Intranasal vaccination with murine cytomegalovirus expressing respiratory syncytial virus antigens promotes tissue-resident memory CD8+ T cells

Cytomegalovirus (CMV) vectors are a promising vaccine strategy aimed at eliciting effector CD8+ T cells. CMV-based vaccines have been shown to mediate protection against viral pathogens including simian immunodeficiency virus and Ebola virus, and produce responses that delay tumor growth in melanoma and prostate cancer in animal models. CMV infection generates robust cellular and humoral immune responses. In addition to conventional CD8+ T cell memory responses that contract following acute T cell expansion resulting in a low-level stable memory population, CMV infection generates populations of CD8+ T cells that continue to accumulate throughout latent infection termed “inflationary memory.” These inflationary memory cells have high avidity T cell receptors, an effector phenotype, and remain functional throughout chronic infection providing the basis for highly effective and durable T cell-mediated immunity. For this dissertation, I engineered murine CMV (MCMV) to express the M and M2 proteins of respiratory syncytial virus (RSV). When expressed in MCMV, the M-specific CD8+ T cell population was inflationary and the M2-specific CD8+ T cell population was conventional. Intranasal vaccination generated a robust tissue-resident (TRM) effector and effector memory CD8+ T cell population that was absent in intraperitoneally vaccinated mice. This population of TRM was sustained at a high level through twenty-four weeks post-vaccination in mice vaccinated with the inflationary M epitope (MCMV-M). In contrast, mice vaccinated with MCMV expressing the conventional M2 epitope (MCMV-M2) generated a TRM population that waned over time. Co-administration of MCMV-M and MCMV-M2 had no impact on the M-specific CD8+ T cell response, but did decrease the M2-specific CD8+ T cell response during acute time points. All vaccinations led to decreased viral loads compared to RSV-naïve controls, but intranasal vaccination with MCMV-M elicited sustained TRM resulting in earlier T cell responses, viral clearance, and cytokine secretion after RSV challenge. Our findings using this novel approach of intranasal administration of MCMV emphasizes the importance of vaccination route for the generation of effective immune responses at sites of vulnerability, and the potential value of combining the local induction of TRM with inflationary properties as a general vaccination strategy for pathogens that require CD8+ T cell-mediated immunity

Phenotype and Hierarchy of Two Transgenic T Cell Lines Targeting the Respiratory Syncytial Virus KdM282-90 Epitope Is Transfer Dose-Dependent.

In this study, we compared two lines of transgenic CD8+ T cells specific for the same KdM282-90 epitope of respiratory syncytial virus in the CB6F1 hybrid mouse model. Here we found that these two transgenic lines had similar in vivo abilities to control viral load after respiratory syncytial virus infection using adoptive transfer. Transfer of the TRBV13-2 line resulted in higher levels of IL-6 and MIP1-α in the lung than TRBV13-1 transfer. Interestingly, when large numbers of cells were co-transferred, the lines formed a hierarchy, with TRBV13-2 being immunodominant over TRBV13-1 in the mediastinal lymph node despite no identifiable difference in proliferation or apoptosis between the lines. This hierarchy was not established when lower cell numbers were transferred. The phenotype and frequency of proliferating cells were also cell transfer dose-dependent with higher percentages of CD127loCD62LloKLRG1lo and proliferating cells present when lower numbers of cells were transferred. These results illustrate the importance of cell number in adoptive transfer experiments and its influence on the phenotype and hierarchy of the subsequent T cell response

Quantitative and qualitative deficits in neonatal lung-migratory dendritic cells impact the generation of the CD8+ T cell response.

CD103+ and CD11b+ populations of CD11c+MHCIIhi murine dendritic cells (DCs) have been shown to carry antigens from the lung through the afferent lymphatics to mediastinal lymph nodes (MLN). We compared the responses of these two DC populations in neonatal and adult mice following intranasal infection with respiratory syncytial virus. The response in neonates was dominated by functionally-limited CD103+ DCs, while CD11b+ DCs were diminished in both number and function compared to adults. Infecting mice at intervals through the first three weeks of life revealed an evolution in DC phenotype and function during early life. Using TCR transgenic T cells with two different specificities to measure the ability of CD103+ DC to induce epitope-specific CD8+ T cell responses, we found that neonatal CD103+ DCs stimulate proliferation in a pattern distinct from adult CD103+ DCs. Blocking CD28-mediated costimulatory signals during adult infection demonstrated that signals from this costimulatory pathway influence the hierarchy of the CD8+ T cell response to RSV, suggesting that limited costimulation provided by neonatal CD103+ DCs is one mechanism whereby neonates generate a distinct CD8+ T cell response from that of adults

Memory Inflation Drives Tissue-Resident Memory CD8+ T Cell Maintenance in the Lung After Intranasal Vaccination With Murine Cytomegalovirus

