A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology

Abstract

CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity.

Fig 1. Numerical dominance of K^dM2₈₂ T cell response is associated with expansion capacity.

(a) Magnitude of D^bM₁₈₇ and K^dM2₈₂ T cell response at 7 dpi. Frequency of D^bM₁₈₇ and K^dM2₈₂ T cells in CD8 T cell population were quantitatively assessed with flow cytometry. Data represent 5 independent experiments (n = 5/group/experiment). (b) The D^bM₁₈₇ and K^dM2₈₂ T cells infiltrate into lung parenchyma following RSV infection. Lung lymphocytes were isolated from RSV- infected mice at 7 dpi. T cells in vasculature were pre-labeled by intravenous anti-Thy1.2 staining prior to euthanizing mice. Proportion of D^bM₁₈₇ and K^dM2₈₂ T cells in lung parenchyma were quantitatively assessed with flow cytometry. Data represent 3 independent experiments (n = 5/group/experiment). (c) Lymphocytes isolated from spleens at 7 and 9 dpi were studied for Ki-67 expression by flow cytometry. The frequencies of Ki-67(+) cells in D^bM₁₈₇ and K^dM2₈₂ T cell subsets are shown. Data represent 3 independent experiments (n = 4/group/experiment). All data are shown as mean with independent data point and compared by Student t-test. Each symbol represents one mouse.

Fig 2. K^dM2₈₂ T cells dominate bulk cytotoxicity but D^bM₁₈₇ T cells have superior individual cytotoxicity.

(a) Bulk cytotoxicity of D^bM₁₈₇ and K^dM2₈₂ T cells in vivo. The epitope peptide-loaded and fluorochrome-labeled targets, as well as OVA₂₅₇ peptide-loaded and fluorochrome-labeled controls, were co-transferred into RSV-infected mice at 7dpi, and recovered 3 hours later. The recovery ratio were assessed with flow cytometry and compared with recovery ratio from naïve recipients to calculate epitope-specific lysis. Data represent 5 independent experiments (n = 5/group/experiment). (b) Cytotoxicity of individual D^bM₁₈₇ and K^dM2₈₂ T cells. Ratio of specific lysis of donor targets in (a) was divided by frequency of endogenous D^bM₁₈₇ and K^dM2₈₂ T cells respectively to quantitatively express arbitrary “Killing Unit” of individual cells. Data represent 5 independent experiments (n = 5/group/experiment). All data are shown as mean with independent data point and compared by Student t-test. Each symbol represents one mouse.

Fig 3. The D^bM₁₈₇ T cells express high avidity TCR and signaling pathways promoting cytotoxic function.

(a) The TCR avidity was assessed by dissociation of D^bM₁₈₇ and K^dM2₈₂ from CD8 T cells. CD8 T cells were labeled with pMHCs and assessed cell-bound median fluorescence intensity (MFI) at indicated time point by flow cytometry. The MFI at 0 min was defined as the maximum measurement (100%). Data were analyzed with one-phase exponential decay using nonlinear regression, and shown at mean ± SEM of three independent experiments (n = 5/group/experiment). (b) Transcriptional expression of genes that are up-regulated after RSV infection and associated with conventional signaling pathways. The D^bM₁₈₇, K^dM2_82, and bulk CD8 T cells were sorted from spleen lymphocytes by FACS at 7 dpi. The mRNAs were isolated, amplified and labeled, then hybridized onto Illumina Mouse Chips. The quantitative gene expression were analyzed and normalized. Genes with Log₂ Fold Change (FC) > 1.3, p < 0.05 and FDR < 0.25 (listed on left side of the chat) and associated signaling pathways were shown (Pathways 1: Altered T Cell and B Cell Signaling in Rheumatoid Arthritis; 2: T Helper Cell Differentiation; 3: Dendritic Cell Maturation; 4: Type I Diabetes Mellitus Signaling; 5: Roe of NFAT in Regulation of the Immune Response; 6: Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses; 7: IL-10 Signaling; 8: Role of CHK Proteins in Cell Cycle Checkpoint Control; 9: TREM1 Signaling; 10: Communication between Innate and Adaptive Immune Cells; 11: CD40 Signaling; 12: Production of Nitric Oxide and Reactive Oxygen Species in Macrophages; 13: Cell Cycle Control of Chromosomal Replication; 14: Acute Phase Response Signaling; 15: CD28 Signaling in T Helper Cells; 16: PKC Signaling in T Lymphocytes; 17: IL-12 Signaling and Production in Macrophages.). Data were pooled from 10 or 11 individual mice in each group.

