SLE and Tregs What we know and what we would like to know

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SLE and TregsWhat we know and what we would like
to know

• Rheum Rounds
• March 21st 2008
• Amit Golding MD, PhD
• JHH Rheum Fellow Year 3

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Amit Golding MD PhD
Disclosures
No Relevant Financial Relationships with
Commercial InterestsNo Discussion of
Non-FDA-Approved Uses of Medications
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Objectives

• I. Review published literature on Tregs in SLE10
  primary studies from 2003-2008
• A. Various Parameters
• -Treg Numbers
• -Treg Function
• -Foxp3 Expression
• B. Some Details from two best papers

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Objectives

• II. Set out my current questions of focus
  regarding Tregs in SLE and other Autoimmune
  Diseases
• A. Possible Mechanisms for a reduction in Tregs
• B. Basic explanation of our system for studying
  Treg expansion
• C. Summary of preliminary data

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• First, A Few Basic Definitions
• (For Review)

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What are Regulatory T cells (Tregs)?
Early Ideas

• Postulated to exist by Paul Ehrlich over 100
  years ago
• Observed as a population of Thymic-derived cells
  that arise a few days later than effector
  cellsday 3 thymectomized mice develop a
  multi-organ autoimmune disease due to unregulated
  runaway effector T cells

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What are Regulatory T cells (Tregs)?
CD25 Marks Tregs

• CD25 IL-2 Receptor alpha subunit
• CD25 can be expressed on all T cells, however in
  a resting population the T cells with the highest
  levels of CD25 on their surface have suppressor
  activity, i.e. they are Tregs
• CD25 can be used to enrich for or deplete Tregs
  from a resting population of T cells

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What are Regulatory T cells (Tregs)?
Foxp3 is the Master Treg Gene

• FoxP3 is a DNA-binding protein that regulates
  many other genes
• The Foxp3 gene marks the T cell lineage that is
  committed to becoming Tregs
• Using intracellular staining, the amount of Foxp3
  protein can be quantitated in resting and
  expanded Tregs

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What are Regulatory T cells (Tregs)?
IL-2
CD25
Reg. T Cell
STAT5b
NFAT
Effector T Cell
-
TGF
B

SMAD
FoxP3
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The Treg Axis and Human Disease

• CD25 Deficiency causes an IPEX-like syndrome with
  enteropathy, endocrinopathy, eczema, hemolytic
  anemia, hepato-splenomegaly, and lymphadenopathy
• STAT5 deficiency causes Lymphopenia and
  Lymphocytic interstitial pneumonia
• FoxP3 deficiency IPEX syndrome of
  immunodysregulation,polyendocrin-opathy,
  enteropathy, X linked

IL-2
CD25
STAT5b

FoxP3
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Objective I. 10 Studies 2003-2008
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Objective I. 10 Studies 2003-2008
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Objective I. 10 Studies 2003-2008
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The Treg Axis and Lupus
Suppressive Function of cells with the Brightest
CD25 Staining is Maintained
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The Treg Axis and Lupus
CD25bright Tregs that retain full suppressive
function are reduced in SLE patients with active
disease
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The Treg Axis and Lupus
The number of CD25bright, fully suppressive Tregs
correlates with disease activity and also
longitudinally in SLE patients
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The Treg Axis and Lupus
The number of FoxP3 Cells in a LN from an active
SLE patient is markedly reduced
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The Treg Axis and Lupus
The of Tregs that are Foxp3 are reduced in SLE
patients with more active disease

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The Treg Axis and Lupus
The ability of CD25High Treg cells to function as
suppressors is reduced in SLE patients with more
active disease
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A Paradox?

• The two studies are actually two sides of the
  same coin
• Study one If you only capture and quantitate the
  purest Tregs that have retained normal full
  suppressive function, you will find less of them
  in Active SLE patients eg. 0.5 of total CD4 T
  cells are CD25bright compared to 1.4 for normal
  controls.
• Study two If you capture the top 2 highest
  CD25CD4 T cells and confirm that they are
  true Tregs based on a number of accepted
  markers (other than CD25 and Foxp3), you will
  find that some of them have lost normal Foxp3
  expression and also lost normal suppressive
  function.

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Objective II. We are still left with more
Questions than Answers

• 1 Why are Treg numbers reduced in Active SLE?
• Decreased Thymic production?
• Increased Destruction?
• Diminished Peripheral Expansion/Ineffective
  Homeostasis?
• 2 What is the mechanism for reduced Foxp3
  expression in SLE Tregs?

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• 1 Why are Treg numbers reduced in Active SLE?
• Decreased Thymic production?Unlikely since
  studies suggest the problem is not intrinsic to
  the Tregs themselves, eg. Valencia et al showed
  recovery of suppressor function by Tregs after
  ex-vivo culture.
• Increased Destruction?Compelling evidence for
  many years for the existence of anti-lymphocyte
  antibodies including anti-Tac which targets the
  CD25 molecule and could selectively target Tregs
  (we have been pursuing this actively).

