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Immune Activation in HIV-treated Subjects and the Role
of Age and Discordant Phenotype. A Cross-sectional
Study
Anna Bonjoch1, Julià Blanco2,3, Jordi Puig1, Núria Pérez-Álvarez1,4, Elisabeth Gómez-Mora2, Cecilia Cabrera2, Marta
Massanella5, Patricia Echeverria1, Bonaventura Clotet1,2,3, and Eugènia Negredo1,3
1 Lluita contra la SIDA Foundation, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de
Barcelona, Badalona, Spain
2 IrsiCaixa-IGTP, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Barcelona,
Spain
3 Universitat de Vic-Universitat Central de Catalunya (UVIC-UCC), Barcelona, Spain
4 Statistics and Operation Research Department, Technical University of Catalonia, Barcelona, Spain 5 Centre de Recherche du Centre Hospitalier de l’Universite de Montreal (CRCHUM), Montreal, Canada
E-mail de correspondencia: abonjoch@flsida.org
ABSTRACT Background
Discordant immunologic pattern is a considerable in a proportion of the HIV infected subjects under successful treatment. It is unclear whether age has a stronger impact on immune parameters than the extent of CD4 reco-very.
Methods
We performed a subanalysis from a cross-sectional study in which we characterized the mechanisms underlying poor immune response in 229 virologically suppressed subjects (<50 copies/mL >2 years); 96 were discordant (D, defined as CD4 T-cell counts <350 cells/µL) and 133 were concordant (C).Levels of immunological markers were compared between 4 subsets(D>50 years [n=28]; D≤50 years[n=68]; C>50 years[n=34]; and C≤50 years[n=99]). Continuous variables were compared using nonparametric tests (Mann-Whitney and Kruskal-Wal-lis); categorical variables were compared using the χ2 or Fisher exact as appropriate.
Results
Comparison of the 4 groups revealed lower thymic function (CD45RA+CD31+) and higher naïve CD4 T-cell pro-liferation (CD31–% of CD45RA+CD4+) in older patients, irrespective of the extent of immune recovery. When C>50 years and D<50 years were compared, both factors had a similar impact on thymic output, while the discordant phenotype had a higher impact on CD4 and CD8 T-cell activation than age.
Conclusions
The immunodiscordant phenotype is associated with more frequency of activation markers than age, while age is linked to a reduced thymic function.
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Activación inmune en sujetos tratados con VIH
y el papel de la edad y el fenotipo discordante.
Un estudio transversal
RESUMEN Introducción
Los patrones inmunológicos discordantes son un problema en una proporción considerable de personas infec-tadas por el VIH, aunque se hallen bajo un tratamiento antirretroviral supresor. Permanecen en controversia si la edad tiene un impacto más importante en los parámetros inmunológicos que el grado de recuperación de CD4.
Métodos
Realizamos un subanálisis de un estudio transversal en el que caracterizamos los mecanismos que subyacen a la respuesta inmune deficiente en 229 sujetos suprimidos virológicamente (<50 copias /ml> 2 años); 96 fue-ron discordantes (D, definida como recuentos de células T CD4 <350 células / µl) y 133 fuefue-ron concordantes (C). Los niveles de marcadores inmunológicos se compararon entre 4 subgrupos (D> 50 años [n = 28]; D≤50 años [n = 68]; C> 50 años [n = 34]; y C≤50 años [n = 99] ). Las variables continuas se compararon mediante pruebas no paramétricas (Mann-Whitney y Kruskal-Wallis), las variables categóricas se compararon utilizando el χ2 o el Fisher exacto según fuera apropiado.
Resultados
La comparación entre los 4 grupos reveló una función tímica más baja (CD45RA+ CD31+) y una mayor
prolife-ración de células T CD4 naive (CD31-% de CD45RA+ CD4+) en los pacientes de mayor edad,
independiente-mente de la extensión de la recuperación inmunitaria. Cuando se compararon C> 50 y D <50 años, ambos factores tuvieron un impacto similar en la producción tímica, mientras que el fenotipo discordante tuvo un impacto mayor en la activación de las células T CD4 y CD8 que en la edad.
