Differential Functional Avidity of Dengue Virus-Specific T-Cell Clones for Variant Peptides Representing Heterologous and Previously Encountered Serotypes

ABSTRACT Proinflammatory cytokines secreted by memory CD8+ and CD4+ T cells are thought to play a direct role in the pathogenesis of dengue virus infection by increasing vascular permeability and thereby inducing the pathophysiologic events associated with dengue hemorrhagic fever and dengue shock syndrome. Severe disease is frequently observed in the setting of secondary infection with heterologous dengue virus serotypes, suggesting a role for cross-reactive memory T cells in the immunopathogenesis of severe disease. We used a large panel of well-characterized dengue virus-specific CD8+ T-cell clones isolated from Pacific Islanders previously infected with dengue virus 1 to examine effector memory function, focusing on a novel dominant HLA-B*5502-restricted NS5329-337 epitope, and assessed T-cell responses to stimulation with variant peptides representing heterologous serotypes. Variant peptides were differentially recognized by dengue virus 1-specific effector CD8+ cytotoxic T lymphocytes (CTL) in a heterogeneous and clone-specific manner, in which cytolytic function and cytokine secretion could be enhanced, diminished, or abrogated compared with cognate peptide stimulation. Dengue virus-specific CTL stimulated with cognate and variant peptides demonstrated a cytokine response hierarchy of gamma IFN (IFN-γ) > tumor necrosis factor alpha (TNF-α) > interleukin-2 (IL-2), and a subset of clones also produced IL-4 and IL-6. Individual clones demonstrated greater avidity for variant peptides representing heterologous serotypes, including serotypes previously encountered by the subject, and IFN-γ and TNF-α secretion was enhanced by stimulation with these heterologous peptides. Altered antiviral T-cell responses in response to stimulation with heterologous dengue virus serotypes have implications for control of virus replication and for disease pathogenesis.

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Robust Anti-Leukemia CD4+ and CD8+ T Cell Responses in CML Patients Treated with Imatinib Mesylate.

Blood ◽

10.1182/blood.v108.11.2199.2199 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2199-2199

Author(s):

Christiane I.-U. Chen ◽

Alexandre Johannsen ◽

Wesley Witteles ◽

Holden T. Maecker ◽

Peter P. Lee

Keyword(s):

T Cell ◽

T Cell Responses ◽

Cd8 T Cell ◽

Leukemic Cells ◽

T Cell Clones ◽

Cell Responses ◽

Cell Clones ◽

Tnf Α ◽

Ifn Γ ◽

Cytogenetic Remission

Abstract Imatinib mesylate, a selective inhibitor of the bcr/abl tyrosine kinase, has revolutionized the treatment of CML and is the first-line therapy for most patients. Most CML patients in chronic phase achieve hematologic remission under treatment with imatinib, with up to 70% also achieving cytogenetic remission. However, imatinib therapy is not curative, as patients who discontinue the drug invariably relapse. Thus, the need to find alternate, potentially curative therapies remains. Low levels of CD8+ T cell responses to certain HLA restricted peptides have been detected in CML patients after successful treatment. To determine, if CML patients in remission on imatinib develop and sustain anti-leukemia CD4+ or CD8+ T cell responses, blood samples from patients before and several time points after treatment were collected and analyzed. Pre-treatment samples were utilized as sources of autologous leukemic cells to detect anti-leukemia T cell responses in post-treatment remission samples. Autologous leukemic samples alone and remission samples alone served as controls. In 7 of the 14 patients investigated, significant IFN-γ responses (p<0.01) in ELISPOT assay were detectable when patients achieved cytogenetic remission, and peaked at 10–15 months (median 36 SFCs, range 26–71 SFCs), before they slowly decreased (up to 46 months post-treatment) to levels similar to those from leukemic samples alone and remission samples alone (6 SFCs, range 0–13 SFCs). These results correlated with T cell responses in cytokine flow cytometry (TNF-α 1.4–40%, IFN-γ 1.0–6.6%, p<0.05), and showed a predominance for CD4+ T cells. Furthermore, TNF-α and IFN-γ production (p<0.05) was confirmed in CD4+ and CD8+ T cell clones generated in a remission sample from one patient using multiplex cytokine assay: 6.43–37.90 pg/ml for TNF-α, 14.90–110.42 pg/ml for IFN-γ. CD8+ T cell clones (HLA-A0201+) were tested for reactivity against HLA-A0201 restricted peptides derived from leukemia associated antigens proteinase 3, Wilms tumor 1 and bcr/abl, as well as tumor associated antigens p53, p68, and human telomerase (hTERT) in IFN-γ ELISPOT assays. No IFN-γ responses were detected in the CD8+ T cell clones when stimulated with the peptides compared to stimulation with an irrelevant peptide (0–15 SFCs, p=0.55), suggesting that these clones react to as yet to be defined leukemia associated antigens. Together, these data show that CD4+ T cells play an important role in anti-leukemia immune responses in patients in remission (sustained over time) and might contribute to killing of leukemic cells, possibly via TNF-α. Banked autologous leukemic cells could be used for vaccination of CML patients in order to enhance anti-leukemia T cell responses and may, in combination with imatinib, lead to eradication of residual leukemic cells with a durable cure.

