The forkhead/winged helix transcription factor (Foxp3) is expressed as two different isoforms in humans: the full-length isoform (Foxp3FL) and an alternative-splicing product lacking the exon 2 (Foxp3DeltaE2). We here studied the cellular distribution of Foxp3 isoforms by quantitative PCR and evaluated the functional outcome of retroviral transduction of Foxp3FL and Foxp3DeltaE2 genes into CD4(+)CD25(-) cells. In PBMC, both isoforms were preferentially expressed in CD4(+)CD25(hi) cells. In single-cell-sorted and expanded Treg, both Foxp3 isoforms were expressed simultaneously but without a fixed ratio. Forced expression of Foxp3FL or Foxp3DeltaE2 genes in CD4(+)CD25(-) T cells induced bona fide Treg that not only displayed Treg phenotype but also were anergic and mediated significant suppressive activity against CD3-activated CD4(+)CD25(-) cells. GFP(-) nontransduced cells or cells transduced with an empty vector showed no Treg phenotype, anergy or suppressive activities. In conclusion, our results reveal that both Foxp3 isoforms possess similar capacities to induce Treg; however, unnaturally high expression levels are required to convey Treg functions to CD4(+)CD25(-) cells. As both Foxp3 isoforms appear to be expressed in an independent fashion, studies aiming at quantification of Treg in peripheral blood or in tissue samples can benefit from determination of total Foxp3 levels rather than one of the isoforms.
In the last decade, mumps virus (MuV) causes outbreaks in highly vaccinated populations. Sub-optimal T cell immunity may play a role in the susceptibility to mumps in vaccinated individuals. T cell responses to mumps virus have been demonstrated, yet the quality of the MuV-specific T cell response has not been analyzed using single cell immunological techniques. Here we developed an IFNγ ELISPOT assay to assess MuV-specific T cell responses in peripheral blood mononuclear cells (PBMC) of healthy (vaccinated) donors and mumps patients. Various in vitro MuV-specific stimulation methods of PBMC were compared, using either live or inactivated MuV alone or MuV-infected autologous antigen presenting cells, i.e. Epstein Barr Virus-transformed B lymphoblastoid cell lines (EBV-BLCL) or (mitogen pre-activated) PBMC, for their ability to recall IFNγ-producing responder cells measured by ELISPOT. For the detection of MuV-specific T cell responses, direct exposure (24h) to live MuV was the preferred stimulation method when assay sensitivity and practical reasons were considered. Notably, flowcytometric confirmation of data revealed that primarily T cells and NK cells produce IFNγ upon live MuV stimulation. Depleting PBMC from CD56(+) NK cells prior to stimulation with live MuV led to the enumeration of MuV-specific T cell responses by ELISPOT. Our assay constitutes a tool to evaluate memory MuV-specific T cell responses in MuV vaccinated or infected persons. Furthermore, this study provides evidence that live MuV not only induces IFNγ production by T cells, but also by NK cells.
With the introduction of measles-mumps-rubella (MMR) vaccination, the mumps incidence decreased dramatically.1Dayan G.H. Quinlisk M.P. Parker A.A. Barskey A.E. Harris M.L. Schwartz J.M.H. et al.Recent resurgence of mumps in the United States.N Engl J Med. 2008; 358: 1580-1589Crossref PubMed Scopus (310) Google Scholar Mumps vaccination has been assumed to provide lifelong immune protection, as seems to be the case after natural infection. However, several countries reported mumps outbreaks among vaccinated young adults, indicating less long-lived protection than anticipated.1Dayan G.H. Quinlisk M.P. Parker A.A. Barskey A.E. Harris M.L. Schwartz J.M.H. et al.Recent resurgence of mumps in the United States.N Engl J Med. 2008; 358: 1580-1589Crossref PubMed Scopus (310) Google Scholar, 2Kaaijk P. Gouma S. Hulscher H.I. Han W.G. Kleijne D.E. van Binnendijk R.S. et al.Dynamics of the serologic response in vaccinated and unvaccinated mumps cases during an epidemic.Hum Vaccin Immunother. 2015; 11: 1754-1761Crossref PubMed Scopus (14) Google Scholar Waning of anti-MuV antibody levels has been attributed to the resurgence of mumps,3Kaaijk P. van der Zeijst B. Boog M. Hoitink C. Increased mumps incidence in the Netherlands: review on the possible role of vaccine strain and genotype.Euro Surveill. 