Understanding New Regulatory Network to Develop Novel Immunotherapy - - PowerPoint PPT Presentation

Palermo, October 24, 2011

Valentina Di Caro, PhD

Fondazione Ri.Med University of Pittsburgh School of Medicine

Dendritic Cell - B Regulatory Cell Crosstalk: Understanding New Regulatory Network to Develop Novel Immunotherapy

Type 1 Diabetes

(Mathis D, et al. Nature, 2001)

Reprogramming the immune system to stop the inflammation: Co-stimulation blockade as a strategy to prevent diabetes and to reverse new onset disease

Dendritic Cells

After sampling the environment, they move to the closest lymph node(s) Are the body’s sentinels Inside the lymph node, they initiate communication with T-cells (mostly) They sense “tissue states” as they migrate throughout the body Signal can be ACTIVATING

• r SUPPRESSIVE

Signals between DC and T Cell

Tolerogenic dendritic cells can attenuate T cell mediated immune responses by deleting, anergizing or changing the effector function of antigen-specific T cells

Dendritic cell, engineered to maintain low levels

• f CD40, CD80 and CD86 costimulatory

molecules, can prevent type 1 diabetes mellitus and reverse new-onset disease in the non-obese diabetic mouse model

(Machen et al. J of Immunology, 2004)

NIH-funded, IRB- and FDA-approved Safety Study

• 5. Immunological, biochemical, physiologic monitoring to establish safety
• 4. Administer to volunteer intradermally
• 2. Engineer DC towards a “diabetes-suppressive”

capacity under GMP/GLP conditions; provide mixture of AS-ODN (CD40/CD80/CD86)

• 3. Test potency, sterility and divide into

individual administration aliquots

Giannoukakis et al. Ped Diabetes, 2008

To confirm that intradermal administration of autologous diabetes suppressive dendritic cells (DC) is safe, non-toxic and without side-effects.

• 1. Obtain leukocytes via apheresis

B Cells in Phase I Clinical Trial Patients

B Cell Changes in Phase I Clinical Trial 10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Blood Draw B220+ CD11c- Cells (%)

1 2 4 6 8 9 10 12

(Giannoukakis N et al, Diabetes Care 2011)

(Mizoguchi, A. Journal of Immunology, 2006)

Hypothesis

Can AS-ODN DC interact with specific B cell subpopulations, with immune regulatory functions, and promote peripheral tolerance?

Collect Bone Marrow Generate DC’s

AS-ODN CN

Inject Mouse Inject Mouse Perform Flow Cytometry B220+CD19+L10+ Harvest Spleen 3 Days Later Overnight LPS Treatment

Experimental plan

CN AS-ODN IL10+ Cells 36.9 +/- 3.9 47.5 +/- 5.6

Breg cell populations 3 days post DC injection

• Two populations of

IL10+ B220+ CD19+

• 30% increase with

AS-ODN DC treatment

• n = 3 mice p = 0.054

B220+ CD19+ IL10+ Bregs isolated from AS-ODN treated mice are suppressive

T = T-cells S = Irradiated Allo-Spleenocytes B = 1/10 Mix of B Cells B = 1/1 Mix of B Cells

* ** *** **

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 T S B TS TSB 1:10 TSB 1:1

No DC CN DC AS-ODN DC

Controls Co-cultures

0.2 0.4 0.6 0.8 1 1.2 1.4

T S B TS TS+IL-10 antibody TSB TSB+IL-10 antibody

* **

B220+ CD19+ Bregs suppressive activity is not dependent on IL10 secretion

T = CD4+ T-cells S = Irradiated Allo-Spleenocytes B = 1/10 Mix of B Cells IL10 = IL10 Antibody

T = CD4+ T-cells S = Irradiated Allo-Spleenocytes B = 1/10 Mix of B Cells IL10 = IL10 Antibody

To be suppressive B220+ CD19+ Bregs need to physically interact with T cells

Transwell plate

MLR with B220+ CD19+IL10+ Bregs isolated from buffy coat of human blood

****

0.2 0.4 0.6 0.8 1 1.2 T S B TS TSB

T = CD4+ T-cells S = stimulators B = 1/10 Mix of B Cells

Cytokine production

20 40 60 80 100 1 2 3

TNFalpha Production

50 100 150 200 250 1 2 3

INFgamma Production

20 40 60 80 100 120 1 2 3

IL-2 Production

Breg suppression in vitro is associated with impaired production of Th1 type pro-inflammatory cytokines

20 40 60 80 100 120 140 160 180 200 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 Up Down

Fold induction (log) n# genes Control DC AS-ODN DC

Mouse transcriptome analysis

385 up 395 down

DC



T cell

• 10
• 8
• 6
• 4
• 2

• 10
• 8
• 6
• 4
• 2

P1 1296 P8 1196 Control 751 153

Fold induction Fold induction

Human transcriptome analysis

n# genes

1 2 3 4

1 DC control sample1 2 AS-ODN DC sample1 3 DC control sample 2 4 AS-ODN DC sample2

Fold of induction

1 DC control 2 AS-ODN DC

ALDH1A2 expression

Autologous DC can upregulate the frequency of B220+ CD11c- B lymphocytes; This B220+ CD11c- B cell population contain a novel suppressive B regulatory cell subpopulation phenothipically characterized as CD220+CD19+IL10+; To be suppressive this B regulatory cell needs to physically interact with T cell and DC; Tolerogenic DC produce high level of rate-limiting enzyme for retinoic acid, ALDH1.

Still to demonstrate how DC can regulate Breg

Conclusion

Thanks…..

 Massimo Trucco MD  Nick Giannoukakis PhD  Brett Phillips PhD  Carl Engman  Alexis Styche  Bob Lakomy

Funding

• NIH
• Fondazione Ri.Med