Faculty Opinions recommendation of Cytosolic localization of Listeria monocytogenes triggers an early IFN-gamma response by CD8+ T cells that correlates with innate resistance to infection.

Lyt-2+ T cell clones were established from Listeria monocytogenes-infected mice. The clones secreted IFN-gamma after stimulation with spleen cells from L. monocytogenes-infected mice plus IL-2. Spleen cells from normal mice were not stimulatory. Furthermore, cloned T cells lysed L. monocytogenes-infected macrophages. Cytolysis was antigen-specific and H-2K-restricted. These findings suggest a role for specific cytotoxic T cells in the immune response to intracellular bacteria.

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The role of CD4 + and CD8 + T-cells in host morbidity and innate resistance to Angiostrongylus cantonensis in the mouse

Parasitology Research ◽

10.1007/s004360050363 ◽

1997 ◽

Vol 84 (2) ◽

pp. 91-99 ◽

Cited By ~ 8

Author(s):

Mikiko Aoki ◽

Hiroko Sugaya ◽

Kazuto Ishida ◽

Kentaro Yoshimura

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Angiostrongylus Cantonensis ◽

Innate Resistance

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In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Blood ◽

10.1182/blood.v114.22.4079.4079 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4079-4079

Author(s):

Lei Bao ◽

Mindy M Stamer ◽

Kimberly Dunham ◽

Deepa Kolaseri Krishnadas ◽

Kenneth G Lucas

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Lymphocytes ◽

Cell Therapy ◽

Cytotoxic T Lymphocytes ◽

Cd8 T Cells ◽

Target Cells ◽

Ifn Gamma ◽

Healthy Donors

Abstract Abstract 4079 Poster Board III-1014 MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures: No relevant conflicts of interest to declare.

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Automated Generation of Antigen-Specific T Cells for Adoptive T Cell Therapy.

Blood ◽

10.1182/blood.v116.21.1177.1177 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1177-1177

Author(s):

Melanie Fahrendorff ◽

Nanette von Oppen ◽

Georg Rauser ◽

Mario Assenmacher ◽

Juergen Schmitz ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cd8 T Cells ◽

Allogeneic Stem Cell Transplantation ◽

Closed System ◽

Isolated Cells ◽

Ifn Gamma ◽

Cell Processing

Abstract Abstract 1177 The adoptive transfer of antigen-specific T cells can be a powerful tool for immunotherapy of malignant diseases or infectious complications after allogeneic stem cell transplantation (Riddell et al., 1992; Heslop et al. 2010). Human adenovirus (AdV), Epstein-Barr virus (EBV) or cytomegalovirus (HCMV) infections are frequent and often life-threatening complications post allogeneic stem cell transplantation. To reduce the time required to isolate antigen-specific T cells for adoptive transfer we have developed a method to isolate IFN-gamma-secreting CD4+ as well as CD8+ T cells after antigen-specific restimulation, the Cytokine Capture System IFN-gamma (CCS). Several preclinical studies demonstrate the efficient enrichment of functional CD4+ and CD8+ T cells specific for HCMV (Rauser et al., 2004), EBV (Hammer et al., 2007) or AdV (Feuchtinger et al. 2008) using the CCS. First clinical data of adoptively transferred HCMV-, EBV- or AdV-specific T cells into patients post allogeneic stem cell transplantation are very encouraging (Feuchtinger et al., 2010; Moosmann et al., 2010; Feuchtinger et al., 2006) with low T cell doses infused varying from 1–97×10e3 cells/kg. We have now developed a cell processing device for the automation of the CCS procedure. Antigen-specific stimulation, labeling with the CCS reagents, washing steps, cytokine capture, magnetic enrichment and potentially expansion of the isolated cells are performed fully automated in a closed system. At the beginning of the procedure all components including the cellular starting product, antigen(s), reagents, buffer, and media are connected to a sterile single-use closed system processing set in the device. Due to usage of sterile filters and sterile docking the whole process runs under sterile conditions. The cellular end product can be obtained in the medium/buffer of choice, with the desired cell concentration and volume. The cellular starting product can be leukapheresis or bone marrow and the yield of antigen-specific T cells depends on the frequency of IFN-gamma producing cells. When starting with 1×10e9 cells 1–20×10e5 HCMV-specific T cells could be isolated. Cell processing is possible overnight and the isolated cells might be used directly after enrichment or after a phase of in vitro expansion. Using this cell processing device, IFN-gamma secreting HCMV-specific T cells were enriched to the same purity (>80% IFN-gamma secreting CD4+ and CD8+ T cells) as with the semi-automated procedure. Cell loss during the procedure is markedly reduced, leading to an increased yield of IFN-gamma positive cells. An improved viability was observed resulting in better expansion rates. In conclusion, the automation in a closed system enables the fast and robust generation of antigen-specific T cells for adoptive therapy and will reduce clean room requirements. Disclosures: Fahrendorff: Miltenyi Biotec GmbH: Employment. von Oppen:Miltenyi Biotec GmbH: Employment. Rauser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Biehl:Miltenyi Biotec GmbH: Employment. Miltenyi:Miltenyi Biotec GmbH: Membership on an entity's Board of Directors or advisory committees.