2008; 13PubMed Google Scholar, 4Kontio M. Jokinen S. Paunio M. Peltola H. Davidkin I. Waning antibody levels and avidity: implications for MMR vaccine-induced protection.J Infect Dis. 2012; 206: 1542-1548Crossref PubMed Scopus (119) Google Scholar but failing of cellular immune responses may be occurring as well. Mumps-specific IFN-γ responses have been observed in adults, vaccinated or naturally infected during childhood, after stimulating PBMCs with mumps-virus (MuV) antigen,5Jokinen S. Osterlund P. Julkunen I. Davidkin I. Cellular immunity to mumps virus in young adults 21 years after measles-mumps-rubella vaccination.J Infect Dis. 2007; 196: 861-867Crossref PubMed Scopus (63) Google Scholar, 6Hanna-Wakim R. Yasukawa L.L. Sung P. Arvin A.M. Gans H.A. Immune responses to mumps vaccine in adults who were vaccinated in childhood.J Infect Dis. 2008; 197: 1669-1675Crossref PubMed Scopus (52) Google Scholar and flow cytometric analysis revealed low frequencies of MuV-specific IFN-γ–producing CD4+ T cells.6Hanna-Wakim R. Yasukawa L.L. Sung P. Arvin A.M. Gans H.A. Immune responses to mumps vaccine in adults who were vaccinated in childhood.J Infect Dis. 2008; 197: 1669-1675Crossref PubMed Scopus (52) Google Scholar Clearly, these studies indicated that the cellular response to MuV includes T cells and IFN-γ production, but a detailed characterization of the cellular immune response against mumps is lacking. More knowledge on cell-mediated immunity is needed to understand mumps-vaccine failure and to be able to develop interventions. Here, we aimed to analyze in detail the magnitude and phenotype of MuV-specific cellular responses in young adults, taking cellular responses after natural mumps infections as a benchmark and compare those with aged-matched healthy vaccinees. In addition, immune responses in adults were compared with those in recently vaccinated children. MuV-specific T-cell responses were determined by IFN-γ ELISpot from a cohort of 23 mumps cases and 20 age-matched healthy vaccinees (see Table E1 in this article's Online Repository at www.jacionline.org). In accordance with our previous data,7Han W.G. Emmelot M.E. Jaadar H. Ten Hulscher H.I. van Els C.A. Kaaijk P. Development of an IFNgamma ELISPOT for the analysis of the human T cell response against mumps virus.J Immunol Methods. 2016; 431: 52-59Crossref PubMed Scopus (7) Google Scholar besides T cells, CD56+ natural killer (NK) cells contributed to the MuV-specific IFN-γ response in this assay (see Fig E1 in this article's Online Repository at www.jacionline.org). Therefore, NK cells were depleted to measure exclusively MuV-specific T-cell responses. The frequency of MuV-specific T cells 1 to 2 months after disease onset was approximately 9-fold higher than in healthy vaccinees (Fig 1, A). Over time (7-10 months), the frequency of MuV-specific T cells from mumps cases decreased, but was still elevated compared with that in healthy vaccinees. No significant differences were observed between vaccinated and unvaccinated mumps cases, although the number of subjects in each subgroup is low (data not shown). From 7 mumps cases, 3-year follow-up samples could be obtained. The frequencies of MuV-specific T cells after 3 years were comparable to those 7 to 10 months after disease onset, and still significantly higher compared with those in healthy vaccinees (Fig 1, A). This implies the presence of a long-lived T-cell response after mumps disease. The MuV-specific IFN-γ response after in vitro stimulation with live MuV was analyzed in more detail by multiparametric flow cytometry of PBMC samples from 7 mumps cases and 5 healthy vaccinees. Both CD3+ T cells and CD56+ NK cells participated in the MuV-specific IFN-γ response (Fig 1, B-E; see Table E2 in this article's Online Repository at www.jacionline.org). Early (1-2 months) after disease onset, most IFN-γ+ cells appeared to be CD3+ T cells (66%), of which most were CD8+ (69%) (Fig 1, B; see Table E2). NK cells participated relatively less (22%) (Fig 1, B). Estimations on the absolute number of IFN-γ+ NK cells showed no change at convalescence, that is, 7 to 10 months and 3 years after disease (Fig 1, F). The absolute number of IFN-γ+ CD8 T cells declined 7 to 10 months after infection, indicating contraction of the T-cell response (Fig 1, G). However, up to 3 years after infection, the number of IFN-γ+ CD8 T cells