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

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.

Introduction

Respiratory syncytial virus (RSV) induces robust CD8 T cell responses that play a critical role in controlling virus replication and determining the progression of disease in animal models and infected humans. For example, autopsies of children with fatal RSV infections show a relative deficiency of CD8 T cell responses[1]; recipients of allogeneic bone marrow and lung transplants have difficulty in controlling RSV replication and often have fatal outcomes as a consequence of syncytium-forming pneumonia[2, 3]; and in patients with severe combined immunodeficiency (SCID), RSV infection results in persistent virus shedding that can be controlled with T cell reconstitution[4, 5]. Adoptive transfer of effector CD8 T cells can clear persistent RSV shedding in immunodeficient mice[6]; and depletion of T cells in mice results in persistence of the virus[7, 8]. However, the effect of CD8 T cell responses is not always beneficial. While T cell reconstitution reduces RSV load in SCID patients, it causes significant pulmonary inflammation[5]. In mice, passive transfer of a large amount of CD8 T cells in the setting of RSV infection results in hemorrhagic pneumonia[6], while depletion of CD8 T cells reduces disease severity[7]. Achieving an acceptable balance between protective immunity and immunopathology has been an elusive goal for RSV vaccine development and is a key objective for programs developing preventive and therapeutic strategies for pathogens requiring T cell-mediated immunity.

CD8 T cells are a heterogeneous population with phenotypically and functionally diverse subsets, and viral infection often induces a broad spectrum of CD8 T cell responses[9, 10]. Most studies report bulk CD8 T cell responses, or are focused on CD8 T cells targeting a relatively small number of epitopes. Those immunodominant epitopes are often discovered and selected as endpoints in evaluation of vaccination and immune intervention. Quantitative assessment of bulk CD8 T cell responses has shown little correlation with control of virus replication, and numerically subdominant T-cell responses have been demonstrated to play important roles in immunity against selected viral infections[11, 12], particularly in settings where multiple epitopes are recognized or where immune escape mutations are common[13]. Therefore, it is important to characterize CD8 T cell responses not only by magnitude, but also by functional properties, breadth, location, durability, and contribution to viral clearance and immunopathology.

We took advantage of the CB6F1 hybrid mouse model to characterize CD8 T cell subsets and their role in response to RSV infection. Although semi-permissive, mice infected by intranasal inoculation of RSV generate robust T cell and antibody responses, and pulmonary inflammation. The kinetics of virus replication and progression of the disease is highly reproducible[14-16]. In CB6F1 mice, the dominant CD8 T cell responses of each parent strain assume a distinct hierarchy, in which response to H2-Db-restricted M187-195 epitope (DbM187) is numerically subdominant to response to the H2-Kd-restricted M282-90 epitope (KdM282)[17]. The numerically dominant KdM282 T cell response lyses RSV-infected cells and reduces viral load, but is also associated with infection-induced immunopathology and severe illness[18, 19]. Mutating this epitope leads to reduced disease and pathology[20]. This observation suggested that this high magnitude and easily measurable response may do more harm than good, and that other subdominant responses could effectively clear virus with less immunopathology. Here, we defined the attributes of the numerically subdominant DbM187 T cell response that targets the RSV matrix protein. We compared the cytotoxicity and control of virus replication, as well as the associated lung pathology and illness to that associated with KdM282 T cells to help define properties that should be targeted by vaccines designed to elicit immunity mediated by CD8 T cells.

Results

RSV-specific CD8 T cell responses have epitope-dependent expansion capacity

Following RSV infection, expanded DbM187 and KdM282 T cells were quantified by fluorochrome-conjugated epitope peptide-MHC class I complexes (pMHCs) with flow cytometry (). We previously reported that DbM187 and KdM282 T cells assumed a numerical hierarchy in lung[17], the primary site of RSV infection. Here, we show a similar hierarchy in spleen at relatively low magnitude (, p < 0.001 and p = 0.001, DbM187 and KdM282 T cells respectively). Using a well-developed in vivo labeling procedure to distinguish cells in lung parenchyma from those in vasculature[21, 22], we found both DbM187 and KdM282 T cells preferentially infiltrated into lung tissue (). Since these two subsets have similar precursor frequency in naïve mice and similar infiltrating capacity into the inflammatory site, there are other factors that determine the hierarchy[23]. The Ki-67 expression showed that KdM282 T cells were more proliferative than DbM187 T cells (), consistent with the numerical dominance.

Numerical dominance of KdM282 T cell response is associated with expansion capacity.

(a) Magnitude of DbM187 and KdM282 T cell response at 7 dpi. Frequency of DbM187 and KdM282 T cells in CD8 T cell population were quantitatively assessed with flow cytometry. Data represent 5 independent experiments (n = 5/group/experiment). (b) The DbM187 and KdM282 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 DbM187 and KdM282 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 DbM187 and KdM282 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.

