Immunodominant Dengue Virus-Specific CD8+ T Cell Responses Are Associated with a Memory PD-1+ Phenotype

Abstract

Dengue disease is a large public health problem that mainly afflicts tropical and subtropical regions. Understanding of the correlates of protection against dengue virus (DENV) is poor and hinders the development of a successful human vaccine. The present study aims to define DENV-specific CD8(+)T cell responses in general and those of HLA alleles associated with dominant responses in particular. In human blood donors in Nicaragua, we observed a striking dominance of HLA B-restricted responses in general and of the allele B*35:01 in particular. Comparing these patterns to those in the general population of Sri Lanka, we found a strong correlation between restriction of the HLA allele and the breadth and magnitude of CD8(+)T cell responses, suggesting that HLA genes profoundly influence the nature of responses. The majority of gamma interferon (IFN-γ) responses were associated with effector memory phenotypes, which were also detected in non-B*35:01-expressing T cells. However, only the B*35:01 DENV-specific T cells were associated with marked expression of the programmed death 1 protein (PD-1). These cells did not coexpress other inhibitory receptors and were able to proliferate in response to DENV-specific stimulation. Thus, the expression of particular HLA class I alleles is a defining characteristic influencing the magnitude and breadth of CD8 responses, and a distinct, highly differentiated phenotype is specifically associated with dominant CD8(+)T cells. These results are of relevance for both vaccine design and the identification of robust correlates of protection in natural immunity.

Importance: Dengue is an increasingly significant public health problem as its mosquito vectors spread over greater areas; no vaccines against the virus have yet been approved. An important step toward vaccine development is defining protective immune responses; toward that end, we here characterize the phenotype of the immunodominant T cell responses. These DENV-reactive T cells express high levels of the receptor programmed death 1 protein (PD-1), while those from disease-susceptible alleles do not. Not only does this represent a possible correlate of immunodominance, but it raises the hypothesis that PD-1 might be a regulator that prevents excessive damage while preserving antiviral function. Further, as this study employs distinct populations (Nicaraguan and Sri Lankan donors), we also confirmed that this pattern holds despite geographic and ethnic differences. This finding indicates that HLA type is the major determinant in shaping T cell responses.

FIG 1

HLA coverage, allele frequency, and DENV-specific CD8⁺ T cell reactivity in the Nicaraguan population. (A) The HLA frequency in the Nicaraguan population and percentage of responders for each HLA allele are shown. (B) The number of epitopes per allele and the total magnitude of exact HLA-matched IFN-γ responses (spot-forming cells [SFC]/10⁶ cells) are shown. The magnitude (C) and breadth (D) of responses were compared for HLA alleles that were expressed in both populations and for which responses were detected (n = 11 [A*02:01, A*02:06, A*03:01, A*24:02, A*33:01, A*68:01, B*07:02, B*15:01, B*35:01, B*40:01, and B*44:03]).

FIG 2

Phenotypic analysis of DENV-specific CD8⁺ T cells (A) Gating strategy for evaluation of IFN-γ-producing and tetramer-binding CD8⁺ T cells. (B) DENV-specific IFN-γ-producing CD8⁺ T cells were measured in donors expressing the B*35:01 and non-B*35:01 HLA alleles. (C) The IFN-γ response of sorted central (CCR7⁺ CD45RA⁺) and effector memory (CCR7⁻ CD45RA⁻ and CCR7⁻ CD45RA⁺) T cells was measured by ICS after DENV-specific peptide stimulation. (D) IFN-γ-producing cells were stained for phenotypic markers, including CD28, CD62L, and PD-1. (E) Binding of B*35:01- and A*24:02-restricted tetramers was evaluated in donors seropositive for DENV. (F) Tetramer binding in central memory and the two effector memory T cell subsets (as defined in panel B). (G) B*35:01 or A*24:02 tetramer-positive CD8⁺ T cells were stained for phenotypic markers, including CD28, CD62L, and PD-1.

FIG 3

Expression of PD-1 correlates with the magnitude of response. (A) Representative staining of PD-1⁺ and IFN-γ⁺ B*35:01 CD8⁺ T cells. (B) DENV-specific IFN-γ-producing PD-1⁺ cells for each individual donor plotted against the total DENV-specific IFN-γ⁺ CD8⁺ population. (C) Representative staining of PD-1⁺ and B*35:01 tetramer⁺ CD8⁺ T cells. (D) PD-1 expression on DENV-specific tetramer⁺ CD8⁺ T cells plotted against the total DENV-specific tetramer-positive CD8⁺ population.

FIG 4

PD-1-expressing cells express low levels of other coinhibitory molecules and retain proliferative capacity. (A) Peptide pools specific for DENV and EBV were used to stimulate donor PBMCs (at 1 μg/ml per individual peptide), and corresponding IFN-γ⁺ responses were characterized. IFN-γ⁺⁺ PD-1⁺ and IFN-γ⁺⁺ PD-1⁻ responses were analyzed for the coexpression of TIM3 (B), CTLA4 (C), KLRG1 (D), and 2B4 (E). The proliferative capacity of DENV-specific cells was assessed by expansion of tetramer-positive cells (F), expression of Ki-67 (G), and CFSE assays (H) after 6 days of stimulation with DENV-specific peptide pools. (I) PD-1 expression was measured on antigen-specific tetramer-positive CD8⁺ T cells in the presence or absence of antigen. (J) Expression of CD127 on tetramer-positive and -negative T_CM and T_EM/T_EMRA subsets. (K and L) PD-1⁺ and PD-1⁻ central memory (CCR7⁺ CD45RA⁻) and effector memory (CCR7⁻ CD45RA^+/−) cells were sorted and incubated with antigen-presenting cells labeled with DENV-specific peptides. The difference in absolute numbers of APCs cocultured with either of these subsets compared to naive T cells was then expressed as the percentage of cytolytic activity.