still remained significantly higher than in healthy vaccinees (Fig 1, G). Interestingly, in healthy vaccines, the MuV-specific IFN-γ response was dominated by NK cells (Fig 1, E-G). MuV-specific IFN-γ+ T-cell subsets were further defined on the basis of CD45RO/CCR7 expression (Fig 1, B-F; see Table E2). CD8+ T cells consisted mainly of effector memory RA (Temra; CD45RO−CCR7−; 49%-62%) and effector-memory (Tem; CD45RO+CCR7−; 24%-27%) cells, whereas most CD4+ T cells had a Tem phenotype (41%-58%). In healthy vaccinees, the MuV-specific IFN-γ–producing CD4+ and CD8+ T cells were mainly Tem (60%). To further characterize the MuV-specific CD8+ T-cell response, the activation marker CD137 (4-1BB) was used.8Wolfl M. Kuball J. Ho W.Y. Nguyen H. Manley T.J. Bleakley M. et al.Activation-induced expression of CD137 permits detection, isolation, and expansion of the full repertoire of CD8+ T cells responding to antigen without requiring knowledge of epitope specificities.Blood. 2007; 110: 201-210Crossref PubMed Scopus (305) Google Scholar CD137 proved to be an excellent marker for the identification of activated MuV-specific T cells with different functional features (see Figs E2 and E3 in this article's Online Repository at www.jacionline.org). Similar to the dynamics of IFN-γ+ ELISpot responses (Fig 1, A), the enhanced frequency MuV-specific CD137+CD8+ T cells early after infection declined at convalescence, but remained higher than in healthy vaccinees (Fig 2, A). Temra and Tem dominated the MuV-specific CD8+ T-cell response, even 3 years after infection (see Fig E4 in this article's Online Repository at www.jacionline.org), indicating the induction of long-lived memory T cells. Immunological control of virus infections has been associated with the presence of polyfunctional T cells showing multiple effector functions.9Seder R.A. Darrah P.A. Roederer M. T-cell quality in memory and protection: implications for vaccine design.Nat Rev Immunol. 2008; 8: 247-258Crossref PubMed Scopus (892) Google Scholar Therefore, multiple effector parameters within the MuV-specific CD137+CD8+ T-cell population were analyzed. Up to 3 years after disease, most MuV-specific CD137+CD8+ T cells produced IFN-γ (45%) and/or displayed a cytotoxic phenotype by expression of CD107a (40%) (see Fig E5 in this article's Online Repository at www.jacionline.org). A considerable proportion (33%-40%) showed polyfunctionality, simultaneously expressing 2 or more of the functional markers IFN-γ, CD107a, TNF, and IL-2 (Fig 2, B-D; see Fig E6 in this article's Online Repository at www.jacionline.org). Despite the reduced frequencies of the activated CD137+CD8+ T cells over time, their polyfunctionality remained high up to 3 years. Vaccinated and unvaccinated mumps cases showed comparable polyfunctionality (see Fig E7 in this article's Online Repository at www.jacionline.org). Strikingly, only a minor fraction of CD137+CD8+ T cells was polyfunctional (5%) in age-matched healthy vaccinees (Fig 2, E). In both mumps cases and healthy vaccinees, a proportion of the MuV-specific CD137+CD8+ T cells did not display any of the measured effector parameters (Fig 2, B-E). This was not due to exhaustion, as expression of PD-1 and Tim3 was absent (data not shown), but other factors might be involved that were not tested, or the functional markers were not detected because of timing after stimulation. Because the healthy vaccinees received their second and last MMR vaccination more than 10 years ago, waning of an initially polyfunctional vaccine-induced mumps-specific CD8+ T-cell response might have occurred. Alternatively, MMR vaccination may not induce a polyfunctional T-cell response at all. To discriminate between these possibilities, from another clinical study longitudinal blood samples of 9-year-old children who recently received a second MMR vaccination were investigated. The frequencies of MuV-specific IFN-γ−producing cells in PBMCs in these children were low (0.1%), before (see Fig E8 in this article's Online Repository at www.jacionline.org) as well as 1 month (Fig 2, F) and 1 year (Fig E8) after vaccination. Before and after vaccination, 50% to 60% of the IFN-γ–producing cells were NK cells, and 16% to 23% were T cells, similar to healthy adult vaccinees (>10 years postvaccination; Fig 1, E). The MuV-specific CD137+CD8+ T cells observed in recently vaccinated children lacked polyfunctionality (Fig 2, G; see Fig E9 in this article's Online Repository at www.jacionline.org). Thus, in contrast to mumps infection, childhood vaccination with live-attenuated MuV does not induce a polyfunctional CD8+ T-cell response. Other live-attenuated viral vaccines have been shown to induce polyfunctional CD8+ T cells; not only host-virus interactions but also viral load may underlie these differences.9Seder R.A. Darrah P.A. Roederer M. T-cell quality in memory and protection: implications for vaccine design.Nat Rev Immunol. 