Age-associated changes of the CD4+ T-cell response induced by yellow fever vaccination

Das menschliche Immunsystem unterliegt komplexen, altersassoziierten Veränderungen, die als Grundlage für die im höheren Lebensalter steigende Inzidenz von Infektions-, Tumor- und Autoimmunerkrankungen diskutiert werden. Der Gelbfieberimpfstoff YFV-17D enthält attenuiertes Gelbfieber-Virus und erzeugt nach Impfung eine Virusinfektion im menschlichen Organismus. So wird die hervorgerufene antivirale Immunantwort dem Untersucher zugänglich. In der hier vorliegenden Studie wurde die Immunantwort nach Gelbfieber-Erstimpfung bei 24 Probanden in zwei altersgetrennten Teilnehmerkohorten (23-28 Jahre und 55-68 Jahre) mithilfe von Analysen der induzierten Impfvirämie und Antikörperantwort im Serum sowie hochdimensionaler Durchflusszytometrie charakterisiert. Den Fokus bildeten hier der Phänotyp und die Funktionalität des CD4+ T Zellkompartiments vor und im Verlauf nach Impfung. CD4+ T Zellen üben einen wichtigen koordinativen Einfluss auf verschiedene Komponenten des Immunsystems aus: So unterstützen sie die Antikörperproduktion durch B Zellen, die Aktivität von zytotoxischen CD8+ T Zellen und agieren auch regulatorisch, um Immunantworten effizient zu begrenzen. CD4+ T Zellen durchlaufen entscheidende Selektionsschritte im Thymus, einem lymphatischen Organ, dessen Funktionalität mit zunehmendem Lebensalter abnimmt. Dies geht mit einem schrumpfenden Angebot und einer eingeschränkten Diversität neu gebildeter T Zellen einher mit bislang unzureichend geklärtem Einfluss auf die Erstantwort gegen ein neues Pathogen. Die serologische Analyse der YFV-17D-Virämie mittels RT-qPCR zeigte, dass ältere Probanden den Höhepunkt der Virämie signifikant später erreichten, also vermutlich schlechter die Replikation des Impfvirus eingrenzen konnten. Eine Verzögerung zeigte sich ebenfalls in der Bildung Gelbfieber-spezifischer neutralisierender Antikörper mit niedrigeren Titern der älteren Gruppe an Tag 14. Die Zusammensetzung des peripheren T-Zellpools vor Impfung wurde mithilfe von Zelloberflächenmarkern bestimmt. Hier zeigte sich, dass ältere Personen über signifikant weniger naive CD4+ T Zellen verfügten und insbesondere der Anteil der kürzlich dem Thymus entstammenden CD4+ T Zellen verringert war. Dies korrelierte mit niedrigeren Zahlen Gelbfieber-spezifischer CD4+ T Zellen in der Expansionsphase der Antwort. Probanden, mit niedrigeren Frequenzen dieser aktivierten Zellen, zeigten einen späteren Höhepunkt ihrer Serumvirämie und niedrigere Titer neutralisierender Antikörper an Tag 14. Das Zytokinprofil der mittels CD40L identifizierten YFV- 17D-spezifischen CD4+ T Zellen wurde nach in vitro Impfstoff-Stimulation untersucht und zeigte eine robuste, polyfunktionale Th1-dominierte Antwort ohne signifikante Differenzen zwischen beiden Altersgruppen. Alle Probanden entwickelten ungeachtet ihres Alters bis Tag 28 eine als protektiv einzustufende Impfantwort, welche auch bis zum Folgetermin nach 20-35 Monaten weitestgehend stabil blieb. Altersassoziierte Einschränkungen der Immunkompetenz ließen sich also vorrangig in der Akutphase der Impfantwort nachweisen. Dies könnte zusammengefasst als Folge des bei älteren Menschen verkleinerten Repertoires naiver CD4+ T Zellen bei erhaltener Funktionalität der einzelnen T Zelle interpretiert werden.The human immune system is subject to complex, age-associated changes, which are being discussed as causes for the increasing incidence of infectious, malignant and autoimmune diseases at higher age. The Yellow Fever vaccine YFV- 17D contains attenuated Yellow fever virus and induces upon vaccination a viral infection in the human organism. Thus, an antiviral immune response becomes accessible to the investigator. In this study, the immune responses after first-time YFV-17D-vaccination was monitored by serological analyses of serum viremia and antibody titers as well as multidimensional flow cytometry (FACS) in 24 healthy volunteers belonging to two age-sperated cohorts (23-28 years and 55-68 years). A focus was set on the phenotype and functionality of the CD4+ T-cell-compartment before and after vaccination. CD4+ T Cells act as important coordinators of various arms of the immune system, supporting antibody production by B cells, enhancing the performance of cytotoxic CD8+ T cells and furthermore regulating immune responses to an optimal level. During their formation, CD4+ T cells pass through selection in the thymus, a lymphoid organ loosing functionality with increasing age. This involves a shrinking and less diverse pool of newly formed T cells with so far unclear impact on the primary response against a new pathogen. The serological analysis of YFV-17D- viremia by RT-qPCR showed, that elderly participants reached their maximum of viremia significantly later, suggesting an impaired capability to control viral replication. A delay was also observed in the production of Yellow- fever-specific neutralizing antibodies in elderly donors with lower median titers at day 14. The composition of the peripheral CD4+ T-cell-pool prior to vaccination was assessed with the help of cell surface markers. Elderly donors possessed significantly less naive CD4+ T cells with a particularly strong reduction in cells having recently emigrated from the thymus. These lower numbers correlated with fewer Yellow-fever-specific CD4+ T cells during expansion-phase of the response. Donors, displaying smaller numbers of this activated subset, showed a delayed peak in serum -viremia and lower titers of neutralizing antibodies at day 14. The cytokine-profile of YFV-17D-specific CD4+ T cells, identified according to their surface-expression of CD40L after in vitro vaccine-stimulation, showed a robust, polyfunctional Th1-dominated immune response without significant deviations between age groups. Until day 28, all donors displayed protective immune responses regardless their age, which remained stable in terms of antigen-specific CD4+ T cells and antibody titers also until the follow up at 20-35 months after vaccination. Age-related impairments of the immune competence could therefore be observed predominantly in the acute phase of the immune response. The initial delay suggests a skewed repertoire of naive CD4+ T cells in the elderly with preserved functionality of the individual cell

