Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity - PubMed (original) (raw)

Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity

Daniela Weiskopf et al. Proc Natl Acad Sci U S A. 2015.

Abstract

Dengue virus (DENV) is a rapidly spreading pathogen with unusual pathogenesis, and correlates of protection from severe dengue disease and vaccine efficacy have not yet been established. Although DENV-specific CD8(+) T-cell responses have been extensively studied, the breadth and specificity of CD4(+) T-cell responses remains to be defined. Here we define HLA-restricted CD4(+) T-cell epitopes resulting from natural infection with dengue virus in a hyperepidemic setting. Ex vivo flow-cytometric analysis of DENV-specific CD4(+) T cells revealed that the virus-specific cells were highly polarized, with a strong bias toward a CX3CR1(+) Eomesodermin(+) perforin(+) granzyme B(+) CD45RA(+) CD4 CTL phenotype. Importantly, these cells correlated with a protective HLA DR allele, and we demonstrate that these cells have direct ex vivo DENV-specific cytolytic activity. We speculate that cytotoxic dengue-specific CD4(+) T cells may play a role in the control of dengue infection in vivo, and this immune correlate may be a key target for dengue virus vaccine development.

Keywords: CD4; HLA DR; T cell memory; cytotoxic; dengue virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Subset-specific CD4 T-cell responses after in vitro restimulation were determined by HLA restriction and DENV infection history. PBMC were thawed, and CD4+ T cells were isolated by magnetic bead negative selection. CD45RA+ and CD45RA− cells were subsequently isolated by magnetic bead selection and cocultured with autologous APCs and with DENV-specific pools (averaging 20 peptides per pool). On day 14, cells were harvested and screened for reactivity against individual DENV-specific peptides in an IFN-γ ELISPOT. (A) Eight DENV-negative donors were stimulated with HLA-matched peptide pools and tested for reactivity against individual peptides. The reactivity of seronegative individuals that was >99% of all individual responses (SFC = 320; dashed line) was used to define a threshold value for positivity corresponding to a P value of 0.01 for false positives. (B_–_G) CD45RA+ (red bars) and CD45RA− (black bars) CD4+ T cells from donors expressing the DRB1*0701 (B, n = 4; and C, n = 7), DRB1*0401 (D, n = 5; and E, n = 6), or DRB1*0801 (F, n = 2; and G, n = 6) allele and have experienced either primary (1°) or secondary (2°) infection with DENV were stimulated with HLA-matched peptide pools and tested for reactivity against individual peptides. Error bars represent mean ± SEM. (H) The sum of responses adjusted by the number of donors tested for each HLA is shown as a function of infection history. Significance of CD45RA+ (red bars) and CD45RA− (black bars) responses was compared in a two-tailed Mann–Whitney test.

Fig. S1.

Fig. S1.

MHC binding of predicted peptides. Binding affinity of all predicted peptides to the respective HLA allele was measured by using a high-affinity radiolabeled peptide in a competition assay as described in Materials and Methods. The concentration of peptides yielding 50% inhibition of the binding of the radiolabeled peptide was calculated (IC50). The dashed line indicates the threshold of 1,000 nM, which corresponds to the biological threshold for efficient binding to a MHC class II molecules (24). Black bold lines indicate the geometric mean for all peptides tested for each allele (n = 132, 148, and 142 for DRB1*0401, *0702, and *0802, respectively).

Fig. 2.

Fig. 2.

DENV-specific responses and memory T-cell subsets change as a function of infection history and restricting HLA alleles. (A) PBMCs from the Sri Lanka cohort (n = 37) were stimulated with HLA-matched peptides for 6 h, and the IFN-γ responses were measured by ICS. Responses are shown as a function of the donors’ exposure to the dengue virus [DENV-negative (n = 4) and primary (1°; n = 11) and secondary (2°, n = 22) DENV infection]. (B) Representative staining of the memory CD4+ T cells subsets and IFN-γ–producing cells of a secondary donor is shown. Memory subsets were defined as naïve T cells (CCR7+CD45RA+), central memory T cells (CCR7+CD45RA+), and the two effector memory subsets (CCR7−CD45RA− and CCR7−CD45RA+), according to their expression of these markers. (C) The IFN-γ response of each memory T-cell subset was measured by ICS after stimulation with HLA-matched peptides (n = 23). (D) Representative FACS plots of CD4+ T-cell subsets in donors with a DENV-negative, (1°) primary or (2°) secondary DENV infection history are shown. (E_–_G) Distribution of CD4+ memory T-cell subsets in negative, primary, and secondary donors are shown (E, CCR7−CD45RA+; F, CCR7−CD45RA−; G, CCR7+CD45RA+, n = 28). (H) Percentage of total CD4+ T cells that produce IFN-γ upon stimulation with HLA-matched peptides in donors carrying the DRB1*0802, DRB1*0701, or DRB1*0401 allele. (I and J) The ability of CCR7−CD45RA+ (I) and CCR7−CD45RA− (J) subsets to produce IFN-γ upon peptide stimulation was measured for donors expressing DRB1*0802, DRB1*0701, and DRB1*0401 alleles (n = 24). (K and L) The size of the total CD4+ CCR7−CD45RA+ (K) and CCR7−CD45RA− (L) subsets were also measured for each allele. Error bars represent mean ± SEM. Statistical significance was determined by using the two-tailed Mann–Whitney test.

Fig. 3.

Fig. 3.

