T‐cells producing multiple combinations of IFNγ, TNF and IL10 are associated with mild forms of dengue infection

Marcela Helena Gonçalves Pereira; Maria Marta Figueiredo; Camila Pereira Queiroz; Télcia Vasconcelos Barros Magalhães; Adriana Mafra; Lilian Martins Oliveira Diniz; Último Libânio da Costa; Kenneth J Gollob; Lis Ribeiro do Valle Antonelli; Helton da Costa Santiago

doi:10.1111/imm.13185

. 2020 Apr 6;160(1):90–102. doi: 10.1111/imm.13185

T‐cells producing multiple combinations of IFNγ, TNF and IL10 are associated with mild forms of dengue infection

Marcela Helena Gonçalves Pereira ¹, Maria Marta Figueiredo ², Camila Pereira Queiroz ¹, Télcia Vasconcelos Barros Magalhães ³, Adriana Mafra ⁴, Lilian Martins Oliveira Diniz ⁵, Último Libânio da Costa ⁶, Kenneth J Gollob ⁷, Lis Ribeiro do Valle Antonelli ², Helton da Costa Santiago ^1,^8,^✉

PMCID: PMC7160666 PMID: 32128816

Summary

Multifunctional interleukin 10 (IL10)⁺Th1 cells have been implicated in favorable evolution of many infectious diseases, promoting an efficacious immune response while limiting immunopathology. Here, we investigated the presence of multifunctional CD4⁺ and CD8⁺ T‐cells that expressed interferon gamma (IFNγ), IL10 and tumor necrosis factor (TNF), or its combinations during dengue infection. Peripheral blood mononuclear cells (PBMCs) from outpatients with dengue (mild dengue forms) and hospitalized patients (or patients with dengue with warning signs and severe dengue) were cultured in the presence of envelope (ENV) or NS3 peptide libraries of DENV during critical (hospitalization period) and convalescence phases. The production of IFNγ, IL10 and TNF by CD4⁺ and CD8⁺ T‐cells was assessed by flow cytometry. Our data show that patients with mild dengue, when compared with patients with dengue with warning signs and severe dengue, presented higher frequencies of multifunctional T‐cells like NS3‐specific IFNγ/IL10‐producing CD4⁺ T‐cells in critical phase and NS3‐ and ENV‐specific CD8⁺ T‐cells producing IFNγ/IL10. In addition, NS3‐specific CD8⁺ T‐cells producing high levels of IFNγ/TNF and IFNγ/TNF/IL10 were also observed in the mild dengue group. We observed that multifunctional T‐cells produced higher levels of cytokines as measured by intracellular content when compared with single producer T‐cells. Importantly, multifunctional CD4⁺ and CD8⁺ T‐cells producing IFNγ, TNF and IL10 simultaneously displayed positive correlation with platelet levels, suggesting a protective role of this population. The presence of IL10⁺Th1 and IL10⁺Tc1 multifunctional cells was associated with mild dengue presentation, suggesting that these cells play a role in clinical evolution of dengue infection.

Keywords: dengue, IL10⁺Tc1, IL10+Th1, immunoregulation, multifunctional T‐cells

Multifunctional IL10‐producing Tc1 or Th1 cells are important to provide effective immune responses balanced with regulatory mechanism in the process of infectious disease control. We identified DENV‐specific multifunctional T‐cells producing IFNγ, TNF and/or IL10 that were associated with mild dengue evolution and showed positive correlation with platelets. These data suggest a role of this T‐cell subpopulation on clinical evolution of DENV infection.

graphic file with name IMM-160-90-g005.jpg

Abbreviations

DENV: dengue virus
ENV: envelope
IFNγ: interferon gamma
IL10: interleukin 10
NS3: non‐structural protein 3
Tc: T cytotoxic cells
Th: T helper cells
ws+: positive for warning signs

Introduction

Dengue fever is a mosquito‐borne viral infection that is mainly characterized by a self‐limited acute fever, which resolves spontaneously in most cases. ¹ The World Health Organization (WHO) classifies clinical dengue as dengue without warning signs (mild dengue), dengue with warning signs (ws+) and severe dengue. ¹ The mechanisms by which the host develops mild or severe forms of dengue are unclear and may be associated with extrinsic factors, such as the virulence of the dengue virus (DENV), ² or intrinsic host factors, especially the host's immunity status. Previously exposed individuals with heterotypic infection present higher chances of developing severe dengue, ³ associated to a phenomenon called antibody‐dependent enhancement (ADE) in which heterotypic non‐neutralizing antibodies opsonize mature and immature virus particles, facilitating infection of mononuclear phagocytes and promoting virus replication. ³

Interestingly, individuals that develop secondary dengue also present an increased chance of asymptomatic dengue presentation during heterologous infection. ⁴ , ⁵ Such resistance was associated with the presence of an efficient response of DENV‐specific cross‐reactive cytotoxic T lymphocytes producing interferon gamma (IFNγ). ⁵ , ⁶ Indeed, active CD8⁺ T‐cells against DENV can reduce viral load and prevent ADE in mice, ⁷ , ⁸ suggesting that lymphocyte activation is critical to DENV resistance. On the other hand, some authors propose that T‐cell hyperactivation can be associated with the development of hemorrhagic manifestations. ⁹ , ¹⁰

While details of the mechanisms that trigger severe dengue development are unclear, there is a consensus that hyperinflammatory status either from innate ¹¹ or possibly adaptive immunity ¹⁰ activation is important. The pathways that lead to immune hyperactivation have been the subject of several investigations and speculations. In contrast, the mechanisms that regulate inflammation during dengue infection are poorly addressed. Although the activation and function of regulatory T‐cells (Treg) in dengue infection have not been carefully investigated, it has been demonstrated that, although not functionally impaired during infection, the number of peripheral Tregs may be decreased in relation to effector cells ¹² in severe cases of dengue. On the other hand, while transforming growth factor β levels have been associated with favorable evolution of dengue, ¹³ the role of another major regulatory cytokine, interleukin 10 (IL10), is still controversial. High serum levels of IL10 are found in severe dengue patients during early moments of clinical manifestations. ¹⁴ , ¹⁵ , ¹⁶ On the other hand, T‐cell‐derived IL10 has also been implicated in favorable evolution of several virus infections. ¹⁷ , ¹⁸ A very interesting study concluded that polymorphisms associated with low IL10 production can increase the chances for severe dengue presentation. ¹⁹ Importantly, there is evidence showing loss of coordinate regulation between the anti‐inflammatory cytokine IL10 and the inflammatory cytokines IL6 and IL8 in patients with more severe disease. ²⁰

Interleukin 10 has been implicated in preventing hyperinflammation during various infectious diseases, ¹⁷ especially if produced by multifunctional T‐cells. For example, pathogen‐specific IL10⁺IFNγ⁺ CD4⁺ T‐cells are associated with favorable evolution of malaria, ²¹ , ²² toxoplasmosis, ²³ Chagas disease ²⁴ , ²⁵ and influenza. ²⁶ In contrast, these multifunctional T‐cells are associated with impaired effector responses to leishmaniasis ²⁷ and tuberculosis, ²⁸ but still considered an important mechanism to limit pathology to these same diseases.

