Human T cell responses to Dengue and Zika virus infection compared to Dengue/Zika coinfection

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Abstract

 

Figure 1

Chemokine receptor expression on CD4⁺ and CD8⁺ T cell populations in acute DENV, ZIKV, and DENV/ZIKV patients. Peripheral blood mononuclear cells (2 × 10⁵) were stained with mAb against surface markers CD3, CD8, CD4, CCR5, CX3CR1, and CXCR3. The expression of CCR5, CX3CR1, and CXCR3 on CD4⁺ (a) and CD8⁺ T cells (e) was showed in contour plots from one representative exposed donor, DENV‐, ZIKV, and DENV/ZIKV‐patients by flow cytometry. Frequency, median, 25th and 75th percentile of CCR5⁺ (b), CX3CR1⁺ (c), and CXCR3⁺ (d) CD4⁺ T cells from acute viral patients were compared between them and with those in exposed healthy controls. The same strategy was used for CD8⁺ T cells (f–h). Patient 2 in late acute phase (22 days of illness) was show as open squares. Statistical significance of differences between groups was determined by using two‐tailed Mann–Whitney test, where p < 0.05 were considered significant (*p < 0.05; **p < 0.01).

CCR5, CX3CR1, and CXCR3 were detected in higher numbers of circulating CD8⁺ T cells in all three groups of infected patients in comparison to those exposed donors (Fig. ​(Fig.11f–h).

Therefore, regardless of acute viral infection, CD4⁺ and CD8⁺ T cells would be readily able to migrate and perform effector function.

Frequency of cytokine‐producing T CD4⁺ and CD8⁺ are differentially regulated in DENV, ZIKV, and DENV/ZIKV infection.

To assess the effector function of T cells by means of cytokine produced, we also evaluated the frequency of cells spontaneously producing TNF, IL2, and IFNγ.

As shown in Figure ​Figure2,2, even without antigenic in vitro stimulation, we observed low, but detectable, frequencies of IFNγ⁺ (0.1–1.6%, minimum to maximum), TNF⁺ (0.06–1.3%) and IL2⁺ (0.3–1.8%) producing T cells from acute patients and in healthy DENV‐exposed donors’ T cells (IFNγ, 0.1–0.6%; TNF, 0.1–0.7%; IL2, 0.5–4.9%).

 

 

 

Figure 2

Frequency of IFNγ‐, TNF‐, and IL2‐producing CD4⁺ and CD8⁺ T cells in acute DENV, ZIKV, and DENV/ZIKV‐patients and exposed donors. Cultures of 2 × 10⁵ PBMCs were stimulated with PMA/Ionomycin or unstimulated (medium) for 6 h in presence of brefeldin in the last 4 h. Then, cells were stained with mAb against surface markers CD3, CD8, CD4, and mAb against intracellular IFNγ, TNF, and IL2. Frequency of IFNγ, TNF, and IL2 on CD4⁺ (a) and CD8⁺ T cells (f) was exhibited in counter plots from one representative exposed donor, DENV‐, ZIKV, and DENV/ZIKV‐patients by flow cytometry for both conditions. Frequency, median, 25th and 75th percentile of IFNγ⁺ (b), TNF⁺ (c), and IL2⁺ (d) CD4⁺ T cells in unstimulated condition from acute viral patients were compared between them and with those in exposed healthy controls. The same strategy was used (g–i). Patient 2 in late‐acute phase (22 days of illness) was show as open squares. Bars represent the median of frequency of CD4⁺ T cells (e) and CD8⁺ T cells (j) expressing each of the seven possible combinations of IFNγ, TNF, and IL2 among the studied groups in unstimulated condition. Statistical significance of differences between groups and comparisons among the multifunctional populations were determined by using two‐tailed Mann–Whitney test and represented by lines. Values of p < 0.05 were considered significant (*p < 0.05; **p < 0.01).

Initially, we evaluated separately the frequency of IFNγ, TNF, IL2 producer CD4⁺ and CD8⁺ T lymphocytes, regardless of their simultaneous production (Fig. ​(Fig.2b–d,2b–d, g–i). First, we observed a trend toward an increased frequency of total IFNγ⁺CD4⁺ T cells in DENV (exception of patient 2, open squares) compared to exposed donors. DENV/ZIKV‐patients and exposed healthy donors had similar frequencies of total IFNγ⁺CD4⁺ T cells (Fig. ​(Fig.2b).2b). Exposed donors, DENV‐ and ZIKV‐patients had a similar frequency of total TNF⁺CD4⁺ T cells, while DENV/ZIKV‐patients had significantly decreased frequencies of total TNF⁺CD4⁺ T cells compared to exposed donors and DENV‐patients (Fig. ​(Fig.2c).2c). A trend toward decreased frequencies of total IL2⁺CD4⁺ T cells was seen in all acute patients compared to exposed donors (Fig. ​(Fig.22d).

