Identification of human regulatory T cells in the setting of T cell … · 2011. 12. 23. · 5 4. In the setting of T cell activation, LAP permits accurate identification and isolation

1

Identification of human regulatory T cells

in the setting of T cell activation and anti-CTLA-4 immunotherapy based on

expression of latency associated peptide (LAP)

Jingjing Sun1, Derek Ng Tang1 Tihui Fu1 and Padmanee Sharma1,2

Authors’ Affiliations: 1The University of Texas M. D. Anderson Cancer Center,

Departments of Genitourinary Medical Oncology and 2Immunology

Corresponding Author: Padmanee Sharma, The University of Texas, M. D. Anderson Cancer Center,

Box 0018-7, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713.792.2830;

Fax: 713.745.1625; Email: [email protected].

Acknowledgments

This work was supported in part by a UTMDACC Physician-Scientist Program Award, an

American Cancer Society Mentored Research Grant (MRSG-09-185-01-LIB), a Cancer

Research Institute Clinical Investigator Award and a Doris Duke Charitable Foundation

Clinical Scientist Development Award (all to P.S.).

Conflict of Interest Disclosure: P.S. has served on BMS advisory boards and received

honorariums for her services.

Research. on July 20, 2021. © 2011 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 27, 2011; DOI: 10.1158/2159-8290.CD-11-0236

http://cancerdiscovery.aacrjournals.org/

2

ABSTRACT

Effector and regulatory T cells (Tregs) share multiple markers that make it difficult to

discern differences in these populations in humans. The transcription factor FoxP3 has

been shown to identify Tregs. However, detection of FoxP3 requires cell

permeabilization thereby preventing isolation of viable Tregs. Subsequently, the

extracellular marker CD127 was established for the identification of Tregs. However,

these studies were not conducted in the setting of immunotherapy. Here, we conducted

studies to analyze CD127 and FoxP3 expression on T cells before and after in vitro

activation as well as in the setting of patients treated with anti-CTLA-4 immunotherapy.

We show that latency-associated peptide (LAP), as opposed to CD127, was capable of

identifying Tregs after in vitro activation as well as after treatment with anti-CTLA-4.

Therefore, we propose that LAP should be used as a marker of Tregs for immune

monitoring studies in patients treated with active immunotherapy such as anti-CTLA-4.

SIGNFICANCE

Regulatory T cells (Tregs) play an important role in human diseases, including cancer

and autoimmunity; however, it has been difficult to study these cells due to lack of an

appropriate marker. Here, we propose latency-associated peptide (LAP) as a marker

that can be used to identify Tregs in patients treated with immunotherapy, thereby

permitting isolation of these cells for functional studies and for ex vivo expansion.




3

INTRODUCTION

Regulatory T cells (Tregs) play an important role in the maintenance of self-tolerance

and the control of immune homeostasis (1, 2); however, these cells have been co-opted

by tumors to aid in immune suppression and evasion (3). Cancer immunotherapy

agents will need to overcome the immunosuppressive activity of Tregs in order to

provide clinical benefit. In order to asses the impact of cancer immunotherapy agents on

human Tregs, it is critically important to establish appropriate cell-surface markers that

accurately identify these cells.

In a landmark paper, investigators described a population of IL-2 receptor α-chain

(CD25)-expressing CD4 T cells that were capable of suppressing immune responses (1).

The use of CD25 as a marker for Tregs was quickly adopted by many researchers but,

CD25 cannot accurately distinguish Tregs from activated conventional/effector T cells,

particularly in human samples, in which up to 30% of CD4 T cells can express CD25 (4).

Further investigations into Treg cell biology led to the identification of the X

chromosome-encoded gene FoxP3. A loss-of-function mutation in the FoxP3 gene

leads to an early onset lymphoproliferative immune-mediated disease that is fatal (5). In

subsequent studies, the transcription factor FoxP3 was found to be stably expressed in

Tregs and was required for Treg cell differentiation and immunosuppressive function (6,

7). Together, these studies established a central role for FoxP3 in defining the Treg cell

lineage.

Given that Foxp3 is an intracellular molecule, it requires fixation and permeabilization of

cells in order to allow for detection. Therefore, although FoxP3 remains the best and

most specific marker of Tregs to date, it cannot be used to isolate viable Treg

populations for functional studies or ex vivo expansion. Furthermore, although stable

high expression of FoxP3 is restricted to Treg cells in both mice and humans, it has

been shown in humans (but not in mice) that FoxP3 expression can be induced after

stimulation of conventional T cells (8, 9), thereby making it difficult to distinguish Tregs

from activated effector T cells (Teff) in the setting of patients who are treated with active

immunotherapy agents.