There is little information about multifunctional T‐cells in dengue infection, ²⁹ , ³⁰ , ³¹ especially with regard to IL10⁺Th1 and IL10⁺Tc1 cells. In this work, we aimed to investigate the presence of these cells in different clinical forms of dengue infection. Because IL10⁺Th1 may also express other cytokines besides IL10 and IFNγ, ³² we decided also to investigate the production of tumor necrosis factor (TNF), a cytokine that possesses anti‐viral properties ³³ and has also been found in a subpopulation of T‐cells producing IFNγ simultaneously during DENV infections. ³⁰ , ³¹ Therefore, in this work, we evaluated dengue patients for the presence of DENV‐specific T‐cells producing IFNγ, TNF and/or IL10 at defervescence (critical) and convalescence phases of infection. We found that the IFNγ⁺IL10⁺, IL10⁺TNF⁺ and IFNγ⁺TNF⁺IL10⁺, in addition to IFNγ⁺TNF⁺, multifunctional CD4⁺ and CD8⁺ T‐cells were associated with mild dengue presentation. These results suggest that IL10⁺ multifunctional T‐cells may play a role in clinical evolution of dengue.

Materials and methods

Ethical statement

This study was carried out in Belo Horizonte, Minas Gerais, in a dengue‐endemic area with seasonal transmission between 2013 and 2015. The study was reviewed and approved by the Institutional Review Board from Instituto René Rachou, Fiocruz‐MG (CAAE: 30492014.9.0000.5091). All adult candidates or legal guardians (in the case of an underage participant) signed a consent form prior to their enrollment in the study. Underage teenagers signed an assent form. Outpatients were recruited at Primary Care Center Jardim Montanhês and Santo Ivo Hospital. Inpatients were recruited in Odilon Behrens Metropolitan Hospital, Santa Casa Hospital and João Paulo II Hospital. Healthy volunteers were recruited in the community.

Clinical information, sample collection and processing

Classification between different clinical forms of dengue was performed according to WHO guidelines ¹ by the attending physician at the moment of consenting, which was also responsible to collect illness symptoms and evolution and blood work results. Classification was reviewed retrospectively by an independent research physician. Dengue cases were classified in this study into two groups: (i) mild dengue (outpatients), cases of mild dengue treated at home; and (ii) ws+/severe dengue (inpatients), the combination of dengue with warning signs (ws+) and severe dengue groups (ws+/severe), which were treated at the hospital. The clinical information and platelet levels were retrieved from the patients' charts. Some patients did not have platelet level information available. Volunteers donated blood (40–60 ml) in heparinized vacuum tubes during hospitalization or time‐matched control for mild cases (i.e. 6–12 days post‐fever onset) and at convalescence (more than 30 days after fever onset). On the same day of collection, plasma and peripheral blood mononuclear cells (PBMCs) were separated by centrifugation using Ficoll‐Hypaque gradient (SIGMA, San Luis, MI). Plasma samples were stored in −80° freezer. Cells were frozen in fetal bovine serum (FBS; Cultilab, Campinas, SP, Brazil) in the presence of 10% dimethyl sulfoxide (DMSO; SIGMA) at −80° for 24 hr, and then transferred to liquid nitrogen (−196°) until use.

Dengue diagnostics, inclusion criteria and secondary infection

Serological tests for dengue were performed using IgM and IgG PanBio ELISA kit (Inverness Medical Innovations Australia Pty, Brisbane, QLD, Australia) according to the manufacturer's instructions.

RNA from plasma/serum was obtained using PureLink Viral RNA/DNA Kits (INVITROGEN, Carlsbad, CA). Amplifications were performed by quantitative polymerase chain reaction (qPCR) using SuperScript III Platinum One‐Step Quantitative RT‐PCR System with ROX (INVITROGEN) according to manufacturer's instructions in the presence of primers and probes described previously (Table S1). ³⁴ We added 1 U/µl of heparinase when necessary to avoid polymerase‐inhibiting properties of heparin. ³⁵

Individuals were included in this study if positive for dengue infection when IgM at the first 7 days of symptoms or PCR were positive for dengue. Moreover, healthy controls were symptom‐free and negative for dengue‐specific IgM and IgG. Individuals with positive dengue diagnostic presenting IgG during the first 7 days of symptoms were considered pre‐exposed and having secondary infection.

Peptide library

Peptide libraries containing sequential peptides of 15aa and 5aa offset spanning envelope (ENV) and non‐structural protein 3 (NS3) of DENV1 (NP_059433.1) were purchased from Mimotopes (Melbourne, VIC, Australia). The libraries of DENV1 ENV (NP_722460.2) and NS3 (NP_722463) contained 97 and 123 peptides, respectively, and the list of peptides in each library is listed in Table S2. The peptides were reconstituted in DMSO (20 mg/ml) and used at final concentration of 2 μg/ml in each assay.