Highly Predictive Model for a Protective Immune Response to the A(H1N1)pdm2009 Influenza Strain after Seasonal Vaccination

Understanding the immune response after vaccination against new influenza strains is highly important in case of an imminent influenza pandemic and for optimization of seasonal vaccination strategies in high risk population groups, especially the elderly. Models predicting the best sero-conversion response among the three strains in the seasonal vaccine were recently suggested. However, these models use a large number of variables and/or information post- vaccination. Here in an exploratory pilot study, we analyzed the baseline immune status in young

Age dependent differences in the kinetics of Y delta T cells after influenza vaccination

Immunosenescence is a hallmark of the aging immune system and is considered the main cause of a reduced vaccine efficacy in the elderly. Although γδ T cells can become activated by recombinant influenza hemagglutinin, their age- related immunocompetence during a virus-induced immune response has so far not been investigated. In this study we evaluate the kinetics of γδ T cells after vaccination with the trivalent 2011/2012 northern hemisphere seasonal influenza vaccine. We applied multi-parametric flow cytometry to a cohort of 21 young (19–30 years) and 23 elderly (53–67 years) healthy individuals. Activated and proliferating γδ T cells, as identified by CD38 and Ki67 expression, were quantified on the days 0, 3, 7, 10, 14, 17, and 21. We observed a significantly lower number of activated and proliferating γδ T cells at baseline and following vaccination in elderly as compared to young individuals. The kinetics changes of activated γδ T cells were much stronger in the young, while corresponding changes in the elderly occurred slower. In addition, we observed an association between day 21 HAI titers of influenza A and the frequencies of Ki67+ γδ T cells at day 7 in the young. In conclusion, aging induces alterations of the γδ T cell response that might have negative implications for vaccination efficacy

Age dependent differences in the kinetics of γδ T cells after influenza vaccination

<div><p>Immunosenescence is a hallmark of the aging immune system and is considered the main cause of a reduced vaccine efficacy in the elderly. Although γδ T cells can become activated by recombinant influenza hemagglutinin, their age-related immunocompetence during a virus-induced immune response has so far not been investigated. In this study we evaluate the kinetics of γδ T cells after vaccination with the trivalent 2011/2012 northern hemisphere seasonal influenza vaccine. We applied multi-parametric flow cytometry to a cohort of 21 young (19–30 years) and 23 elderly (53–67 years) healthy individuals. Activated and proliferating γδ T cells, as identified by CD38 and Ki67 expression, were quantified on the days 0, 3, 7, 10, 14, 17, and 21. We observed a significantly lower number of activated and proliferating γδ T cells at baseline and following vaccination in elderly as compared to young individuals. The kinetics changes of activated γδ T cells were much stronger in the young, while corresponding changes in the elderly occurred slower. In addition, we observed an association between day 21 HAI titers of influenza A and the frequencies of Ki67⁺ γδ T cells at day 7 in the young. In conclusion, aging induces alterations of the γδ T cell response that might have negative implications for vaccination efficacy.</p></div

Hierarchical network representation of immune cell-subset counts at baseline with respect to A(H1N1)pdm09 protection in the pilot study.