4

As a result of the limitations that arise with the use of FoxP3 as a marker of Tregs,

investigators have attempted to identify cell-surface markers that correlate with FoxP3

expression. Different groups have reported that surface expression of CD127, the α-

chain of the IL-7 receptor, in combination with CD25 can distinguish between human

regulatory and conventional CD4 T cells (10, 11). However, these studies were not

conducted on samples from patients treated with active immunotherapy agents such as

anti-CTLA-4, which can impact T cell activation thus affecting the expression of multiple

markers on both Teff and Tregs.

Anti-CTLA-4 therapy (ipilimumab, Bristol-Myers Squibb) was FDA-approved in March

2011 as a standard-of-care treatment for patients with metastatic melanoma after it was

shown to improve survival of treated patients in a randomized Phase III clinical trial (12).

Since anti-CTLA-4 targets a T cell specific molecule, as opposed to a tumor cell specific

molecule, it is being investigated as a potential therapeutic agent in multiple

malignancies (13). Anti-CTLA-4 targets a T cell molecule known as cytotoxic

lymphocyte antigen-4 (CTLA-4). CTLA-4 functions to limit T cell responses (14). In the

setting of T cell activation, which occurs as a result of engagement of T cell receptor and

CD28 co-stimulation, intracellular signals lead to trafficking of CTLA-4 to the

immunologic synapse, whereby CTLA-4 outcompetes CD28 for ligand binding thus

acting as an intrinsic “off” switch to restrict T cell activity. Anti-CTLA-4 acts to remove

this “off” switch thus allowing for enhanced Teff function and anti-tumor responses (15).

CTLA-4 is also expressed by Tregs (16); however, the impact of anti-CTLA-4 on human

Tregs has been difficult to assess due to the fact that both Teff and Tregs express

markers that can be affected by T cell activation.

In an attempt to identify specific extracellular markers of Tregs in the setting of T cell

activation, we conducted flow cytometric studies with CD25, FoxP3, and CD127 on in

vitro activated human peripheral blood mononuclear cells (PBMCs) and PBMCs derived

from patients treated with anti-CTLA-4 immunotherapy. Here, we report that in the

setting of in vitro activation, CD4+CD25+CD127low cells failed to represent Tregs but, a

different extracellular marker, latency-associated peptide (LAP), could be used to identify

Tregs. LAP was previously shown to be associated with TGF-β, which is expressed on

Tregs (17-19). Furthermore, our data indicate that LAP, as opposed to CD127, is a

better marker for identification of Tregs after patients have been treated with anti-CTLA-




5

4. In the setting of T cell activation, LAP permits accurate identification and isolation of

Tregs for functional studies.

RESULTS

CD4+CD25+CD127low cells correlate with Tregs in the absence of in vitro activation

Due to the fact that staining for FoxP3 requires cellular permeabilization, which leads to

cell death thereby prohibiting isolation of FoxP3+ T cells for functional studies, many

investigators rely on CD127low staining as a surrogate marker for FoxP3+ T cells (10, 11).

In our studies, we found that ~ 6% of unactivated human CD4 T cells from normal

healthy donors were CD25+FoxP3+ (Fig. 1A, representative individual and Fig. 1B,

summary data of 8 healthy donors). Similarly, ~7% of unactivated human CD4 T cells

from normal healthy donors were CD25+CD127low (Fig. 1C, Left Panel), with a

predominant proportion (~78%) demonstrating FoxP3 expression, consistent with a Treg

phenotype (Fig. 1C, Right Panel). Summary data from 8 healthy donors are also shown

(Fig. 1C, Lower Panels). To determine whether the CD4+CD25+CD127low T cells from

unactivated samples were capable of functioning as Tregs, we conducted in vitro

suppression assays. We isolated CD4+CD25+CD127low T cells from unactivated human

PBMCs and found that these cells were capable of suppressing proliferation of

autologous CD4+CD25- T cells (Fig. 1D). Increasing numbers of CD4+CD25+CD127low

suppressor (S) cells led to decreased proliferation of responder (R) cells with significant

suppression (p<0.01) at a R:S ratio of 1:1, which was consistent with the idea that the

CD4+CD25+CD127low cells functioned as Tregs.

CD4+CD25+CD127low cells do not correlate with Tregs in the presence of in vitro

activation

Although there was an observed correlation between CD4 T cells that were FoxP3+ and

CD127low in unactivated human PBMCs, this correlation was not observed in the setting

of activated human PBMCs. Upon in vitro activation with anti-CD3 and anti-CD28, we

observed an ~2-fold increase in the frequency of CD4+CD25+FoxP3+ cells (Fig. 2A as

compared to Fig. 1A), which is consistent with previously published results indicating an

increase in FoxP3 expression upon T cell activation (8, 9). Summary data from 8

healthy donors are shown in Figure 2B. Upon T cell activation, there was also an

increase in the frequency of CD4+CD25+CD127low cells (Fig. 2C, Right Panel) but,

interestingly, ~67% of these cells were FoxP3- and ~33% of these cells were FoxP3+ (Fig.




