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Janus Kinase Inhibitor Tofacitinib Shows Potent Efficacy in a Mouse Model of Autoimmune Lymphoproliferative Syndrome (ALPS)

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Purpose: Autoimmune lymphoproliferative syndrome (ALPS) is a non-malignant genetic disorder of lymphocyte homeostasis with defective Fas-mediated apoptosis. Current therapies for ALPS primarily target autoimmune manifestations with non-specific immune suppressants with variable success thus highlighting the need for better therapeutics for this disorder. Methods: The spectrum of clinical manifestations of ALPS is mirrored by MRL/lpr mice that carry a loss of function mutation in the Fas gene and have proven to be a valuable model in predicting the efficacy of several therapeutics that are front-line modalities for the treatment of ALPS. We evaluated the potential efficacy of tofacitinib, an orally active, pan-JAK inhibitor currently approved for rheumatoid arthritis as a single agent modality against ALPS using MRL/lpr mice. Results: We demonstrate that a 42-day course of tofacitinib therapy leads to a lasting reversal of lymphadenopathy and autoimmune manifestations in the treated MRL/lpr mice, Specifically, in treated mice the peripheral blood white blood cell counts were reversed to near normal levels with almost a 50 % reduction in the TCRαβ(+)CD4(-)CD8(-)T lymphocyte numbers that coincided with a parallel increase in CD8(+) T cells without a demonstrable effect on CD4(+) lymphocytes including FoxP3(+) regulatory T cells. The elevated plasma IgG and IgA levels were also drastically lowered along with a significant reduction in plasmablasts and plasmacytes in the spleen. Conclusion: On the basis of these results, it is likely that tofacitinib would prove to be a potent single agent therapeutic modality capable of ameliorating both offending lymphadenopathy as well as autoimmunity in ALPS patients.
Panel aTofacitinib treatment dramatically reduces secondary lymphoid organ enlargement in MRL/lpr mice. Ten-week old mice were either sham treated with formulation vehicle or tofacitinib for a period of 42 days. Mice were sacrificed when they reached 7 months of age and their secondary lymphoid organs harvested for evaluation. Organs are representative of five such animals. Panel b Assessment of the impact of tofacitinib treatment on serum immunoglobulin subtypes in MRL/lpr mice. Serum samples were collected just prior to initiating treatment when mice were 10 weeks old and the second serum samples were collected just prior to euthanasia for organ harvest at 7 months of age (n = 5). Serum immunoglobulin subtypes were determined by sub-type specific ELISA. For comparison, blood samples from age matched Balb/c were also included in the assay. Y-axis denotes OD readings at 450 nm. Panel c tofacitinib treatment results in reduction of CD3⁻B220brightCD138⁺plasmablasts and CD3⁻B220dimCD138⁺ differentiated plasma cells in the spleen. Twenty five-week old MRL/lpr mice were treated as described in the legend to Fig. 1 and control mice were identically sham-treated with the vehicle formulation. Lymphocytes were purified from harvested spleens 10 weeks post treatment. Ficoll gradient isolated lymphocytes were further subjected to a round of negative selection using anti-CD43 microbeads to eliminate contaminating T lymphocytes. Flow cytometric analysis was performed using a BD FACSCanto II workstation with Flo Jo data analysis software. Data shown is representative of five similarly treated and five untreated mice
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ORIGINAL ARTICLE
Janus Kinase Inhibitor Tofacitinib Shows Potent Efficacy
in a Mouse Model of Autoimmune Lymphoproliferative
Syndrome (ALPS)
Seiji Yokoyama
1
&Pin-Yu Perera
2
&Seigo Terawaki
1
&Nobumasa Watanabe
1
&
Osamu Kaminuma
1
&Thomas A. Waldmann
3
&Takachika Hiroi
1
&Liyanage P. Perera
3
Received: 4 August 2015 /Accepted: 24 September 2015 /Published online: 9 October 2015
#Springer Science+Business Media New York (outside the USA) 2015
Abstract
Purpose Autoimmune lymphoproliferative syndrome
(ALPS) is a non-malignant genetic disorder of lymphocyte
homeostasis with defective Fas-mediated apoptosis. Current
therapies for ALPS primarily target autoimmune manifesta-
tions with non-specific immune suppressants with variable
success thus highlighting the need for better therapeutics for
this disorder.
Methods The spectrum of clinical manifestations of ALPS is
mirrored by MRL/lpr mice that carry a loss of function muta-
tion in the Fas gene and have proven to be a valuable model in
predicting the efficacy of several therapeutics that are front-
line modalities for the treatment of ALPS. We evaluated the
potential efficacy of tofacitinib, an orally active, pan-JAK in-
hibitor currently approved for rheumatoid arthritis as a single
agent modality against ALPS using MRL/lpr mice.