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• 1 Why are Treg numbers reduced in Active SLE?
• Diminished Peripheral Expansion/Ineffective
  Homeostasis?
• IL-2, a general growth factor for T cells, is
  particularly critical for Treg homeostasis.
• CD25 IL-2 Receptor alpha
• SLE long known to be a state of reduced IL-2
  production and T cells from SLE patients also
  shown to have reduced responsiveness to IL-2

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• 2 What is the mechanism for reduced Foxp3
  expression in SLE Tregs?
• IL-2 is implicated here as well--
• IL-2?STAT5?turns on Foxp3 gene
• Possible defects in TCR signaling
• Maintenance of Foxp3 expression in Tregs is
  critical to maintaining their identity
• Therefore, sustained Foxp3 expression and Treg
  expansion/homeostasis are intertwined.

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Foxp3 DefinesTreg Identity Must keep identity
while expanding to keep up with Teffector cells
in an evolving immune response Sustained Foxp3

• A recent mouse model of partially inactivated
  Foxp3 (hemizygous in males) led to a disease
  resembling rapidly progressing SLE
• By 4 weeks mice had scaly skin and blepharitis.
• By 3 months mice had enlarged spleen/LNs,
  infiltration of non-lymphoid organs, eg. kidney,
  liver, lungs, GI hyperactive CD4/CD8 high titer
  ANA and dsDNA antibodies.

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Regulatory T-cell functions are subverted
andconverted owing to attenuated Foxp3
expressionYisong Y. Wan1 Richard A.
FlavellNature 2007 Foxp3 expression drops off
by 5-10 less than normal and Tregs lose their
identity.
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Our Purpose

• To account for reduced numbers of functional
  Tregs in active SLE patients
• a) due to the possible mechanism of faulty Treg
  expansion in SLE
• And b) due to reduced Foxp3 expression in SLE
• We need a system that recapitulates production of
  new bona-fide Tregs from natural Treg precursors
  and which also requires new Foxp3 expression in
  Tregs.

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Our Aims

• 1. To test whether Tregs from SLE patients are
  able to expand normally and retain their
  identity, including sustaining a high level of
  FoxP3 expression during expansion.
• 2. To test whether the Lupus Environment can
  disrupt the normal pathways that lead to FoxP3
  expression during T cell activation and expansion.

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• A system to study the normal expansion of Tregs,
  including a stereotypical up-regulation of Foxp3
  that can be easily followed on a per-cell basis.

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Methods Experimental System to Study the
Expansion of Regulatory T Cells in PBMCs
NON- T Cells
CD4CD25- T Cells
CD4CD25 T Cells
SolubleAnti-CD3 heatinactivated Serum Variables
Normal Plasma vs. SLE Plasma (or in combination)
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Methods Flow Cytometry Markers
CD25
CD25
FoxP3
INFg
FoxP3
Teff
Treg
CD4
CD4
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Depletion of CD25 cells from PBMC abolishes
induction of CD25hiFoxP3hi cells
Fresh Whole PBMC
Whole PBMC-d3
CD25FoxP3HI
CD25HIFoxP3 Natural Treg
3.2
4.1
CD25HIFoxP3 Natural Treg
CD25FoxP3HI
0.2
0.3
Fresh CD25- Depleted PBMC
CD25-Depleted PBMC Plus 5 pooled norm plasma-d3
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Depletion of CD25high Tregs from PBMCs Reduces
Inhibition of Early and Late Proliferation of
Effector CD25- T cells
CD25-Depleted PBMC
Whole PBMC
4.2
2.1
Day 4
10.8
7.0
Day 6
20.5
11.1
Day 9
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Preliminary Data

• Aim 1To test whether Tregs from SLE patients are
  able to expand normally and retain their
  identity, including sustaining a high level of
  FoxP3 expression during expansion--Recruiting
  Patients
• Aim2
• SLE Plasma disrupts the normal expansion of
  Foxp3highCD25highTregs from NORMAL donors
• IFN alpha also reduces the normal expansion of
  these cells and may be inhibiting Foxp3
  expression (to be determined).

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Testing of IFN alpha with TitrationModerate
Supression of expansion of FoxP3 cells
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Summary

• Published Observations in SLE
• Tregs Quantitatively DOWN
• Tregs Qualitatively DOWN
• FoxP3 Expression DOWN
• Our System
• Recapitulates Treg Expansion
• Foxp3 Up-regulation in Tregs
• Our Initial Observations
• SLE Plasma Interferes with normal Treg Expansion
• IFNalpha similarly Interferes with normal Treg
  expansion
• Our Current Experiments
• Does IFN alpha directly inhibit Foxp3 expression
  in purified CD4 T cells
• Do Anti-lymphocyte Abs in SLE target Tregs

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Acknowledgements

• Felipe AndradePrimary Research Mentor, funded by
  Lupus Foundation
• Antony RosenDivision Director and general
  research guidance
• ALL members of Andrade and Rosen Lab!
• Allan GelberProgram Director
• Lisa Christopher-Stine AND Clifton
  BinghamFaculty mentors