Round 23: SLE and Tregs: What we know and what we would like to know

By: Amit Golding, MD, PhD
Johns Hopkins University School of Medicine

Dr. Golding has no significant financial interest or relationships to disclose.

Release Date: TBD
Expiration Date: January 1, 2011

For CME credit,TAKE POST-TEST & EVALUATION

Objectives

Participants will be able to:

  1. Discuss regulatory T cells (Tregs) in systemic lupus erythematosus (SLE) — 10 primary studies from 2003 to 2008:
    1. Various Parameters
      1. Treg Numbers
      2. Treg Function
      3. Foxp3 Expression
    2. B. Details from two best papers
  2. Describe Tregs in SLE and other Autoimmune Diseases:
    1. Possible Mechanisms for a reduction in Tregs
    2. Basic explanation of our system for studying Treg expansion
    3. Summary of preliminary data

What are regulatory T cells?

Tregs were initially observed as a population of Thymic-derived cells that arise a few days later than effector cells—day 3 thymectomized mice develop a multi-organ autoimmune disease due to unregulated runaway effector T cells. This is due to the fact that later than day 3, another subset of regulatory T cells develop in and leave the thymus as the separate subset of Tregs.

The CD25 is one of the key markers of Tregs, it’s also the 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 are Tregs. CD25 can be used to enrich for or deplete Tregs from a resting population of T cells.

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.

In the basic scenario of a Treg regulating the effector T cell, there are a number of ways in which the gene Foxp3 is turned on.

Figure 1

On day 0, at the beginning of an immune response, an antigen simulation and effector T cell will respond.  At the same time, a Treg in that same environment will be present, and both of them will be able to expand. Both of them will continue to expand, and the Treg will be able to regulate the effector cell so that the effector cell stops expanding––the immune response can be regulated in normal fashion and go back to baseline. Without the Tregs, the unregulated effector T cells continue to expand in a runaway fashion.

The Treg axis and human disease

Again, looking at one access of control of Foxp3 expression, and making sure we keep ourselves based in focusing on human disease, a number of genes in controlling the master regulator of Tregs, and that are critical to the existence of Tregs, have been found to be associated with severe autoimmune disease in humans.

  • 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

Summary of literature, 2003 to 2008

Author Year Tregs CD25High Defined # of Pts. Overall Quality
Crispin et al 2003 CD25High Only 30 SLE (10 Active)
10 Healthy Controls
Fair
Liu et al 2004 CD25High Only 94 SLE
50 Controls
Fair
Miyara et al 2005 CD25High + Marker +
Suppressor Assay
107 SLE (45 Active)
82 Controls
Best
Lee et al 2006 CD25High + Marker + 27 SLE (17 Active)
25 Controls
High
Mellor-Pita et al 2006 CD25High Only 33 SLE (26 Active)
14 Controls
Good
Suarez et al 2006 CD25High + Marker + 110 SLE
56 Controls
Good
Alvarado-Sanchez et al 2006 CD25High + Foxp3 +
Suppressor Assay
23 SLE (19 Active)
27 Controls
Good
Valencia et al 2007 CD25High + Foxp3 +
Suppressor Assay
25 SLE (17 Active)
40 Controls
High
Bonelli et al 2007 CD25High + Foxp3 + 17 SLE (6 Active)
8 Controls
Good
Yan et al 2008 CD25High + Foxp3 +
Suppressor Assay
25 SLE (15 Active)
15 Controls
Good

There are different ways of defining Tregs in these studies.  The most basic way is simply to say that a cell that has the highest expression of the CD25 marker at baseline in resting cells when taken from the patient.  Defined, for example, with an antibody, the highest CD25 expressing cell is a regulatory T cell.  That’s somewhat simplistic because an activated cell will also up-regulate CD25.  So, that’s not the best way to determine if it’s definitely a Treg. A suppression assay can solidly determine true Tregs as opposed to a surrogate.  The best quality studies to date are those in which they use:

  • A combination of a number of markers, especially including Foxp3, which only became available during the last 5 to 6 years and,
  • Suppression assays.

