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Inflammation, HIV and Aging
Savita Pahwa, MDUniversity of Miami Miller School of Medicine
HIV DART, Miami28 NOVEMBER 2018
The HIV infected population is aging
Why focus on Older Age Group?
In the US, Florida has the highest percentage of elderly people (19.4%)
Aging population is increasing worldwide
Chronologic Aging - associated with• Inflammation---termed inflammaging –driver
of comorbidities • Immune senescence- declining immunity –
increased susceptibility to infections
Chronic HIV (on ART) - associated with• Inflammation and immune activation • Earlier development of comorbidities-
?Premature aging• Immune compromised state-
?Premature aging
Features of Natural Aging and of HIV
Immunity in HIV and Aging
Aging
InflammationHIV
Immune activation
? ?
?
Prevailing questions:
A. What are independent and combined effects of each state (i.e. aging and HIV) on measurements of IA and inflammation?
B. How does each state (and combined) affect immune competence?
Immunity in HIV and Aging Project
R01 AI108472
HIV+ (154)Virally suppressed (plasma virus load < 50 copies/ml) on cART for >1 year prior to enrollment
HIV negative (161) healthy controls
(HC)
Age Groups HC HIVYoung (Y), 18-39 years 48 34Middle (M), 40-59 years 62 70Old (O), 60-80 years 51 50
Study Participants
Immune signatures ofA. AgeB. Immune Competence
Evaluation of Immunologic Biomarkers
Immune Activation/ Immune Checkpoint • CD38, HLA-DRPD-1, Ki-67, etc
Immune Cell Distributions• T cell subset distribution (CD4 and
CD8 absolute counts, Naïve, Memory, pTfh
• B cell subsets• Monocyte subsets
Soluble Plasma Biomarkers• Pro-Inflammatory (TNFa, IL-6)• Soluble Receptors (sTNFR,
IL2R)• Microbial Translocation (sCD14,
LPS)
Immune senescence
Immune exhaustionPlasma Biomarkers
HIV vs HC
Young HIV (age <40 years) show most differences from HC
Plasma Biomarkers: Correlation with Age in HIV and HC
Red box: Common for HIV and HC; Green Box: only in HC
Lesley de Armas
Immune senescence
Immune exhaustionPlasma Biomarkers
HIV vs HC
Young HIV (age <40 years) show most differences from HC
Plasma Biomarkers: Correlation with Age in HIV and HC
Red box: Common for HIV and HC; Green Box: only in HC
Lesley de ArmasAging is associated with inflammation in HC and HIV, with more cytokines associated with healthy aging. However differences between HC and HIV more evident in young age.
0 2 0 4 0 6 0 8 0 1 0 00
5
1 0
1 51 53 04 5
C D 4 + C D 3 8 + H L A D R +
A g e (y rs )
% o
f C
D4
H C n s
H IV n s
H C v s . H IV :p < 0 .0 0 0 1
C o rre la tio n w ith A g e
Immune activation
0 1 0 2 0 3 0 4 00
2 0
4 0
6 0
8 0
1 0 0
P D 1 (% o f C D 4 )
Ag
e E
ntr
y (
Ye
ars
) H C n sH IV p = 0 .0 0 0 4
Immune exhaustion
0 2 0 4 0 6 0 8 00
2 0
4 0
6 0
8 0
1 0 0
5 7 + 2 8 - (% o f C D 4 )
Ag
e E
ntr
y (Y
ea
rs) H C n s
H IV p = 0 .0 0 5 4
Immune senescence
de Armas et al, JCI insight, 2017
Immune Activation: Correlation with Age in HIV and HC
Frequency of cells co-expressing >1 immune activation markers is greater in HIV than in HC
(CD38, HLA-DR, PD-1, ICOS, Ki-67)
0 2 0 4 0 6 0 8 0 1 0 00
5
1 0
1 51 53 04 5
C D 4 + C D 3 8 + H L A D R +
A g e (y rs )
% o
f C
D4
H C n s
H IV n s
H C v s . H IV :p < 0 .0 0 0 1
C o rre la tio n w ith A g e
Immune activation
0 1 0 2 0 3 0 4 00
2 0
4 0
6 0
8 0
1 0 0
P D 1 (% o f C D 4 )
Ag
e E
ntr
y (
Ye
ars
) H C n sH IV p = 0 .0 0 0 4
Immune exhaustion
0 2 0 4 0 6 0 8 00
2 0
4 0
6 0
8 0
1 0 0
5 7 + 2 8 - (% o f C D 4 )
Ag
e E
ntr
y (Y
ea
rs) H C n s
H IV p = 0 .0 0 5 4
Immune senescence
de Armas et al, JCI insight, 2017
Immune Activation: Correlation with Age in HIV and HC
Frequency of cells co-expressing >1 immune activation markers is greater in HIV than in HC
(CD38, HLA-DR, PD-1, ICOS, Ki-67)
HIV is associated with far greater immune activation than HC, most of which is age-independent
Immunity in HIV and AgingEvaluation of Immune Competence
Aging
InflammationHIV
Immune activation
Immune Response to InfluenzaVaccination
?
