Bachelor of Science, Unlisted School (2006)
Master of Science, Unlisted School (2008)
Doctor of Philosophy, Karolinska Institutet (2015)
Emmanuel Mignot, Postdoctoral Faculty Sponsor
Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.
Journal of immunotherapy (Hagerstown, Md. : 1997)
; 39 (2): 81–89
Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-γ production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR Vβ was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8 T cells. CD3CD8, CD3CD4, and CD3CD4CD8 [double-negative (DN) T cells] resided predominantly in central (CD45RACCR7) and effector (CD45RACCR7) memory subsets. CD8 TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4 TILs showed only up to 12% LAG-3 staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-α and IFN-γ production. Twelve of 17 of CD8 TILs showed preferential expansion of certain TCR Vβ families (eg, 99.2% Vβ13.2 in CD8 TILs, 77% in the Vβ1, 65.9% in the Vβ22, and 63.3% in the Vβ14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8 Vβ13.2) reacting strongly against autologous tumor defined by INF-γ production or by cytotoxicity. We have optimized methods for generating pancreatic cancer-specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.
View details for DOI 10.1097/CJI.0000000000000111
View details for PubMedID 26849077
IL-7δ5 protein is expressed in human tissues and induces expression of the oxidized low density lipoprotein receptor 1 (OLR1) in CD14+ monocytes.
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases
The 6 exon spanning ́canonical' Interleukin-7 (IL-7c) is a non-redundant cytokine in human T-cell homeostasis that undergoes extensive alternative pre-mRNA splicing. The IL-7 gene variant lacking, exon 5 (IL-7δ5), exhibits agonistic effects as compared to IL-7c. We studied in this report for the first time the protein expression of IL-7δ5 variant in tissues and its role in monocyte activation.We visualized the expression of IL-7δ5 protein by immunohistochemistry in both healthy and malignant (human) tissues and investigated the impact of IL-7δ5 stimulation on CD14+ monocytes using gene expression analysis and flow cytometry.IL-7≏5 is largely expressed by human epithelial cells, yet also by stromal cells in malignant lesions. Gene expression analysis in CD14+ monocytes, induced by the 6-exon spanning IL-7 or IL-7δ5 showed similar changes resulting in a pro-inflammatory phenotype and increased expression of genes involved in lipid metabolism. IL7≏5 was superior in inducing upregulation of the oxidised low density lipoprotein receptor (OLR), measured by flow cytometry, in CD14+ cells.IL-7≏5, produced from non-transformed and transformed cells, may contribute to chronic inflammatory responses and development of 'foamy' cells by increased OLR1 expression that mediates increased oxLDL uptake.
View details for DOI 10.1016/j.ijid.2017.03.001
View details for PubMedID 28279736
Selenite promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells.
Selective targeting of the PML/RARα oncoprotein demonstrates a successful molecular targeted therapy in acute promyelocytic leukemia (APL) with a typical t(15:17) chromosomal translocation. The zinc-thiolate coordination is critical for structural stability of zinc finger proteins, including the PML moiety of PML/RARα. Based on the known interaction of redox-active selenium compounds with thiolate ligands of zinc, we herein have investigated the abrogatory effects of selenite alone or in combination with all-trans retinoic acid on PML/RARα and the possible effects on differentiation in these cells. At pharmacological concentrations, selenite inhibited the proliferation and survival of APL originated NB4 cells. In combination with ATRA, it potentiated the differentiation of NB4 cells without any differentiating effects of its own as a single agent. Concordant with our hypothesis, PML/RARα oncoprotein expression was completely abrogated by selenite. Increased expression of RAR, PU.1 and FOXO3A transcription factors in the combined treatment suggested the plausible basis for increased differentiation in these cells. We show that selenite at clinically achievable dose targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemic cells in combination with ATRA. The present investigation reveals the hitherto unknown potential of selenite in targeted abrogation of PML/RARα in APL cells with prospective therapeutic value.
View details for DOI 10.18632/oncotarget.12531
View details for PubMedID 27732960
T-Cell Therapy: Options for Infectious Diseases.
Clinical infectious diseases
2015; 61: S217-24
The emergence of drug-resistant tuberculosis is challenging tuberculosis control worldwide. In the absence of an effective vaccine to prevent primary infection with Mycobacterium tuberculosis and tuberculosis disease, host-directed therapies may offer therapeutic options, particularly for patients with multidrug-resistant and extensively drug-resistant tuberculosis where prognosis is often limited. CD8(+) and CD4(+) T cells mediate antigen-specific adaptive cellular immune responses. Their use in precision immunotherapy in clinical conditions, especially in treating cancer as well as for prevention of life-threatening viral infections in allogeneic transplant recipients, demonstrated safety and clinical efficacy. We review key achievements in T-cell therapy, including the use of recombinant immune recognition molecules (eg, T-cell receptors and CD19 chimeric antigen receptors), and discuss its potential in the clinical management of patients with drug-resistant and refractory tuberculosis failing conventional therapy.
