Influence of the innate immune system

We also hypothesized that early response signatures of the innate immune system might affect the YF-specific adaptive immune responses. For this, we developed a FACS panel that allowed a comprehensive analysis of major peripheral innate cell types over the entire study course. Our analysis included: Three different DC subsets, such as plasmacytoid DCs (pDCs), myeloid type 1 DCs (CD11c⁺ mDCs) and myeloid type 2 DCs (CD141⁺ mDCs), as well as three monocyte subsets: CD14⁺⁺/CD16^- classical, CD14⁺⁺/CD16⁺ intermediate and CD14⁺/CD16⁺ nonclassical monocytes (Chap. 2.2.9.2) and basophils (Fig. 2.2). In addition we assessed neutrophils and eosinophils in 18 of 23 vaccinees by complete blood count (Chap. 2.2.6).

We observed distinct innate response patterns after YF vaccination (Fig. 3.12A-I). Neutrophils, pDCs, CD11c⁺ mDCs and CD14⁺⁺/CD16^- classical monocytes were characterized by a significant and transient increase of cell numbers around day 2/4, followed by a drop towards day 7/10 and a secondary increase at the late phase of the response (Day 14 - 21). Other innate cell types, such as eosinophils, basophils and CD141⁺ mDCs did not show this pronounced primary peak at day 2/4 but a decrease in numbers around day 7/10 and a subsequent re-appearance or even slight peak in the late phase of the response. In contrast to the aforementioned cell types, CD14⁺⁺/CD16⁺ intermediate and CD14⁺/CD16⁺ nonclassical monocytes peaked at day 7 after vaccination and subsequently converged to baseline levels. We furthermore found remarkable age-differences in some innate cell subsets: Elderly had highly significantly fewer pDCs and CD11c⁺ mDCs particularly at the primary peak at day 4. Conversely, they possessed significantly more CD14⁺⁺/CD16⁺ intermediate monocytes at day 10 and also higher numbers of CD14⁺/CD16⁺ nonclassical monocytes from day 10 onwards, indicating deferred kinetics of both monocyte subsets in elderly individuals. Interestingly, calculating ratios of classical to nonclassical monocytes revealed very significant differences at all study days between the age groups (Day 4, Fig. 3.12K). Thus, the relative proportion of CD14⁺/CD16⁺ nonclassical monocytes was generally elevated in the monocyte compartment of the elderly.

We applied multivariate analysis to our multiplex innate immune data of day 4 and generated complex, systemic views of our vaccinees (Chap. 2.2.10). The automated cluster analysis (ACA) revealed a relatively continuous distribution of innate immune states among our subjects that could only approximately segregate between different clusters (Fig. 3.13A). However we

RESULTS

observed an accumulation of young vaccinees in clusters 3 and 4 characterized by mostly higher numbers of CD11c⁺ mDCs, pDCs, basophils and partly lower numbers of CD16⁺/CD14⁺ nonclassical monocytes (cluster 3). In contrast, clusters 1 and 2 were dominated by older vaccinees with intermediate to high levels of CD16⁺/CD14⁺ nonclassical monocytes and lower numbers of CD11c⁺ mDCs, pDCs and basophils. Exceptional and separated from all other

Figure 3.12: Innate immunity during YF vaccination

(A-I) Absolute counts of classical (A), non-classical (B), intermediate (C) monocytes, pDCs (D), CD11c⁺ mDCs (E), CD141⁺ mDCs (F), basophils (G), neutrophils (H) and eosinophils (I). P-values are listed in suppl. table 1. (K) Age comparison of the ratio classical/non-classical monocytes at day 4. p=0.01 Lines indicate the median with interquartile error bars. Young: filled circles; elderly:

open circles

RESULTS

Figure 3.13: Multivariate analysis of innate immunity

(A) Automated cluster analysis of innate immunity at day 4 based on whole blood counts of nine major innate cell subsets. Values are scaled and normalized. (C) Below, normalized YF response parameters are assigned to the cluster analysis. Dark blue indicates low, red high values, empty fields data not available (B) PCA of innate immunity at day 4 based on whole blood counts of seven innate cell subsets. Vector arrows indicate contribution of innate components on PCA (2fold magnified). Young: filled circles; elderly: open circles

vaccinees was donor 28, who had very high amounts of nonclassical and transitional monocytes, basophils and eosinophils. Additionally, we assessed our innate dataset by PCA.