DbM187 T cells have superior cytolytic activity

To functionally evaluate DbM187 and KdM282 T cell cytotoxicity in vivo, we co-transferred M187 or M282 peptide-loaded naïve spleen cells as targets with OVA257 peptide-loaded controls into RSV-infected and naïve recipients, and recovered the donor cells 3 hours later. The donor target and control cells were identified and distinguished by fluorochrome spectra and fluorescence intensity (). Recovery ratio of live targets to controls from the infected recipients was compared to recovery ratio from naïve recipients to calculate specific cytotoxicity[24]. We found that the bulk cytotoxic hierarchy correlated with overall numerical hierarchy, as more M282 peptide-loaded targets were lysed in RSV-infected recipients than M187 peptide-loaded targets (). Although lung had higher DbM187 and KdM282 T cell frequency and counts than spleen (), there were more targets lysed in spleen than in lung (p = 0.0047 and p = 0.0016, M187 and M282 peptide–loaded respectively). The data suggest that cytotoxic potency may differ at the individual effector cell level.

KdM282 T cells dominate bulk cytotoxicity but DbM187 T cells have superior individual cytotoxicity.

(a) Bulk cytotoxicity of DbM187 and KdM282 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 DbM187 and KdM282 T cells. Ratio of specific lysis of donor targets in (a) was divided by frequency of endogenous DbM187 and KdM282 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.

To evaluate individual cell cytotoxicity, we divided lysed ratio of donor targets by frequency of endogenous CD8 T effector cells with relevant pMHC specificity, and expressed cytotoxic potency of individual effector cells in “killing” units. This value represents a relative frequency of targets lysed by individual CD8 T effector cells. We found that individual DbM187 T cells lysed more targets than KdM282 T cells (), and both subsets more efficiently lysed targets in spleen than they did in lung (p < 0.0001 for both DbM187 and KdM282 T cells).

DbM187 T cells have high avidity TCR and enhanced effector-function signaling pathways

Since T cell cytotoxic potency has been associated with TCR avidity, we studied dissociation of pMHCs from cells as a surrogate measurement of TCR avidity. By measuring half-life (T) of maximum median fluorescence intensity (MFI), we found that DbM187 tetramers were retained on CD8 T cells longer (59.62 and 405.7 min., lung and spleen respectively) than KdM282 tetramers (25.07 and 28.51 min., lung and spleen respectively) (). The dissociation rate (k) was calculated using Equation of Dissociation Kinetics in Prism 6 (Version 6.0f), and demonstrated that DbM187 tetramer fell off CD8 T cells at lower rates (0.012 and 0.002, lung and spleen respectively) than KdM282 tetramer (0.028 and 0.024, lung and spleen respectively). The data suggest that TCRs recognizing DbM187 have higher avidity than those recognizing KdM282, and that avidity correlates with the individual cell cytotoxic potency. In addition, the data also indicate that CD8 T cells in spleen have higher TCR avidity than those in lung, suggesting that the binding capacity may be diminished in the setting of high antigen load and high level of T cell activation, or TCR may be down regulated.

The DbM187 T cells express high avidity TCR and signaling pathways promoting cytotoxic function.

(a) The TCR avidity was assessed by dissociation of DbM187 and KdM282 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 DbM187, KdM282, 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.

Since TCR expression levels may have an effect on the expansion of antigen-specific cells, we assessed expression of TCR Vβ on CD8 T cells and found that KdM282 T cells have slightly higher level of TCR expression than DbM187 T cells in lung, but not in spleen (). This difference may provide a partial explanation for the numerical hierarchy.

Transcriptional profiling of 8365 immune response-related genes revealed a distinct pattern in DbM187 and KdM282 T cells (). Among the top 25 up-regulated genes in each subset, genes related to cytotoxic function and effector-memory differentiation (eg. H2, cd40, and ccr6) were up-regulated in DbM187 T cells, while genes related to cell division (eg. cdk2 and cdc6) were up-regulated in KdM282 T cells (). Analyzing those with fold change (FC) ≥ Log21.3, p < 0.05 and FDR < 0.25, the up-regulated gene sets in DbM187 T cells were related to effector-function pathways, such as cytotoxicity, cytokine and chemokine production, memory differentiation, co-stimulatory signaling and cell survival signaling, while those in KdM282 T cells were related to promoting cell cycle and clonal expansion (). The expression of genes encoding cell-division cycle proteins, cell division cycle-associated proteins, mini-chromosome maintenance proteins, histones and cyclin-dependent kinases () is consistent with post-transcriptional expression of Ki-67, which supports the findings that numerical hierarchy is associated with proliferative capacity.