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An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

Infection and Immunity ◽

10.1128/iai.66.10.4981-4988.1998 ◽

1998 ◽

Vol 66 (10) ◽

pp. 4981-4988 ◽

Cited By ~ 33

Author(s):

Irina Lyadova ◽

Vladimir Yeremeev ◽

Konstantin Majorov ◽

Boris Nikonenko ◽

Sergei Khaidukov ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Antimycobacterial Activity ◽

Enzymatic Digestion ◽

Interleukin 4 ◽

T Cell Clones ◽

Cell Clones ◽

Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

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Diversity of HLA Class I and Class II Restricted Minor Histocompatibility Antigens in Graft-Versus-Leukemia Reactivity.

Blood ◽

10.1182/blood.v114.22.4084.4084 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4084-4084

Author(s):

Marieke Griffioen ◽

M. Willy Honders ◽

Anita N. Stumpf ◽

Edith D. van der Meijden ◽

Cornelis A.M. van Bergen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Hematopoietic Cells ◽

Cd4 T Cell ◽

Cell Recognition ◽

T Cell Clones ◽

T Cell Recognition ◽

Cell Clones ◽

T Cell Clone ◽

Ifn Γ

Abstract Abstract 4084 Poster Board III-1019 Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T-cells recognizing minor histocompatibility antigens (mHags) encoded by single nucleotide polymorphisms (SNPs) on malignant cells of the recipient. Donor T-cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues. The aim of this study was to investigate the specificity and diversity of mHags recognized by T-cells in Graft-versus-Leukemia (GvL) reactivity. Activated (HLA-DR+) CD8+ and CD4+ T-cell clones were isolated from a patient successfully treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. GvL reactivity in this patient was accompanied with mild GvHD of the skin. Isolated T-cell clones were shown to recognize 13 different mHags. CD8+ T-cell clones were specific for HA-1 and HA-2 in HLA-A*0201, one unknown mHag in B*0801 and 4 unknown mHags in B*4001. CD4+ T-cell clones were specific for one unknown mHag in HLA-DQ and 5 unknown mHags in DR. By screening plasmid (class I) and bacteria (class II) cDNA libraries, we identified a mHag in HLA-DQ encoded by the PI4K2B gene (Griffioen et al., PNAS 2008), 4 mHags in HLA-DR encoded by the PTK2B, MR-1, LY75 and MTHFD1 genes (Stumpf et al., Blood 2009) and a mHag in B*4001 encoded by the TRIP10 gene. For the 3 T cell clones recognizing unknown mHags in B*4001, we performed Whole Genome Assocation scanning (WGAs). A panel of 60 EBV-LCL was retrovirally-transduced with B*4001 and tested for T-cell recognition. In parallel, genomic DNA was isolated and more than one million single nucleotide polymorphisms (SNPs) were determined by the Illumina beadchip array. Statistical analysis revealed significant association between T-cell recognition of EBV-LCL and the presence of coding SNPs in the SON DNA-binding protein and SWAP-70 genes. To get more insight into the role and potential use of the mHags in GvL reactivity and GvHD, all T-cell clones were analyzed in detail for reactivity against hematopoietic and non-hematopoietic cells. Hematopoietic cells included peripheral blood cells (monocytes, B-cells and T-cells), professional antigen presenting cells (APC) and leukemic cells (CML, ALL and AML). All CD8+ T-cell clones recognized (subsets of) peripheral blood cells as well as CML cells, except for the T-cell clone for TRIP10. Recognition of (subsets of) peripheral blood cells was also observed for all CD4+ T-cell clones, but CML cells were differentially recognized. CML cells were strongly recognized by the T-cell clones for MTHFD1 and the unknown mHag in HLA-DR, whereas no or low reactivity was observed for all other CD4+ T-cell clones. All CD8+ and CD4+ T-cell clones strongly recognized professional APC, including monocyte-derived dendritic cells and in vitro differentiated CML cells with APC phenotype. All T-cell clones were also capable of recognizing AML and ALL, except for the T-cell clone for TRIP10, which showed restricted recognition of AML-M4 and -M5 of monocytic origin. As non-hematopoietic cells, patient-derived fibroblasts were cultured with and without IFN-γ and tested for T-cell recognition. In the absence of IFN-γ, all T-cell clones failed to recognize fibroblasts, except for the T-cell clone for the unknown mHag in B*0801. After treatment with IFN-γ, additional reactivity was observed for the T-cell clones for SON DNA-binding protein and the unknown mHag in B*4001. Our data showed the specificity and diversity of mHags recognized by T-cells induced in a patient successfully treated with DLI for relapsed CML. The T-cell response was directed against 13 different mHags, of which 10 mHags in HLA class I and class II have now been identified by different techniques. Detailed analysis of T-cell recognition of hematopoietic and non-hematopoietic cells provides evidence that the mHags played different roles in the onset and execution of GvL and GvHD. Moreover, only one of the 10 identified mHags was expressed on fibroblasts after treatment with IFN-γ, indicating the characterization of mHags with potential relevance for T-cell based immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Functional Characterization and Modulation of Cytokine Production by CD8+ T Cells from Human Immunodeficiency Virus-Infected Individuals

Blood ◽

10.1182/blood.v89.10.3672.3672_3672_3681 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3672-3681 ◽

Cited By ~ 3

Author(s):