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Immunomodulatory Drugs Restore Effector Cell Immune Functions In Myeloma Patients With Low Disease Burden After Autologous Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.3214.3214 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3214-3214 ◽

Cited By ~ 1

Author(s):

Karel Fostier ◽

Jurgen Corthals ◽

Carlo Heirman ◽

Joeri L. Aerts ◽

Kris Thielemans ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Disease Burden ◽

Effector Cell ◽

Culture Medium ◽

Tnf Alpha ◽

Dendritic Cell Vaccination ◽

Immunomodulatory Drugs ◽

Ifn Gamma

Abstract The micro-environment in multiple myeloma (MM) is highly immunosuppressive with increased numbers of regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) favoring tumorcell survival and hampering immunotherapeutic strategies such as dendritic cell vaccination. Immunomodulatory drugs (IMiDs) are known to enhance T- and NK-cell function. In this study we evaluated the effects of low dose (0.5 microM) lenalidomide (Len) and pomalidomide (Pom) on the functionality of CD8+ and CD4+ T cells, MDSCs, Tregs and ex-vivo generated mononuclear derived dendritic cells (moDCs) obtained from MM patients after first autologous stem cell transplantation (ASCT). Peripheral blood mononuclear cell fractions were obtained by leukapheresis from 9 MM patients (age 29-62 years), in very good partial response (4/9) or complete response (5/9) after ASCT. The magnitude of cytokine release (mean +/- standard error of the mean, in ng/ml) by purified CD8+ T cells after 144 hours stimulation with anti-CD3/anti-CD28 coated microbeads was significantly increased after addition of Len and Pom to the culture medium, respectively : IFN-gamma (217.5 +/- 62.1 and 437.1 +/- 137.1** vs 66.4 +/- 21.0) , TNF-alpha (21.4 +/- 5.4 and 44.9 +/- 9.4*** vs 4.9 +/- 1.7) and IL-2 (5.3 +/- 2.7 and 12.7 +/- 6.6 vs 1.9 +/- 1.7 ng/ml) (** p< 0.01, *** p< 0.001). We also evaluated the number of different types of cytokines/chemokines on a per cell basis by intracellular flow cytometry staining for IFN-gamma, TNF-alpha, IL-2 and MIP-1beta and observed increased polyfunctionality of CD8+ and CD4+ T cells. After 72 h of stimulation with anti-CD3/CD28 microbeads the number of single, double, triple or quadruple functional CD8+ T cells increased from 5.96 %, 2.82 %, 0.1 %, 0 % (culture medium alone) to 9.68 %, 7.57 %, 0.41%, 0.03 % (Len) and 12.57 %, 8.96 %, 0.81 %, 0.03 % (Pom), respectively. A similar observation was made for CD4+ T cells. A significant percentage, median 5.7 % (4.0-7.2 %) of CD4+ CD25high CD127low (Tregs) was found in the CD4+ T cell population in 8 out of 9 patients, demonstrating the highly suppressive immune environment in myeloma patients even with low disease burden. Effector T cells (Teffs) were stimulated with anti-CD3/CD28 microbeads and cocultured at varying ratios with purified Tregs. After 144 h of coculture, Len and Pom reduced the suppressive effects of Tregs on Teffs proliferation and IFN-gamma and TNF-alpha production (see figure). A similar effect was observed for MDSC but did not reach statistical significance (data not shown). TriMix DCs (moDCs matured by electroporation with mRNA encoding TLR4, CD40L and CD70) and cytokine matured moDCs were cocultured with autologous CD4+ and CD8+ T cells and anti-CD3 microbeads. Adding IMiDs resulted in more polyfunctional CD4+ and CD8+ T cells with both types of DCs but effects were most pronounced with the TriMix variant. Our study shows that Len and Pom restore effector cell functions in myeloma patients with low tumor burden after ASCT. These findings provide a immunomechanistic explanation for IMiD-based maintenance therapy. They also offer a rationale to combine IMiD-based maintenance with immunotherapeutic approaches such as dendritic cell vaccination in this particular setting. Disclosures: No relevant conflicts of interest to declare.

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CD11a Regulates Effector CD8 T Cell Differentiation and Central Memory Development in Response to Infection with Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.00749-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1140-1151 ◽

Cited By ~ 27

Author(s):

Tina O. Bose ◽

Quynh-Mai Pham ◽

Evan R. Jellison ◽

Juliette Mouries ◽

Christie M. Ballantyne ◽

...

Keyword(s):

T Cells ◽

Listeria Monocytogenes ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

T Cell Response ◽

Central Memory ◽

Secondary Response ◽

Cellular Interactions ◽

Deficient Mice

ABSTRACTβ2 (CD18) integrins with α-chains CD11a, -b, -c, and -d are important adhesion molecules necessary for leukocyte migration and cellular interactions. CD18 deficiency leads to recurrent bacterial infections and poor wound healing due to reduced migration of leukocytes to inflammatory sites. CD8 T cells also upregulate CD11a, CD11b, and CD11c upon activation. However, the role these molecules play for CD8 T cellsin vivois not known. To determine the function of individual β2 integrins, we examined CD8 T cell responses toListeria monocytogenesinfection in CD11a-, CD11b-, and CD11c-deficient mice. The absence of CD11b or CD11c had no effect on the generation of antigen-specific CD8 T cells. In contrast, the magnitude of the primary CD8 T cell response in CD11a-deficient mice was significantly reduced. Moreover, the response in CD11a−/−mice exhibited reduced differentiation of short-lived effector cells (KLRG1hiCD127lo), although cytokine and granzyme B production levels were unaffected. Notably, CD11a deficiency resulted in greatly enhanced generation of CD62L+central memory cells. Surprisingly, CD8 T cells lacking CD11a mounted a robust secondary response to infection. Taken together, these findings demonstrated that CD11a expression contributes to expansion and differentiation of primary CD8 T cells but may be dispensable for secondary responses to infection.

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