Then, we evaluated the fractions of each multifunctional cell population expressing all three, any combination of two or single production of cytokines (Fig. ​(Fig.2e2e and j). Among T CD4⁺ lymphocytes, the least prevalent populations with two functions were IFNγ⁺TNF⁺ and IL2⁺TNF⁺ in DENV/ZIKV‐patients compared to exposed donors. Higher frequency of IFNγ⁺TNF⁺ CD4⁺ T cells was observed in DENV‐ compared to DENV/ZIKV‐patients. Finally, we observed increased frequency of single IFNγ⁺CD4⁺ T cell population in ZIKV‐patients compared to exposed donors (Fig. ​(Fig.22e).

A similar analysis was applied to CD8⁺ T cells. We detected a significant increase in total IFNγ⁺CD8⁺ T cells frequency in DENV/ZIKV‐patients compared to exposed healthy donors. A trend toward increased frequency of total IFNγ⁺CD8⁺ T cells was observed in ZIKV and in DENV patients (except for patient 2, open squares) (Fig. ​(Fig.2g).2g). Exposed donors, DENV, ZIKV, and DENV/ZIKV‐patients had similar frequency of total TNF⁺CD8⁺ T cells (Fig. ​(Fig.2h).2h). A decreased frequency trend of total IL2⁺CD8⁺ T cells, similarly to total IL2⁺CD4⁺ T cells, was detected in all acute patients compared to exposed donors (Fig. ​(Fig.2i).2i). Regarding the multifunctional analysis, we only detected a higher frequency of single IFNγ⁺CD8⁺ T cells in DENV‐ and DENV/ZIKV‐patients compared to exposed donors (Fig. ​(Fig.2e).2e). Therefore, we suggest that DENV/ZIKV coinfection may influence differently CD4⁺ and CD8⁺ T cells responses, an effect mainly observed in the frequencies of CD4⁺IFNγ⁺TNF⁺, CD4⁺TNF⁺IL2⁺, and CD8⁺ total IFNγ⁺ populations.

DENV‐specific response targeting NS1 proteins was rarely detectable in acute patients

We used DENV NS1 protein to evaluate DENV‐specific response in acute patients because ZIKV and DENV NS1 share 53–56% of amino acid identity 33. Moreover, NS1 has gained considerable attention for early dengue diagnostic tests. All healthy donors and 75% of acute patients in our cohort had indications of previous dengue, thus it was expected that they would be great responders of NS1 DENV. Considering this, we assessed DENV‐specific T cell response against a pool of NS1 from all four serotypes in PBMCs isolated from acute patients from DENV‐exposed donors by IFNγ ELISPOT assay (Fig. ​(Fig.33a).

 

Figure 3

DENV‐specific cells targeting DENV 1–4 NS1 protein in acute DENV, ZIKV, and DENV/ZIKV‐patients, and donors experiencing dengue. Peripheral blood mononuclear cells (2 × 10⁵ in 0.1 mL) were incubated with recombinant mammalian (rm) NS1 (0.1 μg/mL) derived from four different DENV serotypes or PHA for 20 h and IFNγ production was measured by ELISPOT assay. (a) Representative IFNγ production from one representative exposed donor, DENV, ZIKV, and DENV/ZIKV‐patients are shown against rmNS1. (b) The values obtained of IFNγ production, expressed as spot‐forming cells (SFC) relative to 10⁶ PBMC, is shown for the rmNS1 and PHA were compared between them and with those in exposed healthy controls. Graphs show the median, 25th and 75th percentile from acute‐patients and DENV‐exposed ZIKV naïve donors. Statistical significance was determined by using the two‐tailed Mann–Whitney test, where p < 0.05 were considered significant (*p < 0.05; **p < 0.01).

Our initial objective was to compare NS1 DENV‐specific memory response from dengue‐exposed donors with dengue‐infected individuals. Our data indicate that DENV NS1 memory response had the highest frequencies and magnitude in healthy dengue‐exposed donors compared to those with active DENV infection. Among DENV ingle‐infected patients, the 22‐days DENV patient 2 showed the best response to NS1, indicating that latter phases of dengue could be a better time point for evaluating specific cell responses through ELISPOT. Our second goal was to determine whether active ZIKV infection could affect the NS1 DENV‐specific memory response in two different situations: in DENV‐exposed patients or in those who have current ZIKV or DENV/ZIKV infections. Our data showed that DENV NS1 was not able to induce a response in any acute active ZIKV‐patients, even the samples being positive for Dengue IgG ELISA test. Two out five DENV/ZIKV‐patients responded with above 50 IFNγ SFC following DENV NS1 stimulation, while the other three responded with below 20 IFNγ SFC after stimulation (Fig. ​(Fig.3b).3b). Finally, the magnitude of IFNγ SFC response was not statistically different among the groups of acute patients. In order to understand the specificity of this response, we intend to evaluate healthy donors exposed to Zika in the future.