DbM187 T cells express an effector- and central-memory phenotype

Comparing gene expressions related to cell differentiation, DbM187 T cells had elevated expression of bcl6, ccr6, ccr7, il7r, fos, myc, sell and tcf7 () that have been associated with effector-memory differentiation[25, 26]. In contrast, KdM282 T cells had elevated expression of eomes, id-2, klrg-1, roar and tbx21, factors associated with terminal differentiation[25, 27, 28]. Phenotyping by flow cytometry confirmed the transcriptional profiles, in that more than 50% of DbM187 T cells were CD62L(-) and CD127(+), a phenotype of effector memory, compared to 37% of KdM282 T cells in lung, and 23% of DbM187 T cells were CD62L(+) and CD127(+), a phenotype of central-memory, in spleen, while only 9% of KdM282 T cells expressed both memory markers ( and ). We assessed epitope-specific CD8 T cell populations and their phenotype at 8 weeks post RSV infection. Our data showed that DbM187 T cells have a higher frequency of cells with central memory phenotype (CD62L+ & CD127+) than KdM282 T cells, and the central memory cell counts are the same although KdM282 T cell have a greater total cell count denominator than DbM187 T cells at peak of response (). These data provide additional evidence that DbM187 T cells are more likely to differentiate towards a central memory phenotype and survive after virus clearance.

The DbM187 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 KdM282 –Log2 DbM187. Positive values indicate a specific gene expression was up-regulated in the KdM282 subset, while negative values indicate up-regulated gene expression in the DbM187 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.

Activated KdM282 T cells are susceptible to inhibitory regulation and apoptosis

T cell function is regulated by inhibitory receptors. Transcriptional profiling revealed that KdM282 T cells up-regulated expression of multiple inhibitory receptor genes (), such as cd160, cd223, cd152, ctla-4 and sosc2[29]. This is consistent with our phenotype study that showed PD-1, CD160 and CD223 expression was more common on KdM282 T cells than on DbM187 T cells (). Although DbM187 T cells elevated cd272 (btla) expression, another inhibitory receptor gene, the post-transcription level was similar to that of KdM282 T cells (). We also found that cells isolated from lung were more likely to express inhibitory receptors than those isolated from spleen, including CD152, Tim1 and Tim3, although the difference in Tim3 expression was not statistically significant (). The transcriptional profile and phenotype of inhibitory receptors inversely correlate with individual DbM187 and KdM282 T cell cytotoxic potency and suggest that the KdM282 T cells are more susceptible to inhibitory regulation than the DbM187 T cells.

Activated KdM282 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.

TCR ligation triggers activation-induced cell death that also negatively regulates T cell function. The expression of pro-apoptotic genes, such as bcor, bcorl1, apitd1, bik, casp7, sival, aimf2, pdcd4 and pdcd10[30-38], were up-regulated in KdM282 T cells, while those associated with anti-apoptosis, such as bcl11a, bcl6 and faim3[, were up-regulated in DbM187 T cells (). The bcl11a was also one of the top 25 up-regulated genes in DbM187 T cells (). Flow cytometry analysis showed that KdM282 T cells were more likely to be stained with Annexin V than DbM187 T cells, which is an early indication of apoptotic change (. In contrast, DbM187 T cells were more likely to express Bcl-2 than KdM282 T cells. Both CD8 T cell subsets from lung were more apoptotic than those from spleen (p < 0.0001, DbM187 and KdM282 T cells). The pattern of pro- and anti-apoptotic molecule expression at both transcriptional and protein levels suggests that a large proportion of the CD8 T cells, particularly KdM282 T cells, progress towards apoptosis at the site of inflammation.

Activated KdM282 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.

DbM187 T cells efficiently control viral replication with limited pulmonary inflammation and disease severity

To evaluate capacity of controlling virus replication in vivo, we sorted live DbM187, KdM282, and bulk (with neither specificity) CD8 T cells by FACS from the spleen of RSV-infected mice and transferred equal numbers into naive recipients. The recipients were then challenged with RSV one day later. At 4 days post infection (4 dpi), we detected 0.9 ± 0.3% DbM187 and 0.8 ± 0.5% KdM282 T cells (mean ± SD) in recipient’s lung CD8 T cell population respectively. The epitope-specific CD8 T cells were not detectable above background (≤ 0.05% of CD8 T cells) in mice that received bulk CD8 T cells or CD8 T cells with irrelevant pMHC specificity (). This indicates that virtually all the epitope-specific CD8 T effector cells in lung at 4 dpi are donor origin. This is consistent with previous studies that have shown endogenous RSV-specific CD8 T cells are rarely detectable at 4 dpi of primary infection. Assessing anti-viral activity of isolated lung samples in vitro, we found that DbM187 T cell recipients had significantly less viral replication in lungs than KdM282 or bulk CD8 T cell recipients (). KdM282 T cell recipients had similar viral replication in lungs as bulk CD8 T cell recipients (p = 0.115). We assessed IFN-γ in lung tissue homogenates, and found no significant difference among the recipient groups (). Although DbM187 T cells have more efficient effector cytokine production at the individual cell level than KdM282 T cells, the impact of cytokines from adoptively transferred donor cells is likely to be masked by high levels of endogenous cytokine production.