Results We demonstrate that a 42-day course of tofacitinib
therapy leads to a lasting reversal of lymphadenopathy and
autoimmune manifestations in the treated MRL/lpr mice,
Specifically, in treated mice the peripheral blood white blood
cell counts were reversed to near normal levels with almost a
50 % reduction in the TCRαβ
+
CD4
CD8
T lymphocyte
numbers that coincided with a parallel increase in CD8
+
T
cells without a demonstrable effect on CD4
+
lymphocytes
including FoxP3
+
regulatory T cells. The elevated plasma
IgG and IgA levels were also drastically lowered along with
a significant reduction in plasmablasts and plasmacytes in the
spleen.
Conclusion On the basis of these results, it is likely that
tofacitinib would prove to be a potent single agent therapeutic
modality capable of ameliorating both offending lymphade-
nopathy as well as autoimmunity in ALPS patients.
Keywords Autoimmunity .lymphoproliferation .ALPS .
tofacitinib .SLE
Abbreviations
ALPS Autoimmune lymphoproliferative syndrome
DN Double negative
SLE Systemic lupus erythematosus
Introduction
Autoimmune lymphoproliferative syndrome (ALPS) is a non-
malignant disorder of defective lymphocyte homeostasis and
is characterized by massive accumulation of lymphocytes
resulting in splenomegaly, and lymphoadenopathy [1,2].
The majority of ALPS patients harbor heterozygous germline
mutations in the death-receptor encoding TNF receptor super-
family, member 6 gene (TNFRSF6 also known as Fas or
CD95) that are inherited in an autosomal dominant manner
[3,4]. The presence of a large number of TCRαβ
+
CD4
CD8
Electronic supplementary material The online version of this article
(doi:10.1007/s10875-015-0203-z) contains supplementary material,
which is available to authorized users.
*Takachika Hiroi
hiroi-tk@igakuken.or.jp
*Liyanage P. Perera
pereral@mail.nih.gov
1
Department of Genome Medicine, The Tokyo Metropolitan
Institute of Medical Science, 2-1-6, kamikitazawa,
Setagaya-ku, Tokyo 156-8506, Japan
2
Veterans Affairs Medical Center, Washington, DC 20422, USA
3
Lymphoid Malignancies Branch, National Cancer Institute,
Bldg 10 4N114, Bethesda, MD 20892-1374, USA
J Clin Immunol (2015) 35:661667
DOI 10.1007/s10875-015-0203-z
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Supplementary resource (1)

... Tofacitinib is a non-selective JAK inhibitor, and is approved by the Food and Drug Administration (FDA) for RA and psoriasis [6,13]. Previous studies showed remarkable therapeutic efficacies of tofacitinib on mice models of certain autoimmune diseases such as SKG mice (an animal model of RA), interstitial lung disease in SKG mice, autoimmune lymphoproliferative syndrome (ALPS), and murine lupus [13][14][15][16][17]. ...
... Tofacitinib is the first generation jakinib that blocks multiple JAKs targeting a wide range of cytokines and has widespread immunosuppressive activity for T helper cells, antibody-producing B cells, antigen-presenting macrophages, DCs, and other mononuclear cells [14][15][16][17]. Tofacitinib is also an FDA-approved drug for RA and psoriasis; however, little is known about its effect on scleroderma [6,13]. ...
... Tofacitinib ameliorated arthritis in SKG mice by the inhibition of CD4 + T cell proliferation in vivo and in vitro [15]. In contrast, CD4 + T cells were not affected by tofacitinib treatment in murine lupus and ALPS [14,17]. In addition, in a mouse model of ALPS, tofacitinib reduced the proportion of TCR αβ + CD4 À CD8 À doublenegative T cells with a concomitant robust increase in CD8 + T cells [17]. ...
Article
Background Janus kinase (JAK)-signal transducer and activator of transcription (STAT) was hyperactivated in biopsies from patients with systemic sclerosis (SSc) and in several autoimmune disease models. Tofacitinib, a pan-JAK inhibitor, blocks the downstream signaling of multiple cytokines and has exhibited therapeutic efficacy in various autoimmune diseases, although its immunomodulating property in scleroderma is unclear. Objective To evaluate the effect of tofacitinib on the modulation of cytokine-producing T and B cells, and proinflammatory cells in a mouse model of SSc. Methods Bleomycin (BLM) -induced SSc was induced by intradermal injection of BLM or PBS for control. Mice received intraperitoneal tofacitinib (20 mg/kg) or vehicle 3 times per week from day 0 to 28. Mice were sacrificed at day 28 after the last BLM/PBS injection. Results Tofacitinib administration significantly alleviated fibrosis of the skin and lungs in scleroderma mouse model. Furthermore, tofacitinib suppressed adaptive and innate immune responses by reducing splenocytes, total lymphocytes, CD4⁺ T helper cells (especially Th2 and Th17 subtypes), IL-6-producing effector B cells, PDCA-1⁺ dendritic cells in the spleen, and infiltration of F4/80⁺, CD206⁺ and CD163⁺ macrophages in the skin and lungs. Conversely, tofacitinib increased the proportions of splenic regulatory T and B cells. The mRNA expression of extracellular matrix proteins and fibrogenic cytokines was downregulated by tofacitinib in both the skin and lungs. Conclusion These observations suggest JAK inhibition as a therapeutic approach for the treatment of inflammatory and fibrotic diseases, and highlight the potential of tofacitinib as a promising candidate for treating patients with scleroderma.
