Functions of hetIL-15 hetIL-15 Effects in Lymphocytes in Blood Histo-cytometry: Increased Infiltration of B Cell Follicles by GrzB+ CD8 Cells

hetIL-15 Treatment Increases SIV-specific CD8+ T Cells in Peripheral Blood and LN

Functional Assays for CM9-induced SIV-specific responses

Functional Assays for CM9-induced SIV-specific responses (continued)

hetIL-15 Treatment Reduces Viral RNA In LN

hetIL-15 Effects in Mucosal Effector Sites

Accumulation of Ki67+ and GrzB+CD8+ T cells in Follicular Areas upon hetIL-15 Treatment

2- or 3-week Administration of SC hetIL-15Similar to the clinical schedule

hetIL-15 Increases the Number of Cytotoxic Effector Lymphocytes and NK cells in

Lymph Nodes

Background: Heterodimeric interleukin-15 (hetIL-15) is a native stable form of the cytokine that activates and expands cytotoxic T and NK cells. Based on its properties and extensive preclinical data, hetIL-15 is currently evaluated in humans for the treatment of cancer (NCT02452268). We study the effects of hetIL-15 in infected macaques to evaluate its use in HIV infection and especially in the reduction of SIV/SHIV reservoir towards a functional cure.

Methods: Rhesus macaques, either chronically infected by SHIV or uninfected received injections of hetIL-15 over 2 weeks using increasing doses of cytokine (step-dosing). At the end of the treatment, the animals were sacrificed and the hetIL-15 effects on different lymphocyte populations isolated from tissues collected at necropsy were monitored by multi-parametric flow cytometry and quantitative multiplexed confocal microscopy (histo-cytometry). Cell-associated viral RNA and plasma viral load was measured by quantitative PCR.

Results: This protocol was safe in rhesus macaques and resulted in systemic expansion of CD8+ T lymphocytes and NK cells with higher granzyme B content. These expanded cell populations were found in both effector sites, such as liver, vagina and rectum, and secondary lymphoid tissues. Importantly, a significant increase in cytotoxic effector memory CD8+ T cells was found in lymph nodes (LN) from all hetIL-15-treated macaques. CM9 tetramer staining demonstrated that the increase of CD8+ effector T cells in lymphoid organs included actively proliferating SIV-specific T cells with higher granzyme content. Imaging analysis by histo-cytometry revealed that these effector CD8+ T cells infiltrated the B cell follicles where chronically infected follicular helper CD4+ T cells are located. Following hetIL-15 treatment, cell-associated RNA was decreased in LN and plasma viral load was also decreased. Treatment of macaques under Antiretroviral Therapy (ART) with this regimen was also safe and induced cytotoxic CD8+ accumulation in LN follicles.

Conclusions: Step-dose administration of hetIL-15 is a well-tolerated regimen that results in systemic activation and expansion of cytotoxic leukocytes that infiltrate areas where chronic HIV-infected cells reside. These results suggest that hetIL-15 could be useful in disrupting sanctuary sites within the B cell follicles and reducing long-term viral reservoirs in HIV-1 infected individuals, thus contributing to a functional cure of the infection.

(published Watson, D.C., et al. PLoS Pathogen, Feb 2018)

http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006902

IL-15 is a heterodimeric cytokine (hetIL-15)

hetIL-15 is A Unique Immune System Agonist For Cancer and AIDS Immunotherapy

Heterodimeric IL-15 treatment increases cytotoxic lymphocytes in LN follicles and reduces SHIV RNA

Dionysios C. Watson1, Eirini Moysi1, Antonio Valentin1, Cristina Bergamaschi2, Santhi Devasundaram2, Sotirios P. Fortis1, Claire Deleage3, Jacob D. Estes3, Elena Chertova3, Jeffrey D. Lifson3, Constantinos Petrovas5, Barbara K. Felber2, and George N. Pavlakis1

1Human Retrovirus Section, National Cancer Institute, Frederick; 2Human Retrovirus Pathogenesis Section;Frederick; 3AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick; 4Biostatistics and Data Management Section, CCR, NCI, Rockville; and 5Vaccine Research Center, NIAID, Bethesda