View details for DOI 10.1093/cid/civ615
View details for PubMedID 26409284
Evaluation of pretransplant influenza vaccination in hematopoietic SCT: a randomized prospective study.
Bone marrow transplantation
2015; 50 (6): 858-864
Pretransplant influenza vaccination of the donor or allogeneic hematopoietic SCT (HSCT) candidate was evaluated in a randomized study. One hundred and twenty-two HSCT recipients and their donors were assigned to three randomization groups: no pretransplant vaccination (n=38), donor pretransplant vaccination (n=44) or recipient pretransplant vaccination (n=40). Specific IgG was assessed by both hemagglutinin inhibition (HI) and, in 57 patients, by an indirect influenza-specific ELISA at specified times after HSCT. Vaccinated donors had seroprotective HI titers for Ags H1 and H3 (P<0.001) compared with the other groups at the time of donation. The titers against H1 (P=0.028) and H3 (P<0.001) were highest in the pretransplant recipient vaccination group until day 180 after transplantation. A significant difference was found in the specific Ig levels against pandemic H1N1 at 6 months after SCT (P=0.02). The mean IgG levels against pandemic H1N1 and generic H1N1 and H3N2 were highest in the pretransplant recipient vaccination group. We conclude that pretransplant recipient vaccination improved the influenza-specific seroprotection rates.
View details for DOI 10.1038/bmt.2015.47
View details for PubMedID 25798680
H1N1 viral proteome peptide microarray predicts individuals at risk for H1N1 infection and segregates infection versus Pandemrix(®) vaccination.
2015; 145 (3): 357–66
A high content peptide microarray containing the entire influenza A virus [A/California/08/2009(H1N1)] proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the haemagglutinin from the [A/South Carolina/1/1918(H1N1)] strain, was used to gauge serum IgG epitope signatures before and after Pandemrix(®) vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu-specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251-265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor-binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using peptide microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine.
View details for DOI 10.1111/imm.12448
View details for PubMedID 25639813
Increased β-haemolytic group A streptococcal M6 serotype and streptodornase B-specific cellular immune responses in Swedish narcolepsy cases.
Journal of internal medicine
Type 1 narcolepsy is a neurological disorder characterized by excessive daytime sleepiness and cataplexy associated with the HLA allele DQB1*06:02. Genetic predisposition along with external triggering factors may drive autoimmune responses, ultimately leading to the selective loss of hypocretin-positive neurons.The aim of this study was to investigate potential aetiological factors in Swedish cases of postvaccination (Pandemrix) narcolepsy defined by interferon-gamma (IFNγ) production from immune cells in response to molecularly defined targets.Cellular reactivity defined by IFNγ production was examined in blood from 38 (HLA-DQB1*06:02(+) ) Pandemrix-vaccinated narcolepsy cases and 76 (23 HLA-DQB1*06:02(+) and 53 HLA-DQB1*06:02(-) ) control subjects, matched for age, sex and exposure, using a variety of different antigens: β-haemolytic group A streptococcal (GAS) antigens (M5, M6 and streptodornase B), influenza (the pandemic A/H1N1/California/7/09 NYMC X-179A and A/H1N1/California/7/09 NYMC X-181 vaccine antigens, previous Flu-A and -B vaccine targets, A/H1N1/Brisbane/59/2007, A/H1N1/Solomon Islands/3/2006, A/H3N2/Uruguay/716/2007, A/H3N2/Wisconsin/67/2005, A/H5N1/Vietnam/1203/2004 and B/Malaysia/2506/2004), noninfluenza viral targets (CMVpp65, EBNA-1 and EBNA-3) and auto-antigens (hypocretin peptide, Tribbles homolog 2 peptide cocktail and extract from rat hypothalamus tissue).IFN-γ production was significantly increased in whole blood from narcolepsy cases in response to streptococcus serotype M6 (P = 0.0065) and streptodornase B protein (P = 0.0050). T-cell recognition of M6 and streptodornase B was confirmed at the single-cell level by intracellular cytokine (IL-2, IFNγ, tumour necrosis factor-alpha and IL-17) production after stimulation with synthetic M6 or streptodornase B peptides. Significantly, higher (P = 0.02) titres of serum antistreptolysin O were observed in narcolepsy cases, compared to vaccinated controls.β-haemolytic GAS may be involved in triggering autoimmune responses in patients who developed narcolepsy symptoms after vaccination with Pandemrix in Sweden, characterized by a Streptococcus pyogenes M-type-specific IFN-γ cellular immune response.