... In some cases, insufficient effectiveness of antitumor remedies can be explained by specific vital functions in the transformed cellular elements and pronounced differences in the development of various tumor types [1][2][3][4][5]. The recent studies on molecular biology and genetics of tumor cells revealed novel and highly specific molecular targets for chemotherapy [2,7,[10][11][12][13]. These fundamental findings made it possible to develop a number of original targeted drugs to treat some tumors that are resistant to the action of routine cytostatics. ...
... Modern pharmacology hopes to solve a number of tasks in antitumor therapy by investigation of intracellular signaling in the transformed cells [2,13,15]. At present, it advanced and effectively employed the drugs targeted on individual signal molecules implemented in realization of vital functions of various tumor cells (cyclin-dependent kinases, protein kinases, etc.) including the suppressors of JAK family tyrosine kinases [2,7,[10][11][12]. ...
... For instance, while JAK3 is a specific kinase in some limited number of cells types of adult organism (certain cells of immune system, hematopoietic progenitors, etc.) [9], it also plays an important role in determining the proliferation and differentiation potential of the tumor cells of various origin. Taking into consideration the potentially wide spectrum of the side effects and complications of chemotherapy, the selective inhibition of JAK3 in treatment of malignant tumors seems to be safer than the use of pan-JAK inhibitors [10,13]. ...
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... In vitro experiments indicated that the effects of tofacitinib were mediated through suppression of IL-17 and IFN-γ production and proliferation of CD4 T cells, presumably Th1 and Th17. In an autoimmune lymphoproliferative (ALPS) mouse model, tofactinib treatment decreased expression of TCRαβ(+)CD4(-)CD8(-)T lymphocyte numbers while it increased CD8( +) T cells without a remarkable effect on CD4(+) lymphocytes including FoxP3(+) regulatory T cells 13 . ...
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Tofactinib is a JAK inhibitor approved for ulcerative colitis in humans. Despite of its’ proven effectiveness in humans, mechanistic data are scarce on the effectiveness of Tofactinib in experimental colitis in mice. We induced experimental colitis by transfer of CD4+CD25− isolated T cells into RAG2−/− (T and B cell deficient) mice and treated these mice with tofacitinib for 5–6 weeks either with a dosage of 10 or 40 mg/kg body weight immediately after CD4+ transfer or started treatment after first symptoms of disease for several weeks. While treatment with tofacitinib immediately after transfer resulted in an enhanced expansion of CD4+ T cells and did not prevent occurrence of colitis, treatment after start of symptoms of colitis ameliorated disease activity on a clinical basis and in histological analyses. Tofacitinib is effective in the treatment of murine experimental T cell transfer colitis, however does not prevent occurrence of disease.
... In vitro experiments indicated that the effects of tofacitinib were mediated through suppression of IL-17 and IFN-γ production and proliferation of CD4 T cells, presumably Th1 and Th17. In an autoimmune lymphoproliferative (ALPS) mouse model, tofactinib treatment decreased expression of TCRαβ(+)CD4(-)CD8(-)T lymphocyte numbers while it increased CD8(+) T cells without a remarkable effect on CD4(+) lymphocytes including FoxP3(+) regulatory T cells 13 . ...
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Introduction: Tofactinib is a JAK inhibitor approved for ulcerative colitis in humans. Despite of its’ proven effectiveness in humans, mechanistic data are scarce on the effectiveness of Tofactinib in experimental colitis in mice. Methods: We induced experimental colitis by transfer of CD4 + CD25- isolated T cells into lymphopenic RAG2-/- mice and treated these mice with tofacitinib for 5–6 weeks either with a dosage of 10 or 40 mg/kg body weight immediately after CD4 + transfer or started treatment after first symptoms of disease for several weeks. Results: While treatment with tofacitinib immediately after transfer resulted in an enhanced expansion of CD4 + T cells and did not prevent occurrence of colitis, treatment after start of symptoms of colitis ameliorated disease activity on a clinical basis and in histological analyses. Discussion: Tofacitinib is effective in the treatment of murine experimental T cell transfer colitis, however does not prevent occurrence of disease.