Conclusiones
El fenotipo inmunodiscordante se asocia con más marcadores de activación que la edad, mientras que la edad está vinculada a una función tímica reducida.
Lista de abreviaturas: VIH: virus de inmunodeficiencia humana; CD: grupo de diferenciación; D: grupo
discordante; C: grupo concordante; SIDA: síndrome de inmunodeficiencia adquirida; HLA-DR+: antígenos de
leucocitos humanos; PD-1+: Muerte-ligando programado 1; IQR: rango intercuartil; SPSS: Paquete
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METHODS
We performed a subanalysis of a cross-sectional study (EO code: EO-07–024) to analyze the role of thymic output and immune activation in 230 patients who were attended in our HIV Outpatient Unit while receiving antiretroviral therapy and had sustained viral suppression for at least 2 years. Discordant patients were defined as individuals with a CD4 cell count al-ways below 350 cells/µL; concordant patients were defined as individuals with a CD4 cell count above 400 cells/µL8. The study was approved by the
Institu-tional Review Board. All patients provided their writ-ten informed consent. A single blood extraction was obtained to determine immunological parameters.8
To clarify the role of age and immune status on immune activation, we evaluated the percentage of thymic output (CD45RA+CD31+), naïve CD4 T cells
(CD45RA+), memory CD4 T cells (CD45RA–), CD4
and CD8 immune activation (HLA-DR+, CD45RA–
CD38+), naïve CD4 T-cell proliferation (CD31–% of
CD45RA+CD4+), and CD4 T-cell exhaustion (PD-1+).
Spontaneous cell death was assessed in cultures of PBMC as described elsewhere.2 The parameters
were compared between 4 groups: discordant>50 years (n=28); discordant≤50 years (n=68); con-cordant>50 years (n=34); and concordant≤50 years (n=99).
Statistical analysis
Continuous variables were expressed as the median (IQR) and compared using nonparametric tests (Mann-Whitneyand Kruskal-Wallis) because they were not normally distributed. Discrete variables were de-scribed as the number of individuals (percentage) and compared using the χ2 or Fisher exact test, as
appro-priate. Multiple testing corrections were developed using the Benjamini and Hochberg approach, the ad-justed p-values are reported. (R software, p.adjust; Package: stats).
INTRODUCTION
One of the challenges in the actual situation of the HIV- infected subjects under antiretroviral therapies is the poor immune recovery in a proportion of people despite the successful treatment. This pattern, called immuno-discordant or immuno-discordant phenotype, presents high lev-els of immune activation and inflammation markers.
Marked intrinsic CD4 T-cell apoptosis is consid-ered the principal mechanism of cell destruction and the cause of the discordant immune response1, 2. The
main predictors of a poor immune response to viro-logical suppressed antiretroviral therapy include ad-vanced age3, low nadir CD4 T-cell count4, presence of
AIDS-defining conditions, and coinfection with virus-es such as hepatitis C virusvirus-es5.
Aging of the HIV-infected population means that age-related complications are more prevalent and ap-pear earlier in persons living with HIV infection6. In this
sense, the effects of aging on the immune system (“inflamm-aging”) are well defined7 and closely
asso-ciated with immune exhaustion and incomplete im-mune recovery.
Age and discordant phenotype may have an inde-pendent and significant impact on immune activation in HIV-infected persons. While the deleterious conse-quences of both factors are well known, it is unclear whether age has a stronger impact on immune pa-rameters than the extent of CD4 T-cell recovery. A po-tentially synergistic effect of both factors (age and discordant phenotype) on immune activation markers has not been investigated.
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RESULTS
Of the original 230 patients included in the main study2,
one was excluded from the subanalysis owing to missing data.