Immunophenotyping and intracellular cytokine assessment

Peripheral blood mononuclear cells were thawed in RPMI 1640 (Sigma‐Aldrich) containing 10% FBS and 20 U/ml benzonase nuclease (Novagen, Madison, WI), washed in phosphate‐buffered saline (PBS) and cultured at the concentration of 1·0 × 10⁶ cells/well. After a period of ambience of 2 hr in a humidified incubator with 5% CO₂ at 37°C, the cells were cultured for another 10 hr in four different experimental conditions: (i) absence of stimulus; (ii) stimulation with DENV1 ENV peptide library; (iii) stimulation with DENV1 NS3 peptide library; and (iv) stimulation with anti‐CD3 (1·0 μg/ml) (BD, Franklin Lakes, NJ) and anti‐CD28 (0·5 μg/ml) antibodies (BD). Brefeldin A (1·0 μg/ml; BioLegend) was added in all culture conditions. After the incubation period, cells were washed in PBS, and stained with Violet Live/Dead (Invitrogen), and with monoclonal antibodies CD3 (Invitrogen), CD4 (BD) and CD8 (Invitrogen). PBMCs were washed, fixed and permeabilized using FoxP3 staining buffer Set (eBioscience, San Diego, CA) according to the manufacturer's instructions. Cells were then incubated with antibodies against IFNγ (BD), TNF (Invitrogen) and IL10 (eBioscience), washed, resuspended in FACS buffer and acquired on an LSR‐FORTESSA (BD).

Analyses of T‐cell subpopulations and cytokines production

Data from cell acquisition were analyzed using Flow Jo X program (LLC, Ashland, OR). The populations of lymphocytes, singlets and time (exclusion of interruptions in the flow) were selected as in Fig. S1. Then, to obtain the intersection of these three initial gates, the Boolean Gate tool ‘Make and Gate’ was used, resulting in the dot plot from which the live CD3⁺ (CD3⁺ViViD⁻) population was selected. The subpopulations of CD4⁺ and CD8⁺ T lymphocytes were selected from the ViViD⁻CD3⁺, and staining of IFNγ, TNF and IL10 cytokines were determined within the gate of ViViD⁻CD3⁺CD4⁺CD8⁻ or ViViD⁻CD3⁺CD4⁻CD8⁺ cells. Single (IFNγ⁺TNF⁻IL10⁻, IFNγ⁻TNF⁺IL10⁻ and IFNγ⁻TNF⁻IL10⁺), double (IFNγ⁺TNF⁺IL10⁻, IFNγ⁺TNF⁻IL10⁺, IFNγ⁻TNF⁺IL10⁺) and triple (IFNγ⁺TNF⁺IL10⁺) cytokine‐producing CD4⁺ or CD8⁺ T‐cells were determined with Boolean gating (Make and Gate). Samples with less than 20 000 events on ViViD⁻CD3⁺ gate were excluded from analysis.

Statistical analysis

Statistical analysis was performed using GraphPad Prism V6.05 software. The results were analyzed using the statistical tests: Kruskal−Wallis statistical test, Mann−Whitney test and Wilcoxon test as indicated in the figure legends. The differences were considered statistically significant when P ≤ 0·05. The outliers were analyzed using the ROUT (Q = 2·0%) method of GraphPad Prism V6.05 software.

Results

Study population

Dengue patients (n = 39) were recruited between 2013 and 2015 (before Zika epidemic in Brazil), and were grouped into mild dengue group (patients that were treated at home as outpatients) and ws+/severe dengue group (inpatient cases that met WHO and Brazilian Ministry of Health for hospitalization). The blood was collected during the critical phase (period when patients were hospitalized or their time match controls) and at convalescence. Unfortunately, only 25 participants accepted to return for second blood collection. Importantly, all participants were contacted by telephone, and had their outpatient or inpatient treatment confirmed by study personnel. Demographic characteristics and laboratory data available are shown in Table 1. Groups were comparable for age, sex distribution, and the proportion of individuals presenting secondary dengue infection was not significantly different between mild and ws+/severe dengue (Table 1). Inpatients (ws+/severe) displayed higher frequencies of platelets under 50 000/mm³ (up to 43%) and lower platelet nadir level (Table 1). All the patients with PCR positive reaction were infected by DENV1, data that were confirmed by the local health authorities stating that DENV1 was the predominant serotype circulating in Belo Horizonte in the years of sample collection. Four patients with DENV4 infection found in our cohort were not included in this study.

Table 1.

Demographics and laboratory characteristics of study population from control, mild dengue (outpatients) and ws+/severe (inpatients) groups during seasonal transmission 2013−2015

Characteristics and diagnostic of study population	Control (n = 15)	Mild dengue (n = 22)	ws+/severe dengue (n = 17)
Age¹	29 (21–42) ¹	36 (13–62)	32 (13–79)
Gender (F)	53% (8/15)	73% (16/22)	65% (11/17)
RT‐PCR (n)	0% (0/15)	68% (15/22)	65% (11/17)
ELISA IgM (n)	0% (0/15)	77% (17/22)	82% (14/17)
ELISA IgG (> 30 d) (n)	0% (0/15)	65% (13/20)	80% (4/5)
Secondary infection (n)	—	73% (16/22)	71% (12/17)
Laboratory characteristics of dengue patient groups
Erythrocytes (×10⁶/mm³) ²		4·8 (4·3–5·4)	4·1 (3·3–5·2)^**
Hematocrit (%) ²		42·7 (35·8–47·4)	37·2 (28·2–51·5)^**
Platelet < 50 000/mm³ (n)		0% (0/12)	43% (6/14)^* ^, ³
Lowest platelet count (× 10³/mm³) ²		106·6 (60·0–224·0)	62·6 (16·9–228·0)^**

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^¹

Geometric mean (min−max).

^²

Geometric mean (confidence interval).

^³

Platelets were not available for all patients as the request of this exam was at attending physician discretion.

Fisher test P < 0·005.

^**

Mann−Whitney test P < 0·002; age: Kruskal−Wallis test P> 0·13; gender: Fisher test P > 0·5.