HIV and Aging have independent and combined effects on IA and inflammation
How does each state (and combined) affect immune competence?
Influenza Vaccination* (2012-2015)
Trivalent Influenza vaccine (TIV):
• H1N1/09*• H3N2• B
Sample (T0)• PBMC• Plasma• Serum
Sample (T1)• PBMC• Plasma• Serum
Day 0 (T0) Day 7 (T1) Day 21-28 (T2)
Sample (T2)• PBMC• Plasma• Serum
Month 5-7 (T3)
Sample (T3)• PBMC• Plasma• Serum
FLORAH Project (FLu Responses Of people in relation to Age and HIV): Study Design
*H1N1 strain included in vaccine since 2009 epidemic
HIV+ (154)Virally suppressed (plasma virus load < 50 copies/ml) on cART for >1 year prior to enrollment
HIV negative (161) healthy controls
(HC)
Age Groups HC HIVYoung (Y), 18-39 years 48 34Middle (M), 40-59 years 62 70Old (O), 60-80 years 51 50
HIV and Aging affect Serologic Response to Flu vaccine
T 0 T 1 T 2 T 33 2
6 4
1 2 8
2 5 6
5 1 2
1 0 2 4
2 0 4 8
T im e P o in ts
H1
N1
Ge
om
etr
ic m
ea
n T
ite
r
4 0
Y-Y+O-O+
H1N1 Ab
Vaccine Responders (VR): > 4-fold increase in titers from pre-vaccination (T0) to post-vaccination (T2)
Vaccine Non-Responders (VNR): < 4-fold increase in titers from T0 to T2.
H1N1, H3N2, B Ab
Y o u n gO
ld
Y o u n gO
ld0
2 0
4 0
6 0
8 0
1 0 0
% o
f In
div
idu
als
V RV N RO th e r
H e a lth y H IV
* * ** *
Y-: Young, HIV- O-: Old, HIV-Y+: Young, HIV+ O+: Old, HIV+
Suresh Pallikkuth
Pallikkuth, AIDS 2018
HIV and Aging affect Serologic Response to Flu vaccine
T 0 T 1 T 2 T 33 2
6 4
1 2 8
2 5 6
5 1 2
1 0 2 4
2 0 4 8
T im e P o in ts
H1
N1
Ge
om
etr
ic m
ea
n T
ite
r
4 0
Y-Y+O-O+
H1N1 Ab
Vaccine Responders (VR): > 4-fold increase in titers from pre-vaccination (T0) to post-vaccination (T2)
Vaccine Non-Responders (VNR): < 4-fold increase in titers from T0 to T2.