The characteristics of the participants are sum-marized in Table 1. The median age in patients aged >50 and ≤50 years was 55 and 44 years, re-spectively. No differences in time from HIV infec-tion were found between the 4 groups. Current and nadir CD4 counts in the concordant and discor-dant groups were 633, 217, 252, and 71 cells/µL, respectively.
TABLE 1.CHARACTERISTICS OF THE POPULATION.
Concordant Discordant
≤50 years
N=99 >50 years N=34 ≤50 years N=68 ≤50 years N=68
Time with HIV infection, years [8.8,16.1]12.6 [10.1,17.9]13.9 [6.8,19.1]13.3 [8.1,20.1]10.5
Age, years [39,46]43 [52,60]55 [43,48]45 [53,60]56
Nadir CD4+ T-cell count (cells/µl) 234
[116,330] [130,293]201 [28,137]66 [55,148]83
CD4+ T- cell count (cells/µl) 668
[497,801] [472,683]546 [202,295]247 [194,332]285
CD4+ % 31
[27,38] [24,34]27 [14,22]19 [15,26]19
CD8+ T- cell count (cells/µl) 827
[639,1102] [703,1212]870 [509,940]682 [476,921]710
CD8+ % 40
[34,48] [39,49]44 [45,58]51 [44,56]50
Thymic output
CD45RA+CD31+ (% of CD4+ T cells) [11.9,26.8]19.6 [7.4,20.9]14.1 [6.5,22.0]12.1 [2.4,12.9]6.8
CD4 Naïve
CD45RA+ (% of CD4+ T cells) [19.5,39.3]29.4 [17.5,31.4]24.3 [10.7,31.5]20.5 [9.4,18.8]13.8
CD4 Memory
CD45RA- (% of CD4+ T cells) [60.7,80.6]70.6 [68.6,82.5]75,8 [68.5,89.3]79.5 [81.2,90.6]86.3
CD4 Naïve proliferation
CD31- (% of CD45RA+CD4+ T cells) [22.5,45.4]33.6 [26.4,58.9]43.7 [22.7,42.7]32.1 [33.1,67.2]51.7
CD4 activation
HLADR+ (% of CD4+ T cells) [4.9,9.9]6.9 [6.7,13.9]7.5 [9.2,32.5]14.6 [9.0,29.7]13.2
CD4 Exhaustion
PD1+ (% of CD4+ T cells) [4.4,10.2]6.9 [6.6,11.4]8.4 [6.8,14.6]9.6 [8.0,15.7]11.1
CD8 activation
CD45RA-CD38+ (% of CD8+ T cells) [6.8,17.9]11.3 [5.6,15.3]7.8 [9.7,22.8]14.7 [7.8,28.4]13.4
CD38+ (% of CD45RA- CD8+ T cells) 20.1
[12.8,30.8] [9.5,24.3]14.5 [18.4,45.3]24.0 [16.2,42.4]28.9
HLADR+ (% of CD8+ T cells) 15.3
[8.5,22.8] [10.4,22.5]16.7 [10.8,34.8]20.2 [15.0,30.1]21.2
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e 2018 The comparison of immune activation parameters between the 4 different groups is summarized in Table 1. Briefly, older patients had a lower percentage of
naïve CD4 T cells (CD45RA+) and reduced thymic
function (CD45RA+CD31+) and a higher percentage of
naïve CD4 T-cell peripheral proliferation (CD31– % of
CD45RA+CD4+) and memory cells (CD4+CD45RA–),
ir-respective of the extent of immune recovery (concor-dant or discor(concor-dant).
The effects of age and immune recovery were compared between concordant patients aged >50 years and discordant patients aged ≤50 years. Both factors had a similar impact on thymic output, while the impact of the discordant phenotype on CD4+ and
CD8+ T-cell activation was more pronounced than that
of age.