6

2C, Left Panel), which was a marked difference when compared to FoxP3 expression in

these cells from unactivated samples (Fig. 1C). Summary data from 8 healthy donors

are also shown (Fig. 2C, Lower Panels). In addition, CD4+CD25+CD127low T cells that

were isolated from activated human PBMCs did not function as Tregs since they were

unable to suppress proliferation of autologous CD4+CD25- T cells (Fig. 2D). Therefore,

CD4+CD25+CD127low cells correlate with a functional Treg population in the setting of

unactivated human T cells but, in the setting of in vitro activation, CD4+CD25+CD127low

cells do not correlate with a functional Treg population.

CD4+CD25+LAP+ cells correlate with Tregs in the presence of in vitro activation

To determine whether LAP could act as a surrogate marker for Tregs, we analyzed LAP

and FoxP3 expression in unactivated and activated human PBMCs. We found that the

frequency of CD4+CD25+LAP+ cells was low (~0.3%) in unactivated samples (Fig. 3A,

representative individual and Fig. 3B, summary data from 8 healthy donors). However,

upon in vitro activation of PBMCs, the frequency of CD4+CD25+LAP+ cells increased to

~12% (Fig. 3C, Right Panel), with ~82.5% of these cells expressing FoxP3 (Fig. 3C, Left

Panel). Summary data from 8 healthy donors are also shown (Fig. 3C, Lower Panels).

Similarly, in activated CD4 T cells, FoxP3+ T cells are predominantly LAP+ (Fig. 3D, Left

and Right Panels). Summary data of 8 healthy donors for frequency of CD4+CD25+

FoxP3+ cells (Fig. 2B) and LAP expression in CD4+CD25+ FoxP3+ cells (Fig. 3D, Lower

Panel) after in vitro activation are also shown. In addition, CD4+CD25+LAP+ T cells from

activated human PBMCs functioned as Tregs that were capable of significantly

suppressing in vitro proliferation of CD4+CD25- T cells (p<0.01) as observed by both

thymidine incorporation (Fig. 3E) and CFSE assays (Fig. 3F).

CD4+CD25+LAP+ cells, as opposed to CD4+CD25+CD127low cells, correlate with

Tregs in the setting of anti-CTLA-4 therapy

We previously reported on a clinical trial whereby 12 patients with bladder cancer were

treated with anti-CTLA-4 (ipilimumab) in the pre-surgical setting (20-22). We and others

have reported changes in the frequency of FoxP3+ T cells after treatment with anti-

CTLA-4 (21, 23). However, in humans, changes in FoxP3 expression may represent T

cell activation rather than reflect the frequency of Tregs. Here, we assessed FoxP3,

CD127 and LAP expression in pre-and post-therapy samples from patients treated with

anti-CTLA-4. In the peripheral blood of 12 patients, we observed that 6 patients had an




7

increase in the frequency of CD4+CD25+FoxP3+ T cells while 6 patients did not (21 and

unpublished data). For the 6 patients who did not demonstrate significant changes in

CD4+CD25+FoxP3+ T cells after anti-CTLA-4 therapy, we noted that the frequency of

CD4+CD25+CD127low and CD4+CD25+LAP+ cells also did not significantly change

between pre- and post-therapy samples (data not shown). In patients who had an

increase in the frequency of CD4+CD25+FoxP3+ T cells after treatment with anti-CTLA-4

(n=6), we observed a concomitant increase in the frequency of CD4+CD25+CD127low

cells. However, we observed that the frequency of CD4+CD25+LAP+ cells did not

increase in three patients’ samples (Fig. 4A, representative patient and Fig. 4B summary

data of three patients) but did increase in three different patients’ post-therapy samples

(Fig. 4C, representative patient and Fig. 4D summary data of three patients). In addition,

CD4+CD25+CD127low cells from post-therapy samples were incapable of suppressing

proliferation of autologous CD4+CD25- T cells while CD4+CD25+LAP+ T cells functioned

as Tregs to significantly suppress the proliferation of CD4+CD25- T cells (p<0.01) (Fig.

4E). These data indicate that although FoxP3 and CD127low expression may change in

patients after treatment with immunotherapy, these markers may not accurately reflect a

predominantly functional Treg population. However, increased LAP expression

correlates with a predominantly functional Treg population in the setting of in vitro

activation as well as after treatment with active immunotherapy such as anti-CTLA-4.

Therefore, for the purposes of immune monitoring of patients treated with active

immunotherapeutic agents such as anti-CTLA-4, CD4+CD25+LAP+ cells, as opposed to

CD4+CD25+CD127low cells, allow for appropriate identification and isolation of functional

Tregs.