Enrico Maggi ◽

Roberto Manetti ◽

Francesco Annunziato ◽

Lorenzo Cosmi ◽

Maria Grazia Giudizi ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

T Cell Clones ◽

Cell Clones ◽

Immunodeficiency Virus ◽

Ifn Γ ◽

Hiv Seropositive

CD8+ T-cell clones were generated from peripheral blood mononuclear cells (PBMC) of three human immunodeficiency virus (HIV)-seronegative individuals and six HIV-seropositive individuals and assessed for their cytokine secretion profile, cytolytic potential, and chemokine production. While the great majority of CD8+ T-cell clones generated from HIV-seronegative individuals produced interferon (IFN)-γ, but not interleukin-4 (IL-4), that is a type 1 cytotoxic (Tc1) profile, high numbers of CD8+ T-cell clones generated from HIV-seropositive individuals produced IL-4 in addition to IFN-γ or IL-4 alone, thus showing a type 0 cytotoxic (Tc0)- or a type 2 cytotoxic (Tc2) profile, respectively. Tc0/Tc2 cells displayed lower cytolytic activity than Tc1 cells, including a reduced ability to lyse autologous targets pulsed with HIV or HIV peptides. By contrast, the production of chemokines RANTES and macrophage inflammatory protein-1α was comparable in Tc1, Tc0, and Tc2 clones irrespective of whether they were derived from HIV-seronegative or HIV-seropositive individuals. When CD8+ T-cell clones were generated from PBMC cultures of HIV-seronegative individuals conditioned with IL-4 plus an anti–IL-12 antibody (Ab), a shift towards the Tc0/Tc2-like profile was observed. Conversely, the addition to PBMC cultures of IL-12 plus an anti – IL-4 Ab shifted the differentiation of CD8+ T cells from HIV-infected individuals towards the Tc1-like profile, whereas IL-12 or anti–IL-4 Ab alone had a lower Tc1-promoting effect. Irradiated PBMC from HIV-infected individuals, used as feeder cells, shifted the differentiation of CD8+ T cells from a healthy HIV-seronegative individual towards the Tc0/Tc2-like profile. On the other hand, a shift towards the Tc1-like profile was noted in CD8+ T-cell clones generated from the skin specimens of two HIV-seropositive patients with Kaposi's sarcoma, successfully treated with IFN-α, in comparison to CD8+ clones generated from the same skin areas before treatment. The IFN-α–induced Tc1 shift could be prevented by the incubation of skin-infiltrating CD8+ T cells with IL-4 before cloning. Taken together, these data indicate that both defective production of IL-12 and abnormal IL-4 production in bulk PBMC populations of HIV-infected individuals may contribute to the development of high numbers of CD8+ T-cell clones showing a Tc0/Tc2-like phenotype and reduced cytolytic potential against HIV itself. They also suggest that the cytokine profile of CD8+ T-cell clones can be modulated by cytokines (or anticytokine Ab) both in vitro and in vivo.

