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.

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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

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Immune exhaustion

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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

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Immune activation

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Immune exhaustion

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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

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ld

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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

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1 2 8

2 5 6

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2 0 4 8

T im e P o in ts

H1

N1

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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

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ld

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% 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

Immunologic Components of an Antibody Response

ICOSLMHCII

IL-12R

IL21

Tfh

Ascl2BCL-6MAF

IL4

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

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Tfh

(% o

f p

Tfh

)

***

H C V R H C V N R H IV V R H IV V N R0

5

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4 0

6 0

8 0

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CD

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+p

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(% o

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) * * * *

* * ** * * *

Frequencies of Antigen specific pTfh at T2 post-vaccination are greater in VR

Molecular interactions of Tfh and B Cells in the GC: Role of ICOS

Nutt SL, Nat Immunol 2011

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

Thank you