Nucleus

2 forms of bioactive hetIL-15

Membrane-boundHeterodimer

SolubleHeterodimer

hetIL-15

ER

IL-1

5

IL-1

5 Rα

Lymphocyte Expansion and DifferentiationNK, CD8, CD4, γδ T cells

- Important for both Innate and Adoptive Immune System Homeostasis

MigrationRapid extravasation of lymphocytes to tissues• Increases CD8 Effectors in tumors

Enhanced Activation, CytotoxicityIncreases expression of granzymes, perforinEnhances ADCC

Day 1 Day 3 Day 5 Day 8 Day 10 Day 12

Blood, LN, tissue collectionBEFORE

Blood, LN, tissue collectionAFTER

Day 15

3354

13

1052

37

1.9 0.7

4.493

25.2 49.4

6.918.5

Ki67

CD28

CD95

GrzB

+hetIL-15Pre

pre +hetIL-15 pre +hetIL-15

p=0.008

p=0.016

uninfected SHIV+

**

**

**

*

*

****

Effector memory CD8+ T cells

p=0.004p=0.016

Grz

B+C

D8+

T ce

lls (%

)

pre +hetIL-15 pre +hetIL-15

uninfected SHIV+

CD8+GrzB+ T cells

Ki67

+ C

D8+

T c

ells

(%)

uninfected SHIV+

p=0.004 p=0.016

pre +hetIL-15 pre +hetIL-15

Cycling (Ki67+) CD8+ T cells

EM C

D8+

T ce

lls (%

)

p=0.0005

p=0.0005p=0.0005

% C

D8+

Ki67

+ T

cells

naive CM EMhetIL-15 - + - + - +

CD8:p=0.0005

p=0.0063

% C

D8+

T ce

lls

naive CM EMhetIL-15 - + - + - +

hetIL-15 - + - + - +

p=0.0034 p=0.001

% C

D4+

Ki67

+ T

cells

naive CM EM

CD4: p=0.0425

% C

D4+

T ce

lls

naive CM EM

p=0.021p=0.021

CD4/

CD8

pre +hetIL-15

p=0.0024

% G

rzB+

T ce

lls

p=0.009

p=0.027

CD4+ CD8 +

hetIL-15: - + - +

% K

i67+

NK c

ells

+hetIL-15

NK:

p=0.0005

pre +hetIL-15 pre

% N

K ce

lls

p=0.042

CD8+ Ki67+

T lymphocytes

NK (CD3-CD16+GrzB-/+):Ki67 expression Frequency

CD8+ subsets

CD4+ Ki67+

T lymphocytesCD4+ subsets

CD4/CD8 ratioGrzB content of

CD4 and CD8 cells

Ki67

+lym

phoc

ytes

(% o

f CD

8+ce

lls)

Rectum

hetIL-15: - + - + - + - +

p=0.0161 p=0.0269 p=0.0049 p=0.0049

- + - + - +

Ki67

+lym

phoc

ytes

(% o

f CD4

+ce

lls)

p=0.0492 p=0.0431

Ki67

+lym

phoc

ytes

(% o

f CD

8+ce

lls)

Vaginap=0.0039 p=0.0039 p=0.0098

p=0.002

hetIL-15: - + - + - + - +

Ki67

+lym

phoc

ytes

(% o

f CD4

+ce

lls)

p=0.0278

p=0.0393

- + - + - +

P921 R902

P572 P090

Uninfected (N=4)

Data 7

P921 P902

P917 5787 P934

57265751

P572

P995

P090

SHIV+ (N=6)

CD3+

CD4-

Grz

B+ce

lls/m

m2 of

follic

le

hetIL-15: - + - +

CD8+GrzB+ In Follicles:

uninfected SHIV+

* *

CD8

pre +hetIL-15pre +hetIL-15

Macaque P082 Macaque P090

PBMC4.4 2.12 1.3

LN

0.3 2.40.5 1.2

A

9M

C gaG VIS

B

pre+hetIL-15

GrzBKi67

C

9M

C gaG

+ C

D8

+ CMBP ni sllec T

2602661

18145047

PBMC

9M

C gaG

+ C

D8

+

NL ni sllec T

3825889

8476407

LN

Perforin

554773

5121064

D

gag DNA vaccinated Mamu A*01+ macaques

CD10

7a

0 3 hrs

1

1

1

89

93

2

CM9

CD107

CM9

IFN

-γ

0 3 hrs0

0

0

84

82

0

IFN-γ

LN(Gag CM9)