Mild dengue patients presented higher frequencies of DENV‐specific single functional T‐cells positive for TNF and IL10, but not IFNγ, when compared with ws+/severe dengue patients

To evaluate the cytokine production by T‐cells during dengue infection, we cultured PBMCs in the presence of ENV or NS3 peptide libraries of DENV1. Because we focused on the production of IFNγ, TNF and IL10, we first investigated if the peptide libraries would stimulate single cytokine producers T‐cells. We observed that, when considering only these cytokines, the frequencies of single cytokine‐producing T‐cells by dengue patients rarely were different from background control levels, indicated by the dashed line (Fig. 1). Only the frequency of ENV‐stimulated IL10‐producing CD8⁺ T‐cells during the critical phase was significantly above background (as indicated by the # sign; Fig. 1b). Interestingly, despite poor stimulation of TNF and IL10 single producers CD4⁺ or CD8⁺ T‐cells by ENV or NS3 (Figs S3 and S4), we could observe that patients with ws+/severe dengue, when compared with the mild dengue group, presented decreased frequencies of T‐cells producing only TNF or IL10 during critical and convalescence phases upon stimulation with peptide libraries for ENV (Fig. 1a−c) and NS3 (Fig. 1e,f). In general, CD4⁺ T‐cells were generally poor single cytokine responders (Figs S2 and S3) upon stimulation with peptide libraries when compared with unstimulated cultures (Fig. S3). Although the frequency of IFNγ‐producing DENV‐specific T‐cells was not different between dengue groups (Fig. 1), CD8⁺ T‐cells from mild dengue at convalescent phase responded to NS3 displaying increased IFNγ‐positive population after stimulation, while T‐cells were refractory in most ws+/severe dengue group (Figs S2 and S4). In contrast, we observed that CD4⁺ T‐cells of ws+/severe patients group showed decreased IFNγ production upon ENV stimulation during the critical phase when compared with unstimulated cultures (Fig. S3). In addition, ENV‐specific IL10 and NS3‐specific TNF in CD8⁺ T‐cell compartments were also inhibited by peptide library stimulation in ws+/severe group (Fig. S4).

Individuals with mild dengue presented more dengue virus (DENV)‐specific T‐cell single producers of tumor necrosis factor (TNF) and interleukin (IL)10, but not interferon gamma (IFNγ), than ws+/severe dengue subjects. Peripheral blood mononuclear cells (PBMCs) from DENV‐infected patients with mild (dark gray squares) and severe (white triangle) forms were stimulated with peptide libraries for envelope (ENV; a–d) and non‐structural protein 3 (NS3; e–h) during critical and convalescence phases of infection. Frequencies of IFNγ, TNF or IL10 production by CD4⁺ and CD8⁺ T‐cells were evaluated by flow cytometry among CD3⁺ live gated cells. The horizontal solid lines represent the geometric mean. Dotted lines represent the geometric mean of the non‐infected control group (N = 11–15). Differences between DENV‐infected groups were analyzed by Mann–Whitney statistical test and are indicated by asterisks (*) for P < 0·05. Comparisons between non‐infected control and dengue groups were performed by Kruskal−Wallis test, and are indicated by the hash sign (#) for P < 0·05. N was 20–22 for mild dengue and 14–16 for ws+/severe dengue cases during the critical phase, and 19–20 for mild dengue and 4–5 for ws+/severe dengue cases during convalescence.

Individuals with mild dengue presented high frequencies of multifunctional T‐cells

We evaluated if T‐cells were able to produce simultaneously IFNγ, TNF and/or IL10 upon stimulation. Multifunctional T‐cells producing multiple cytokines emerged in response to both antigens, ENV and NS3, although triple cytokine producers were induced only by NS3 (Figs 2, 3 and S5–S7). These cell populations, i.e. multifunctional DENV‐specific T‐cells, were more often different from the control non‐exposed group (as indicated by the # sign) when compared with single cytokine‐producing T‐cells.

Individuals with mild dengue presented higher frequencies of interleukin (IL)10⁺ double producer CD4⁺ and CD8⁺ T‐cells. Peripheral blood mononuclear cells (PBMCs) from dengue virus (DENV)‐infected patients with mild and severe clinical presentations were stimulated with peptide libraries of envelope (ENV; a–d) and non‐structural protein 3 (NS3; e–h) during acute (top) and convalescence (bottom) phases of infection. Frequencies of double producer CD4⁺ and CD8⁺ T‐cells were evaluated by flow cytometry in CD3⁺ gated cells. The horizontal lines represent the geometric mean. The dotted lines represent the geometric mean of the non‐infected control group (N = 10–15). Differences between groups were analyzed by Mann−Whitney statistical test and are indicated by asterisks (*) for P < 0·05. Comparisons between non‐infected control and dengue groups were performed by Kruskal−Wallis test and are indicated by the hash (#) sign for P < 0·05. N was 20–22 for mild dengue and 14–16 for ws+/severe dengue cases during the critical phase, and 19–20 for mild dengue and 4–5 for ws+/severe dengue cases during convalescence.

Individuals with mild dengue display higher frequencies of non‐structural protein 3 (NS3)‐specific triple producer CD4⁺ and CD8⁺ T‐cells. Peripheral blood mononuclear cells (PBMCs) from dengue virus (DENV)‐infected patients with mild and severe clinical forms were stimulated with peptide libraries of envelope (ENV; a–d) and NS3 (e–h) during critical (top) and convalescence (bottom) phases of infection. Frequencies of interferon gamma (IFNγ)/tumor necrosis factor (TNF)/interleukin (IL)10‐producing CD4⁺ and CD8⁺ T‐cells were evaluated by flow cytometry in CD3⁺ gated cells. The horizontal lines represent geometric mean. The dotted lines represent geometric mean of the non‐infected control group (N = 11–14). Differences between mild dengue and ws+/severe groups were analyzed by Mann−Whitney statistical test and are indicated by asterisks (*) for P < 0·05. Comparisons between non‐infected control and dengue groups were performed by Kruskal−Wallis test and are indicated by the hash (#) sign for P < 0·05. N was 20–22 for mild dengue and 14–16 for ws+/severe dengue cases during the critical phase, and 19–20 for mild dengue and 4–5 for ws+/severe dengue cases during convalescence.