DISCUSSION

The identification of Tregs and the major role that they play in controlling immune

responses has made it possible to envision effective cancer immunotherapy strategies

that can both enhance effector T cell responses as well as overcome

immunosuppression induced by Tregs. However, the current markers that are being

used to define Tregs in patients may not be adequate in allowing for appropriate

distinctions between Tregs and activated effector cells in patients treated with

immunotherapy. In addition, the gold-standard marker for identifying Tregs is an

intracellular marker, FoxP3, which prevents the use of this marker for isolation of viable




8

cells for functional studies. Furthermore, FoxP3 has been shown to increase in

conventional non-Tregs after T cell activation, which limits the use of this marker to

define the impact of certain immunotherapy agents on Tregs. In a previous publication,

it was shown that in vitro activated human T cells acquired FoxP3 expression and after

the cells were rested for a period of time, the cells from some donors had a decrease in

FoxP3 expression while cells from other donors retained stable FoxP3 expression as

well as suppressive function (9). Therefore, it remains unclear whether increased FoxP3

expression in T cells from patients treated with active immunotherapy agents is

representative of a transient occurrence or a legitimate Treg population.

Anti-CTLA-4 immunotherapy has been reported to enhance T cell responses that lead to

anti-tumor responses. Anti-CTLA-4 (Ipilimumab, Bristol Myers-Squibb) was FDA

approved in March 2011 as a standard-of-care treatment for patients with metastatic

melanoma. Investigations are ongoing to understand the immunologic mechanisms

elicited by anti-CTLA-4 that lead to clinical benefit. We previously documented that anti-

CTLA-4 (ipilimumab) therapy led to an increased frequency of ICOS+ T cells that

consisted of a population of effector T cells (Teff) in both tumor tissues and peripheral

blood (20-22). We also documented that ipilimumab led to a decreased frequency of

FoxP3+ Tregs in tumor tissues, thereby leading to a shift in the Teff (ICOS+)/Treg

(FoxP3+) ratio within tumor tissues as a result of treatment (20-22). However, when we

attempted to calculate the Teff/Treg ratio in peripheral blood using the same T cell

markers, we found that the change between pre-therapy and post-therapy values for the

frequency of FoxP3+ T cells was inconsistent with the changes observed in tumor

tissues. Since FoxP3 expression can increase transiently in activated T cells, it is

possible that FoxP3 expression in T cells from peripheral blood of patients treated with

anti-CTLA-4 does not accurately reflect a predominantly functional Treg population.

Therefore, we examined CD127 expression as a surrogate marker of Tregs in peripheral

blood. However, as we report here, upon in vitro activation of PBMCs from healthy

donors or after patients are treated with anti-CTLA-4 immunotherapy,

CD4+CD25+CD127low cells do not accurately represent a predominantly functional Treg

population (Figures 2 and 4).

Latency-associated peptide (LAP) binds to the cytokine TGF-β in order to form a latent

complex, which is inactive. The complex of LAP plus TGF-β can be expressed on the




9

membrane of many cell types, including megakaryocytes, platelets, immature dendritic

cells and Tregs (18-20). TGF-β is a pleiotropic cytokine and has been reported to play

an important role in the function of Tregs (24). There are multiple mechanisms that can

lead to the activation of TGF-β, which requires release from LAP. In this study, we show

that LAP can be used as a marker to accurately identify a population of predominantly

functional Tregs. More importantly, due to the fact that LAP is an extracellular marker, it

can be used to isolate viable cells for additional studies. We found that CD4+CD25+LAP+

T cells were capable of suppressing immune responses in vitro. In both settings of in

vitro activation of PBMCs and in vivo anti-CTLA-4 therapy, LAP was capable of

identifying Tregs from peripheral blood. Based on these data, we propose that LAP be

included as a marker of Tregs for immune monitoring of patients receiving treatment with

immunotherapy agents such as anti-CTLA-4. We are conducting studies to determine

whether the Teff/Treg ratio as measured by ICOS and LAP can serve as a biomarker for

clinical responses after treatment with anti-CTLA-4.

It should be pointed out that there are extensive investigations ongoing to define Tregs,

which may fall into multiple categories including natural and adaptive Tregs. The

different populations that are encompassed by the term Treg are evolving as

researchers gain a better understanding of how Tregs develop. FoxP3 expression has

been reported in CD4+CD25low/- cells and even in CD8 T cells (25). In our studies,

CD4+CD25+LAP+ cells do not encompass 100% of FoxP3+ T cells nor do

CD4+CD25+FoxP3+ cells encompass 100% of LAP+ T cells, which raises the question of

whether a subset of CD4+CD25+LAP- cells consist of FoxP3+ Tregs or conversely

whether a subset of CD4+CD25+LAP+ cells are FoxP3- Tregs. Additional studies will

need to be conducted to answer these questions. However, based on our current studies,

we propose that LAP is an appropriate marker to be used for the identification of

functional Tregs in patients treated with active immunotherapy agents.