2008; 8: 247-258Crossref PubMed Scopus (892) Google Scholar Whether MuV-induced polyfunctional CD8+ T cells are required for optimal protection remains to be elucidated. Vaccination with live-attenuated MuV may induce other specific T-cell functions, for example, differentiated toward B-cell help, because the vaccine-induced antibody response has been proven to protect against mumps disease. Waning of vaccination-induced antibodies has been implied to increase the risk of vaccine failure over time. However, based on this study, it is tempting to speculate that the induction of a suboptimal CD8+ T-cell response may also play a role. Taking these data together, we show here that MuV-specific IFN-γ responses are dominated by CD8+ T cells after disease, but not after vaccination. Moreover, MuV-specific CD8+ T cells in mumps cases show a persistent polyfunctionality, which is not seen after vaccination. A prospective study in a mumps outbreak setting, investigating the MuV-specific T-cell responses before and after MuV exposure, could provide evidence whether the long-lived polyfunctional T-cell profile could serve as a correlate of protection. We are grateful to Kina Helm for blood sample handling, and Hinke ten Hulscher for advice in generating MuV stocks. We thank Alina Nicolaie for assistance with statistical analyses. Peripheral blood from 23 mumps laboratory-confirmed cases was collected during the mumps epidemic 2009-2012 in the Netherlands as part of an observational clinical study VAC-263 (NL37852.094.11) performed between November 2011 and May 2013 by the National Institute for Public Health and the Environment. Of these 23 cases, 16 received 2 doses of MMR vaccination during childhood. Samples were collected at 1 to 2 months and (at convalescence) 7 to 10 months after disease onset. Seven cases were included in a follow-up clinical study IMMfact (NL4679.094.13) for a 3-year longitudinal sample. In the VAC-263 study, 20 age-matched healthy individuals showing no clinical signs and no serological evidence of a history of mumps infection were included as controls (hereafter named healthy vaccinees), of which 18 received 2 doses of MMR vaccination during childhood. Demographic characteristics of included mumps cases and healthy vaccinees are described in Table E1. As part of the KIM study (NTR4089),E1van der Lee S. Hendrikx L.H. Sanders E.A.M. Berbers G.A.M. Buisman A.-M. Whole-cell or acellular pertussis primary immunizations in infancy determines adolescent cellular immune profiles.Frontiers in Immunology. 2018; 9Crossref PubMed Scopus (47) Google Scholar peripheral blood was collected from 6 children (2 males, 4 females) prevaccination, 1 month postvaccination, and 1 year postvaccination at the age of 9 years. The KIM study was performed between 2013 and 2014, and participants received their regular MMR-2 vaccination, together with a Tdap-booster vaccination. All studies were approved by the medical ethical committee and were performed according to European Union Good Clinical Practice guidelines and the principles outlined in the Declaration of Helsinki. Information about MMR vaccination history was available from all subjects, and from all participants written informed consent was obtained; for children included in the KIM study, written informed consent was obtained from both parents or legal representatives. Generation of MuV stocks (Jeryl Lynn strain; Mumpsvax, Merck, Darmstadt, Germany) was described before.E2Seder R.A. Darrah P.A. Roederer M. T-cell quality in memory and protection: implications for vaccine design.Nat Rev Immunol. 