Fig 4. The D^bM₁₈₇ T cells differentiate toward to an effector- and central- memory phenotype.

(a) Transcriptional expressions that are associated with cell differentiation were analyzed and normalized. Relative expression was calculated and presented as Log₂ Fold Change (Log₂FC). The Log₂FC = Log₂ K^dM2₈₂ –Log₂ D^bM₁₈₇. Positive values indicate a specific gene expression was up-regulated in the K^dM2₈₂ subset, while negative values indicate up-regulated gene expression in the D^bM₁₈₇ subset. Genes with expression differences of FC > 1.3, p < 0.05 and FDR < 0.25 are listed. Data were pooled from 10 or 11 individual mice in each group. (b) Post-transcriptional expression of CD62L and CD127. The CD62L(-)CD127(+) and CD62L(+)CD127(+) frequencies at 7 dpi were assessed by flow cytometry and are shown as mean with independent data point and compared by Student’s t-test. Data represent 5 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.

Fig 5. Activated K^dM2₈₂ T cells up-regulate expression of inhibitory receptors.

(a) Transcriptional expression of genes encoding inhibitory receptors with FC > ±1.3, p < 0.05 and FDR < 0.25 were listed. Data were pooled from 10 or 11 individual mice in each group. (b) Post-transcriptional expression of inhibitory receptors at 7 dpi was assessed by flow cytometry. The frequencies are shown as mean with independent data point and compared by Student’s t-test. Data represent 5 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.

Fig 6. Activated K^dM2₈₂ T cells are apoptotic.

(a) Transcriptional expression of genes encoding pro- and anti-apoptosis molecules with FC > ±1.3, p < 0.05 and FDR < 0.25 were listed. Data were pooled from 10 or 11 individual mice in each group. (b) Apoptotic cells were identified by Annexin V staining, and post-transcriptional expression of Bcl-2 was identified by monoclonal antibody with flow cytometry at 7 dpi. The frequencies are shown as mean with independent data point and compared by Student’s t-test. Data represent 3 or 4 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.

Fig 7. The D^bM₁₈₇ T cells efficiently control viral replication.

(a) Adoptive transfer of pMHC-specific donor cells increases precursor of CD8 T effector cells during early infection. Live D^bM₁₈₇, K^dM2₈₂ and bulk (with neither specificity) CD8 T cells from spleen lymphocytes of RSV-infected mice at 7dpi were sorted with FACS, and transferred into naive recipients respectively. The recipients were then challenged with RSV next day, and were evaluated for the donor D^bM₁₈₇ and K^dM2₈₂ T cell frequencies in the right lung at 4 dpi by flow cytometry. (b) Viral activity in RSV challenged recipients. Left lungs of the RSV-challenged recipients were assessed for virus replication. The virus titers are expressed as log₁₀ PFU/gram of lung tissue. Data are shown as mean with independent data point and compared by Student’s t-test. Data represent 3 independent experiments (n = 4 or 5/group/experiment). Each symbol represents one mouse.

Fig 8. The D^bM₁₈₇ T cells provide protection with limited pulmonary inflammation and illness of the recipients.

The D^bM₁₈₇, K^dM2₈₂ and bulk CD8 T cell recipients were challenged with RSV (a) Inflammatory cells were assessed at 7 dpi with flow cytometry. The absolute number and frequency of CD3 (+) cells and CD8 (+) T cells are expressed as mean with independent data point and are compared by Student’s t-test. Data represent 3 independent experiments (n = 5/group/experiment). Each symbol represents one mouse. (b) Pathology of lung. Left lung of the virus-challenged recipients were studied for histological change at 7 dpi. Microscopic study represents 3 independent experiments (n = 5/group/experiment). (c) Severity of illness. The virus-challenged recipients were weighed daily, from 0 to 7 dpi. Percentage represents retained proportion of pre-infection weight (mean ± SEM). Data represent 3 independent experiments (n = 5/group/experiment) and are compared by Student t-test.