362

Defining Efficacious HIV-Specific CTL Responses Using Saporin-Conjugated Tetramers Leitman EM1, Sims S2, Payne RP1, Chen F3, Riddell L4, Buus S5, Deeks S6, Walker BD7, Matthews PC8, Goulder JR1

1 Department of Paediatrics, University of Oxford, Oxford, UK; 2Department of BSE, ETH Zurich, Basel, Switzerland; 3Royal Berkshire Hospital, Reading, UK; 4Northamptonshire Healthcare NHS Trust, Northampton, UK; 5Laboratory of Experimental Immunology, University of Copenhagen, Copenhagen, Denmark; 6Department of Medicine, UCSF, CA, USA; 7Ragon Institute of MGH, MIT and Harvard, Charlestown, MA, USA; 8Nuffield Department of Medicine, University of Oxford, Oxford, UK

Background

Methods

References

Conclusions Results

Photo credit: Greg Smolonski

Email: ellen.leitman@st-hughs.ox.ac.uk

In vitro killing of HIV-infected cells by cytotoxic T lymphocytes (CTL) is a surrogate marker of CTL antiviral efficacy1. Tetrameric peptide-MHC complexes (tetramers) enable identification of antigen-specific CD8+ T cells in a mixed population (the terms ‘CTL’ and ‘CD8+ T cells’ are used interchangeably)2. Due to their rapid internalization by cognate T cells, tetramers are an effective delivery vehicle of any coupled moiety to target specific CTL. Novel tetramers conjugated to saporin (SAP), a potent toxin causing cell death through ribosome-inactivation, have been used in vivo to deplete murine diabetogenic T cells, but no human reports exploiting this technology exist to date3. Here, we used toxic tetramers to rapidly eliminate or ‘zap’ human CTL of different specificities in order to compare their anti-HIV potency.

•  Human PBMC were treated with HLA-matched SAP-coupled tetramers (tet-SAP) or controls.

•  Specific targeting, internalization3 and depletion assessed by FACS.

•  Antiviral efficacy evaluated in inhibition assays4, using tet-SAP- or control-treated CTL (effectors) and HLA-matched NL4-3-infected targets; NL4-3 was labeled with GFP to use as a marker of infection.

1.  Saez-Cirion et al. PNAS. 104:6776-6781 (2007). 2.  Altman et al. Science. 274:94-96 (1996). 3.  Hess et al. Blood. 109:3300-3307 (2007). 4.  Saez-Cirion et al. Nat Protoc. 5:1033-1041 (2010).

1. Antigen-specific CTL recognize and rapidly internalize cognate tet-PE and tet-SAP

Internal tet-PE

0 30 60 90 120 150 1800

1000

2000

3000

4000

4°C37°C

Time (min)

MFI

A

B

C

Figure 1: Recognition and kinetics of internalization of cognate tetramers by CD8+ T cells. PBMC from HIV+ subject stained with fluorescent tetramer (tet-PE) (A) or tet-SAP (B) of the same specificity. Full internalization of tetramer occurs by 2h of incubation at 37°C (C). Here and further numbers in FACS plots show %tet+ cells (of CD8+); gated on live CD3+ cells.

2. Tet-SAP selectively eliminates human tet-specific CTL

0 20 40 60 800

20

40

60

80

100

UntreatedTet-SAP txMismatch-SAP txFree SAP tx

Time post-treatment (h)

% o

f liv

e C

D8+

tet-P

E+

cells

3. Tet-SAP-mediated CTL elimination is exclusive to desired specificity

Untre

ated

KK10

-SAP

txKY

9-SA

P tx

Untre

ated

KK10

-SAP

txKY

9-SA

P tx

0

20

40

60

80

100

KK10 stainKY9 stain

Nor

mal

ized

% te

t+ c

ells

4. Zapping across specificities and pathogens •  We have optimized a novel method to eliminate human CTL of different

specificities.

•  This technique allows us to measure and compare the individual contribution of different CTL specificities to HIV suppression.

•  This represents a rapid, cost-/labor-saving method, applicable across HLA types and pathogens.

•  The approach enables us to evaluate the quality of different responses and discriminate between the most efficacious and futile CTL.

HIV A*02:01-GL9+ subject

0 20 40 60 800

20

40

60

80

100 UntreatedTet-SAP tx

Time post-treatment (h)

% o

f liv

e C

D8+

HIV

A*

02:0

1-G

L9-P

E+ c

ells

EBV B*07:02-RL9+ subject

0 20 40 60 800

20

40

60

80

100 UntreatedTet-SAP tx

Time post-treatment (h)

% o

f liv

e C

D8+

EB

V

B*0

7:02

-RL9

-PE

+ ce

lls

EBV A*02:01-GL9+ subject

0 20 40 60 800

20

40

60

80

100 UntreatedTet-SAP tx

Time post-treatment (h)

% o

f liv

e C

D8+

EB

VA

*02:

01-G

L9-P

E+

cells

CMV B*07:02-TM10+ subject

0 20 40 60 800

20

40

60

80

100 UntreatedTet-SAP tx

Time post-treatment (h)

% o

f liv

e C

D8+

CM

V

B*0

7:02

-TM

10-P

E+

cells

HIV HLA-A*02:01 CMV HLA-B*07:02

EBV HLA-B*07:02 EBV HLA-A*02:01

Peter Medawar Building for Pathogen Research

Figure 2: Representative time course of selective tet-SAP-mediated CTL depletion. PBMC from HIV+ subject treated for 2h with HLA-B*27:05-KK10-SAP or controls; at indicated times targeted cell removal was assessed by KK10-PE staining.

F i g u re 3 : Tet - S A P -mediated removal of one CTL specificity is highly selective and does not affect other specificities.

Figure 4: Zapping CD8+ T cells of different specificities from subjects of different HLA infected with HIV, CMV or EBV. Representative examples are shown; for clarity only untreated or tet-SAP-treated cells are shown.

3 5 70

20

40

60

+ KK10-PE-treated CD8+ KK10-SAP-treated CD8

Infected targets no CD8+ untreated CD8

+ Mismatch-SAP-treated CD8

Days post-infection

%G

FP

+ ce

lls

6. Functional application: HIV-suppressive capacity of zapped CTL

Bulk

KK10-za

pped

KK10-P

E trea

ted

Mismatc

h-za

pped

0

1

2

3

4

5

Sup

pres

sive

cap

acity

(lo

g fo

ld d

ecre

ase

in %

GFP

+ ce

lls)

Figure 6: HIV-suppressive capacity of zapped CTL. CD8+ T cells zapped of the dominant Gag-KK10 response (confirmed in A) lose their capacity to inhibit viral replication in vitro (B). Suppressive capacity (log fold decrease in % infected GFP+ cells) shown in C.

A

B C

2 4 6 80

5

10

15

20

25

+ KK10-zapped CD8+ KK10-bead-depleted CD8

Infected targets no CD8+ Bulk CD8

Days post-infection

%G

FP+

cells

5. Method validation: zapping vs bead depletion

Bulk

KK10-za

pped

KK10-be

ad-de

pleted

HIV- H

LA-m

atche

dBulk Bulk

0

1

2

3

4

HLA-matched targets

HLA-mismatched targets

Supp

ress

ive

capa

city

(lo

g fo

ld d

ecre

ase

in %

GFP

+ ce

lls)A B C

Figure 5: Removal of specific CTL by novel zapping or conventional bead-depletion method side by side. HLA-B*27:05-Gag-KK10-depleted CD8+ T cells by either method (A) perform similarly in viral inhibition assay (B, C).