2008; 8: 247-258Crossref PubMed Scopus (1201) Google Scholar Briefly, supernatant of MuV-inoculated Vero cells was harvested at peak cytopathic effect, centrifuged (485g), and filtered (5 μm). Virus stocks were aliquoted and stored at −80°C until use. Supernatant of uninfected Vero cells was used as mock control after freeze-thawing cycles to lyse cells analogous to MuV-infected cells. PBMCs were isolated by centrifugation on a Ficoll-Hypaque gradient (Pharmacia Biotech, Amsterdam, The Netherlands) and cryopreserved at −135°C until use. For IFN-γ ELISpot assays, PBMCs were thawed and depleted of CD56+ cells by magnetic sorting using CD56-Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's protocol (purity >98%). As described before,E2Seder R.A. Darrah P.A. Roederer M. T-cell quality in memory and protection: implications for vaccine design.Nat Rev Immunol. 2008; 8: 247-258Crossref PubMed Scopus (1201) Google Scholar multiscreen filtration ELISpot plates (Millipore, Merck, Darmstadt, Germany) were prewetted with 35% ethanol for less than 1 minute and washed with sterile water. Plates were coated with 5 μg/mL anti-human IFN-γ antibodies (1-D1K, Mabtech, Nacka Strand, Sweden) in PBS, overnight at 4°C. Plates were extensively washed with PBS and pretreated with AIM-V (Lonza, Basel, Switzerland) supplemented with 2% human AB serum (Sigma-Aldrich, Zwijndrecht, The Netherlands), 10 U/mL penicillin, 10 μg/mL streptomycin, and 29.2 μg/mL l-glutamine (Gibco, Merck, Darmstadt, Germany) for 1 hour. PBMCs, untreated or depleted of CD56+ cells, were incubated with live MuV (Jeryl Lynn or genotype G strain) at multiplicity of infection (MOI) 2 or mock control and were seeded on ELISpot plates (1 × 105 cells/well). Cells were incubated at 37°C, 5% CO2, for 20 hours. For biosafety purposes, plates were exposed to ultraviolet C-light irradiation (254 nm, 5 minutes at 1.0 J/cm2) to inactivate MuV after 20 hours of culture. Subsequently, plates were washed with PBS and incubated for 2 hours with biotinylated 1 μg/mL anti-human IFN-γ detection antibody (7-B6-1, Mabtech) in PBS with 0.5% FBS (Lonza). Plates were washed with PBS and incubated with ExtrAvidin-Alkaline Phosphatase (Sigma) for 1 hour. After washing, plates were developed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate (Sigma). Spots were analyzed by ELISpot software (A.EL.VIS; version 6.1). Activated T cells were determined after in vitro stimulation using fluorochrome-labeled anti-human CD3, CD4, CD8, CD45RO, CCR7, and fixable viability stain (all BD Bioscience, Erembodegem, Belgium) to exclude dead cells. To investigate MuV-specific responses, 2 × 106 total PBMCs were incubated for 24 hours with MuV as described above and cultured at 37°C, 5% CO2, in the presence of anti-human CD107a (BioLegend, Koblenz, Germany). Monensin and Brefeldin A (BD Bioscience) were added for the last 4 hours. Cells were washed with PBS/0.5% BSA/2 mM EDTA and stained for anti-human CD3, CD4, CD8, CD56, and fixable viability stain (BD Bioscience). After fixation and permeabilization, using FoxP3/Transcription Factor Staining Buffer Set (eBioscience, Thermo Fisher Scientific, Waltham, Mass) cells were stained intracellularly for antihuman IFN-γ, CD137, IL-2 (BD Bioscience), and TNF (eBioscience). Data were acquired on a FortessaX20 analyzer (BD) and analyzed using FlowJo (V10, Tree Star, Ashland, Ore). Absolute numbers of IFN-γ–producing cells was calculated on the basis of the absolute number of isolated PBMCs per milliliter of whole blood and the frequencies of IFN-γ+ cell subsets (as measured by flow cytometry) within this leucocyte population. CD45RO+CCR7+ were defined as central memory T cells (Tcm), CD45RO+CCR7− as Tem, CD45RO−CCR7− as Temra, and CD45RO−CCR7+ as cytokine-producing cells with a naive phenotype (Tn-like). Data were analyzed with SPSS (version 22), using paired Wilcoxon signed-rank tests to compare longitudinal samples, or unpaired Mann-Whitney U tests to compare mumps cases with healthy vaccinees. Significances are shown as ****P < .0001, ***P < .001, **P < .01, and *P < .05.Fig E2MuV-specific CD8+ T cells display CD137 and IFN-γ. Total PBMCs derived from a mumps case 1 to 2 months, 7 to 10 months, or 3 years after disease onset were stimulated with mock or live Jeryl Lynn MuV for 24 hours. Brefeldin A and monensin were added for the last 4 hours. Activation (CD137) and IFN-γ production within CD8+ T cells were determined by flow cytometry. Representative plots are shown from 1 of 7 mumps cases.