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Hantavirus-Specific CD8+-T-Cell Responses in Newborn Mice Persistently Infected with Hantaan Virus

Journal of Virology ◽

10.1128/jvi.77.15.8408-8417.2003 ◽

2003 ◽

Vol 77 (15) ◽

pp. 8408-8417 ◽

Cited By ~ 31

Author(s):

Koichi Araki ◽

Kumiko Yoshimatsu ◽

Byoung-Hee Lee ◽

Hiroaki Kariwa ◽

Ikuo Takashima ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cytotoxic Activity ◽

Hantaan Virus ◽

Virus Inoculation ◽

Newborn Mice ◽

Persistently Infected ◽

Cell Responses ◽

Tnf Α ◽

Ifn Γ

ABSTRACT The relationship between virus-specific CD8+-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNV). We first established a simple method for measuring levels of virus-specific CD8+ T cells by flow cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.1 50% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8+ T cells producing gamma interferon (IFN-γ). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8+ T cells, which have the ability to produce IFN-γ and tumor necrosis factor alpha (TNF-α) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-γ-producing HTNV-specific CD8+ T cells were detected on day 15 after virus inoculation. However, TNF-α production and the cytotoxic activity of these specific CD8+ T cells were impaired and HTNV was not removed. Almost all of these specific CD8+ T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8+ T cells are important for clearance of HTNV.

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Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Clinical and Vaccine Immunology ◽

10.1128/cvi.00162-15 ◽

2015 ◽

Vol 22 (7) ◽

pp. 778-788 ◽

Cited By ~ 31

Author(s):

Mardi C. Boer ◽

Corine Prins ◽

Krista E. van Meijgaarden ◽

Jaap T. van Dissel ◽

Tom H. M. Ottenhoff ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mycobacterium Bovis ◽

Regulatory T Cell ◽

Cell Response ◽

T Cell Response ◽

Bcg Vaccination ◽

Cell Responses ◽

Tnf Α ◽

Ifn Γ

ABSTRACTMycobacterium bovisbacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ+) interleukin 2-positive (IL-2+) tumor necrosis factor α-positive (TNF-α+) polyfunctional CD4+T cells concurrent with CD4+IL-17A+and CD8+IFN-γ+T cells or, in contrast, virtually absent cytokine responses with induction of CD8+regulatory T cells. Significant induction of polyfunctional CD4+IFN-γ+IL-2+TNF-α+T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8+T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8+regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4+T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.