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Discussion

Although T lymphocytes are not infected by DENV and appear not to be infected by ZIKV 34, the impact of each one and of the DENV/ZIKV coinfection seem to affect their function. To address this investigation, we attempted to analyze some features of CD4⁺ and CD8⁺ T cell response in PBMC from patients in a cohort of DENV only, ZIKV only and DENV/ZIKV coinfected patients. Our work began with extensive fieldwork in the Midwest region of Brazil in 2016. There, 134 suspected cases of dengue or zika with an acute febrile illness were recruited, including those with by at least two of the signs and symptoms (headache, myalgia or arthralgia, rash, pruritus, retro‐orbital pain, and prostration). Methods of serology and molecular biology have been designed to diagnose the etiologic agent. Of the suspected cases, 6% of them confirmed ZIKV alone, 50% DENV, 22% were coinfected DENV/ZIKV, the others chikungunya, coinfections or not confirmed cases. At this point we realize that we did not have a larger number of samples with zika alone (Azeredo et al. data in submission). Therefore, we analyzed small numbers of individuals in each studied group to match ZIKV‐infected patients’ number, even though aware of the possible impact on dispersion of variables such as age, gender, and others because of a reduced number of donors. Despite that, we could report for the first time some aspects of DENV/ZIKV coinfection and compare then to DENV and ZIKV only infections, as well as healthy donors. Herein, our data reported from 17 well‐characterized patients infected by DENV, ZIKV or DENV/ZIKV and 4 healthy donors.

Our research evaluated the expression of chemokine receptors on T cells. Chemokine receptors have been useful for dissecting T‐cell subsets with distinct migratory capacity and effector function 35. We assessed the effector as well as memory T cells by an increased expression of CCR5, CX3CR1, or CXCR3, in contrast to their T naïve precursors. CCR5 and CXCR3 are closely linked to Th1 function on activated CD4⁺, memory/activated CD8⁺ T cells, and NK cells 36, 37, 38. Our data confirmed an increased expression of CCR5 on T cells from acute dengue patients 39, 40. CCR5 expression would be promoting an enhanced T cell recruitment into the liver, a hypothesis that was corroborated by a high frequency of CCL5⁺ cells in hepatic tissue from dengue fatal cases 40. It is widely accepted that CCR5 is part of host's immune response in the dengue, its role, mediating the traffic of immune cells from blood to target tissues or acting directly on the antiviral response, is still unknown. A recent study demonstrated CX3CR1‐based transcriptome and proteome‐profiling defined a core signature of memory CD8⁺ T cells with cytotoxic effector function 41. Highly polarized CX3CR1⁺ cytotoxic CD4⁺ T cells was specifically expanded in exposed donors and, in particular, in those donors carrying an HLA allele associated with protection from severe dengue 42. By the fact that an increased frequency of CD4 and CD8 T cells expressing CX3CR1 in patients with DENV and, for the first time, in patients with ZIKV and DENV/ZIKV, our data could indicate a potential cytotoxic capacity of T cells from these in acute patients. The protective role of CXCR3 against DENV infection has been demonstrated in experimental models since CXCR3^−/− mice infected by DENV presented a higher mortality rates than the wild‐type mice. Moreover, brains of CXCR3^−/− mice showed higher viral loads and quantitatively fewer CD8⁺ T cells than those of wild‐type mice 43. We hypothesize that an increase in the frequency CD4⁺ and CD8⁺ T cells expressing chemokine receptors would contribute to regulate virus progression through a precise control of inflammatory cells targeting the affected tissue. Thus, chemokine receptors would play a more immunoprotective role once T cells could exert an antiviral, effector, cytotoxic, and migratory activities.

The frequency of IFNγ‐producing T cells has been the most used parameter to assess an effective immune or vaccine‐induced response. Similarly to IFNγ, TNF is capable of mediating the killing of a variety of intracellular infectious viruses, bacteria, and parasites 44, 45. Although IL2 has little direct effector function, it promotes the expansion of CD4⁺ and CD8⁺ T cells, amplifying any potential effector T cell responses. Importantly, it has been demonstrated that frequency of cytokine‐producing T cells alone is not sufficient to predict protection. To provide prospective evidence of the quality of T cell response, vis‐à‐vis multifunctional T cells is required 23. Regarding dengue, compelling evidence of the importance of multifunctional T‐cells to mediate protection was found in Flavivirus‐naïve volunteers vaccinated with live, attenuated DENV‐1 vaccine, rDEN1Δ30. The authors observed that multifunctional T cells increased significantly in non‐viremic subjects tended to have a higher frequency of multifunctional T cells compared to viremic subjects. Therefore, the presence of multifunctional T cells following rDEN1Δ30 vaccine safely and effectively prompt immune responses associated with control of infection and protection from re‐infection 46.