PBMC(Gag CM9)

PBMC (vif)

A B

vif GagCM9

vif GagCM9

pre hetIL-15

7,3 esapsaC

%+

D

IFN-γ

9MC

+ IF

N-γ+

Time (hrs)

TNFα

CM

9+ TN

Fα+

Time (hrs)

CD107

CM9+

701DC+

Time (hrs)

GrzB

Grz

B(M

FI)

Time (hrs)

Perforin

Perfo

rin (M

FI)

Time (hrs)

C

vif GagCM9

vif GagCM9

pre hetIL-15

7,3 esapsaC

%+

D

IFN-γ

9MC

+ IF

N-γ+

Time (hrs)

TNFα

CM

9+ TN

Fα+

Time (hrs)

CD107

CM9+

701DC+

Time (hrs)

GrzB

Grz

B(M

FI)

Time (hrs)

Perforin

Perfo

rin (M

FI)

Time (hrs)

C

A

RNA

DNA

T416SHIV-1157ipd3N4

T422SHIV-1157ipd3N4

R591SHIV SF162P3

T605SHIV CH505

/seipoc AN

R VIS 106

cells

(log

)/seipoc A

ND VIS 10

6ce

lls (l

og)

pre +hetIL-15 pre +hetIL-15 pre +hetIL-15 pre +hetIL-15

B

C

oitar AN

D/A

NR

pre +hetIL-15

p=0.0001

Co-expression of IL-15 and IL-15Rα produces a stable heterodimer §Natural way of cytokine production in vivo

§High doses of hetIL-15 able to increase total body lymphocytes can be delivered without toxicity § Important for combination of hetIL-15 with other drugs

Individual follicles were analyzed upon hetIL-15 treatment in LN from 4 uninfected and 6 SHIV+ macaques. A range of 2 to 14 follicles were analyzed/ animal. The numbers of CD3+CD4–GrzB+ cells per mm2 of area are in individual follicles for each animal are shown. Values of 0 were entered as 1 for the graph display only. Bars indicate average values. p values are calculated by two-way ANOVA for the SHIV+ versus uninfected and pre- versus post-hetIL-15 effects, with random effects to account for the clustered values by animal.

Dot plots showing changes in the frequency of Gag CM9-specific CD8+ T cells in axillary LN and PBMC upon hetIL-15 treatment of macaques P082 and P090. These animals received a SIV gag DNA vaccine. The CM9-tetramer responses are expressed as % of total T cells.

(B, C, D) Histogram overlays show increased levels of Ki67 (B), GrzB (C) and perforin (D) in Gag CM9-specific T cells upon hetIL-15 treatment in LN and PBMC. pre, blue line, +hetIL-15, red line. Numbers within the histogram overlays show the MFI for the specific markers.

Dot plots show production of IFN-γ (A) and degranulation CD107 (B) by CD8+ T cells specific for the MamuA01 restricted Gag CM9 epitope upon specific stimulation of the TCR with the CM9 peptide. The plots show the functional responses at 0 and 3 hours by lymphocytes from LNMC and PBMC of macaque P082. Similar data were obtained from macaque P090.

Pre-treatment is shown in the upper panels and +hetIL-15 in lower panels

(A) Confocal images showing the distribution of CD20 (blue), CD3 (red), Ki67 (yellow) and GrzB (cyan) positive cells in peripheral LN from an uninfected macaque (R902) before and after hetIL-15 treatment. Follicles are defined as CD20hi-dim areas. (B) Higher magnifications from A of areas indicated by squares. CD20 (blue), CD3 (cyan), CD4 (red) and GrzB (yellow). Follicular areas defined by CD20 (left panel) show increased presence of CD8+ cells (defined as CD3+CD4-) (middle panel) and GrzB+ cells (right panel) upon hetIL-15 treatment. (C) Higher magnification of B shows presence of CD3+ CD4- GrzB+ (GrzB+CD8+) cells upon hetIL-15 treatment (white arrowheads). (D) Distribution of CD20 (blue), Ki67 (purple), CD3 (red) and GrzB (yellow) positive cells in a representative follicle. Pre (upper panels) and +hetIL-15 (lower panels) from an SHIV+ macaque (5787).