DISCUSSION
Our study reveals low thymic function and naïve CD4+count in the case of elderly subjects. In contrast,
high percentages of memory cells were seen in these individuals. The discordant phenotype was closely associated with higher CD4 T-cell exhaustion andC-D4+and CD8+ T-cell activation marker levels.
Com-pared with the discordant phenotype, age seems link a different but synergistic immunological pattern.
Immune activation and inflammation play a crucial role in elderly non–HIV-infected persons by promoting a gradual age-related decline in immune function, leading to “immunosenescence.”9 The impact of aging
on immune function is described mainly as thymic in-volution contributing to decreased naïve CD4+count
and a generalized chronic inflammatory state. Immune activation and chronic inflammation also affect HIV-in-fected persons, even when they are receiving antiret-roviral therapy and have suppressed viral load, and its multifactorial, involving host and viral characteristics as well comorbidities or coinfections. Therefore, both HIV infection and aging share common detrimental pathways that lead to earlier immunosenescence10.
The poorer thymic function we observed in older individuals was more accentuated in discordant pa-tients8. However, the lack of differences between
con-cordant patients aged >50 years and discon-cordant pa-tients aged ≤50 years highlights the specific impact of age on this parameter. In addition, and consistent with previous results, older patients exhibited low percentages of naïve CD4+ and high memory T cell.
Significant differences were also observed when pro-liferation of CD31– naïve cells in the CD4+
compart-ment in older individuals, with higher percentages but non-significant differences between discordants and concordant subjects, thus indicating an additive ef-fect of the discordant phenotype and age.
The discordant phenotype is characterized by high levels of activated CD4+ and CD8+T cells, lower naïve
CD4+ T-cell production, higher spontaneous exvivo
CD4 T-cell death, and higher plasma levels of soluble CD148. We also found high levels of CD4+ and CD8+
T-cell activation in the discordant group, irrespective of age.
Despite the number of parameters analyzed, we have not been able to evaluate the entire range of fac-tors that may have an impact on the immune status, and that involve the host, the virus and external fac-tors. In this sense, as a study of Saison et al demon-strated, the regulatory T cells (Tregs) was higher in older subjects and in case of discordant phenotype.11
Our group performed a substudy also evaluating the Tregs in the context of discordant phenotype. We evaluated a subgroup of 56 subjects (33 concordant versus 23 discordant individuals).The results showed a higher frequency and proliferation of Tregs in discor-dant than the concordiscor-dant subjects, but in this case there was not differences in age between groups.12
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ACKNOWLEDGEMENTS
We are grateful to Thomas O’Boyle for language revi-sion and editorial assistance. No funding was re-ceived for this study.
AB has received fees from ViiV, Jansen Cilag, Abb-Vie, and Roche; JP has received fees from AbbVie; PE has received fees from ViiV, Jansen Cilag, and Abb-Vie; EN and BC have received fees from ViiV, Merck, Jansen Cilag, Bristol-Meyers Squibb, Gilead, and Ab-bVie. All other authors: none to declare.
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Our study is limited by the lack of consensus on the definition of the discordant phenotype, and higher differences may have been found if the discordant group had included more severely immunosup-pressed individuals (<200 cells/µL) for comparison with more immunocompetent patients (>500 cells/ µL). Similarly, a wider range between the median age of groups than ≤50 years and >50 years would help to better define immune patterns. Other concern of the study is that other factors with inflammatory impact are not collected for the analyses due to the retro-spective design of the study: we are unable to collect illicit drugs, co morbidities, as well medication that reduce the inflammation, as statins,
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9. Franceschi C, Monti D, Barbieri D, Salvioli S, Grassilli E, Capri M , et al. Successful immunosenescence and the remodelling of immune responses with ageing. Nephrol Dial Transplant. 1996; 11 (Suppl. 9):18-25. 10. Desai S, Landay A. Early immune senescence in HIV
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