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Characterization of T-Cell Responses to SMX and SMX-NO in Co-Trimoxazole Hypersensitivity Patients Expressing HLA-B*13:01

Frontiers in Immunology ◽

10.3389/fimmu.2021.658593 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jirawat Pratoomwun ◽

Paul Thomson ◽

Kanoot Jaruthamsophon ◽

Rawiporn Tiyasirichokchai ◽

Pimonpan Jinda ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Antigen Processing ◽

T Cell Responses ◽

Stevens Johnson Syndrome ◽

Antigen Specificity ◽

T Cell Clones ◽

Cell Responses ◽

Cell Clones ◽

Ifn Γ

HLA-B*13:01-positive patients in Thailand can develop frequent co-trimoxazole hypersensitivity reactions. This study aimed to characterize drug-specific T cells from three co-trimoxazole hypersensitive patients presenting with either Stevens-Johnson syndrome or drug reaction with eosinophilia and systemic symptoms. Two of the patients carried the HLA allele of interest, namely HLA-B*13:01. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones were generated from T cell lines of co-trimoxazole hypersensitive HLA-B*13:01-positive patients. Clones were characterized for antigen specificity and cross-reactivity with structurally related compounds by measuring proliferation and cytokine release. Surface marker expression was characterized via flow cytometry. Mechanistic studies were conducted to assess pathways of T cell activation in response to antigen stimulation. Peripheral blood mononuclear cells from all patients were stimulated to proliferate and secrete IFN-γ with nitroso sulfamethoxazole. All sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones expressed the CD4+ phenotype and strongly secreted IL-13 as well as IFN-γ, granzyme B and IL-22. No secretion of IL-17 was observed. A number of nitroso sulfamethoxazole-specific clones cross-reacted with nitroso dapsone but not sulfamethoxazole whereas sulfamethoxazole specific clones cross-reacted with nitroso sulfamethoxazole only. The nitroso sulfamethoxazole specific clones were activated in both antigen processing-dependent and -independent manner, while sulfamethoxazole activated T cell responses via direct HLA binding. Furthermore, activation of nitroso sulfamethoxazole-specific, but not sulfamethoxazole-specific, clones was blocked with glutathione. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones from hypersensitive patients were CD4+ which suggests that HLA-B*13:01 is not directly involved in the iatrogenic disease observed in co-trimoxazole hypersensitivity patients.

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Vβ1+ Jβ1.1+/Vα2+ Jα49+ CD4+ T Cells Mediate Resistance against Infection with Blastomyces dermatitidis

Infection and Immunity ◽

10.1128/iai.01148-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 193-200 ◽

Cited By ~ 14

Author(s):

Marcel Wüthrich ◽

Hanna I. Filutowicz ◽

Holly L. Allen ◽

George S. Deepe ◽

Bruce S. Klein

Keyword(s):

T Cells ◽

T Cell ◽

Cell Receptor ◽

Blastomyces Dermatitidis ◽

T Cell Clones ◽

Interleukin 5 ◽

Protective Antigens ◽

Cell Clones ◽

A Cell ◽

Ifn Γ

ABSTRACT Immunization with a cell wall/membrane (CW/M) and yeast cytosol extract (YCE) crude antigen from Blastomyces dermatitidis confers T-cell-mediated resistance against lethal experimental infection in mice. We isolated and characterized T cells that recognize components of these protective antigens and mediate protection. CD4+ T-cell clones elicited with CW/M antigen adoptively transferred protective immunity when they expressed a Vα2+ Jα49+/Vβ1+ Jβ1.1+ heterodimeric T-cell receptor (TCR) and produced high levels of gamma interferon (IFN-γ). In contrast, Vβ8.1/8.2+ CD4+ T-cell clones that were reactive against CW/M and YCE antigens and produced little or no IFN-γ either failed to mediate protection or exacerbated the infection depending on the level of interleukin-5 expression. Thus, the outgrowth of protective T-cell clones against immunodominant antigens of B. dermatitidis is biased by a combination of the TCR repertoire and Th1 cytokine production.

