CTLA-4 engagement on CD4+CD25- cells results in the release of suppressive cytokines
TGFβ and IL-10, and prevents proliferation of naïve T cells in vitro
Progress Report
Abstract
It is widely known that the subset of T cells that are CD4+CD25+ regulate the activity of
other T cells. The ability of these Regulatory T cells (Treg) to suppress an immune
response via educating the effector T cells (Teff) is a trait that has potential utility to treat
various autoimmune diseases such as type 1 diabetes mellitus (T1D), systemic lupus
erythematosus (SLE), and experimental autoimmune thyroiditis (EAT) among several
others. However, the function of the CD4+CD25- cells, termed effector T cells (Teff), in
regulating an immune response, has not been fully analysed. Both Treg and Teff cells,
once activated, express the negative costimulatory receptor Cytotoxic T Lymphocyte
Antigen 4 (CTLA-4). CTLA-4 is perhaps the most well documented signalling molecule
involved in the suppression of an immune response by Treg cells. However the
mechanism of CTLA-4 activity and its consequences have not been fully elucidated. The
ability of CD4+CD25+ and CD4+CD25- cells to convert naïve T cells to a regulatory
phenotype via CTLA-4 engagement was studied in vitro using anti-CTLA-4 antibodies in
active populations of CD4+CD25- and CD4+CD25+ cells isolated from normal C57BL/6
(B6) mice. The subsequent changes in cytokine profiles of TGFβ, IL-10 and IFNγ were
tested in each population. The CD4+CD25- (Teff) population showed a cytokine profile
similar to that of the CD4+CD25+ ( Treg) cells. Next, the suppressive function of the
activated Teff cells was compared to that of the Treg population in vitro. The Teff
population suppressed the division of naïve T cells added to the activated population in a
manner similar to that displayed by the Treg cells. Thus cross-linking of CTLA-4 on
activated CD4+CD25- cells causes secretion of immunosuppressive cytokines, and
suppresses the proliferation of naïve effector T cells, suggesting that naturally occurring
Treg cells have the capacity to cause Teff cells to differentiate into a Treg phenotype in
vitro.
Introduction
Regulatory T cells (Treg) constitute 10-15% of the T cell population, and control other T
cells, suppressing their activity and thereby preventing harmful autoimmune responses to
self Ags. Treg cells have been categorised as those T cells that constitutively express
CD4, CD25 (IL-2R α-chain) and CTLA-4 on the cell surface (1 Zheng et al, 2 Takahashi
et al). On the other hand, effector T cells (Teff) are those that are CD4+CD25-, and do
not constitutively express CTLA-4 (3 Chen et al – add more). These cells are under the
control of the Treg subpopulation, and exhibit pro- or anti- inflammatory effects, either
through humoral or cell-mediated mechanisms depending on the stimulus provided. The
current established notion is that the Treg cells have suppressive functions, and
downregulate an immune response by relaying anti-proliferative signals to the Teff
population (1, 4 Nishimura). Consequently, the Teff cells respond by expressing
immunosuppressive cytokines TGFb, IL-10, costimulatory receptor CTLA-4, CD25, and
downregulating factors such as Smad7, involved in ubiquitination and degradation of
TGFb, thus themselves developing a regulatory phenotype (5 Fantini, 6 Liang et al). A
forkhead-winged-helix transcription factor (FoxP3) has been the subject of great interest
in the development and maintenance of Treg cells. Several groups (4, 7 Ochs et al 8 ADD
ONE 9 Hori et al) show that FoxP3 is not only a specific marker for Treg cells, but is
needed to convert naïve T cells to a Treg phenotype via retroviral transduction of FoxP3
in naïve T cells. However these findings require further investigation, as T cell
phenotypes change drastically as periods of stimulation, activation and anergy affect gene
expression patterns in the same cell over time (10XX, 11XX).
Several mechanisms have been suggested in the suppressive function of Treg cells,
ranging from direct cell-cell contact to release of suppressive cytokines (1, 4-6), and
CTLA-4 has proven to be the most potent costimulatory molecule involved in Treg
mediated suppression of Teff activity. However a specific mechanism of CTLA-4
function and its consequences on Teff phenotype have yet to be elucidated.
