Lymphocytic choriomeningitis virus infection yields overlapping CD4+ and CD8+ T-cell responses

Abstract

Activation of CD4(+) T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4(+) T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2(d). This is quite disparate to the H-2(b) setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2(d) or whether additional CD4(+) T-cell epitopes could be identified in the setting of the H-2(b) background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4(+) T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4(+) epitopes, four of them also stimulate CD8(+) T cells in a statistically significant manner. Furthermore, we assessed these CD4(+) T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4(+) and CD8(+) T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.

FIG. 1.

Identification of LCMV-derived CD4⁺ epitopes by ELISPOT. C57BL/6 (H-2^b) mice were infected i.p. with 2 × 10⁵ PFU of LCMV Armstrong. Eight days postinfection, CD4⁺ T-cells were purified and tested against each of the eight peptides contained in each positive pool at a concentration of 10 mg/ml per peptide. Responses against individual peptides were considered positive if they exceeded the threshold of double the mean negative control wells (effectors plus APCs without peptide) and exceeded a threshold of 200 SFCs/10⁶ CD4⁺ cells. Each positive peptide is labeled by its proteomic location.

FIG. 2.

CD4⁺ versus CD8⁺ IFN-γ production as measured by intracellular cytokine staining. C57BL/6 (H-2^b) mice were infected i.p. with 2 × 10⁵ PFU of LCMV Armstrong. Eight days postinfection, splenocytes were purified and tested in ICCS assays measuring IFN-γ production and surface staining for CD4 (A) and CD8 (B) against 10 peptides identified as positive in the ELISPOT assays.

FIG. 3.

Identification of CD4⁺ responses after chronic infection. C57BL/6 (H-2^b) mice were infected retroorbitally with 2 × 10⁶ PFU of LCMV clone 13. Eight days postinfection, CD4⁺ T cells were purified and tested against each of the six epitopes at a concentration of 10 mg/ml per peptide. Responses against individual peptides were considered positive if they exceeded the threshold of double the mean negative control wells (effectors plus APCs without peptide) and exceeded a threshold of 200 SFCs/10⁶ CD4⁺ cells. Each peptide is labeled by its proteomic location.

FIG. 4.

Identification of CD4⁺ responses during memory stage and secondary infection of LCMV Armstrong. C57BL/6 (H-2^b) mice were infected i.p. with 2 × 10⁵ PFU of LCMV Armstrong. Thirty-five days postinfection, three mice from each group were used in ELISPOT assays and three mice from each group were challenged with a second (2°) infection of LCMV Armstrong. Eight days postinfection, CD4⁺ T cells were purified and tested against each of the six epitopes at a concentration of 10 mg/ml per peptide. Responses against individual peptides were considered positive if they exceeded the threshold of double the mean negative control wells (effectors plus APCs without peptide) and exceeded a threshold of 200 SFCs/10⁶ CD4⁺ cells. Each positive peptide is labeled by its proteomic location.