10

MATERIALS AND METHODS

Healthy donors’ and patients’ samples

Peripheral blood sample were obtained from 8 healthy adult donors after individuals

gave appropriate informed consent on M.D. Anderson Cancer Center (MDACC) IRB-

approved lab protocol 2005-0027. Peripheral blood mononuclear cells (PBMCs) were

isolated from whole blood by density gradient centrifugation using Lymphocyte

Separation Medium (Mediatech, Inc., Herndon, VA) and Leucosep tubes (Greiner Bio-

one, Germany). Blood samples were obtained from bladder cancer patients treated with

anti-CTLA-4 after appropriate informed consent was obtained as per IRB-approved

protocol 2006-0080, as previously published (20-22).

In vitro activation of T cells

25cm2 cell culture flasks (Corning incorporated, Corning, NY) were coated with 33ul

αCD3 antibody (BD Biosciences, San Jose , CA., clone HIT3a) plus 6.6ml coating buffer

(1 Carbonate Bicarbonate buffer capsules, Sigma, St. Louis, MO, in 100 ml PBS) for a

final αCD3 concentration of 5μg/ml. Flasks were left overnight at 4oC . The flasks were

then washed with PBS. Freshly isolated PBMCs were then placed into the flasks for 48h.

Each flask contained 30 million PBMCs in RPMI 1640 with 10% human AB serum

( Invitrogen, Carlsbad, CA ) and 2μg/ml of αCD28 antibody ( BD Biosciences).

Flow cytometry

Antibodies used for flow cytometry consisted of : CD4 AmCyan and CD25-PECy7( BD

Biosciences); CD127 Pacific Blue and FOXP3 PerCP-Cy 5.5 (eBioscience, San Diego,

CA); and LAP-APC (R&D, Minneapdis, MN ). Freshly isolated PBMCs and in vitro

activated PBMCs were stained as per manufacturers’ instructions. Samples were

analyzed using the FACS Canto II (Becton Dickinson, San Jose, CA, USA). Data was

analyzed using BD FACSDIVA software. Gates were set according to appropriate

isotype control.

Suppression assays

96 well U-bottom plates (Corning incorporated, Corning, NY) were coated with 10ug/ml

αCD3 overnight at 4oC. The plates were then washed with PBS and CD4+CD25-

responder (R) 5 x 104 cells plus CD4+CD25+CD127low or CD4+CD25+LAP+ cells were

added at indicated ratios. Cells were left in culture for 48 hours in RPMI 1640 plus 10%




11

human AB serum. 1uCi/well tritiated thymidine (Morawek Biochemicalis, Brea CA.) was

added for the last 18 hours of culture. Thymidine incorporation was measured using

TopCount NXT microplate scintillation & luminescence counter (Packard, Meriden, CT).

For CFSE assays, PBMCs were labeled with 0.5μΜ CFSE (Molecular probes/Invitrogen,

Carlsbad, CA). CFSE-labeled CD4+CD25-, CD4+CD25+CD127low and CD4+CD25+LAP+

cells were then sorted. 5 x 104 cells were co-cultured for a 1:1 ratio of Treg/target cells

per well of a 96-well plate, which was previously coated with 10ug/ml αCD3. Culture

medium was RPMI 1640 with 10% human AB serum. Three days later, CFSE dilution

was analyzed by flow cytometry.

Statistical analysis

All group results are expressed as mean plus or minus SD, if not stated otherwise. The

paired Student t test was used for comparison of group values and discriminatory

parameters, where appropriate. P values less than 0.05 were considered significant.




12

REFERENCES

1. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance

maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25).

Breakdown of a single mechanism of self-tolerance causes various autoimmune

diseases. J Immunol 1995;155:1151-64.

2. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in

immunological tolerance to self and non-self. Nat Immunol 2005;6:345-52.

3. Quezada SA, Peggs KS, Simpson TR, Allison JP. Shifting the equilibrium in cancer

immunoediting: from tumor tolerance to eradication. Immunol Rev 2011;241:104-18.

4. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD4+CD25high regulatory cells in

human peripheral blood. J Immunol 2001;167:1245-53.

5. Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA, et al.

Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal

lymphoproliferative disorder of the scurfy mouse. Nat Genet 2001;27:68-73.

6. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function

of CD4+CD25+ regulatory T cells. Nat Immunol 2003;4:330-6.

7. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the

transcription factor Foxp3. Science 2003;299:1057-61.

8. Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, et

al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human

CD4+CD25- T cells. J Clin Invest 2003;112:1437-43.

9. Wang J, Ioan-Facsinay A, van der Voort EI, Huizinga TW, Toes RE. Transient

expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur J Immunol

2007;37:129-38.

10. Lin W, Haribhai D, Relland LM, Truong N, Carlson MR, Williams CB, et al. Regulatory T

cell development in the absence of functional Foxp3. Nat Immunol 2007;8:359-68.

11. Hartigan-O'Connor DJ, Poon C, Sinclair E, McCune JM. Human CD4+ regulatory T cells

express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent

identification and sorting of live cells. J Immunol Methods 2007;319:41-52.

12. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved

survival with ipilimumab in patients with metastatic melanoma. N Engl J Med.

2010;363:711-23.




13

13. Calabrò L, Danielli R, Sigalotti L, Maio M. Clinical studies with anti-CTLA-4 antibodies in

non-melanoma indications. Semin Oncol 2010;37:460-7.

14. Krummel MF, Allison JP. CD28 and CTLA-4 have opposing effects on the response of T

cells to stimulation. J Exp Med 1995;182:459-65.

15. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4

blockade. Science 1996;271:1734-6.

16. Sansom DM, Walker LS. The role of CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA-

4) in regulatory T-cell biology. Immunol Rev 2006;212:131-48.

17. Nakamura K, Kitani A, Fuss I, Pedersen A, Harada N, Nawata H, et al. TGF-beta 1 plays

an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both

humans and mice. J Immunol 2004;172:834-42.

18. Tran DQ, Andersson J, Hardwick D, Bebris L, Illei GG, Shevach EM. Selective

expression of latency-associated peptide (LAP) and IL-1 receptor type I/II

(CD121a/CD121b) on activated human FOXP3+ regulatory T cells allows for their

purification from expansion cultures. Blood 2009;113:5125-33.

19. Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, Shevach EM. GARP

(LRRC32) is essential for the surface expression of latent TGF-beta on platelets and

activated FOXP3+ regulatory T cells. Proc Natl Acad Sci U S A 2009;106:13445-50.

20. Liakou CI, Kamat A, Ng Tang DS, Chen H, Sun J, Troncoso P, et al. CTLA-4 blockade

increases IFNgamma-producing CD4+ICOShi cells to shift the ratio of effector to

regulatory T cells in cancer patients. Proc Natl Acad Sci U S A 2008;105:14987-92.

21. Chen H, Liakou CI, Kamat A, Pettaway C, Ward JF, Ng Tang DS, et al. Anti-CTLA-4

therapy results in higher CD4+ICOShi T cell frequency and IFN-gamma levels in both

nonmalignant and malignant prostate tissues. Proc Natl Acad Sci U S A 2009;106:2729-

34.

22. Carthon BC, Wolchok JD, Yuan J, Kamat A, Ng Tang DS, Sun J, et al. Preoperative

CTLA-4 blockade: tolerability and immune monitoring in the setting of a presurgical

clinical trial. Clin Cancer Res 2010;16:2861-71.

23. Kavanagh B, O'Brien S, Lee D, Hou Y, Weinberg V, Rini B, et al. CTLA-4 blockade

expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent

fashion. Blood 2008;112:1175-83.

24. Lawrence DA. Latent-TGF-beta: an overview. Mol Cell Biochem 2001;219:163-70.

25. Ziegler SF. FOXP3: of mice and men. Annu Rev Immunol 2006;24:209-26.




14

Figure Legends

Figure 1. Frequency of CD4+CD25+FoxP3+ and CD4+CD25+CD127low T cells in

unactivated PBMCs from healthy donors. Frequency of FoxP3 expression in

CD4+CD25+ T cells from one representative individual (A) and summary data from 8

healthy donors (B); Frequency of CD127low cells in CD4+CD25+ T cells from one

representative individual (C, Right Upper Panel)) with summary data from 8 healthy

donors (C, Right Lower Panel) and frequency of FoxP3+ T cells in the

CD4+CD25+CD127low population from one representative individual (C, Left Upper Panel)

with summary data from 8 healthy donors (C, Left Lower Panel); Representative

thymidine incorporation assay, which measured cell proliferation in the presence of

media only, CD4+CD25- responder (R) cells plus CD4+CD25+CD127low suppressor (S)

cells at R:S ratios of 0:1, 1:0, 1:0.1, 1:0.5 and 1:1 (D). Each experiment was performed

in triplicate and suppression assays were repeated with a minimum of three different

donor samples. Bars represent mean + SEM.

Figure 2. Frequency of CD4+CD25+FoxP3+ and CD4+CD25+CD127low T cells after in

vitro activation of human PBMCs. Frequency of FoxP3 expression in CD4+CD25+ T

cells from one representative individual (A) and summary data from 8 healthy donors (B);

Frequency of CD127low cells in CD4+CD25+ T cells from one representative individual (C,

Right Upper Panel)) with summary data from 8 healthy donors (C, Right Lower Panel)

and frequency of FoxP3+ T cells in the CD4+CD25+CD127low population from one

representative individual (C, Left Upper Panel) with summary data from 8 healthy donors

(C, Left Lower Panel); Representative thymidine incorporation assay, which measured

cell proliferation in the presence of media only, CD4+CD25- responder (R) cells plus

CD4+CD25+CD127low cells at R:S ratios of 0:1, 1:0, 1:0.1, 1:0.5 and 1:1 (D). Each

experiment was performed in triplicate and suppression assays were repeated with a

minimum of three different donor samples. Bars represent mean + SEM.