H1N1, H3N2, B Ab
Y o u n gO
ld
Y o u n gO
ld0
2 0
4 0
6 0
8 0
1 0 0
% o
f In
div
idu
als
V RV N RO th e r
H e a lth y H IV
* * ** *
Y-: Young, HIV- O-: Old, HIV-Y+: Young, HIV+ O+: Old, HIV+
Suresh Pallikkuth
ewer vaccine responders in HIV+ Difference from HC most prominent in young HIV+
Pallikkuth, AIDS 2018
IL-6IL-6R
IL21STAT3
ICOS
cMAF
T follicular helper cells (Tfh)
T follicular helper cellCD4+CD45RO+CD27+CXCR5+ • Th2: CXCR3negCCR6negCCR4+
• Th1: CXCR3+CCR6negCCR4neg
• Th17: CXCR3negCCR6+CCR4neg
• Th1/Th17: CXCR3+CCR6+CCR4neg
• Tfr: FOXP3+
Tfh subsets
CD4 T cell
TfhTh1Th2Th17Treg
CD4 T cell subsets
CD4 T cell
IL-21R
Mature memory CD4 T cell
B cell
IgMNAIVE
• Somatic Hypermutation
• Class switch Recombination
Antigen-Induced Tfh and B Cell interaction to develop Antibody secreting cells
Adapted from: Moir et al, Immunol Rev 2017, Frasca et al., Curr Opin Immunol. 2009
GC reactionSwitched MemoryB cells
Plasma cell
B cell
Short lived plasmablast
Ab secreting cells
CPG
H1N1 Ag: Vaccine R
H1N1 Ag: Vaccine NR
Experimental Protocol: quantitative and qualitative assessment of Flu antigen-specific CD4 pTfh cells
+/- flu peptide+ αCD28
Brefeldin ALast 4 hours
• Phenotype:Activation/exhaustion, co-stim molecules
• Function (ICS) : IL-21, IL-2, TNF, IL-17
Antigen-induced proliferation of pTfh (cell trace dye reduction)
FC: Identification of antigen-specific pTfh (CD40L+CD69+)
for quantitative and qualitative assessment if Ag specific pTfh
SS
C-A
FSC-A FSC-W
FSC
-H
Aqua
FSC
-H
CD
8
CD4CD3
FSC
-H
CX
CR
5
CD4
CD
40L
CD69
pTfh
Scatter Singlets Live cells CD3
CD
27CD45RO
TN TCM
TEMTE
Ag-specific pTfh
Gating strategy for Flu antigen specific CD4 pTfh cells
V R V N R0
1 0
2 0
3 0
4 0
CD
40
L+C
D6
9+A
g.p
Tfh
(% o
f p
Tfh
)
***
H C V R H C V N R H IV V R H IV V N R0
5
1 0
1 5
2 02 0
4 0
6 0
8 0
CD
40
L+
CD
69
+p
Tfh
(% o
f p
Tfh
) * * * *
* * ** * * *
Frequencies of Antigen specific pTfh at T2 post-vaccination are greater in VR
IL-12R
Summary: Immunologic components and determinants of a successful antibody response in
Vaccine responders
Pallikkuth J Immunol 2011; JACI 2011; Blood 2012; Rinaldi Aging 2017de Armas JCI Insight 2018 George AIDS 2018;
B cell features post-vaccination:Increases occur in• Plasmablasts and spontaneous
Ab secreting cells (ASC) at T1• Memory B cells and switch
memory at T2 • IL-21R on memory B cells, T2 • Plasma cells at T2
pTfh cell features post-vaccination:• Expansion of bulk pTfh and
Ag+ pTfh at T1, T2• IL-21 production at T2• Upregulation of ICOS at T1, T2 • Ag-specific IgG in pTfh:B cell
co-culture at T2
Summary: Features of immune cells that negatively influence influenza vaccine responses in Vaccine non-responders
Pallikkuth J Immunol 2011; JACI 2011; Blood 2012; Rinaldi Aging 2017de Armas JCI Insight 2018 George AIDS 2018;
B cells• PDL1 at T0*• DN B Cells at T0*
pTfh phenotype and function: • CD38+HLA-DR at T0• PD-1 high at T0• ICS showing TNF, IL-2,
IL-17 NOT IL-21 at T2.
Monocytes:Inflammatory monocytesCD11b high at T0
• We have identified what constitutes good and bad features of immune cells responding to flu vaccine antigens
• Best vaccine responses are associated with generation of antigen-specific pTfh cells that show: o Proliferation in response to flu antigenso Induction of ICOS and IL-21 secretion post-
vaccinationo Low basal activation (HLADR, CD38, and PD1)o Low to absent secretion of TNF and IL-2
following antigen stimulation
Summary of Cellular Basis of Flu Ab responses
The Effects of Aging and HIV infection on Tfh:B cell Responses to Influenza Vaccination
InflammationImmune activation (B and T cells)High Serum Ab titer FC
“Vaccine Response”
Old HIV+
Old HIV-Young HIV+
Young HIV-
IL-21 IL-2 TNF
Good Bad
HIV infected young individuals resemble old individuals making the differences between young and old less evident
ICOS
University of MiamiMargaret Fischl, MDGordon Dickinson, MDAllan Rodriguez, MDMaria Alcaide, MD
Katherine Klose
Study Participants
AcknowledgementsPahwa Lab
Stefano
Lesley
Celeste
Raj Margie
Suresh
Varghese