Adoptively transferred donor cells increased recipient precursor frequency of relevant pMHC specificity and slightly altered the endogenous hierarchy at the day 7 peak response (). DbM187 T cell recipients had a reduced disparity of CD8 T cell responses (KdM282: DbM187 = 3: 1) compared to bulk CD8 T cell recipients (KdM282: DbM187 = 5:1), while the KdM282 T cell recipients had exaggerated disparity (KdM282: DbM187 = 10:1). Histopathological evaluation of lung tissue revealed DbM187 T cell recipients had decreased frequency and count of total T and CD8 T cells compared to bulk CD8 and KdM282 T cell recipients (). The KdM282 T cell recipients had similar T and CD8 T cell frequencies and total cell counts in lung compared to the bulk CD8 T cell recipients. There was no significant difference in CD4 T cell counts between groups (). Paraffin-embedded sections of formalin-inflated lungs at 7 dpi were stained with hematoxylin-eosin and evaluated by an independent pathologist in a blinded manner. Typically, pathology after primary infection is most severe between 8 and 10 dpi. The histopathology was analyzed on 7 dpi because the transfer of immune competent cells accelerated the time course of response. Mice without RSV infection had few infiltrates with few mononuclear cells present in the interstitium (). After RSV infection, lung shows inflammatory histology. Although the lung pathology of recipient groups did not show significant difference by an over-all pathology score, 13.85±1.10, 12.28±0.79 and 15.43±1.21 for DbM187, KdM282 T cell and bulk CD8 T cell recipients, respectively (mean±SD in a 1–15 scale level), the KdM282 T cell and bulk CD8 T cell recipients showed more peribronchiolar neutrophil infiltration and higher magnitude interstitial inflammation and macrophage infiltration, suggesting a greater non-specific inflammatory response compared to DbM187 T cell recipients. Bulk CD8 T cell recipients had an intermediate phenotype with interstitial infiltrates composed of both mononuclear cells and polymorphonuclear leukocytes (PMNs), but without frank alveolitis (). Recipients of the immunodominant KdM282 T cells had hemorrhagic alveolitis characterized by inflammatory cells and red blood cells (RBCs) frequently found in the alveolar space. The interstitial and alveolar infiltrates were composed primarily of mononuclear cells, but also contained a relative high frequency of PMNs. There was also evidence of epithelial necrosis with more pyknotic nuclei in this group. This is similar to the original report of CD8 transfer in H-2d BALB/c mice causing hemorrhagic alveolitis[6]. The DbM187 T cell recipients had mononuclear cell interstitial infiltrates with rare PMNs and no cells in the alveolar space. This study indicates that the KdM282 T cells cause more tissue destruction and a more severe immunopathology than DbM187 T cells. We also monitored body weight change after virus challenge as a surrogate for severity of illness. All mice lost weight after RSV infection. However, the KdM282 T cell recipients had earlier and greater weight loss than other two groups. The differences were statistically significant from 5 to 7 dpi (, p < 0.05–0.005). The virus clearance, pathology and weight loss data indicate that DbM187 T cell-mediated virus clearance is more efficient and less pathogenic than that mediated by KdM282 T cells.

The DbM187 T cells provide protection with limited pulmonary inflammation and illness of the recipients.

The DbM187, KdM282 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.

Discussion

It has become increasingly evident that magnitude of CD8 T cell response does not necessarily correlate with the extent of protection from infectious pathogens, and that functional properties of T cells may be more predictive of infection outcome[11, 12, 42]. In addition, the analysis of polyclonal T cell responses in bulk masks the functional diversity of T cells responding to specific epitopes. Even within an epitope-specific response, there are clonotype-specific subsets, each with their own functional profiles that collectively determine the characteristics of the overall response[9, 10]. Defining the characteristics of CD8 T cells that can efficiently clear virus-infected cells with minimal immunopathology has obvious implications for developing interventions that provide T cell-mediated immunity.

In the CB6F1 hybrid mouse model of RSV infection, numerically dominant KdM282 T cell response account for the majority of bulk cytolytic activity. But individual KdM282 T cells are relatively deficient in effector functions, and only about half of them produce IFNγ, which is a key molecule in eliminating virus infected cells[17, 43, 44]. Dramatically increasing KdM282 T cells to control RSV replication results in elevated pulmonary inflammation and disease severity[17, 18]. Informed by an earlier finding that virus clearance could be accomplished in mice infected with virus containing a mutated M282 T cell epitope[20], we evaluated numerically subdominant CD8 T cell responses and found that individual DbM187 T cells were more efficient at lysing infected cells and controlling virus replication. CD8 T effector cells pre-existing in mucosal tissue have been reported to eliminate infection at the point of initial virus replication[45]. Adoptive transfer of DbM187 T cells increased pre-existing pulmonary CD8 T effector cells and controlled the virus replication during early infection. Prompt control of virus replication resulted in a mild infection-induced lung inflammation, with fewer infiltrating T cells, mononuclear cells and PMNs, and no hemorrhagic alveolitis or epithelial necrosis displayed in histological sections. As a consequence of the limited damage to lung tissue and function, the DbM187 T cell-mediated protection resulted in less illness than KdM282 T cell-mediated viral clearance. The efficiency of protection against virus challenge is an intrinsic property of DbM187 T cell cytolytic function, and is not associated with differences in migration, since KdM282 T cells have similar or higher presence in host lung[46].