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3MuV-specific CD137+CD8+ T cells produce IFN-γ and TNF and display cytotoxic activity. Total PBMCs longitudinally derived from mumps cases or healthy vaccinees were stimulated with live Jeryl Lynn MuV for 24 hours. Expression of activation marker CD137, intracellular cytokines (IFN-γ and TNF), and CD107a within CD8+ T cells was determined by flow cytometry. Representative plots are shown for longitudinal samples from 1 of 7 mumps cases and 1 of 5 vaccinees.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4MuV-specific activation is mainly displayed by Temra and Tem CD8+ cells. Total PBMCs derived from 7 mumps cases were stimulated with live Jeryl Lynn MuV for 24 hours. Expression of activation marker CD137 within CD8+ T cells was determined by flow cytometry. The proportions of cells with a naive (Tn-like; CD45RO−CCR7+), central memory (Tcm; CD45RO+CCR7+), Tem (CD45RO+CCR7−), and Temra (CD45RO−CCR7−) phenotype within the activated, CD137-expressing CD8+ T cells were determined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5IFN-γ, CD107a, TNF, and IL-2 expression by MuV-specific CD8+ T cells. Total PBMCs derived from mumps cases or healthy vaccinees were stimulated with live MuV for 24 hours. Cytokine production (IFN-γ, TNF, and IL-2) and CD107a expression within activated CD137+CD8+ T cells were determined by flow cytometry. Data are from 6 to 7 mumps cases and 6 healthy vaccinees.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6IFN-γ, CD107a, TNF, and IL-2 expression from MuV-activated CD137+CD8+ T cells. Total PBMCs derived from mumps cases 1 to 2 months, 7 to 10 months, or 3 years after disease onset, or healthy vaccinees were stimulated with live Jeryl Lynn MuV for 24 hours. Cytokine production (IFN-γ, TNF, and IL-2) and CD107a expression within activated CD137+CD8+ T cells were determined by flow cytometry. Data shown are mean (as indicated) + SD from responses at longitudinal time points from 6 mumps cases and from 6 vaccinees.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E7MuV-specific CD8+ T cells show comparable polyfunctionality. Total PBMCs derived from previously vaccinated (left panels; n = 3) or unvaccinated (right; n = 3) mumps cases were stimulated with live MuV for 24 hours. Cytokine production (IFN-γ, TNF, and IL-2) and CD107a expression within activated CD137+CD8+ T cells were determined by flow cytometry. Frequencies of CD137+CD8+ cells expressing 1 or more of 4 markers are presented in bar graphs and combined in pie charts (insert).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E8MuV-specific IFN-γ production from vaccinated children. Total PBMCs derived from 5 children receiving a second, booster MMR vaccination were stimulated with live MuV for 24 hours and IFN-γ–producing cells were determined by intracellular cytokine staining. The 1-month postvaccination data are depicted in Fig 2, G. Because of the low number, Tem/Temra could not be identified.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E9MuV-specific CD8+CD137+ T cells show no polyfunctionality after MMR-2 vaccination in children. Total PBMCs derived from 4 children receiving their second MMR vaccination were stimulated with live MuV for 24 hours and cytokine production (IFN-γ, TNF, and IL-2) and CD107a expression within activated CD137+CD8+ T cells were determined by flow cytometry. Frequencies of CD137+CD8+ cells expressing 1 or more of 4 markers are presented in bar graphs and combined in pie charts (insert).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Demographic and other characteristics of included mumps cases and healthy adult vaccineesIDAge (y)SexMMR vaccineSample 1 (mo)Sample 2 (mo)Sample 3 (mo)SymptomsMumps cases 263-0124M2 doses1939Orchitis, swollen neck glands, abdominal pain, vomiting, fever 263-0521M2 doses1938Parotitis, swollen neck glands, fever, cold, cough 263-0721F2 doses1838Parotitis, swollen neck glands, cold 263-1122FNone21038Parotitis, swollen neck glands, cough, sore throat 263-1723MNone21037Swollen neck glands, fever, loss of appetite, fatigue 263-1859MNone21037Orchitis, swollen neck glands, fever, cough, otitis 263-1953FNone21037Swollen neck glands, fever, cough, vertigo, temporary deafness 263-0226FNone19NAParotitis, swollen neck glands, abdominal pain, cold, otitis 263-0319M2 doses19NAParotitis, swollen