View details for DOI 10.1111/joim.12355
View details for PubMedID 25683265
Immunogenicity of virosomal adjuvanted trivalent influenza vaccination in allogeneic stem cell transplant recipients.
Transplant infectious disease : an official journal of the Transplantation Society
2015; 17 (3): 371–79
Influenza vaccination is generally recommended to hematopoietic stem cell transplant (HSCT) recipients. However, the seasonal subunit vaccination response is frequently suboptimal, and alternate more efficient vaccination systems must be examined. We compared the immunogenicity of an adjuvanted virosomal influenza and subunit vaccine in HSCT recipients.The immunogenicity after a single dose (0.5 mL) of adjuvanted trivalent virosomal vaccination was evaluated in a study cohort of 21 HSCT recipients and compared to a control cohort of 30 HSCT recipients who received a single dose (0.5 mL) of non-adjuvanted seasonal trivalent subunit vaccination over 4 seasons from 2010 to 2014. Whole blood interferon-gamma (IFN-γ) release assays were tested, both before and 30 days after vaccination, in response to influenza pandemic (pdm) H1N1, H3N2, and B antigens. HLA-A*02 dextramers, to gauge for the absolute number of antigen-specific CD8(+) T-cells, and pdm 2009 hemagglutinin inhibition (HI) assays, to test for neutralizing antibodies, were used as immunological readouts.The pdm HI titers were poor in both cohorts with only 23% (5/21) after virosomal vaccination and 13.3% (4/30) in the seasonal vaccine cohort having protective titers (≥40). The delta change of IFN-γ production in response to influenza pdm H1N1 (P = 0.005) and influenza B antigens (P = 0.01) were significantly elevated in blood from individuals who received the virosomal as compared to the seasonal vaccine. The IFN-γ response to pdm H1N1 was stronger (P < 0.001), as compared to seasonal vaccination, in patients vaccinated >6 month post HSCT. We detected a significant increase in the frequency of matrix 1 (GILGFVTL) dextramer-specific CD8(+) T-cells after the virosomal vaccine (P = 0.01). No differences were seen in the hemagglutinin-specific CD8(+) T-cells between the 2 cohorts.Vaccination using a virosomal delivery system is beneficial in eliciting robust cellular immune responses to pdm H1N1 influenza in SCT recipients.
View details for DOI 10.1111/tid.12382
View details for PubMedID 25817044
TCR(+)CD4(-)CD8(-) T cells in Antigen-specific MHC Class I-restricted T-cell Responses After Allogeneic Hematopoietic Stem Cell Transplantation
JOURNAL OF IMMUNOTHERAPY
2014; 37 (8): 416-425
Human TCRαβ(+) CD4(-)CD8(-) double-negative (DN) T cells represent a minor subset in peripheral blood, yet are important in infectious diseases and autoimmune responses. We examined the frequency of DN T cells in 17 patients after allogeneic hematopoietic stem cell transplantation (aHSCT) at 1, 2, 3, 6, and 12 months post-aHSCT and show that these cells increase early after aHSCT and decrease with time after aHSCT. DN T cells reside in the terminally differentiated effector (CD45RA(+)CCR7(-)) T-cell population and are polyclonal, determined by T-cell receptor Vβ CDR3 analysis. Gene expression analysis of ex vivo sorted DN T cells showed a distinct set of gene expression, including interleukin-8, as compared with CD4(+) or CD8(+) T cells. DN T cells contributed to MHC class I-restricted EBV-directed immune responses, defined by antigen-specific cytokine production and by detection of HLA-A*02:01-restricted EBV BMLF-1 (GLCTLVAML), LMP-2A (CLGGLLTMV), and HLA-A*24:02-restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA3 (RYSIFFDY)-specific T cells. We created retroviral-transfected Jurkat cell lines with a Melan-A/MART-1-specific TCR(+) and the CD8α chain to study TCR(+) DN T cells in response to their nominal MHC class I/peptide ligand. We show that DN T cells exhibit increased TCRζ chain phosphorylation as compared with the TCR(+)CD8(+) transgenic T-cell line. DN T cells contribute to antigen-specific T-cell responses and represent an effector T-cell population that may be explored in immunotherapeutic approaches against viral infections or transformed cells.