High TCR avidity has been suggested as a critical factor for induction of CD8 T cell effector function in viral infections[47, 48]. Our previous studies revealed that DbM187-specific TCRs recognize a more complex epitope structure and tend to have private clonotypes, presumably with a greater surface area of interaction with the epitope than KdM282-specific TCRs[49]. Here, our transcriptional profiling showed that the engagement of the high avidity DbM187-specific TCR activated pathways to promote effector function, survival, and effector-memory differentiation. The association of cytolytic potency with enhanced co-stimulation signaling, cytokine and chemokine production has been well studied and demonstrated to be a critical factor in anti-viral immunity[50]. The effector-memory phenotype is a subpopulation of CD8 T cells with particularly good functional activity. For example, CD8 T cells expressing an effector-memory phenotype were shown to be critical for protection from mucosal SIV challenge[51] and could clear virus-infected cells at an early stage of infection[52]. Our transcriptional, post-transcriptional, and long-term survival data suggest that DbM187-specific TCR engagement elicits a program driving cells toward effector-memory differentiation with higher cytotoxic potency than terminally differentiated cells driven by KdM282-specific TCR engagement.

Replication capacity accounts for the numerical dominance of KdM282 T cell response. In some studies, precursors determine the magnitude of response[53, 54]. Our early study has showed that the frequency of DbM187 and KdM282 T cells are about the same in naïve mice[23]. Here, we showed that moderately increasing precursors of the subdominant subset by adoptive transfer influenced but did not reverse the hierarchy pattern. Difference in TCR level may provide partial explanation to the formation of numerical hierarchy but is not likely to be the major determinant. Recent studies reported that the level of TCR expression had minimal effect on the magnitude of antigen-specific CD8 T cells expansion[55, 56]. We also found that the differences in TCR expression levels were not proportional to differences in response magnitudes, and there is no significant difference in TCR expression levels on splenocytes. Variation in antigen dose may affect CD8 T cell expansion. But in our previous experiment using viral vector to express M and M2 antigens as a fusion protein, in which both epitopes were equally available and processed, we showed similar CD8 T cell response hierarchy as observed in RSV infection[23]. We propose that the extreme proliferation of KdM282 T cells is presumably driven by activation signals derived from the pMHC-TCR interaction. KdM282 T cells express TCRs that recognize a relatively flat and featureless epitope, and have a highly public clonotype[10]. These characteristics determine that KdM282 T cells have distinct activation thresholds for proliferation and cytolytic activity or cytokine production. It has been reported that some epitope-specific CD8 T cells can be expanded by cognate peptide in the absence of helper cell-derived signals, although their effector functions might not be triggered[57]. Another study based on mathematic modeling concluded that rapid expanding T cells were likely to be deficient in effector function and short-lived[52]. It is possible that the rapidly dividing population has a diminished TCR avidity due to down-regulation. The type of exaggerated activation that leads to the numerical dominance of KdM282 T cell response may be selected by immunogenicity testing based solely on magnitude of response, but is not the preferred adaptive immune response from the host perspective.

Our previous study reported that the KdM282 T cells were selectively susceptible to regulatory CD4 T cells[20, 58]. Here, we showed that the KdM282 T cells were subject to inhibitory signals, by elevated expression of PD-1, CD160, CD223 and other inhibitory receptors. These inhibitory receptors have a negative impact on T cell effector function and control of viral replications in chronic[29] and acute infections[59]. RSV infection has been reported to induce PD-L1 expression on bronchial epithelial cells, which resulted in an inhibition of CD8 T cell antiviral activity[60]. The PD-1 and other inhibitory signals can also suppressed memory formation and directed CD8 T cells to terminal differentiation[29]. Being apoptotic has been shown to negatively impact effector function. The extremely activated and robustly proliferating KdM282 T cells up-regulate genes promoting apoptosis, but have reduced expression of genes encoding anti-apototic molecules. The inhibitory receptor expression and pro-apoptotic changes are consistent with the functional deficiency of KdM282 T cells, and with previous observations that only a fraction of the KdM282 T cells produce IFN-γ, while almost all DbM187 T cells are IFN-γ-producing[17, 44].

The tissue environment may also have impact on CD8 T cell effector function. Terminal differentiation and lack of cytolytic capacity have been previously demonstrated to be a characteristic of lung-derived CD8 T cells in response to viral infections[57]. Terminally differentiated CD8 T cells in the lung had limited cytolytic activity and were unable to control influenza and Sendai virus infection[61]. Here, we found that DbM187 and KdM282 T cells isolated from lung, where they are needed to clear virus-infected cells, were less cytotoxic than cells isolated from spleen. Our data suggest that cells in the lung are vulnerable to suppressive regulation by elevating expression of inhibitory receptors, and susceptible to activation-induced cell death by down-regulating anti-apoptotic molecules. Terminal differentiation and loss of effector function has been associated with prolonged TCR ligation and inflammatory cytokine-driven differentiation[62-64]. Persistent engagement of TCR can trigger down-regulation of CD127 on CD8 T cells in lung[65], and adversely affect control of viral replication[66]. Thus, a balanced and modulated inflammatory response promotes optimal CD8 T effector function.