Figure 3. Frequency of CD4+CD25+LAP+ T cells before and after in vitro activation

of human PBMCs. Frequency of LAP expression in CD4+CD25+ T cells from

unactivated human PBMCs in one representative individual (A) and summary data from

8 healthy donors (B); Frequency of LAP expression in CD4+CD25+ T cells after in vitro

activation of human PBMCs in one representative individual (C, Right Upper Panel)) with

summary data from 8 healthy donors (C, Right Lower Panel) and frequency of FoxP3+ T




15

cells in the CD4+CD25+LAP+ population from one representative individual (C, Left

Upper Panel) with summary data from 8 healthy donors (C, Left Lower Panel);

Frequency of FoxP3 expression in CD4+CD25+ T cells after in vitro activation of human

PBMCs in one representative individual (D, Right Upper Panel)) and frequency of LAP+ T

cells in the CD4+CD25+FoxP3+ population from one representative individual (D, Left

Upper Panel) with summary data from 8 healthy donors (D, Left Lower Panel);

Representative thymidine incorporation assay, which measured cell proliferation in the

presence of media only, CD4+CD25- responder (R) cells plus CD4+CD25+ LAP+

suppressor (S) cells at R:S ratios of 0:1, 1:0, 1:0.1, 1:0.5 and 1:1 (E); Representative

CFSE assay with CD4+CD25- responder cells alone (F, Right Panel) and CD4+CD25-

responder cells plus CD4+CD25+LAP+ cells at R:S ratio of 1:1 (F, Left Panel). Each

experiment was performed in triplicate and suppression assays were repeated with a

minimum of three different donor samples. Bars represent mean + SEM.

Figure 4. Frequency of CD4+CD25+FoxP3+, CD4+CD25+CD127low and

CD4+CD25+LAP+ T cells before and after treatment with anti-CTLA-4. Representative

patient who had an increase in frequency of CD4+CD25+FoxP3+ and

CD4+CD25+CD127low T cells but not in CD4+CD25+LAP+ T cells after treatment with anti-

CTLA-4 (post-therapy) as compared to before treatment (pre-therapy) (A); Summary

data from three patients who had an increase in frequency of CD4+CD25+FoxP3+ and

CD4+CD25+CD127low T cells but not in CD4+CD25+LAP+ T cells after treatment with anti-

CTLA-4 (post) as compared to before treatment (pre) (B); Representative patient who

had an increase in frequency of CD4+CD25+FoxP3+, CD4+CD25+CD127low and

CD4+CD25+LAP+ T cells after treatment with anti-CTLA-4 (post-therapy) as compared to

before treatment (pre-therapy) (C); Summary data from three patients who had an

increase in frequency of CD4+CD25+FoxP3+, CD4+CD25+CD127low and CD4+CD25+LAP+

T cells after treatment with anti-CTLA-4 (post) as compared to before treatment (pre) (D);

Representative thymidine incorporation assay, which measured cell proliferation in the

presence of media only, CD4+CD25+LAP+ T cells only with a ratio of R:S of 0:1,

CD4+CD25+CD127low cells only with a R:S ratio of 0:1, responder CD4+CD25- cells only

with a ratio of R:S of 1:0, a mixture of R:S (CD4+CD25+CD127low) at a ratio of 1:1 and a

mixture of R:S (CD4+CD25+LAP+ cells) at a ratio of 1:1 (E). Each experiment was

performed in triplicate and suppression assays were repeated with three different donor

samples. Bars represent mean + SEM.