The well established two signal theory states that TcR engagement with its respective
antigen-MHC complex and CD28 engagement with its ligands B7.1 and B7.2 are
required for T cell activation.
Is it direct cell-cell contact between Treg and Teff necessary for
1. define what regulatory T cells are
2. define what effector T cells are
3. describe the 2 signal theory needed to activate T cells
a. TcR (CD3 complex) – MHC on APC
b. CD28 – B7 on APC
4. Describe the function of CTLA-4
a. CD28 homologue
b. Binds B7 with higher affinity
c. Is negative regulator of T cell activity
5. Studies:
a. Show that inhibition of CTLA-4 activity results in rapid
lymphoproliferative disorder in vivo (XX,XX,XX)
b. Show that inhibition of CD28 activity results in T cell hyporesponsiveness
and anergy (XX, XX, XX)
c. Show that cytokines TGFβ and IL-10 cause a suppression of immune
response
6. So far the ability of Treg cells to educate effector T cells to develop suppressive
activity has been studied. But CD4+CD25- cells, once activated, also express
CTLA-4.
a. Do these cells function in the same manner as Treg cells?
b. If so, what is the mechanism of suppression of immune responses?
c. Is this mechanism similar to CD4+CD25+ mechanism?
7. To answer these questions, we compared:
a. CTLA-4 expression levels in activated and inactivated CD4+CD25+ and
CD4+CD25- cells
b. Cytokine profiles of activated and inactivated CD4+CD25+ and
CD4+CD25- cells
c. The ability of activated and inactivated CD4+CD25+ and CD4+CD25cells to suppress naïve T cell proliferation
8. We show that CD4+CD25- cells behave in a manner similar to Treg cells, and
have suppressive activity as well.
Materials and Methods
Mice
Normal 6-8 week old C57BL/6 (B6) mice were purchased from Jackson Laboratories
(Bar Harbor, ME). All mice were housed at the St. Michael’s Hospital animal facility and
treated in accordance with institutional guidelines for animal care and handling.
Abs and cytokines
The following Abs were used: anti-CD3, anti-CD28, anti-CTLA-4, and respective mouse
IgG isotype controls (company, location). Abs used for cell surface staining were PEconjugated anti-CD25, PE-conjugated anti-CTLA-4 and FITC-conjugated anti-B7.1, and
respective control conjugated IgG mAbs (company, location). For cell isolation,
biotinylated anti-CD8, CD11b, CD45R, Ly-6G(Gr-1) and TER119 were used as
components of a CD4+ T cell enrichment cocktail, along with anti-biotin-anti-dextran
tetrameric antibody complexes and dextran coated magnetic nanoparticles. Plate bound
anti-CD3 and anti-CD28 mAbs were used for primary T cell stimulation; plate bound
anti-CD3 and anti-CTLA-4 were used for secondary T cell stimulation.
Cell isolation
Spleens were removed from 6-8 week old B6 mice, and the separated splenocytes were
suspended in serum-free AIMV medium. The CD4+ cells were isolated using an EasySep
negative selection mouse CD4+ T cell enrichment cocktail. The CD25+ subset was
isolated using an EasySep positive biotin selection kit and biotinylated anti-CD25
antibodies. The prepared cell suspensions were stained with PE-anti-CD25-mAb
(company, no.). Purity of CD4+CD25+ and CD4+CD25- cells were >92% and >95%
respectively.
Flow cytometry – CD25+ purity
Single-cell suspensions of splenocytes were washed (PBS, 2% FBS) and stained with PEconjugated anti-CD25 or matched IgG isotype control antibodies at 4oC for 15 min,
washed and analyzed in a Flow Cytometer (name, company, location). A minimum of
10000 cells were analyzed for each sample. All experiments were repeated 4 times.
Flow cytometry – CTLA-4 and B7.1 expression
Procedures were repeated as above, but single cell splenocyte suspensions were washed
and stained with PE-conjugated anti-CTLA-4 and FITC-conjugated anti-B7.1 or matched
IgG isotype control antibodies.