Finally, we studied the magnitude of IFNγ response to DENV NS1. Gideon's team did not observe differences in IFNγ induction between peptide pools and recombinant proteins in overnight ELISPOT assay using PBMC from the same donors 47. A higher magnitude of IFNγ response to DENV NS1 was found in experienced dengue donors, but a hyporesponsiveness was found in any acute viruses, even experiencing dengue. In 2001, our group described a reduced frequency of CD4⁺ and CD8⁺ T lymphocytes and a poor ability for T‐lymphocyte proliferation in response to mitogens and dengue antigens in acute DENV‐infected patients, but re‐established in convalescence phase 48. Another study found that in vitro exposure to DENV‐2 of T lymphocytes isolated from healthy donors reduces the lymphoproliferative capacity in response to mitogen, suggesting that DENV‐2 can inhibit T cell‐mediated immunity, bypassing monocytes and dendritic cells, classical DENV cell targets 49.

Briefly, mono‐ and coinfections similarly induced expression of CCR5, CX3CR1, and CXCR3 on CD4⁺ and CD8⁺ T cells. This could promote functional and migratory similarities of these cells, regardless of the infecting virus. However, DENV/ZIKV coinfection decreased the ability of CD4⁺ T cells to produce IFNγ⁺, TNF⁺, TNF⁺IFNγ⁺, and TNF⁺IL2⁺, compared to monoinfections. We suppose two antagonistic scenarios: coinfected people are more immunocompromised than those monoinfected, so coinfection would be the worst scenario for those individuals. Another example of potential adverse effects of this immunosuppression is, that a reduced capacity to activate CD4⁺IFNγ⁺TNF⁺ and CD4⁺TNF⁺IL2⁺ T cell populations might interfere with future development of specific or cross‐reactive memory lymphocytes, leading to a weak response in a subsequent encounter to other potential arbovirus. In the other scenario, coinfected people have a less inflammatory milieu than monoinfected people. This could mean that a reduced inflammatory condition could avoid harmful effects like cytokine storm. Nevertheless, once the clinical outcome of the patients was similar, it was not possible to associate the symptoms and clinical signs with the immune profile of each group.

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Authors' Contributions

JBCdS, JCSA, ELdA, and LMdOP performed experiments, reviewed data, and planned the experimental strategy. MG collected data using a FACS ARIA BD flow cytometer. ABdP, JBCdS, TMAdSEP, LBdS, PCGN, and MdRQL performed all diagnostic tests. RVdC, JBCdS, TMAdS, LBdS, and ELdA collected samples and provided clinical information. JBCdS, JCSA, and LMdOP conceived and directed the study, and wrote the manuscript. All authors have critically read and edited the manuscript.

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Conflicts of Interest

The authors declare no conflicts of interest.

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Supporting information

Additional supporting information may be found in the online version of this article at the publisher's web‐site.

Table S1. Monoclonal antibodies used in this study.

Click here for additional data file.^{(13K, docx)}

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Acknowledgments

We would like to thank the Flow Cytometry and cell sorting core RPT08A and ELISPOT Plataform (PDTIS/FIOCRUZ) for antibody and acquisition support. We gratefully thank the voluntarily participation in this study of healthy individuals at the Clementino Fraga Filho University Hospital (Rio de Janeiro, Brazil) and patients from Walfrido Arruda Emergency Care Unit, Coronel Antonino (Mato Grosso do Sul, Brazil). Also, we thank to Physicians at the Walfrido Arruda Emergency Care Unit, Coronel Antonino evaluated clinical parameters and classified all infected‐patients according to WHO and Brazilian Ministry of Health. This work was financially supported by IOC/FIOCRUZ and FAPERJ. Juan Camilo Sánchez‐Arcila is recipient of Post‐doctoral fellowship FAPERJ E‐26/202.011/2016.

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Notes

Jessica Badolato‐Corrêa and Juan Camilo Sánchez‐Arcila contributed equally to this work.

Funding information

This work was financially supported by IOC/ FIOCRUZ and FAPERJ. Juan Camilo Sánchez‐Arcila is recipient of Post‐doctoral fellowship FAPERJ E‐26/202.011/2016.

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