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Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge

Clinical and Vaccine Immunology ◽

10.1128/cvi.00415-13 ◽

2013 ◽

Vol 20 (10) ◽

pp. 1604-1616 ◽

Cited By ~ 24

Author(s):

Giulia Franzoni ◽

Nitin V. Kurkure ◽

Daniel S. Edgar ◽

Helen E. Everett ◽

Wilhelm Gerner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Classical Swine Fever Virus ◽

T Cell Responses ◽

Classical Swine Fever ◽

Cd8 T Cell ◽

Cell Responses ◽

Tnf Α ◽

Ifn Γ

ABSTRACTVaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3+CD4−CD8hiT cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3+CD4+CD8α+) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44hiCD62L−and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ+CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.

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Functional Characterization of Aspergillus Fumigatus Specific T-Cells Clones.

Blood ◽

10.1182/blood.v106.11.3223.3223 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3223-3223

Author(s):

Thomas Lehrnbecher ◽

Ulrike Koehl ◽

Emmanuel Roilides ◽

Maria Simitsopoulou ◽

Mitra Hanisch ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Aspergillus Fumigatus ◽

Functional Characterization ◽

Cd4 Cells ◽

Hematopoietic Stem ◽

T Cell Clones ◽

Th1 Response ◽

Cell Clones ◽

Ifn Γ

Abstract Invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematological malignancies, in particular in patients who have undergone allogeneic hematopoietic stem cell transplantation (SCT). There is a growing body of evidence that T-cells play an important role in the immunological response to Aspergillus fumigatus. Using the Aspergillus fumigatus antigen extract EC SAB and the IFN-γ secretion assay (Miltenyi Biotec, Germany), we generated Aspergillus fumigatus specific T-cell clones by limiting dilution (n=4). Flow cytometry revealed a cell population of CD3+/CD4+ cells (mean±SEM, 98.2±1.2%). Functional assessment by ICC revealed that an average of 8.7% of these cells (range, 6.6%–18.5%) specifically secreted IFN-γ on stimulation with EC SAB, which supports the TH1 response of the generated cells to Aspergillus fumigatus antigens. The antigenic components of EC SAB are one or more proteins, since the addition of proteinase completely suppressed the stimulating effect of this preparation. The percentage of IFN-γ producing CD3+/CD4+ cells was less than 1% upon activation with antigen extracts from Aspergillus flavus, Aspergillus niger, Alternaria alternata, Mucor racemosus, Penicillium notatum and Candida albicans, indicating that the generated T-cell clones are specific for Aspergillus fumigatus. A strong proliferation of the generated Aspergillus fumigatus specific T-cells was seen after re-stimulation with EC SAB, whereas alloreactivity was reduced compared to CD4+ T-cells of the original fraction. Hyphal damage of Aspergillus fumigatus was assessed by means of an XTT assay. Polymorphonuclear leukocytes (PMNs) showed a similar hyphal damage when tested alone (mean±SEM, 14.2±2.1%), in combination with antigen presenting cells (APCs) (15.1±1.4%), or in combination with Aspergillus fumigatus specific T-cells (15.0±2.0%). A comparable hyphal damage was seen when Aspergillus fumigatus specific T-cells were co-incubated with APCs (14.2±1.7%). In contrast, the combination of APCs and Aspergillus fumigatus specific T-cells with PMNs resulted in a significantly higher hyphal damage compared to all other settings (23.3±2.8%; P<.0001). Interestingly, APCs alone or Aspergillus fumigatus specific T-cells alone showed a weak, but significant capacity to induce hyphal damage (7.4±1.1% and 11.3±1.8%, respectively). Before considering a clinical application, however, further studies need to focus on defining the optimal antigen(s) which reproducibly induce a TH1 response and elicit high antifungal activity, as well as to characterize the subpopulation of patients undergoing allogeneic SCT who ultimately benefit from either a prophylactic or a therapeutic adoptive transfer of Aspergillus fumigatus specific T-cells.

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