Th2 cytokines from malignant cells suppress Th1 responses and enforce a global
Th2 bias in leukemic cutaneous T cell lymphoma
Emmanuella Guenova1,2, Rei Watanabe1,2, Jessica E. Teague1, Jennifer A. Desimone1,
Ying Jiang3, Mitra Dowlatshahi1, Ahmed Gehad1, Christoph Schlapbach1, Knut
Schaekel4, Alain H. Rook5, Marianne Tawa6, David C. Fisher6, Thomas S. Kupper1,6,
and Rachael A. Clark1,6,7
1Department of Dermatology, Brigham and Women’s Hospital and Harvard Medical
School, Boston, MA
2Both authors contributed equally to the work described in this manuscript and should be
considered as co-first authors.
3Temple University School of Medicine, Philadelphia, PA
4Department of Dermatology, University Hospital Heidelberg, Voßstraße 2, Heidelberg
5Department of Dermatology, Hospital of the University of Pennsylvania, Philadelphia,
6Dana Farber/Brigham and Women’s Cancer Center, Boston, MA
7Address correspondence to: Rachael A. Clark, Department of Dermatology, Brigham
and Women’s Hospital, Room 505A, 221 Longwood Avenue, Boston, MA 02115.
Phone (617) 525-8512; Fax (617) 264-5123; Email: firstname.lastname@example.org.
Statement of translational relevance
Patients with leukemic cutaneous T cell lymphoma (L-CTCL) have an average 3-5 year
survival, die most commonly from infection and have clinical abnormalities consistent
with a Th2-driven immunologic process. We find both malignant and benign T cells in L-
CTCL are markedly Th2 biased, demonstrating a global Th2 skewing. Culture of benign
T cells away from the malignant clone reduced Th2 and enhanced Th1 responses but
separate culture had no effect on malignant T cells. Co-culture of healthy T cells with L-
CTCL T cells reduced IFNγ production and neutralizing antibodies to IL-4 and IL-13
restored Th1 responses. In patients, enhanced Th1 responses were observed following
a variety of treatment modalities that reduced malignant T cell burden suggesting that
Th2 cytokines produced by malignant T cells play a critical role in down-regulating Th1
responses in vivo. Results suggest that neutralization of Th2 cytokines may be beneficial
in enhancing immune responses both to pathogens and to the malignancy itself.
Purpose: In leukemic CTCL (L-CTCL) malignant T cells accumulate in the blood and
give rise to widespread skin inflammation. Patients have intense pruritus, increased IgE,
decreased Th1 responses and most die from infection. Depleting malignant T cells while
preserving normal immunity is a clinical challenge. L-CTCL has been variably described
as a malignancy of regulatory, Th2 and Th17 cells.
Experimental design: We analyzed phenotype and cytokine production in malignant
and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T
cells and studied the immunomodulatory effects of treatment modalities in L-CTCL
Results: 12/12 L-CTCL patients overproduced Th2 cytokines. Remaining benign T cells
were also strongly Th2 biased, suggesting a global Th2 skewing of the T cell repertoire.
Culture of benign T cells away from the malignant clone reduced Th2 and enhanced Th1
responses but separate culture had no effect on malignant T cells. Co-culture of healthy
T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to IL-4
and IL-13 restored Th1 responses. In patients, enhanced Th1 responses were observed
following a variety of treatment modalities that reduced malignant T cell burden.
Conclusions: A global Th2 bias exists in both benign and malignant T cells in L-CTCL
and may underlie the infectious susceptibility of patients. Th2 cytokines from malignant
cells strongly inhibited Th1 responses. Our results suggest therapies that inhibit Th2
cytokine activity, by virtue of their ability to improve Th1 responses, may have the
potential to enhance both anti-cancer and anti-pathogen responses.
Cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of non-Hodgkin’s
lymphomas arising from malignant transformation of T cells that home to and populate
the skin (1, 2). In leukemic variants of CTCL (L-CTCL), malignant T cells can accumulate
in the blood and lymph nodes and also produce widespread inflammatory skin lesions.
L-CTCL is often refractory to multiple therapies and patients often ultimately require
hematopoietic stem cell transplantation (3). The median survival for patients with L-
CTCL varies with the extent of malignant T cell burden but is generally 2-5years and
patients die most commonly from infection (4-7).
CTCL has been proposed to be a malignancy of three separate T cell populations:
FOXP3+ Treg, Th2 T cells and Th17 T cells (8-10). Clinically, patients with L-CTCL have
abnormalities suggestive of a Th2 driven immunologic process, including decreased
antigen specific T cell responses, impaired cell mediated cytotoxicity, peripheral
eosinophilia, and elevated levels of serum IgE and IgA (11-13). Prior studies have
shown increased levels of Th2 cytokines and Th2 associated genes in T cells from
patients with L-CTCL (9, 14-17) but a recent report claims that malignant T cells in the
disease are actually Th17 biased (10).
We performed comprehensive analyses of the cytokine production of benign and
malignant T cells from L-CTCL with identifiable malignant T cell clones. We report here
that both malignant and benign T cells in L-CTCL patients were strongly Th2 biased, that
this bias was intrinsic in malignant T cells but extrinsic in benign cells and that inhibition
of Th2 cytokines led to recovery of Th1 responses in benign T cells.
Materials and Methods
Comment [r1]: References 6 and 7 are new
Blood samples. The protocols of this study were performed in accordance with the
Declaration of Helsinki and were approved by the Institutional Review Board of the
Partners Human Research Committee (Partners Research Management, Boston,
MA, USA). Blood from healthy individuals was obtained as discarded tissue following
leukopheresis. Blood and lesional skin from patients with CTCL were obtained from
patients seen at the Dana-Farber/Brigham and Women’s Cancer Center Cutaneous
Lymphoma Program. L-CTCL patients described in this manuscript met the WHO-
EORTC criteria for L-CTCL/SS (18). Patient characteristics are included in Table I;
based on the malignant T cell burden in this cohort of patients, expected survival is
approximately 5 years (4-7). PBMC were isolated by ficoll centrifugation and clonal and
non-clonal CD4+ T cells from CTCL patients were isolated using magnetic bead
separation (Miltenyi Biotec, Auburn, CA) after staining with TCR Vβ-specific antibodies
(Beckman Coulter Inc., Fullerton, CA).
Flow cytometry. Analysis of T cells was performed using directly conjugated
monoclonal antibodies obtained from BD Biosciences (San Jose, CA), eBioscience (San
Diego, CA), Biolegend (San Diego, CA) or R&D Systems (Minneapolis, MN). Vβ staining
was performed using the IOtest Beta Mark TCR V beta Repertoire kit (Beckman Coulter)
as per manufacturer’s instructions. Isotype-matched negative control antibodies were
used to set the gates for positive staining. For analysis of cytokine production, T cells
were stimulated with either control medium or 50 ng/ml PMA (Sigma Aldrich, Allentown,
PA) and 750 ng/ml ionomycin (Life Technologies, Grand Island, NY) plus 10 μg/ml
Brefeldin A (BD) for four hours. Cells were surface stained, fixed, permeabilized, stained
with anti-cytokine antibodies, and examined by flow cytometry. Analysis was performed