<p>We monitored 36 immune cell subpopulations in A(H1N1)pdm09 sero-negative donors and compared donors who became either sero-protected or not at day 21 after vaccination. We observe a number of cell populations for which the counts are significantly different between protected and non-protected donors, specifically on the CD4⁺ T cell axis. The colors indicate the relative median counts of the groups. Significant differences were determined using the Wilcoxon-Test and indicated with * for p<0.05 and ** for p<0.01.</p

Prediction of non-protection to the A(H1N1)pdm09 influenza strain as function of the combination of age, NSSN and CD4⁺ T cells after the validation study.

<p>The logistic regression model combining baseline CD4⁺ T cell counts with age and NSSN is validated with a high ROC-AUC = 0.85, significant p-value = 0.0056 and high accuracy of 85% for the same age groups (<31 and >49 years) as in the pilot study (left panels in A and B). However, the addition of the middle age group (31–49 years) in the validation study somewhat reduces the accuracy of the prediction when using age as a linear function (center panels in A and B), because donors with these ages respond rather like the younger donors. Transformation of age to a sigmoid based function (with a midpoint age of 50 years) gives the best prediction with accuracy 85% and a highly significant p-value = 0.0000004 when combining both studies (right panels in A and B). The multi-factorial risk profile for non-protection (HAI<40) to the A(H1N1)pdm09 influenza strain is clearly seen (C) when combining the sero-negative vaccinees from both studies (N = 80). Donors with high baseline CD4⁺ T cell counts (>860 cells/μL) are all protected (p = 0.02 for NSSN = 3), as well as young (<50 years) donors with low CD4⁺ counts but NSSN = 1–2. Non-protection is only observed for old donors with low CD4⁺ counts (20%, 50% and 64% for NSSN = 1, 2 and 3 respectively) and for young donors with low CD4⁺ counts and NSSN = 3 (24%). Lastly, a prediction model (D) for the probability of non-protection to the California H1N1 strain is obtained by simulating the continuous contribution of age (after logistic function transformation from 20 years young in blue to 80 years old in red), NSSN and baseline CD4⁺ T cell counts, where the combined effect of the 3 variables can be clearly seen.</p

Prediction of serological response to A(H1N1)pdm09 as function of counts of major lymphocyte sub-populations in the pilot study.

<p>A) H1N1 sero-negative individuals that were sero-protected (blue circles) to H1N1 at day 21 post-vaccination had higher counts of CD4⁺ T cells, CD8⁺ T cells or B cells (CD19) at baseline as compared to non-protected individuals (red triangles, P<0.05). However, there is no continuous correlation between the various cell counts and the HAI titer. B) Logistic regression shows that a model combining baseline CD4⁺ T cell counts with age and NSSN, is the best predictor of sero-protection, with a high ROC-AUC = 0.92 and significant p-value = 0.00002. Similar models for CD8⁺ T cells or B cells (CD19⁺) give reasonable albeit lower prediction values. C) The combination of CD4⁺ T cell counts, NSSN and age gives a highly accurate 89% prediction of non-protection (when selecting for a specificity = 100%, or positive predictive value ppv = 100%, in order to capture all non-responders), with a sensitivity of sen = 86% and negative predictive value npv = 69%. The other lymphocyte sub-populations counts give less accurate predictions.</p

Multi-factorial association of serological response to the A(H1N1)pdm09 influenza strain as function of age, NSSN and total, naïve and influenza specific activated CD4⁺ T cells in the pilot study.

<p>A) High CD4⁺ T cell counts give rise to sero-protection (HAI>40) irrespective of age or NSSN. Age still plays a role in vaccinees with NSSN = 3 and low CD4⁺ T cell counts, where 80% of old (red circles) versus only 20% of young (blue circles) are non-protected (p = 0.01). The same trend (p = NS) is also seen for NSSN = 2 (triangles) with low CD4⁺ T cell counts, albeit with better response than NSSN = 3. The only 2 donors with NSSN = 1 are sero-protected even if they are old and have low CD4⁺ T cell counts. B) Naive CD4⁺ T cell counts show a trend (p = NS) for a positive association with serological response in all age groups (NSSN = 2–3). C) Influenza specific activated CD4⁺CD40L⁺ T cell counts are not associated with serological response in any of the age groups (NSSN = 2–3).</p