In the mouse model of RSV infection, a subdominant CD8 T cell response provided adequate control of viral replication during early infection, limited the viral replication, and reduced pulmonary inflammation and disease severity. These data support the concept that induction of T cells with high cytolytic activity are more favorable for protecting the host from viral infection than a high magnitude response with cells exhibiting a more extreme differentiation phenotype. The goal for vaccine-induced CD8 T cell-mediated protection should be to increase the precursor frequency of cells that can respond rapidly based on a pre-existing memory phenotype and localization. They should exhibit potent and efficient cytolytic activity without excessive proliferation that may lead to unwarranted immunopathology[12]. These findings raise the interesting possibility that by selecting for T cell epitopes with more complex structures, it may be possible to elicit T cell responses that have higher TCR-pMHC avidity. These cells would be more likely to achieve the activation threshold for initiating the effector functions needed to rapidly clear virus-infected cells with minimal immunopathology.

Materials and Methods

Mice and virus infection

Pathogen-free CB6F1 female mice between the ages of 8 and 10 weeks were purchased from Jackson Laboratories (Bar Harbor, ME) and cared for in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The NIH Animal Care and Use Committee approved all animal protocols in this study. Experimental groups were age matched. RSV challenge stock was derived from the A2 strain by sonicating HEp-2 cell monolayers as previously described[16]. For infection, mice were inoculated with 1 x 107 PFU live RSV in 10% EMEM intranasally under anesthesia with 3% isoflurane and oxygen.

Ethics statement

Animal use and all procedures (VRC-13-443 and -447) were approved by the Vaccine Research Center, Animal Care and Use Committee (ACUC) in accordance with NIH policy and ARAC guidelines, and is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and in compliance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals. The National Institutes of Health Intramural Research Program OLAW assurance number is A4149-01.

Cell culture and flow cytometry reagents

The cell culture medium was RPMI 1640 from HyClone (Logan, UT), supplemented with 10% heat inactivated fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin. Fluorochrome-conjugated antibodies used to study cell phenotype, differentiation and other expressions with flow cytometry were anti-CD3–cyanine 7 (Cy7) allophycocyanin (APC), anti-Ki67-fluorescein isothiocyanate (FITC), anti-Bcl2-FITC, and Annexin V-FITC that were purchased from BD Biosciences (San Jose, CA). Unconjugated antibodies to CD4, CD8 and CD62L were purchased from Harlan (Indianapolis, IN) and were conjugated with Qdot605, Qdot655 and Alexa Fluor 688 from Invitrogen (Carlsbad, CA). The in-house conjugates were validated by comparison with commercial conjugates. Anti-PD-1-FITC, anti-CD272-phycoerythrin (PE), anti-CD152-PE, anti-Tim1-biotin, anti-Tim3-biotin, anti-CD223-PE and anti-CD127-Cy7PE were purchased from eBioscience Inc (San Diego, CA). Streptavidin (SA)-PE was purchased from ProZyme (Hayward, CA). Violet fluorescent reactive dye (Invitrogen, CA), anti-CD19- and anti-CD16/32-Pacific blue (eBioscience) were used as cell viability and lineage markers to exclude dead and non-T cells from analysis. Fluorochrome-conjugated epitope peptide–MHC class I complexes (pMHC), Db-M187-195-APC (DbM187-APC) and Kd-M282-90-PE (KdM282-PE), were purchased from Beckman Coulter (Fullerton, CA).

In vivo T cell labeling

Naïve or RSV-infected mice were injected intravenously with Cy5PE-conjugated anti-Thy1.2 antibody purchased from BioLegend (San Diego, CA). Mice were sacrificed 10 min. later. Lymphocytes from lung and spleen were isolated and stained for lineage differentiation and pMHC specificity following standard procedures, and then applied to flow cytometry analysis[21, 22].