The mild dengue group presented higher frequencies of DENV‐specific T‐cell double producers of IFNγ and IL10 when compared with controls and the ws+/severe group during critical (ENV‐ and NS3‐specific; Fig. 2a,b,e,f) and convalescence (ENV‐specific) phases (Fig. 2c,d). Although not different from uninfected controls, NS3‐specific IFNγ⁺IL10⁺ CD4⁺ T‐cells were found more frequently in the mild dengue group when compared with ws+/severe patients (Fig. 2e). We also could observe a consistent population of IFNγ⁺TNF⁺ CD8⁺ T‐cells responding to ENV and NS3 during the critical phase in the mild group when compared with ws+/severe patients (Fig. 2b,f), a difference that faded in the convalescence phase. In addition, despite no difference between the mild dengue and ws+/severe dengue groups, frequencies of ENV‐specific IFNγ⁺TNF⁺ CD4⁺ T‐cells were also found to be increased over non‐infected controls during critical phase, for mild dengue group (Fig. 2a), and convalescence, for both mild and ws+/severe groups (Fig. 2c). Furthermore, ws+/severe dengue patients showed decreased frequencies of ENV‐ and NS3‐specific T‐cell double producers of TNF and IL10 when compared with mild dengue and control subjects during convalescence (Fig. 2c,d,g,h). In addition, NS3‐specific, but not ENV‐specific, T‐cell triple producers of IFNγ, TNF and IL10 were found increased in the mild dengue group when compared with controls and ws+/severe dengue at both critical (CD8⁺ T‐cells; Fig. 3g) and convalescence (CD4⁺ and CD8⁺ T‐cells) phases (Fig. 3f,h). Interestingly, our data indicate that NS3 seems a better stimulator of multifunctional T‐cell responses. For example, CD4⁺ T‐cells were able to respond to NS3 with double and triple multifunctional T‐cells, while ENV stimulation could marginally induce these types of responses (Fig. S6).

In order to investigate if these multifunctional T‐cells were associated with objective clinical evolution parameters, we investigated the correlation between the frequencies of T‐cell populations and the lowest levels of platelets measured during the critical phase. Serial platelet measurements during the critical phase were available for most of the inpatients and few outpatients as Brazilian Health Ministry guidelines do not require serial platelet measurement for mild dengue cases. From the available data, we could observe that, during the critical phase, the frequencies of NS3‐specific IFNγ⁺IL10⁺ and IFNγ⁺TNF⁺ producer CD8⁺ T‐cells and triple cytokine producer CD4⁺ and CD8⁺ T‐cells responding to either ENV or NS3 showed positive correlations with the nadir platelet levels observed (Table 2), suggesting a role of these cell populations in clinical evolution.

Table 2.

Correlation of T‐cell frequencies with nadir platelet levels

Cytokine production	Spearman rank r (P)
	ENV‐specific		NS3‐specific
	CD4⁺	CD8⁺	CD4⁺	CD8⁺
IFNγ only	ns	ns	ns	ns
TNF only	ns	ns	ns	ns
IL10 only	ns	ns	ns	ns
IFNγ⁺IL10⁺	ns	ns	ns	0·51 (0·037)
IFNγ⁺TNF⁺	ns	0·62 (0·005)	ns	0·58 (0·010)
TNF⁺IL10⁺	ns	ns	ns	ns
IFNγ⁺TNF⁺IL10⁺	0·47 (0·038)	0·50 (0·029)	0·71 (0·001)	0·53 (0·018)

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ns, not significant.

Multifunctional T‐cells produce higher amounts of IFNγ, TNF and IL10

To gain insight about the ability of multifunctional T‐cells to produce effector cytokines, we compared the mean fluorescence intensity (MFI) of each cytokine in T‐cells producing single or multiple cytokines. Triple producer T‐cells often showed the highest MFI of each cytokine when compared with single or double producers (Figs 4 and S8). For example, the MFI of IFNγ displayed by ENV‐specific IFNγ⁺TNF⁺IL10⁺ CD8⁺ T‐cells was 9739, while IFNγ⁺TNF⁻IL10⁻ CD8⁺ T‐cells presented 872·6, IFNγ⁺TNF⁺IL10⁻ CD8⁺ T‐cells were 1629, and IFNγ⁺TNF⁻IL10⁺ CD8⁺ T‐cells presented 2986 (Fig. 4a). Similar results were observed for TNF‐ and IL10‐producing T‐cells (Fig. 4b,c, respectively), as well as for production of IFNγ, TNF and IL10 by NS3‐specific T‐cells (Fig. 4a–c). The few multifunctional cells found in the control non‐exposed group and in the ws+/severe dengue group, when detected, were also more efficient in producing cytokines (Fig. S8).

Multifunctional T‐cells produce more interferon gamma (IFNγ), tumor necrosis factor (TNF) and interleukin (IL)10 than single producer T‐cells. Peripheral blood mononuclear cells (PBMCs) from mild dengue patients were stimulated with peptide libraries for envelope (ENV; top) and non‐structural protein 3 (NS3; bottom) during the acute phase of infection. Mean fluorescence intensity (MFI) of IFNγ (a) by single (IFNγ⁺TNF⁻IL10⁻), double (IFNγ⁺TNF⁺IL10⁻ and IFNγ⁺TNF⁻IL10⁺), triple (IFNγ⁺TNF⁺IL10⁺) T‐cell producers, TNF (b) by single (IFNγ⁻TNF⁺IL10⁻), double (IFNγ⁺TNF⁺IL10⁻ and IFNγ⁻TNF⁺IL10⁺), triple (IFNγ⁺TNF⁺IL10⁺) T‐cell producers, or IL10 (c) by single (IFNγ⁻TNF⁻IL10⁺), double (IFNγ⁺TNF⁻IL10⁺ and IFNγ⁻TNF⁺IL10⁺), triple (IFNγ⁺TNF⁺IL10⁺) T‐cell producers cells were evaluated by flow cytometry in CD3⁺CD4⁺ and CD3⁺CD8⁺ gated cells. Differences between groups were analyzed by Kruskal−Wallis test when comparing single, double and triple producers, and are indicated by asterisks (*) for P < 0·05. Analysis included only cytokine‐positive T‐cell populations, and N was, respectively, for CD4⁺ and CD8⁺ T‐cells, 21 and 20 for single producers, 18 and 20 double producers, and 13 and 10 for triple producers.

Discussion

Dengue virus infection resistance is highly associated with a robust and sustained production of IFNγ. ³⁶ Although there are few reports analyzing multifunctional T‐cells in dengue, IFNγ⁺TNF⁺IL2⁺‐producing CD4 T‐cells seem to be associated with a robust response to live‐attenuated dengue vaccine. ³⁰ , ³¹ , ³⁷ In this report, we analyzed the composition and quality of CD4⁺ and CD8⁺ T‐cell responses against DENV during natural infection. We found that T‐cells producing only IFNγ are present in similar percentages during both mild and severe disease presentations. Strikingly, the most significant differences in T‐cell populations between mild dengue and ws+/severe dengue patients were found in the double (IFNγ⁺TNF⁺, IFNγ⁺IL10⁺ and TNF⁺IL10⁺) and triple (IFNγ⁺TNF⁺IL10⁺) T‐cell producers, which were often increased in individuals developing mild forms of dengue and rarely upregulated in relation to background in the ws+/severe group.

