Chikungunya virus-specific CD4+ T cells are associated with chronic chikungunya viral arthritic disease in humans

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne virus that can cause chronic chikungunya virus disease (CHIKVD), which is characterized by persistent incapacitating arthralgia. Despite recurring CHIKV outbreaks and recent approval of a vaccine, the breadth and target of T cell responses in CHIKVD remain largely understudied. Here, we tested peripheral blood mononuclear cells (PBMCs) collected from CHIKV-infected individuals against overlapping peptide pools sequentially spanning the entire CHIKV proteome. We detected robust CHIKV-specific CD4⁺, but not CD8⁺, T cell responses in infected individuals. Individuals with chronic arthralgia displayed significantly higher CD4⁺ T cell responses against nsP1, nsP2, and E2 proteins and exhibited a significantly lower Th1 CD4⁺ T cell population, compared to individuals who had recovered. Additionally, CD4⁺ T cells in chronic individuals were marked by a predominant production of tumor necrosis factor alpha (TNF-α). Overall, our work comprehensively characterizes T cell responses in CHIKVD in humans and provides insights into the role of T cells in CHIKVD.

Keywords: CD4(+) T cells; T cells; chikungunya virus; chronic viral infection; human immunology.

Graphical abstract

Figure 1

CHIKV-specific T cell responses in CHIKV-seropositive donors (A) Schematic representation of CHIKV proteome comprising non-structural proteins (nsP1-4) and structural proteins (CP, E3, E2, 6K, and E1). (B) Antigen-specific CD4⁺ T cells quantified by the AIM (OX40⁺CD137⁺) assay after 24-h stimulation with all CHIKV protein megapools (MPs), Cytomegalovirus (CMV) MP, and tetanus-toxoid (TT) MP in CHIKV-seropositive donors (n = 31). The dotted line represents the limit of sensitivity (LOS; 0.02%). (C) Antigen-specific CD8⁺ T cells quantified by the AIM (CD69⁺CD137⁺) assay after 24-h stimulation with all CHIKV protein, CMV, and TT MPs in CHIKV-seropositive donors (n = 31). The dotted line represents the LOS (0.03%). (D) Frequency of AIM+ CD4⁺ T cell responses against all CHIKV proteins. (E) Summary of AIM+ CD4⁺ T cell responses per CHIKV protein MP and protein length. Black bars represent the percentage of magnitude of responses for each CHIKV protein MP; blue bars represent the percentage of responders (n = 31); gray bar represents the number of amino acids present in each CHIKV protein. (F) AIM+ CD4⁺ T cell responses associated with recent activation against CHIKV (blue dots), CMV (brown dots), and TT-specific (black dots). Recent activation was evaluated as the percentage of HLA-DR⁺CD38⁺ from AIM+ (OX40⁺CD137⁺) CD4⁺ T cells for individual samples with AIM > 0.1%. The dotted line indicates the threshold of reactivity (15%). For all graphs, each dot (n) represents the response of an individual donor to an individual MP. All samples were tested in individual experiments. % responders were calculated as the number of responses higher than the LOS divided by the total number of responses tested. Data are represented as mean ± SD or geomean ± geometric SD, as indicated. Data were analyzed for statistical significance using a Kruskal-Wallis test (p > 0.05, non-significant [ns]; ∗p < 0.05; ∗∗p < 0.01) for Figure 1F. See also Figures S1 and S2.