Cytotoxic activity assays

To evaluate cytotoxicity of DbM187 and KdM282 T cells in vivo, we setup an assay based on previous described[24]. The targets were prepared from naïve spleen cells that were loaded with M187-195 (M187) or M282-95 (M282) peptides (Anaspec Inc, San Jose, CA) at a concentration of 10 μg/ml, then stained with 10 uM carboxyfluorescein succinimidyl ester (CFSE) or 10 uM CellTracker Red CMTPX (both from Invitrogen) respectively; controls were prepared from naïve spleen cells that were loaded with OVA257-264 (OVA257) peptide (Anaspec Inc) at a concentration of 10 μg/ml, then stained with either 0.1 uM CFSE or 0.2 uM CellTracker Red CMTPX respectively. These four populations, including M187-CFSEhigh, M282-CMTPXhigh, OVA257-CFSElow and OVA257-CMTPXlow, each at 2 x 106, were co-transferred into RSV-infected or naïve mice via tail vein. Cells from the lung and spleen were collected 3 hours later from the recipients. Donor cells were identified by fluorochromes spectra and distinguished by fluorescence intensity. The specific lysis were calculated using formula: ratio = (percentage fluorochromelow/percentage fluorochromehigh). Specific lysis (%) = [1 − (ratio naive/ratio infected) × 100]. Frequency of pMHC-specific CD8 T cells was assessed simultaneously in the same cell preparation recovered. The cytolytic unit per pMHC specific CD8 T effector cell was calculated by dividing the percent of specific lysis by the frequency of pMHC-specific CD8 T cells.

TCR avidity assessment

The pMHC decay assessment was performed as described previously[67]. Briefly, lung and spleen lymphocytes from RSV-infected mice were stained for lineage differentiation and epitope specificity with fluorochrome-conjugated pMHCs. Cells were washed and then incubated in the presence of anti-H2-Db (12.5 μg/ml) or anti-H2-Kd (2.5 μg/ml) respectively, in culture medium containing 0.1% sodium azide at room temperature. Cells were recovered every 20 minutes, washed and then fixed with 1% paraformaldehyde and applied for flow cytometry to assess the intensity of fluorescence.

Sorting and adoptive transfer of pMHC-specific donor cells

Live DbM187, KdM282 and bulk (with neither specificity) CD8 T cells from spleen lymphocytes of RSV-infected mice at 7dpi were sorted by FACS. From each population, 5 x 105 cells were transferred into naive recipients respectively via tail vein. The recipients were challenged with RSV 24 hours later, and were evaluated for the donor cell frequencies and the virus load.

Viral load assessment

Lung tissue was sampled at indicated times after RSV infection and quickly frozen using ethanol-dry ice in 10% EMEM medium. After grinding and pelleting of tissue debris, serial dilutions of supernatant were inoculated onto 80% confluent HEp-2 cell monolayers in triplicate and overlaid with 0.75% methylcellulose in 10% EMEM. The cultures were incubated at 37°C for 4 days, then fixed with phosphate buffered 10% formalin and stained with hematoxylin and eosin. Plaques were counted and expressed as log10 PFU/gram of lung. The limit of detection is 1.8 log10 PFU/gram of lung.

Histology

At indicated time points after RSV infection, mice were euthanized and the left lung was gently inflated with phosphate-buffered 10% formalin, then removed and placed in the buffered formalin. Thin sections were cut from paraffin-embedded lung tissues, stained with hematoxylin and eosin and examined by light microscopy.

Microarray cDNA hybridization analysis

Splenocytes from RSV-infected mice were isolated and stained for lineage differentiation and pMHC specificities. DbM187 and KdM282 T cells were sorted into RLT buffer (Qiagen, CA) with cell sorter Aria (BDbiosciences, CA). After isolation of mRNA, the total RNA was amplified and labeled using the Illumina TotalPrep RNA Amplification kit. The biotinylated cRNA was hybridized onto Illumina Mouse Chips and quantified using Illumina BeadStation 500GX scanner and Illumina BeadStudio v3 program. Illumina probe data were exported from BeadStudio as raw data and screened for quality. Samples failing chip visual inspection and control examination were removed. Gene expression data were analyzed using Bioconductor, an open-source software library for the analyses of genomic database on R, which is a language and environment for statistical computing and graphics (www.r-project.org). The R software package was used to first filter out probes with intensities below background in all samples, and then to replace minimum (a surrogate replacement policy) values below background using the mean background value of the built-in Illumina probe controls as an alternative to background subtraction (which may introduce negative values) to reduce “overinflated” expression ratios determined in subsequent steps, and finally quantitatively normalize the probe intensities. The resulting matrix showing probes as rows and samples as columns was log2 transformed and used as input for linear modeling using BioConductor Linear models for microarray analysis (LIMMA). The LIMMA package was used to identify differentially expressed genes (pFDR < 0.05).

Accession numbers

Genes and proteins mentioned in the text can be found in the SwissProt data libraries (http://www.uniprot.org/uniprot/) under the following accession numbers: RSV-M (P03419), RSV-M2 (P04545), Ki-67 (Q61769), CD62L (P18337), CD127 (P16872), IFNγ (P01580), PD-1 (Q02242), CD160 (O88875), CD223 (Q61790), CD152 (P09793), Tim 1 (Q5QNS5), Annexin V (P48036), Bcl-2 (P10417).

Identification of DbM187 and KdM282 T cells by flow cytometry.