neck glands 263-0426M2 doses29NAParotitis, swollen neck glands, fever, cough 263-0620F2 doses18NAParotitis, swollen neck glands 263-0840MNone2NANAParotitis, swollen neck glands, headache 263-1040MNone210NAOrchitis, parotitis, swollen neck glands, fever, sore throat 263-1225M2 doses110NAParotitis, swollen neck glands, fever 263-1324F2 doses19NAParotitis, fever 263-1423M2 doses17NAParotitis, swollen neck glands, fever 263-1521F2 doses1NANAParotitis 263-1624F2 doses19NAParotitis, swollen neck glands, fever, nausea 263-2021M2 doses110NAParotitis, swollen neck glands 263-2127M2 doses18NAParotitis, swollen neck glands, fever, painful testicles 263-2226M2 doses17NAOrchitis 263-2522F2 doses17NAParotitis, swollen neck glands, fever 263-2619M2 doses17NAParotitis, abdominal painHealthy vaccinees 263-4321M2 doses———None 263-4623F2 doses———None 263-3021F2 doses———None 263-3325F2 doses———None 263-3521F2 doses———None 263-4425F2 doses———NoneF, Female; M, male; NA, not available. Open table in a new tab Table E2Frequencies within IFN-γ+ cells∗Percentage from parent population (± SD) from 7 mumps cases and 5 healthy vaccinees (Fig 1, B-E).IFN-γ+ cells1-2 mo7-10 mo3 yHealthy vaccineesTotal IFN-γ1.6% ± 0.9%1.0% ± 0.9%0.7% ± 0.9%1.1% ± 0.9% CD3+66% ± 13.7%50% ± 9.9%58% ± 13.0%26% ± 5.0%CD4+15% ± 6.8%21% ± 7.9%25% ± 7.3%23% ± 6.7%Tn24% ± 3.7%41% ± 11.6%27% ± 11.1%19% ± 8.8%Tcm13% ± 3.3%13% ± 3.0%15% ± 3.6%17% ± 10.5%Tem58% ± 4.4%41% ± 12.1%51% ± 12.9%60% ± 12.8%Teff5% ± 1.1%5% ± 1.5%7% ± 1.0%5% ± 3.0%CD8+69% ± 7.0%61% ± 7.6%61% ± 5.7%22% ± 5.0%Tn7% ± 3.4%10% ± 3.8%14% ± 8.4%15% ± 9.6%Tcm4% ± 1.7%6% ± 3.4%11% ± 4.4%13% ± 7.7%Tem27% ± 8.4%24% ± 9.1%26% ± 9.2%60% ± 13.8%Teff62% ± 9.7%60% ± 10.7%49% ± 9.0%12% ± 2.8%CD4−CD8−15% ± 7.2%16% ± 7.4%12% ± 4.0%54% ± 7.7% CD56+22% ± 13.7%37% ± 7.6%29% ± 10.0%62% ± 8.7%CD56dim85% ± 9.1%81% ± 6.3%90% ± 5.0%89% ± 4.9%CD56bright14% ± 9.1%19% ± 6.3%10% ± 5.0%11% ± 4.9% CD3+CD56+4% ± 2.9%7% ± 3.7%7% ± 4.8%8% ± 1.9%Tcm, Central memory T cell; Tn, naive T cell.∗ Percentage from parent population (± SD) from 7 mumps cases and 5 healthy vaccinees (Fig 1, B-E). Open table in a new tab F, Female; M, male; NA, not available. Tcm, Central memory T cell; Tn, naive T cell.
// Laurens E. Franssen 1 , Inger S. Nijhof 1 , Chad C. Bjorklund 2 , Hsiling Chiu 2 , Ruud Doorn 3 , Jeroen van Velzen 3 , Maarten Emmelot 1 , Berris van Kessel 1 , Mark-David Levin 4 , Gerard M.J. Bos 5 , Annemiek Broijl 6 , Saskia K. Klein 7 , Harry R. Koene 8 , Andries C. Bloem 3 , Aart Beeker 9 , Laura M. Faber 10 , Ellen van der Spek 11 , Reinier Raymakers 12 , Pieter Sonneveld 6 , Sonja Zweegman 1 , Henk M. Lokhorst 1 , Anjan Thakurta 2 , Xiaozhong Qian 2 , Tuna Mutis 1 and Niels W.C.J. van de Donk 1 1 Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Translational Development, Celgene Corporation, Summit, NJ, USA 3 Laboratory for Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands 4 Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, The Netherlands 5 Department of Hematology, Maastricht University Medical Center, Maastricht, The Netherlands 6 Department of Hematology, Erasmus Medical Center, Rotterdam, The Netherlands 7 Department of Internal Medicine, Meander Medical Center, Amersfoort, The Netherlands 8 Department of Hematology, St. Antonius Hospital, Nieuwegein, The Netherlands 9 Department of Internal Medicine, Spaarne Hospital, Hoofddorp, The Netherlands 10 Department of Internal Medicine, Rode Kruis Hospital, Beverwijk, The Netherlands 11 Department of Internal Medicine, Rijnstate Hospital, Arnhem, The Netherlands 12 Department of Hematology, University Medical Center Utrecht Cancer Center, Utrecht, The Netherlands Correspondence to: Niels W.C.J. van de Donk, email: n.vandedonk@vumc.nl Keywords: multiple myeloma; immunomodulation; lenalidomide; refractory; cyclophosphamide Received: May 01, 2018 Accepted: September 10, 2018 Published: September 21, 2018 ABSTRACT We recently showed that the outcome of multiple myeloma (MM) patients treated in the REPEAT study (evaluation of lenalidomide combined with low-dose cyclophosphamide and prednisone (REP) in lenalidomide-refractory MM) was markedly better than what has been described with cyclophosphamide-prednisone alone. The outcome with REP was not associated with plasma cell Cereblon expression levels, suggesting that the effect of REP