Multifunctional IL10⁺Th1 or IL10⁺Tc1 cells are able to produce sustained and controlled levels of IFNγ contributing to host resistance to several infections. ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ³⁸ These cells not only contribute to a better effective control of pathogens, but also are able to limit immunopathology. For example, IFNγ/IL10‐producing CD8⁺ T‐cells are absent in mice susceptible to Trypanosoma cruzi infection, ²⁴ , ²⁵ which develop higher levels of parasitemia and increased pathology. Similarly, IL10 production by Th1 cells was found to display potent effector function against Toxoplasma gondii, ²³ contributing to parasite control. These cells were also found to contribute to virus control and limit immunopathology in the lungs of mice infected with influenza virus ²⁶ and in the brain of mice infected with coronavirus. ³⁸ Despite low frequencies of these cells during effector immune response, they are considered very important to promote effector mechanisms against infection and to contain hyperinflammation during disease evolution minimizing pathology. ³⁹

Dengue virus‐specific T‐cells producing IFNγ and TNF can be found in human ⁹ , ²⁹ , ³⁰ , ³¹ , ³⁷ and experimental ⁴⁰ dengue infection. Interestingly, IFNγ⁺TNF⁺ CD8⁺ T‐cells were also found to be increased in patients with a history of Hemorrhagic Dengue, ⁹ suggesting a deleterious role of these cells. On the other hand, this T‐cell population was found to be induced by different immunogenic live‐attenuated dengue vaccines. ³⁰ , ³¹ , ³⁷ In our cohort, we observed that IFNγ⁺TNF⁺ CD8⁺ T‐cell populations were increased in individuals developing mild dengue infection in response to structural (ENV) or non‐structural (NS3) proteins when compared with more severe forms of disease. In addition, the frequency (or amount) of CD8⁺ T‐cells positive for IFNγ and TNF, i.e. IFNγ⁺TNF⁺ and IFNγ⁺TNF⁺IL10⁺ populations, demonstrated positive correlation with higher platelet levels, suggesting association with resistance to DENV infection. Interestingly, Hatch et al. ⁵ found that higher frequencies of T‐cells producing IFNγ, TNF or IL2 were associated with asymptomatic dengue infection, but they did not find multifunction in their observation. Possibly, the type of antigen stimulation, peptide library in the present work versus inactivated virus culture, may have had an impact on the ability to detect multifunctional T‐cells.

The role of IL10 in dengue infection has been a matter of much debate. While some reports associate IL10 with favorable evolution of infections in general, including dengue, ¹⁸ , ³⁹ , ⁴¹ others find IL10 associated with impaired immune responses in dengue and hemorrhagic manifestations. ¹⁶ , ⁴² , ⁴³ , ⁴⁴ We believe that the cell source of IL10 may have important implications in these divergent findings. Most of the evidences linking IL10 to detrimental dengue evolution are from reports that evaluate its levels in plasma during very early phase (mostly febrile period) of infection and, importantly, do not assess the source. On the other hand, most of the evidences linking IL10 to a favorable evolution of infectious diseases evaluate T‐cell‐derived IL10. In our work, we focused on T‐cell‐derived IL10, especially when associated with effective immune response against DENV infection.

T‐cells producing simultaneously IL10 and IFNγ are of especial interest because they have been associated with increased resistance to viral or protozoal infections associated with decreased infection‐related pathology. ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ³⁸ However, a recent observation of an IFNγ⁺IL10⁺ CD4⁺ T‐cell DENV‐specific subpopulation has found them more frequently in severe dengue cases. ³² Interestingly, we found this DENV‐specific CD4+ T‐cell population increased in mild dengue patients, but this population did not show correlation with platelet levels. In contrast, we also found that mild forms of dengue presented higher frequencies of not only IFNγ⁺IL10⁺ CD4⁺ and CD8⁺ T‐cells, but also T‐cells TNF⁺IL10⁺, IFNγ⁺TNF⁺ and IFNγ⁺TNF⁺IL10⁺, especially responding to NS3 antigen, many of which showed positive correlation with platelet levels. Our data show some fluctuations regarding the differences between mild and ws+/severe dengue groups according to the time of infection. While some double producer T‐cell populations were upregulated in mild forms during the critical phase, others were upregulated in the circulation only during convalescence. These data suggest a dynamic T‐cell homeostasis along the infection. Noteworthy, the IFNγ, TNF and IL10 triple producer NS3‐specific T‐cells were observed in our cohort in low frequencies in both groups during the critical phase, but in higher frequencies, up to seven times, in individuals with mild dengue when compared with individuals with ws+/severe dengue. This cell population decreased in the convalescence phase, especially in the ws+/severe dengue group. In convalescence, patients with mild dengue presented 17–22 times more triple producer NS3‐specific CD4⁺ or CD8⁺ T‐cells, suggesting that mild dengue individuals are able to develop and maintain memory of this cell population.

