Diminished acute YF-specific CD8 + T-cell responses in the elderly

CD8⁺ T cells, capable of killing virus-infected cells, represent an important immune effector mechanism essentially limiting virus dissemination in the body. As recently demonstrated in the murine YF model, CD8⁺ T cells complement humoral protection mediated through YF-neutralizing antibodies. In a B-cell knockout situation 25 % of mice survived an otherwise

DISCUSSION

100 % lethal intracerebral YFV injection (Bassi et al., 2015). For tracking YF-specific CD8⁺ T cell in the acute phase, we took advantage of the expression of the activation marker combination CD38 and HLA-DR, which can be measured directly ex vivo and which is applicable to all vaccinees regardless of their HLA types. In this kind of measurement it is unavoidable that some unspecific CD8⁺ T cells are co-detected, as seen in our baseline data, however CD8⁺ T cells expressing CD38⁺/HLADR⁺ in the acute response phase (between day 4 and 28) are almost exclusively YF-specific, as demonstrated in several publications (Miller et al., 2008;

Akondy et al., 2009, 2015; Ahmed and Akondy, 2011; Kohler et al., 2012; Blom et al., 2013;

DeWitt et al., 2015). In accordance with these reports, YF-specific CD38⁺/HLADR⁺ CD8⁺ T cells in our study expanded extensively until about day 17 and returned to basal levels by day 28, which we could demonstrate was kinetically true for both age groups. However, the magnitude of the YF-specific CD8⁺ T cell response was strikingly reduced in the elderly, which is another key finding of our study and confirms for the first time similar observations made in various aged animal models (Murasko and Jiang, 2005; Brien et al., 2009; Nikolich-Žugich et al., 2012) for a human setting. Thus, both major anti-viral effector mechanisms, neutralizing antibodies and specific CD8⁺ T cells, are obviously quantitatively diminished in the elderly, which we assume affects clearance of YF virus.

We furthermore investigated possible qualitative differences in the YF-specific CD8⁺ T-cell response for which we stimulated PBMCs with the very immunodominant HLA-A0201-restricted peptide LLWNGPMAV (Akondy et al., 2009). Only 5 young and 5 old vaccinees were carriers of HLA-A0201 in our study and thus statistics was underpowered. Nevertheless and in agreement with published data (Akondy et al., 2009; Querec et al., 2009), specific cell numbers in the acute phase obtained by peptide stimulation strongly overlapped with our CD38/HLADR measurements, indicating that results of the HLA-A0201⁺ subgroup are probably applicable to all YF vaccinees. Interestingly, YF-specific CD8⁺ T cells in aged vaccinees were characterized by increased expression of Granzyme A and B and decreased amounts of Perforin, while IFNγ, IL2 and CD40L expression was indifferent, which is in contrast to a publication comparing West-Nile-specific CD8⁺ T cells in young and old infected individuals, reporting no qualitative differences at all (Lelic et al., 2012). However, these West-Nile-specific CD8⁺ T cells were measured 3-4 months after disease onset and therefore might not reflect the acute state we observed within the first month after infection. This time difference might also explain why this group could not find any age-difference in the West-Nile-specific CD8⁺

DISCUSSION

response magnitude in an earlier publication (Parsons et al., 2008). Whether the observed age-related qualitative alterations, which certainly require validation in larger cohorts, further deteriorate the already quantitatively reduced YF-specific CD8⁺ T cell response, remains an open question. Future studies might employ ex vivo functional assays, e.g. specific killing assays of peptide-pulsed or infected target cells, which we could not conduct in this study due to the restricted blood volume we were limited to. Such analysis may help to uncover qualitative deficits in YF-specific CD8⁺ T cells in the elderly.

Beside that, it would be interesting to analyze the influence of the individual HLA composition, which we, except for HLA-A0201, did not consider in our study. In acute dengue patients it has been shown that certain HLA alleles correlate with stronger CD8⁺ response magnitudes (Weiskopf et al., 2013). Therefore individual HLA composition might bias our quantitative results and should be included in future studies, though this will require much larger cohorts.

Another aspect is bystander activation, which has been shown to exist in the CD8⁺ compartment in severe infections, such as with Hepatitis B, dengue, adeno-, hanta- and Influenza A virus, where a significant proportion of CD38⁺/HLA-DR⁺ CD8⁺ T cells are CMV or EBV specific (Tuuminen et al., 2007; Sandalova et al., 2010; Rivino et al., 2015). But this has not been observed after experimental YF or smallpox infection (Miller et al., 2008) or naturally-acquired TBE virus infection (Blom et al., 2015). The reason for these conflicting observations might be due to the fact that severe virus infections cause high levels of inflammation, inducing frequencies of more than 20 % of acutely activated CD38⁺/HLA-DR⁺ CD8⁺ T cells. In contrast, the infection with attenuated YF vaccine virus is usually very mild and typically induces CD38⁺/HLA-DR⁺ CD8⁺ T-cell frequencies below 10 %. As we did not determine CMV or EBV specific CD8⁺ T cells in the acute phase and such an analysis has never been done in aged donors, we cannot rule out that bystander CMV or EBV-specific CD8⁺ T cells contributed to the measured CD8⁺ response magnitude in our study. In case this had occurred in our elderly vaccinees, their net CD8⁺ response would have been even lower, which thus would have even worsened virus elimination.

Future studies should also address CD8⁺ T-cell clonality, which has been very recently investigated in YF vaccination in young adults. Based on deep sequencing of the CDR3 locus of the TCRβ chain, DeWitt et al. identified on average 2.000 YF-specific CD8⁺ T-cell clones at the peak of the acute response in each subject (DeWitt et al., 2015). It would be interesting, whether clonality is reduced in aged vaccinees, which could be another factor beside

DISCUSSION

quantitative and qualitative differences influencing vaccination outcome, though quite large amounts of blood are required for proper analysis.

Collectively, the cytotoxic CD8⁺ T-cell response appeared to be considerably reduced in aged YF vaccinees, which might be detrimental in the anti-viral defense.

4.5 Quantitative and qualitative alterations in the acute YF-specific CD4⁺ T cell response