The preponderance of evidence from all of these studies shows that in active SLE disease, the absolute numbers of CD25High Tregs are significantly diminished. This was shown both in comparison to normal controls and when comparing active to inactive SLE patients. Only two studies showed exceptions: one study showed an exception in the patients that were treated with high doses of steroids; and another study showed an exception because they inhibited the T-effector cells approximately 60% whereas the standard suppression assays inhibit the proliferation to 95% or higher (this questions the accuracy of the suppression assays).  Aside form these two studies, the overall conclusion supports that in active SLE, CD25High Tregs are diminished.  Furthermore, in many of these studies, they were able to find statistically significant negative correlations with disease activity.  They found negative correlations between disease activity and absolute numbers of Treg and, in some cases, to find found a negative correlation between disease activity and the ability to suppress. In particular, the studies from Alvarado Sanchez et al and Valencia et al (2006 and 2007) showed a very good correlation negatively in the SLE patients with the most active disease because their Tregs had the poorest ability to suppress T-effector cells.

Miyara et al, 2005

  • Suppressive function of cells with the brightest CD25 staining is maintained. In this particular paper, when they only took the cells with the very highest CD25 expression and asked whether those cells from active SLE patients, when they’re mixed in a 1:1 ratio with T-effector cells, could suppress. In all of the patients, they were able to suppress the T-effector cells in an equal and comparable fashion to both healthy controls and inactive SLE patients.  However, if they quantitated the absolute numbers of these CD25 bright cells from active SLE patients, you can see that there’s a statistically significant decrease in abundance of those truly suppressor cells from the active SLE patients as compared to inactive SLE patients, as compared to controls, and even as compared to Sjögren’s and myositis patients.
  • CD25bright Tregs that retain full suppressive function are reduced in SLE patients with active disease.
  • The number of CD25bright, fully suppressive Tregs correlates with disease activity and also longitudinally in SLE patients. Miyara et al took individual patients who had active disease and followed them longitudinally from an active flare after treatment, and saw that the number of these cells increased in a statistically significant fashion.
  • The number of FoxP3+ Cells in a LN from an active SLE patient is markedly reduced. Using biopsies of lymph nodes stained in situ for the number of CD25 and Foxp3 high-staining cells, which again are the Tregs, as compared to controls, Miyara et al showed that the absolute numbers of Foxp3 high cells in the lymph nodes from active SLE patients was significantly reduced.

Valencia et al, 2006

This study looked at the CD25 positive population and took the top 2% of the CD25 positive cells, gated on them, purified them and tested how well they did in the suppression assay.

  • The percentage of Tregs that are Foxp3+ is reduced in SLE patients with more active disease. What Valencia et al found is that a significant number of Tregs were not Foxp3 high, and that correlated negatively with disease activity. The higher the disease activity, the lower the Foxp3 percentage, and the lower the Foxp3 expression of the CD25 high Tregs.
  • The ability of CD25High Treg cells to function as suppressors is reduced in SLE patients with more active disease.

Why does Miyara et al show that in active SLE the cells that are suppressor cells function just fine, but there are less of them; and Valencia et al show there may be an equivalent number of cells, but they don’t function well?  This is a “two sides of one coin” indicator, not a paradox. In the first paper, by forcing themselves to only look at those Tregs that, by definition, were good suppressors, an extremely low percentage of the total cells was analyzed. It’s not surprising that the number of cells in active SLE patients that met their criteria for being true suppressive cells was lower in active SLE patients.  Whereas, if a higher number of CD25 positive cells is captured, and confirmed as true Tregs by other criteria (which is what was done in the Valencia paper) then it’s evident that some of them have actually lost Foxp3 expression and some of the normal suppressive function.