Interleukin 10 production has been widely reported to control the levels of cytokine production, including IFNγ, but few studies have focused on the ability of cytokine production by IL10⁺ multifunctional T‐cells. On the other hand, it is commonly observed that IFNγ and IL10 double producers display MFI in the highest fluorescence spectrum during intracellular staining. ²⁶ We found that double producers are more efficient cytokine producers than simple producers, and triple producers presented the highest level of intracellular cytokine accumulation. That was true even for the background producers in the control group. We speculate that the DENV response in the control group might be associated with some level of cross‐reactivity between Yellow Fever vaccine, widely used in Brazil, and DENV epitopes, but additional studies are needed to confirm this hypothesis. Nevertheless, we do not believe that such cross‐reactivity may compromise our conclusions, as multifunctional T‐cells are regarded as better cytokine producers in other models. For example, others have demonstrated that IFNγ⁺TNF⁺IL2⁺ multifunctional T‐cells, although in lower numbers, also produce higher levels of cytokines. ⁴⁵ , ⁴⁶ Recently, the biology of IL2⁺ multifunctional T‐cells has been thoroughly investigated, and it was found that their lifetime of multiple cytokine production was shorter than the lifetime of single cytokine producers, but the balance between multiple and single producers was very dynamic with interchanging conversion. ⁴⁷ Similar analysis has not been performed by others for IL10⁺ multifunctional T‐cells, but these data are coherent with our findings that show a decline in frequencies of multifunctional T‐cells from critical to convalescent phases. Interestingly, we did not find major differences in the intracellular accumulation of cytokines when comparing a specific T‐cell subpopulation between mild dengue and ws+/severe patients. This observation suggests that once differentiated, the biology of multifunctional T‐cells may be similar even between different patient groups. The difference between the groups, therefore, seems to be the ability to generate higher frequencies of multifunctional T‐cell populations, especially IFNγ⁺IL10⁺ and IFNγ⁺TNF⁺ CD8⁺ T‐cells, and IFNγ⁺TNF⁺IL10⁺ CD8⁺ and CD4⁺ T‐cells, which displayed positive correlation with platelets. The mechanisms underlying the difference between mild and ws+/severe dengue groups to produce multifunctional T‐cells demand further investigation.

Our findings that multifunctional T‐cells produced more cytokines and were present preferentially in the mild dengue group seems to create tension with the cytokine storm, a high level of cytokine in the blood like TNF, sTNFR, IL2, MIF, IFNγ, and other pro‐inflammatory cytokines, often observed in individuals with complicated dengue. ¹⁰ , ¹⁴ , ⁴³ , ⁴⁸ However, the source of such massive production of cytokine remains elusive. On the other hand, the majority of the data regarding the physiology of T‐cells in dengue infection suggest a protective role, ⁵ , ⁷ , ³⁰ , ³¹ , ⁴⁹ , ⁵⁰ , ⁵¹ , ⁵² but the innate source of this cytokinemia has been poorly investigated. Some data suggest that mast cells ⁵³ , ⁵⁴ , ⁵⁵ and macrophages, ⁵⁶ , ⁵⁷ for example, may contribute to early cytokine production and also to severe dengue presentation. On the other hand, NK cells were found to produce cytokines associated to the cytokine storm, like IFNγ but, while some reports show that NK cells might be associated with resistance to DENV infection, ⁵⁸ , ⁵⁹ , ⁶⁰ NKT cells might be associated with disease severity. ⁶¹ , ⁶² We believe that pathogen‐specific lymphocytes, by acting in a specific manner (antigen‐presenting cells displaying pathogen antigens or infected non‐immune cells), may provide precise and controlled efficacious response to infection. It has been observed that, despite some leakage, most of the cytokines produced by T‐cells will be channeled to the immunological synapse and act primarily and preferentially on the target cells. ⁶³ , ⁶⁴ Conversely, innate immunity sources of IL10, IFNγ or TNF may cause increased collateral damage as their production is not driven by antigen‐specific and target cell‐oriented mechanisms. We have not investigated innate sources of these cytokines, but we do not discharge a role of cytokines coming from innate cells, especially IL10, in ws+/severe dengue, which will demand further investigation to establish.

An important limitation of this study is the reduced number of patients, especially in the ws+/severe dengue group in the convalescent phase. However, despite a weakened statistical analysis at this time point, the main findings are still validated by the consistent differences we found from the IFNγ/IL10 and IFNγ/IL10/TNF double and triple CD4⁺ and CD8⁺ T‐cell producers during critical or convalescent phases from the mild dengue group when compared with the ws+/severe group and also from controls. In addition, the correlations of these populations and platelets indicate a beneficial role of these cells during active dengue infection. Another limitation of this study is that most of our volunteers have a positive history of vaccination for Yellow Fever, which could impact the background levels, especially in the non‐infected group, due to possible cross‐reactive clones. ⁶⁵ Importantly, the cells associated with our main findings, DENV‐specific IL10⁺Th1 (IFNγ⁺IL10⁺TNF^± CD4⁺ T‐cells) and especially IL10⁺Tc1 (IFNγ⁺IL10⁺TNF^± CD8⁺ T‐cells), were statistically increased in mild dengue patients when compared with ws+/severe patients and with non‐infected controls.

This paper shows that multifunctional T‐cells producing IFNγ, TNF and IL10 are present during mild forms of dengue disease. We found a strong association between high frequencies of DENV‐specific IFNγ production by T‐cells, especially when combined with TNF and/or IL10, and mild clinical forms of dengue. These data agree with the literature that IFNγ is necessary for resistance against DENV infection. ³⁶ Importantly, double (IFNγ⁺TNF⁺, IFNγ⁺IL10⁺ and TNF⁺IL10⁺) and markedly triple (IFNγ⁺TNF⁺IL10⁺) cytokine producer CD4⁺ and CD8⁺ T‐cells seem to be important effector players of immune response against DENV as they seem able to produce very high levels of IFNγ, TNF and IL10. These results suggest that type 1 pro‐inflammatory response associated with a regulatory response provided by IL10, produced by multifunctional T‐cells, contributes to an appropriate evolution of dengue infection, possibly balancing effector mechanisms against the virus and preventing pathology.

Author contributions

Conceived and designed the experiments: HCS, LRVA and MHGP. Performed the experiments: MHGP, MMF and CPQ. Analyzed the data: MHGP, HCS and LRVA. Volunteer recruitment and clinical evaluation: HCS, MHGP, TVB, KJG, ULC, AM and LMOD. Wrote the paper: HCS and MHGP. All authors revised and approved the manuscript.

Funding

This work was supported by the CNPq, FAPEMIG, INCT‐Vacinas, the Program for Technological Development in Tools for Health–PDTIS‐FIOCRUZ (Flow Cytometry Platform) and Flow Cytometry core facility of the Pharmacy School of UFMF.

Disclosure

The authors declare no conflict of interest.

Supporting information

Figure S1. Cytokine panel gate strategy.

Figure S2. Dot plots representative for cytokines production by CD4⁺ and CD8⁺ T‐cells in the presence of ENV or NS3 peptide library stimulation.

Figure S3. Dengue patients show poor cytokine production by single producers CD4⁺ T lymphocytes in the presence of DENV‐specific peptides.

Figure S4. Mild dengue patients showed IFNγ and IL10 production by DENV‐specific CD8⁺ T lymphocytes in convalescence phase.