View details for Web of Science ID 000341965500004
View details for PubMedID 25198529
Difference in immune response in vaccinated and unvaccinated Swedish individuals after the 2009 influenza pandemic
BMC INFECTIOUS DISEASES
Previous exposures to flu and subsequent immune responses may impact on 2009/2010 pandemic flu vaccine responses and clinical symptoms upon infection with the 2009 pandemic H1N1 influenza strain. Qualitative and quantitative differences in humoral and cellular immune responses associated with the flu vaccination in 2009/2010 (pandemic H1N1 vaccine) and natural infection have not yet been described in detail. We designed a longitudinal study to examine influenza- (flu-) specific immune responses and the association between pre-existing flu responses, symptoms of influenza-like illness (ILI), impact of pandemic flu infection, and pandemic flu vaccination in a cohort of 2,040 individuals in Sweden in 2009-2010.Cellular flu-specific immune responses were assessed by whole-blood antigen stimulation assay, and humoral responses by a single radial hemolysis test.Previous seasonal flu vaccination was associated with significantly lower flu-specific IFN-γ responses (using a whole-blood assay) at study entry. Pandemic flu vaccination induced long-lived T-cell responses (measured by IFN-γ production) to influenza A strains, influenza B strains, and the matrix (M1) antigen. In contrast, individuals with pandemic flu infection (PCR positive) exhibited increased flu-specific T-cell responses shortly after onset of ILI symptoms but the immune response decreased after the flu season (spring 2010). We identified non-pandemic-flu vaccinated participants without ILI symptoms who showed an IFN-γ production profile similar to pandemic-flu infected participants, suggesting exposure without experiencing clinical symptoms.Strong and long-lived flu-M1 specific immune responses, defined by IFN-γ production, in individuals after vaccination suggest that M1-responses may contribute to protective cellular immune responses. Silent flu infections appeared to be frequent in 2009/2010. The pandemic flu vaccine induced qualitatively and quantitatively different humoral and cellular immune responses as compared to infection with the 2009 H1N1 pandemic H1N1 influenza strain.
View details for DOI 10.1186/1471-2334-14-319
View details for Web of Science ID 000338728900001
View details for PubMedID 24916787
A/H1N1 antibodies and TRIB2 autoantibodies in narcolepsy patients diagnosed in conjunction with the Pandemrix vaccination campaign in Sweden 2009-2010
JOURNAL OF AUTOIMMUNITY
2014; 50: 99-106
Narcolepsy is a lifelong sleep disorder related to hypocretin deficiency resulting from a specific loss of hypocretin-producing neurons in the lateral hypothalamic area. The disease is thought to be autoimmune due to a strong association with HLA-DQB1*06:02. In 2009 the World Health Organization (WHO) declared the H1N1 2009 flu pandemic (A/H1N1PDM09). In response to this, the Swedish vaccination campaign began in October of the same year, using the influenza vaccine Pandemrix(®). A few months later an excess of narcolepsy cases was observed. It is still unclear to what extent the vaccination campaign affected humoral autoimmunity associated with narcolepsy. We studied 47 patients with narcolepsy (6-69 years of age) and 80 healthy controls (3-61 years of age) selected after the Pandemrix vaccination campaign. The first aim was to determine antibodies against A/H1N1 and autoantibodies to Tribbles homolog 2 (TRIB2), a narcolepsy autoantigen candidate as well as to GAD65 and IA-2 as disease specificity controls. The second aim was to test if levels and frequencies of these antibodies and autoantibodies were associated with HLA-DQB1*06:02. In vitro transcribed and translated [(35)S]-methionine and -cysteine-labeled influenza A virus (A/California/04/2009/(H1N1)) segment 4 hemagglutinin was used to detect antibodies in a radiobinding assay. Autoantibodies to TRIB2, GAD65 and IA-2 were similarly detected in standard radiobinding assays. The narcolepsy patients had higher median levels of A/H1N1 antibodies than the controls (p = 0.006). A/H1N1 antibody levels were higher among the <13 years old (n = 12) compared to patients who were older than 30 years (n = 12, p = 0.014). Being HLA-DQB1*06:02 positive was associated with higher A/H1N1 antibody levels in both patients and controls (p = 0.026). Serum autoantibody levels to TRIB2 were low overall and high binders did not differ between patients and controls. We observed an association between levels of A/H1N1 antibodies and TRIB2 autoantibody levels particularly among the youngest narcolepsy patients (r = 0.819, p < 0.001). In conclusion, following the 2009 influenza pandemic vaccination, A/H1N1 antibody levels were associated with young age-at-onset narcolepsy patients positive for HLA-DQB1*06:02. The possibility that TRIB2 is an autoantigen in narcolepsy remains to be clarified. We could verify autoantibody responses against TRIB2 which needs to be determined in larger patient cohorts and control populations.
View details for DOI 10.1016/j.jaut.2014.01.031
View details for Web of Science ID 000336114600013
View details for PubMedID 24485154