Bergamaschi, J Biol Chem. 2008; J Immunol. 2009

Bergamaschi, Blood 2012; Chertova, J.Biol Chem 2013

Thaysen-Andersen Glycoconj J. 2016 33:417

Watson, Biomaterials 2016 105:195

Bergamaschi et al., Cytokine, 2018

https://www.ncbi.nlm.nih.gov/pubmed/29402721

(C) Time course showing the frequency of IFN-γ, TNFα and CD107 positive CD8+ CM9+ T cells in blood, as well as changes in their content of granzyme B and perforin after stimulation with the CM9 peptide in lymphocytes recovered before (black symbols) and after (red symbols) in vivo hetIL-15 treatment. Results in (C) are from a separate experiment with similar results to that shown in panels A and B.

(D) Graph showing the frequency of bone marrow cells (macaque P090) loaded with the CM9 peptide or a peptide pool covering HIV-1 Vif undergoing apoptosis (measured as caspase 3 and 7+ cells) after incubation with LNMC obtained before and after hetIL-15 treatment.

Quantitation of cell-associated viral RNA in axillary and inguinal LN and PBMC of 4 SHIV+ macaques upon hetIL-15 treatment.

The viral RNA determinations in T416 and T422 are from duplicate measurements from purified flash-frozen LNMC and PBMC. The viral RNA determinations in R591 and T605 are from single measurements of flash-frozen LN before treatment and from two independent flash-frozen LN collected after hetIL-15. Dotted and solid lines denote the two independent LN samples. No inguinal LN was available for T605 post treatment.

Axillary LN (blue square), inguinal LN (red triangle) and PBMC (green triangle) data are shown.

(B) Quantitation of viral DNA copies from the samples of the macaques in panel A. (C) RNA/DNA ratio of the measurements shown in panels A and B. T416, filled diamond; R591, open diamond; T422, filled circle; T605, open circle. RNA/DNA ratio are for inguinal LN (red symbols) and axillary LN (blue symbols). p value for the difference before and after treatment determined by Mann-Whitney test.

Changes in Plasma VL of 13 SHIV+ Macaques upon hetIL-15 Treatment

Conclusions

Macaque: T413 T416 T419 T421 T422 T427 5726 5751 5757 5787 P917 R591 T605

SHIV: 1157 1157 1157 1157 1157 1157 327C 327C 327C 327C SF162 SF162 CH505

Plasma VL:

day 0, pre4300 40 350 2300 3600 12 30 880 480 50 160 108 1950

hetIL-15, week 1

1800 25 30 3500 1000 5 20 20 500 60 31 170 450

week 2 (necropsy)

1500 15 20 11000 230 3 10 5 5 7 3 140 120

VL fold drop, day 0 - week 2 3x 3x 18x 0.2x 16x 4x 3x 176x 96x 7x 53x 0.8x 16x

§Decrease in plasma VL upon hetIL-15 treatment in the majority of animals

hetIL-15 is a promising immune system agonist

§ Promotes a great infiltration of cytotoxic cells in B cell follicles and LN

§ Activates Lymphocytes (NK, CD8, CD4) and delivers to areas of tumor

§ May provide a general method to enhance T-cell tumor entry, increasing the success rate of immunotherapy interventions?

§ Homeostatic cytokine: Regulates number of lymphocytes

§ Exogenous administration: Increases total body lymphocytes and replaces the need for lymphodepletion prior to adoptive cell transfer

§ Elimination of the need for lymphodepletion could make more patients eligible for cell-transfer protocols

For AIDS functional cure

§ Cytotoxic T cells and NK entry into the follicle in massive numbers may enhance the elimination of virus expressing cells or may disrupt virus sanctuaries via altered cell interactions

Prepared by Scientific Publications, Graphics & Media Frederick National Laboratory for Cancer Research, Frederick, MD [208787]

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

353LB