Figure S5. Dot plots representative for double production of cytokines by CD4⁺ and CD8⁺ T‐cells in the presence of ENV or NS3 stimuli.

Figure S6. Production of cytokines by polyfunctional DENV‐specific CD4⁺ T lymphocytes from mild dengue patients at defervescence phase.

Figure S7. Production of cytokines by polyfunctional DENV‐specific CD8⁺ T lymphocytes from mild dengue patients at defervescence phase.

Figure S8. The multifunctional T‐cells produce more cytokines than double and simple producer T‐cells.

Click here for additional data file.^{(3.2MB, pdf)}

Table S1. Oligo primers and probes.

Table S2. Sequence of peptides in each library.

Click here for additional data file.^{(186.2KB, pdf)}

Acknowledgements

The authors are grateful to study participants and healthy volunteers for their participation. The authors thank Ana Beatriz Ribeiro de Queiroz, Lorena Junia de Souza Santos, Pedro Augusto Carvalho Costa, Sarah Carvalho Pains, Suemara Fernandes S. Martins and Rafael de Paula Paschoalino for technical assistance. The authors also thank Laila Sampaio Horta, Michele Faria Ramos and Kênia Cristina Magalhães for their help in recruiting volunteers. MHGP and CQP are graduate students of the Programa de Pós‐Graduação em Bioquímica e Imunologia from UFMG. HCS, LRVA and KJG are CNPq fellows (PQ).

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59. Costa VV, Ye W, Chen Q, Teixeira MM, Preiser P, Ooi EE et al Dengue virus‐infected dendritic cells, but not monocytes, activate natural killer cells through a contact‐dependent mechanism involving adhesion molecules. mBio 2017; 8: e00741‐17. [DOI] [PMC free article] [PubMed] [Google Scholar]
60. Laoprasopwattana K, Libraty DH, Endy TP, Nisalak A, Chunsuttiwat S, Ennis FA et al Antibody‐dependent cellular cytotoxicity mediated by plasma obtained before secondary dengue virus infections: potential involvement in early control of viral replication. J Infect Dis 2007; 195:1108–16. [DOI] [PubMed] [Google Scholar]
61. Matangkasombut P, Chan‐In W, Opasawaschai A, Pongchaikul P, Tangthawornchaikul N, Vasanawathana S et al Invariant NKT cell response to dengue virus infection in human. PLoS Negl Trop Dis 2014; 8:e2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
62. Renneson J, Guabiraba R, Maillet I, Marques RE, Ivanov S, Fontaine J et al A detrimental role for invariant natural killer T cells in the pathogenesis of experimental dengue virus infection. Am J Pathol 2011; 179:1872–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
63. Calvo V, Izquierdo M. Imaging polarized secretory traffic at the immune synapse in living T lymphocytes. Front Immunol 2018; 9:684. [DOI] [PMC free article] [PubMed] [Google Scholar]
64. Sanderson NS, Puntel M, Kroeger KM, Bondale NS, Swerdlow M, Iranmanesh N et al Cytotoxic immunological synapses do not restrict the action of interferon‐gamma to antigenic target cells. Proc Natl Acad Sci USA 2012; 109:7835–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
65. Kurane I, Brinton MA, Samson AL, Ennis FA. Dengue virus‐specific, human CD4+ CD8‐ cytotoxic T‐cell clones: multiple patterns of virus cross‐reactivity recognized by NS3‐specific T‐cell clones. J Virol 1991; 65:1823–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Cytokine panel gate strategy.

Figure S2. Dot plots representative for cytokines production by CD4⁺ and CD8⁺ T‐cells in the presence of ENV or NS3 peptide library stimulation.

Figure S3. Dengue patients show poor cytokine production by single producers CD4⁺ T lymphocytes in the presence of DENV‐specific peptides.

Figure S4. Mild dengue patients showed IFNγ and IL10 production by DENV‐specific CD8⁺ T lymphocytes in convalescence phase.

Figure S5. Dot plots representative for double production of cytokines by CD4⁺ and CD8⁺ T‐cells in the presence of ENV or NS3 stimuli.

Figure S6. Production of cytokines by polyfunctional DENV‐specific CD4⁺ T lymphocytes from mild dengue patients at defervescence phase.

Figure S7. Production of cytokines by polyfunctional DENV‐specific CD8⁺ T lymphocytes from mild dengue patients at defervescence phase.

Figure S8. The multifunctional T‐cells produce more cytokines than double and simple producer T‐cells.

Click here for additional data file.^{(3.2MB, pdf)}

Table S1. Oligo primers and probes.

Table S2. Sequence of peptides in each library.

Click here for additional data file.^{(186.2KB, pdf)}

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[imm13185-bib-0065] 65. Kurane I, Brinton MA, Samson AL, Ennis FA. Dengue virus‐specific, human CD4+ CD8‐ cytotoxic T‐cell clones: multiple patterns of virus cross‐reactivity recognized by NS3‐specific T‐cell clones. J Virol 1991; 65:1823–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

T‐cells producing multiple combinations of IFNγ, TNF and IL10 are associated with mild forms of dengue infection

Marcela Helena Gonçalves Pereira

Maria Marta Figueiredo

Camila Pereira Queiroz

Télcia Vasconcelos Barros Magalhães

Adriana Mafra

Lilian Martins Oliveira Diniz

Último Libânio da Costa

Kenneth J Gollob

Lis Ribeiro do Valle Antonelli

Helton da Costa Santiago

Summary

Abbreviations

Introduction

Materials and methods

Ethical statement

Clinical information, sample collection and processing

Dengue diagnostics, inclusion criteria and secondary infection

Peptide library

Immunophenotyping and intracellular cytokine assessment

Analyses of T‐cell subpopulations and cytokines production

Statistical analysis

Results

Study population

Table 1.

Mild dengue patients presented higher frequencies of DENV‐specific single functional T‐cells positive for TNF and IL10, but not IFNγ, when compared with ws+/severe dengue patients

Figure 1.

Individuals with mild dengue presented high frequencies of multifunctional T‐cells

Figure 2.

Figure 3.

Table 2.

Multifunctional T‐cells produce higher amounts of IFNγ, TNF and IL10

Figure 4.

Discussion

Author contributions

Funding

Disclosure

Supporting information

Acknowledgements

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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