Lymphocytes isolated from the lung and spleen of RSV-infected mice at 7 dpi were stained for CD3, CD4 and CD8 lineage differentiation and pMHC-specificities. Dot plots represent strategies to identify DbM187 and KdM282 T cells, by sequentially gating on singlets, lymphocytes, CD3(+) Dump(-), CD8(+) CD4(-), and pMHC(+) populations. The Dump channel included Violet fluorescent reactive dye, fluorochrom-conjugated anti-CD16/32 and anti-CD19 to exclude dead cells, monocytes, B cells, and other non-T cells. Dot plots represent 5 independent experiments (n = 5/group/experiment).

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Quantitative assessment of peptide-loaded and fluorochrome-labeled targets and controls recovered from recipients in cytotoxicity assay in vivo.

After adopting peptide-loaded and fluorochrome-labeled targets and controls, the recipient’s lung and spleen cells were isolated 3 hours later and analyzed by flow cytometry. The M187 and M282 peptide-loaded targets were identified by high intensity of CFSE and CMTPX respectively; the OVA257 peptide-loaded controls were identified by lower intensity of CFSE and CMTPX and used as controls for the same fluorochrome-labeled targets respectively. Histograms show the proportion of cells with distinct fluorochrome labeling and intensity recovered from infected or naïve mice. Dot plots and histograms represent 5 independent experiments (n = 5/group/experiment).

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The DbM187 and KdM282 T cell counts in lung and spleen.

The DbM187 and KdM282 T cells from lung and spleen of infected mice at 7 dpi were quantitatively assessed by flow cytometry. Cell counts were calculated based on total cell counts in the lung and spleen cell preparations and the frequency of individual subsets. Data are shown as mean with independent data point and are compared by Student’s t-test. Data represent 5 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.

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TCR level of DbM187 and KdM282 T cells.

Lymphocytes isolated from the lung and spleen of RSV-infected mice at 7 dpi were stained for CD3, CD4 and CD8 lineage differentiation and pMHC-specificities. The expression of TCR on epitope-specific CD8 T cells was studied using fluorescence-conjugated anti-TCR Vβ (pan Vβ) monoclonal antibody, and the relative level of expression was measured by the medium fluorescence intensity (MFI). Data are shown as mean with independent data point and are compared by Student’s t-test. Data represent 2 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.

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Pattern of Transcriptional expressions of DbM187 and KdM282 T cells.

The DbM187, KdM282 and bulk CD8 T cells were sorted from spleen by FACS at 7 dpi and studied for transcriptional expression of genes associated to immune responses. The quantitative gene expression were analyzed and normalized. Relative expression was calculated and presented as Log2 Fold Change (Log2FC). The Log2FC = Log2 KdM282 –Log2 DbM187. Positive values indicate gene expression was up-regulated in the KdM282 subset, while negative values indicate gene expression was up-regulated in the DbM187 subset. (a) All gene expressions with Log2 FC > ±1.3. (b) Top 25 up-regulated expressions among (a) in each subset. (c) Up-regulated expressions of genes in (a) related to MHC class I molecule and clone expansion. Each column represents data from one mouse.

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Pattern of CD62L and CD127 expression and memory subsets.

Lymphocytes were isolated from the lung and spleen of infected mice at 7dpi and studied for expression of CD62L and CD127 with flow cytometry. Dot plots show the gating strategy and represent 5 independent experiments (n = 5/group/experiment). (b) Lymphocytes were isolated from the lung and spleen of mice at 8 weeks post infection. The DbM187 and KdM282 T cells were studied for their frequency, count, and expression of CD62L and CD127 with flow cytometry. Data are shown as individual data points with horizontal bar representing the mean, and are compared by Student’s t-test. Data represent 2 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.

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Similar expression of CD272 and Tim 3 in DbM187 and KdM282 T cells.

Lymphocytes were isolated from the lung and spleen of infected mice at 7dpi and studied for expression of (a) CD272 and (b) Tim 3 with 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.

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Lung IFN-γ level of recipients after RSV challenge.

Lung supernatant described in Fig 7B were collected and frozen saved. INF-γ level was assessed using LEGENDplex (BioLegend), a bead-based commercially available immunoassay service. The concentration was calculated referring to standard series. Each symbol represents one mouse (n = 11).

Fig 7

The DbM187 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 DbM187, KdM282 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 DbM187 and KdM282 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.

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Adapting donor cells increases frequency of CD8 T cells with relevant specificity after virus challenge.

After virus challenge, the recipients that adopted DbM187, KdM282 and bulk CD8 T cells were evaluated for DbM187 and KdM282 T cell frequencies in the right lung at 7 dpi with flow cytometry. The frequencies 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.

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CD4 T cell counts were not significantly altered in recipients.

The DbM187, KdM282 and bulk CD8 T cell recipients were challenged with RSV. Inflammatory cells were assessed at 7 dpi with flow cytometry. The absolute number and frequency of CD4 (+) 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.

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Histology of naïve lung.

Left lung were isolated from naïve mice and studied for the histology of lung structure. Sections were examined under light microscope by independent blinded investigators. Histology study represents 3 independent experiments (n = 5).

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