treatment may involve mechanisms independent of plasma cell Cereblon-mediated direct anti-tumor activity. We therefore hypothesized that immunomodulatory effects contribute to the anti-MM activity of REP treatment, rather than plasma cell Cereblon-mediated effects. Consequently, we now characterized the effect of REP treatment on immune cell subsets in peripheral blood samples collected on day 1 and 14 of cycle 1, as well as on day 1 of cycle 2. We observed a significant mid-cycle decrease in the Cereblon substrate proteins Ikaros and Aiolos in diverse lymphocyte subsets, which was paralleled by an increase in T-cell activation. These effects were restored to baseline at day one of the second cycle, one week after lenalidomide interruption. In vitro , lenalidomide enhanced peripheral blood mononuclear cell-mediated killing of both lenalidomide-sensitive and lenalidomide-resistant MM cells in a co-culture system. These results indicate that the Cereblon-mediated immunomodulatory properties of lenalidomide are maintained in lenalidomide-refractory MM patients and may contribute to immune-mediated killing of MM cells. Therefore, combining lenalidomide with other drugs can have potent effects through immunomodulation, even in patients considered to be lenalidomide-refractory.
Abstract Purpose: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4+CD25+ regulatory T cells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell–dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2−/−γc−/− mice by i.v. administration of human peripheral blood mononuclear cells (PBMC). Experimental Design: Human PBMC, depleted of or supplemented with autologous CD25+ Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of human T cells, and secretion of human cytokines were monitored at weekly intervals. Results: Depletion of CD25+ cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25+ cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4+ and CD8+ T cells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-γ, suggesting the de novo development of TR1 cells. Conclusions: These results show, for the first time, the potent in vivo capacity of naturally occurring human Tregs to control GvHD-inducing autologous T cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring human Tregs.
Background The development and preclinical testing of novel immunotherapy strategies for multiple myeloma can benefit substantially from a humanized animal model that enables quantitative real-time monitoring of tumor progression. Here we have explored the feasibility of establishing such a model in immunodeficient RAG2−/−γc−/− mice, by utilizing non-invasive bioluminescent imaging for real-time monitoring of multiple myeloma cell growth.Design and Methods Seven multiple myeloma cell lines, marked with a green fluorescent protein firefly luciferase fusion gene, were intravenously injected into RAG2−/−γc−/− mice. Tumor localization and outgrowth was monitored by bioluminescent imaging. The sensitivity of this imaging technique was compared to that of free immumoglobulin light chain -based myeloma monitoring. Established tumors were treated with radiotherapy or with allogeneic peripheral blood mononuclear cell infusions to evaluate the application areas of the model.Results Five out of seven tested multiple myeloma cell lines progressed as myeloma-like tumors predominantly in the bone marrow; the two other lines showed additional growth in soft tissues. In our model bioluminescent imaging appeared superior to free light chain-based monitoring and also allowed semi-quantitative monitoring of individual foci of multiple myeloma. Tumors treated with radiotherapy showed temporary regression. However, infusion of allogeneic peripheral blood mononuclear cells resulted in the development of xenogeneic graft-versus-host-disease and a powerful cell dose-dependent graft-versus-myeloma effect, resulting in complete eradication of tumors, depending on the in vitro immunogenicity of the inoculated multiple myeloma cells.Conclusions Our results indicate that this new model allows convenient and sensitive real-time monitoring of cellular approaches for immunotherapy of multiple myeloma-like tumors with different immunogenicities. This model, therefore, allows comprehensive preclinical evaluation of novel combination therapies for multiple myeloma.