Expanded CD4 T-cell populations in DENV-exposed individuals are associated with a cytotoxic phenotype. (A_–_D) PBMC were stimulated with HLA-matched peptides for 6 h, and the IFN-γ responses were measured by ICS. CD4 effector memory cells (CCR7−CD45RA+ and CCR7−CD45RA−) were defined as Th1 (CXCR3+CCR4−CCR6−), Th2 (CXCR3−CCR4+CCR6−), Th17 (CXCR3−CCR4+CCR6+), Th1/17 (CXCR3+CCR4−CCR6+), and Tfh (CXCR5+) subsets, according to the expression of these surface markers. The chemokine receptor expression for IFN-γ–producing CCR7−CD45RA+ (A) and CCR7−CD45RA− (C) effector memory cells is shown (n = 5). The distribution of CD4+ Th subsets in DENV-negative (filled circles; n = 9) and donors experiencing secondary infection with DENV (2°; open triangles; n = 10) for the effector memory subsets CCR7−CD45RA+ (B) and CCR7−CD45RA− (D) is shown (n = 10). (E_–_J) PBMCs from donors who were found to be dengue-negative (neg.; filled circles) or having experienced a secondary infection (2°; DENV infection; triangles) were stained for memory markers, IFN-γ production, and markers for effector function. Shown are expression of the specific marker in CD4 T-cell subsets (Left), representative FACS plots (Center), and the expression in DENV-specific IFN-γ–producing CD4 T cells (Right). (E) CD226 expression was measured and compared among DRB1*04:01 secondary donors (n = 5), DRB1*08:02 secondary donors (n = 3), and DENV-negative donors (n = 5). Expression was compared between naïve cells and memory subsets, as well as between bulk CD4+ and IFN-γ–producing CD4+ T cells. Similar analysis was carried out for TIGIT (F), CD107a (G), perforin (H), granzyme B (I), and CD8α (J). (K) Transcription factors Tbet and Eomes were stained, and their joint expression was compared among CD4+ subsets, with representative plots shown. Error bars represent mean ± SEM. Statistical significance was determined by using the two-tailed Mann–Whitney test.

Fig. 4.

Fig. 4.

DENV-specific CD4+ T cells express CX3CR1 and mediate direct cytotoxic activity. (A) The relative expression of CX3CR1 was measured in the four T cells subsets in DRB1*0401 donors with a history of secondary DENV infection donors (n = 8). (B) A representative dot plot of total CD4+ (black dots) is overlaid with CX3CR1 expression (red dots). (C) CX3CR1 expression in total CD4+ T cells and DENV-specific IFN-γ–producing cells is shown (n = 5). (D) CX3CR1+ and naïve CD4+ T cells were sorted and cultured with peptide pulsed APCs overnight at 37 °C. Cells were then harvested, stained for HLA-DR, and analyzed by flow cytometry. The difference in absolute numbers of HLA-DR expressing APCs cocultured with either naïve CD4+ T cells or CXCR3+ cells was then expressed as percentage of killing (n = 3). Error bars represent mean ± SEM. Statistical significance was measured by using a two-tailed Mann–Whitney test. (E) Proposed model of cytotoxic CX3CR1+ T-cell differentiation. Naïve CD4+ T cells (CD45RA+CCR7+) get activated during primary infection with DENV (red hexagons) and differentiated into effector T cells (CD45RA−CCR7−). Upon continuous reexposure to heterologous DENV infections (yellow hexagons), effector memory T cells down-regulate CD28, CD45RO, CD127, and TIGIT. CD8α, CD57, CD107, and CD226, as well as perforin, granzyme B and the transcription factors T-bet and Eomes are up-regulated in highly differentiated effector memory T cells (CD45RA+CCR7−).

Fig. S2.

Fig. S2.

Further phenotypic characterization of CD4+ T-cell subsets. PBMCs from donors seronegative for DENV (neg; filled circles) and donors with neutralizing Ab patterns characteristic of multiple DENV infections (2°; open triangles) were stained with mAbs and analyzed by flow cytometry. CD4+ T cells were grouped according their expression of CCR7 and CD45RA. Expression levels for CD28-positive (A), CD57-positive (B), CD45RO-positive (C), and CD127-positive (D) cells in each subset were plotted. Statistical significance was measured by using the Mann–Whitney test. **P ≤ 0.01; ***P ≤ 0.001.

Fig. S3.

Fig. S3.

Phenotypic characterization of CMV-specific CD4+ T-cell subsets. (A) IFN-γ production upon peptide stimulation with CMV-specific peptides was measured for donors expressing DRB1*0401 alleles (n = 7). (B) Representative staining of the memory CD4+ T cells subsets and IFN-γ–producing cells donor is shown. Memory subsets were defined as naïve T cells (CCR7+CD45RA+), central memory T cells (CCR7+CD45RA+), and the two effector memory subsets (CCR7−CD45RA− and CCR7−CD45RA+), according to their expression of these markers. (C and D) The relative distribution of IFN-γ response between the memory T cells subsets (C) and subsets defined by chemokine receptor expression is shown (D). (E_–_K) Expression of CD226 (E), TIGIT (F), CD107a (G), perforin (H), granzyme B (I), CD8α (J), and CX3CR1 (K) was compared between bulk CD4+ and IFN-γ–producing CD4+ T cells. Error bars represent mean ± SEM. Statistical significance was determined by using the one-tailed Mann–Whitney test.

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