More questions than answers

1. Why are the Treg numbers reduced in active SLE?

  • Decreased production in the thymus.  It is rather unlikely that there is decreased thymic production of the Tregs, and it is rather unlikely that it is an intrinsic problem to the Tregs themselves.  Valencia et al were able to take those Tregs that didn’t suppress properly, culture them ex vivo and retrain them to suppress properly.  They regained their ability to suppress the T-effector cells simply by culturing them in the presence of IL-2 and a stimulator for the T-cell receptor.
  • Increased destruction. There is a long history in lupus and a lot of evidence going back to the 70’s of antilymphocyte antibodies, and many of these might target T cells in general and, in particular, Tregs for destruction.  And, this is one avenue that we have been pursuing.
  • Diminished Peripheral Expansion/Ineffective Homeostasis. There is a lot of evidence that in lupus there is both diminished IL-2 production and diminished responsiveness to IL-2. IL-2, a general growth factor for T cells, is particularly critical for Treg homeostasis, and CD25 is an IL-2 Receptor alpha. SLE has been long known to be a state of reduced IL-2 production and T cells.

2. What is the mechanism for reduced Foxp3 expression in SLE Tregs?

  • IL-2 is implicated here as well: IL-2 à STAT5 à 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.

Johns Hopkins study

Purpose

To account for reduced numbers of functional Tregs in active SLE patients

  • due to the possible mechanism of faulty Treg expansion in SLE
  • 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.

Aims

  • To test whether Tregs from SLE patients are able to expand (has not begun).
  • To test whether the lupus environment itself disrupts the normal pathways that lead to Foxp3 expression during T-cell activation and expansion.

Methods

There is system that was first published in 2006, which instead of using just purified Tregs and T effectors in the suppression assay uses total PBMC’s. With polyclonal activation, that system can be used to study the expansion of both Tregs and T effectors as a mini example of an immune response that should be analogous to a response to a foreign antigen, for example.

Figure 2

Schematic of what one has in the tissue culture with the PBMC’s.  There are non-T cells as well as CD4 positive CD25 negative T-effector cells, and CD4 positive CD25 positive Tregs.  And, in the presence of anti-CD3 acting as a T-cell receptor stimulant in a polyclonal fashion, and heat inactivated human serum, there is an activation of this entire population of T cells.  They all expand and divide together.

If the starting population of PBMC’s is removed (CD4+CD25+T cells), and the same expansion is performed, the CD25 high Foxp3 high cells, which represent the expansion of the natural Tregs are no longer upregulated. In the initial population, there is about 3% CD25High Foxp3High natural Tregs at baseline.  After 3 days of stimulation, and removal of the expansion (Tregs CD25High, Foxp3High) there is only 0.2%, the expanded population is no longer there.

What happens to these T-effector cells that are left behind when the normal natural Tregs are removed?  If the effector cells are stained with a dye that allows following cell divisions in the effector cells, and the normal pattern of division in whole PBMC’s that have not been depleted of the Tregs is analyzed, the dye is basically diluted out as the cells divide. In the expanded population of cells there is a significant increase in the number of T-effector cell divisions that occur.

Preliminary data

Using the experimental system for recapitulating the expansion of Tregs and also following Foxp3 expression in those Tregs, shows that the actual level of Foxp3 expression goes up in the natural Tregs as they expand.

  • SLE Plasma disrupts the normal expansion of Foxp3High CD25High Tregs from NORMAL donors.
  • IFN alpha also reduces the normal expansion of these cells and may be inhibiting Foxp3 expression (to be determined).

Summary

Published observations in SLE:

The Tregs in active SLE patients are quantitatively and qualitatively diminished, that Foxp3 suppression is also suppressed.

Experimental system and initial observations:

We have a system that recapitulates Treg expansion and Foxp3 upregulation, and our initial observation suggests that SLE plasma can interfere with normal Treg expansion.  Similarly, that interferon alpha also interferes with normal Treg expansion.

Current experiment:

The focus is on trying to figure out the exact mechanism by which interferon alpha inhibits Foxp3 expression in purified CD4 T cells, and we are still interested in seeing whether antilymphocyte antibodies in SLE may target Tregs.

For CME credit,TAKE POST-TEST & EVALUATION

Updated: August 16, 2012

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