Tissue-resident memory T (TRM) cells provide first-line defense against invading pathogens encountered at barrier sites. In the lungs, TRM cells protect against respiratory infections, but wane more quickly than TRM cells in other tissues. This lack of a sustained TRM population in the lung parenchyma explains, at least in part, why infections with some pathogens, such as influenza virus and respiratory syncytial virus (RSV), recur throughout life. Intranasal (IN) vaccination with a murine cytomegalovirus (MCMV) vector expressing the M protein of RSV (MCMV-M) has been shown to elicit robust populations of CD8+ TRM cells that accumulate over time and mediate early viral clearance. To extend this finding, we compared the inflationary CD8+ T cell population elicited by MCMV-M vaccination with a conventional CD8+ T cell population elicited by an MCMV vector expressing the M2 protein of RSV (MCMV-M2). Vaccination with MCMV-M2 induced a population of M2-specific CD8+ TRM cells that waned rapidly, akin to the M2-specific CD8+ TRM cell population elicited by infection with RSV. In contrast to the natural immunodominance profile, however, coadministration of MCMV-M and MCMV-M2 did not suppress the M-specific CD8+ T cell response, suggesting that progressive expansion was driven by continuous antigen presentation, irrespective of the competitive or regulatory effects of M2-specific CD8+ T cells. Moreover, effective viral clearance mediated by M-specific CD8+ TRM cells was not affected by the coinduction of M2-specific CD8+ T cells. These data show that memory inflation is required for the maintenance of CD8+ TRM cells in the lungs after IN vaccination with MCMV

Cell transfer number affects the Tg T cell phenotype.

<p>Different numbers of a 1:1 mixture of TRBV13-2:TRBV13-1 cells were transferred 1 day prior to RSV infection. On day 6 post-infection, we assessed the phenotype of the transferred Tg T cells based on expression of CD62L, CD127, and KLRG1. The percentage of Tg CD8+T cells of each phenotype in the 10⁴ transfer group are in the lungs (A) and MLN (B) are shown. The CD127^lo, CD62L^lo; KLRG1^lo to CD127^hi, CD62L^lo; KLRG-1 ^lo ratio in the lungs (C) and MLN (D) was determined. Bars represent mean±SEM.</p

Different input TRBV13-2/TRBV13-1 ratios affect the magnitude of TRBV13-2 skewing.

<p>10⁶ or 10⁴ cells were transferred into mice at input ratios of 2:1, 1:1, and 1:2 of TRBV13-2:TRBV13-1 and mice were infected with RSV one day later. (A) Flow cytometry plots showing the input frequency for TRBV13-2 and TRBV13-1 at different ratios for the 10⁶ cell number transfer. (B and C) TRBV13-2/TRBV13-1 ratio on day 6 post-RSV infection in the lungs and MLN of mice who received different TRBV13-2/TRBV13-1 ratios. Dotted line indicates input ratio of TRBV13-2/TRBV13-1.</p

Proliferation is dependent on transfer number, but does not differ between TRBV 13–1 and TRBV13-2.

<p>(A and B) 1:10 dilutions of a 1:1 ratio of TRBV13-1:TRBV13-2 cells were transferred 1 day prior to RSV infection. Four days post-infection, the percentage of proliferating cells in the lung (A) and MLN (B) were identified by <i>in vivo</i> incorporation of BrdU during a two-hour incubation. <b>(</b>C and D) violet labeled TRBV13-1 and TRBV13-2 were co-transferred into CB6F1 mice prior to infection with RSV. Proliferation profiles of TRBV13-1and TRBV13-2 (C) in the MLN were evaluated at day 4 post-infection and percentage of dividing cells was determined (D).</p

Transfer of TRBV 13–2 leads to increased levels of proinflammatory cytokines in the lungs following RSV infection.

<p>10⁶ of TRBV13-1, TRBV13-2 cells, or a 1:1 mixture of both (mix) were transferred into CB6F1 mice one day prior to infection with RSV. On days 4, 5, and 6 post-infection, lungs were removed, homogenized, and analyzed for IFNγ, TNFα, IL-6 and MIP-1α by multiplex ELISA. Bars represent mean ± SEM from 4 mice/group.</p

Transfer of TRBV13-1, TRBV13-2, and 1:1 mix leads to increased specific lysis, decreased viral loads, and increased morbidity after RSV infection.

<p>(A-D) CB6F1 mice received 10⁶ of either TRBV13-1 or TRBV13-2 cells or a 1:1 mixture of both (mix) one day prior to infection with RSV. On day 5 (A) and 6 (B) post-infection lungs were removed and processed to determine viral titer in the lungs by plaque assay. (C) CFSE labeled target cells were transferred by IN instillation into TRBV13-1 and TRBV13-2 recipient mice to evaluate cytolysis in the lung on day 4 post-infection. (D) Morbidity of mice in the different groups was evaluated by calculation of weight loss. * indicates days in which TRBV13-1 and Mix were significantly different than TRBV13-2.</p

Effects of transfer cell number on TRBV13-2/TRBV13-1 ratio and endogenous CD8+T cell response.

<p>The indicted number of a 1:1 mix of TRBV13-1 and TRBV13-2 were transferred into CB6F1 mice one day prior to RSV infection. On <b>(A-C)</b> day 4 and <b>(D-F)</b> day 6, lungs and mediastinal lymph node (MLN) were collected and the percentages of transferred TRBV13-1, TRBV13-2, and endogenous M2-specific CD8+T cells were determined. <b>(C, F)</b> TRBV13-2/TRBV13-1. Dotted line indicates input ratio of TRBV13-2/TRBV13-1. NA indicates that there were not enough events to calculate TRBV13-2/13-1.</p