Figure 2

Immunodominance, recent activation, and memory phenotype of CHIKV-specific CD4⁺ T cells in symptomatic and recovered donors (A) Summary of frequency of CHIKV-specific CD4⁺ T cell response against all CHIKV protein MPs is shown for symptomatic (n = 170; blue dots) and recovered (n = 140; black dots) donors. The dotted line indicates the LOS (0.02%). (B) Frequency of CHIKV-specific CD4⁺ T cells is shown per protein in symptomatic (n = 17; blue dots) and recovered (n = 14; black dots) donors against each of the five non-structural (nsP1, nsP2_1, nsP2_2, nsP3, and nsP4) and five structural (CP, E3, E2, 6K, and E1) protein MPs. The dotted line indicates the LOS (0.02%). (C) Pie charts display the distribution of CHIKV-specific CD4⁺ T cell responses per protein for symptomatic (n = 17) and recovered (n = 14) donors. (D) Comparison of the percentage of CHIKV-specific CD4⁺ T cells with expression of recent activation markers (HLA-DR⁺CD38⁺) in symptomatic (n = 18) and recovered (n = 5) samples, where AIM+ CD4⁺ T cell responses >0.1% were used to evaluate recent activation. The dotted line indicates the threshold of reactivity (15%). (E) CHIKV-specific CD4⁺ T cell memory subsets have been defined in samples from the symptomatic (blue dots) and recovered (black dots) donors based on the expression of CCR7 and CD45RA in AIM+ CD4⁺ T cells as: T naive (CCR7⁺CD45RA⁺), TCM (T central memory; CCR7⁺CD45RA⁻), TEM (T effector memory; CCR7⁻CD45RA⁻), and TEMRA (T effector memory re-expressing CD45RA; CCR7⁻CD45RA⁺). Responses were only evaluated for samples that were AIM+ (OX40⁺CD137⁺). (F) CHIKV-specific CD4⁺ T helper cell (Th) subsets have been defined based on the expression of CXCR3 and CCR6 in AIM+ CD4⁺ T cells in samples from symptomatic (blue dots) and recovered (black dots) donors. Cells have been defined as Th1 (CCR6⁻CXCR3⁺), Th1/Th17 (CCR6⁺CXCR3⁺), Th17 (CCR6⁺CXCR3⁻), or Th2 (CCR6⁻CXCR3⁻) subsets. Responses were only evaluated for samples that were AIM+ (OX40⁺CD137⁺). For all graphs, each dot (n) represents the response of an individual donor to an individual MP. All samples were tested in individual experiments. The percentage of responders was calculated as the number of responses higher than the LOS divided by the total number of responses tested. Data are represented as mean ± SD or geomean ± geometric SD. CHIKV-specific CD4⁺ T cells were analyzed for statistical significance using Mann-Whitney U test (A, B, D, E, and F, p > 0.05. non-significant [ns]; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). See also Figure S2.

Figure 3

Cytokine profile of CHIKV-specific CD4⁺ T cell responses Cytokine expression in AIM+ donors against individual CHIKV protein (nsP1, nsP2_1, nsP2_2, CP, E2, and E1). n represents the number of donors tested, which include both symptomatic and recovered donors. The black bars represent the number of donors that tested positive, and the gray bars represent the number of donors that tested negative for specific cytokine. Positive response was defined by response > LOS (0.02%). CHIKV proteins that were screened more than five times are shown. See also Figure S2.

Figure 4

Multifunctional cytokine profile of CD4⁺ T cells in AIM+ donors (A) Frequency of cytokine+ CHIKV-specific CD4⁺ T cell after stimulation with all CHIKV proteins in samples from symptomatic donors (n = 58; blue dots) and recovered donors (n = 14; black dots). The dotted line indicates the LOS (0.002%). Boolean gating was used to determine the multifunctional capacity of CHIKV-specific CD4⁺ T cells. The pie chart on the right shows the distribution of single-, double-, and triple-positive cytokine-producing CHIKV-specific CD4⁺ T cells and the average percent of specific single-cytokine (IFNγ, TNF-⍺, IL-2, and IL-10)-producing CHIKV-specific CD4⁺ T cells. % positive was calculated as the number of responses higher than the LOS divided by the total number of responses tested. (B) Frequency of the cytokine+ CMV-specific CD4⁺ T cell multifunctional profile in samples from symptomatic donors (blue dots; n = 13) and recovered donors (black dots; n = 5). The dotted line indicates the LOS (0.002%). Boolean gating was used to determine the multifunctional capacity of CMV-specific CD4⁺ T cells. The pie chart on the right shows the distribution of single-, double-, and triple-positive cytokine-producing CMV-specific CD4⁺ T cells and the average percent of specific single-cytokine (IFNγ, TNF-⍺, IL-2, and IL-10)-producing CMV-specific CD4⁺ T cells. % positive was calculated as the number of responses higher than the LOS divided by the total number of responses tested. (C) Comparison between the frequency of single-cytokine-producing CD4⁺ T cells in response to all CHIKV protein MPs and CMV MPs for IFNγ, TNF-α, IL-2, and IL-10 (SPs, single producers) in symptomatic donors. For all graphs, each dot (n) represents the response of an individual donor to an individual MP. All samples were tested in individual experiments. For all graphs, data are represented as mean ± SD or geomean. Data were analyzed for statistical significance using Mann-Whitney U test. (p > 0.05, non-significant [ns]; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). See also Figure S3.