Here, we excluded donor 28 from the analysis as he was disproportionally influencing the scaling of the data. As illustrated in fig. 3.13B, YF vaccinees could be segregated into two large clusters following largely the age criterion with only one old and three young misclassified individuals (two-tailed Fisher’s test: p=0.007). Thereby, PCA produced a similar classification as the automated cluster analysis, i.e. PCA cluster 1 (mostly elderly) corresponded to ACA cluster 1 and 2, whereas PCA cluster 2 (mostly young vaccinees) was congruent with ACA cluster 3 and 4. Altogether, by multivariate analysis we could comprehensively and unbiasedly compare the young and senescent innate immune system. Similar to the univariate analysis, age was identified as the most determining factor manifested e.g. by decreased DC and increased nonclassical monocyte numbers.

RESULTS

We noticed that the innate immune peak at day 4/7 coincided with the occurrence of mild adverse reactions such as fever, myalgia and erythema. Remarkably, young vaccinees were reporting more frequently side effects than elderly vaccinees (young: 64 %, elderly: 33 %).

Grouping vaccinees according to mild adverse symptoms, revealed a clear difference in pDC numbers at day 4, i.e. high numbers of pDCs were characteristic for vaccinees reporting any symptoms (Fig. 3.14A).

Figure 3.14: Influence of DCs on the YF-specific T-cell response

(A) Absolute numbers of pDCs at day 4 in groups with or without occurrence of mild adverse events after vaccination. p=0.005 Without symptoms: filled triangles; With symptoms: open triangles (B-C) Positive correlations of absolute numbers of pDCs (B) or CD11c⁺ mDCs (C) at day 4 with numbers of activated HLA-DR⁺/CD38⁺ CD8⁺ T cells at day 14 after vaccination. For pDCs positive correlations exists also for the CD8⁺ T-cell response at day 17 and 21. (D) Numbers of CD11c⁺ mDCs at day 4 correlate with numbers of Ki-67⁺/CD38⁺ CD4⁺ T cells at day 7. (E-F) Numbers of pDCs at day 4 correlate with qualitative functionality of the YF-specific CD4⁺ T-cell response. Lines indicate the median with interquartile error bars. Young: filled symbols; elderly: open symbols

We next analyzed whether early innate signatures translated into YF-specific adaptive immune responses. We discovered that numbers of pDCs and CD11c⁺ mDCs at the peak of innate immunity (day 4) were highly correlative with the specific T-cell response, e.g. both DC subsets positively correlated with numbers of YF-specific HLA-DR⁺/CD38⁺ CD8⁺ T cells at days 14, 17 and 21 (Day 14, Fig. 3.14, B and C). Moreover CD11c⁺ mDCs also correlated with YF-specific Ki-67⁺/CD38⁺ CD4⁺ T cells in the expansion phase at day 4 and 7 (Day 7, Fig. 3.14D).

In line with this, early DC responses also translated positively into YF-specific CD4⁺ cytokine responses at day 7 and 10 (not shown) and were significantly related to polyfunctionality of

RESULTS

YF-specific CD4⁺ T cells (Fig. 3.14, E and F). We further connected cluster analysis of innate cell subsets at day 4 with major adaptive response parameters (e.g. acutely activated CD4⁺ and CD8⁺ T cells) that were scaled and color-coded for better visualization (Fig. 3.13C). The distribution of some of these parameters amongst the clustered vaccinees followed very interesting patterns: For example YF-specific HLA-DR⁺/CD38⁺ CD8⁺ T cells at day 14 and 17 were clearly higher in clusters 3 and 4 than in cluster 1 and 2. The same could be observed for CD4⁺ cytokine polyfunctionality (3 and 4 functions), whereas the opposite was true for CD4⁺ single functionality. It was of note that these patterns did not solely follow the age criterion, but the true individual innate immune state. For example donor 12 was grouped despite its age to cluster 4 and demonstrated equally high levels of e.g. YF-specific HLA-DR⁺/CD38⁺ CD8⁺ T cells as young vaccinees of cluster 3 and 4. Conversely, two of three young vaccinees (4 and 17), who belonged to cluster 2, showed similar low levels of YF-specific HLA-DR⁺/CD38⁺ CD8⁺ T cells as elderly subjects of cluster 1 and 2. Consistently, we could visualize these associations also in our PCA when we overlaid median-segregated YF-specific response signatures (Suppl. fig. 2A-D). Thus multivariate analysis strongly supported the correlations observed by univariate analysis demonstrating a close connection of innate signatures particularly of pDCs and CD11c⁺ mDCs with quantity and quality of the YF-specific adaptive T-cell response.