6%

25 6 0 %

7.0 %

8 .0 %

7%

5

78%

D25

A B C

FoxP3

CD2

2 .0 %

3 .0 %

4 .0 %

5.0 %

6 .0 %

CD25

CD127

CD

FoxP3

8 0 .0 %

0 .0 %

1.0 %

2 .0 %

CD4+CD25+FoxP3+

3 .0 %

4 .0 %

5.0 %

6 .0 %

7.0 %

8 .0 %

2 0 0 %

3 0 .0 %

4 0 .0 %

50 .0 %

6 0 .0 %

70 .0 %

CD4+CD25+CD127low

0 .0 %

1.0 %

2 .0 %

0 .0 %

10 .0 %

2 0 .0 %

% FoxP3+ cells in CD4+CD25+CD127low population14,000

D

8,000

10,000

12,000

M

4,000

6,000CPM

0

2,000

Media only R:S of 0:1 R:S of 1:0 R:S of 1:0.1 R:S of 1:0.5 R:S of 1:1

Figure 1

Research. on July 20, 2021. ©

2011 Am

erican Association for C

ancercancerdiscovery.aacrjournals.org

Dow

nloaded from

Author m

anuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author M

anuscript Published O

nlineFirst on D

ecember 27, 2011; D

OI: 10.1158/2159-8290.C

D-11-0236


14% 2 0 .0 %

2 5.0 %

40%

5 33%D25

A B CCD

25

10 .0 %

15.0 % CD25

CD127

CD

FoxP3

4 0 0 %5 0 . 0 %

FoxP3

0 .0 %

5.0 %

CD4+CD25+FoxP3+2 0 .0 %

3 0 .0 %

4 0 .0 %

2 0 . 0 %

3 0 . 0 %

4 0 . 0 %

0 .0 %

10 .0 %

0 . 0 %

10 . 0 %

CD4+CD25+CD127low % FoxP3+ cells in CD4+CD25+CD127low population

D

15,000

20,000

5,000

10,000CPM

0Media only R:S of 0:1 R:S of 1:0 R:S of 1:0.1 R:S of 1:0.5 R:S of 1:1

Figure 2


2011 Am



Dow

nloaded from

Author m


Author M


nlineFirst on D

ecember 27, 2011; D

OI: 10.1158/2159-8290.C

D-11-0236


0 . 3 %

0 . 4 %

CD25

12%

CD25 82.5%

A B C

0 . 1%

0 . 2 %CD25

LAP

0.3%

6 0 0 %

8 0 . 0 %

10 0 . 0 %

15 . 0 %

2 0 . 0 %

2 5 . 0 %

LAP FoxP3

0 . 0 %

LAP

CD4+CD25+LAP+0 . 0 %

2 0 . 0 %

4 0 . 0 %

6 0 . 0 %

0 . 0 %

5 . 0 %

10 . 0 %

CD4+CD25+LAP+ % FoxP3+ cells in CD4+CD25+LAP+

populationD

CD25

14%

CD25 79.6%

8,000

10,000

12,000

14,000

16,000

CPM

E

FoxP3

C

LAP

10 0 . 0 %

0

2,000

4,000

6,000

Media only R:S of 0:1 R:S of 1:0 R:S of 1:0.1 R:S of 1:0.5 R:S of 1:1

C

F

4 0 . 0 %

6 0 . 0 %

8 0 . 0 %

F

0 . 0 %

2 0 . 0 %

% LAP+ cells in CD4+CD25+FoxP3+

populationFigure 3 CD4+CD25- CD4+CD25- : CD4+CD25+LAP+

(R:S ratio 1:1)


2011 Am



Dow

nloaded from

Author m


Author M


nlineFirst on D

ecember 27, 2011; D

OI: 10.1158/2159-8290.C

D-11-0236


Pre-therapy CD25

LAP

0.4%

CD25

FoxP3

4%

CD25

CD127

4%

20Patient #1

Patient #2

Post

Post

A B

Post-therapy CD25

LAP

0.4%

CD25

CD127

7%

CD25

FoxP3

8%

10

15

% c

ells

Patient #3

Patient #2

Pre

Pre

Pre

Post

Post

C

5 Pre

Pre

Pre

Pre

Pre Pre Pre

Post

Post

Post Post Post7% 6

Pre-therapy

CD25

FoxP3

7% 6%

CD25

CD127

0.4%

CD25

LAP

C

0CD4+CD25+CD127lowCD4+CD25+FoxP3+ CD4+CD25+LAP+

%

10%

4%

Post-therapy CD25

FoxP3

11% 10%

CD25

CD127

4%

CD25

LAP DE

15

20

s

Patient #4

Patient #6Patient #5

Pre

Post

Post

Post

Post

8 00010,00012,00014,00016,00018,000

CPM

E

5

10% c

ells

Pre

Pre

Pre

Pre

Pre

PostPost

Post

PostPost

02,0004,0006,0008,000C

0CD4+CD25+LAP+CD4+CD25+CD127lowCD4+CD25+FoxP3+

PrePre Pre

Figure 4


2011 Am



Dow

nloaded from

Author m


Author M


nlineFirst on D

ecember 27, 2011; D

OI: 10.1158/2159-8290.C

D-11-0236


Published OnlineFirst December 27, 2011.Cancer Discovery Jingjing Sun, Derek Ng Tang, Tihui Fu, et al. expression of latency associated peptide (LAP)activation and anti-CTLA-4 immunotherapy based on Identification of human regulatory T cells in the setting of T cell

Updated version

10.1158/2159-8290.CD-11-0236doi:

Access the most recent version of this article at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerdiscovery.aacrjournals.org/content/early/2011/12/23/2159-8290.CD-11-0236To request permission to re-use all or part of this article, use this link



http://cancerdiscovery.aacrjournals.org/lookup/doi/10.1158/2159-8290.CD-11-0236

http://cancerdiscovery.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerdiscovery.aacrjournals.org/content/early/2011/12/23/2159-8290.CD-11-0236


Documents

Identification of human regulatory T cells in the setting of T cell … · 2011. 12. 23. · 5 4. In the setting of T cell activation, LAP permits accurate identification and isolation