocyte ◽  
Cell Subset ◽  
Cd8 T Cell ◽  
Cytolytic Activity ◽  
T Cell Subset

Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA−CD45R0+ cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon γ, tumor necrosis factor α, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27− population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-γ and TNF-α. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27− cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

scholarly journals Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis

2017 ◽  
Vol 214 (6) ◽  
pp. 1593-1606 ◽  
Author(s):  
Hossam A. Abdelsamed ◽  
Ardiana Moustaki ◽  
Yiping Fan ◽  
Pranay Dogra ◽  
Hazem E. Ghoneim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Human Memory ◽  
Memory Cd8 T Cells ◽  
Memory Cd8 T Cell ◽  
Cell Effector

Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell–mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (TEM), and longer-lived central memory (TCM) and stem cell memory (TSCM) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7– and IL-15–mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of TCM and TSCM memory cells resulted in phenotypic conversion into TEM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired TEM-associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells.

IL-7 Effects on Thymocyte Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3318-3318
Author(s):  
Nahed El Kassar ◽  
Baishakhi Choudhury ◽  
Francis Flomerfelt ◽  
Philip J. Lucas ◽  
Veena Kapoor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Notch Signaling ◽  
Stromal Cells ◽  
T Cell Development ◽  
Fold Increase ◽  
Cell Development ◽  
B Cell Development ◽  
Over Expression

Abstract IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

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