... Clinically, also effects on Tregs (i.e., frequency) were investigated and a potential association between Tregs and the clinical response was explored. However, the amount of data presented in published studies (i.e., on local and systemic Treg frequency and functionality in mice and men, but also in vitro) was much more extensive than present in the dossier reports, although for one of the JAK inhibitors only one literature study was available (151)(152)(153)(154)(155)(156)(157)(158). ...
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Regulatory T cells (Tregs) have a prominent role in the control of immune homeostasis. Pharmacological impact on their activity or balance with effector T cells could contribute to (impaired) clinical responses or adverse events. Monitoring treatment-related effects on T cell subsets may therefore be part of (pre-)clinical studies for medicinal products. However, the extent of immune monitoring performed in studies for marketing authorisation and the degree of correspondence with data available in the public domain is not known. We evaluated the presence of T cell immunomonitoring in 46 registration dossiers of monoclonal antibodies indicated for immune-related disorders and published scientific papers. We found that the depth of Treg analysis in registration dossiers was rather small. Nevertheless, data on treatment-related Treg effects are available in public academia-driven studies (post-registration) and suggest that Tregs may act as a biomarker for clinical responses. However, public data are fragmented and obtained with heterogeneity of experimental approaches from a diversity of species and tissues. To reveal the potential added value of T cell (and particular Treg) evaluation in (pre-)clinical studies, more cell-specific data should be acquired, at least for medicinal products with an immunomodulatory mechanism. Therefore, extensive analysis of T cell subset contribution to clinical responses and the relevance of treatment-induced changes in their levels is needed. Preferably, industry and academia should work together to obtain these data in a standardised manner and to enrich our knowledge about T cell activity in disease pathogenesis and therapies. This will ultimately elucidate the necessity of T cell subset monitoring in the therapeutic benefit-risk assessment.
... Treatment was initiated after two weeks of tumor growth and continued for four weeks at ~2/3 of the maximal tolerated dose of tofacitinib (21.5 mg/kg/day by subcutaneous infusion). 38 Encouragingly, we found significantly increased murine survival in this cell line model ( Figure Repurposing tofacitinib as anti-myeloma therapy haematologica | 2018; 103(7) Figure 6. Tofacitinib shows synergy with venetoclax only in the coculture setting. ...
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... In this study, we demonstrated significant decreases in anti-dsDNA antibodies, proteinuria, and splenomegaly in TOFA alone and TOFA + DEXA-administered SLEmouse group (Fig. 1) and amelioration of glomerular nephritis in TOFA + DEXA-treated SLE-prone mouse, BWF1 (Fig. 2). Previous report revealed that TOFA is immunologically effective for another SLE-prone mouse, MRL [20] with different genetic background from BWF1. TOFA + DEXA treatment was also clinically effective for MRL (Additional file 1: Figure S1). ...
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... ALPS is general caused by the defects in Fas signalling and is manifested by chronic non-malignant lymphoproliferation and autoimmunity with massive accumulation of lymphocytes, resulting in splenomegaly and lymphadenopathy [13,19,20]. The hallmark feature of the disease is an expanded homogenous population of T cell that is TCRab+CD4ÀCD8À double-negative T (DNT) cell in peripheral blood and lymphoid organs [21,22]. Previous research has shown that the genetical manipulated TCF1 gene and generated mutant mice have shown that TCF1 has an essential role during preT cell receptor (TCR) and TCR-dependent stages of T cell development, and regulates various aspects of T cell development and CD4 and CD8 T cell function, such as the differentiation of mature CD4 T cells into T helper lineages [5]. ...
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... Mutations of JAK or STAT in humans are associated with severe immune/autoimmune dysfunction, revealing the fundamental role of this pathway in the induction and regulation of immune responses [129][130][131]. Notably, a series of JAK-STAT signaling cytokines, especially type I IFNs, TNF-α, IL-10, and IL-6 as well as the hormone prolactin have been implicated in the pathogenesis of SLE [132][133][134]. As previously mentioned, the biological actions of TNF-α are mediated by two cell surface receptors, TNFR-1 and TNFR-2. ...
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Fas (also known as Apo1 and CD95) is a cell surface receptor involved in apoptotic cell death. Fas expression and function were analyzed in three children (including two siblings) with a lymphoproliferative syndrome, two of whom also had autoimmune disorders. A large deletion in the gene encoding Fas and no detectable cell surface expression characterized the most affected patient. Clinical manifestations in the two related patients were less severe: Fas-mediated apoptosis was impaired and a deletion within the intracytoplasmic domain was detected. These findings illustrate the crucial regulatory role of Fas and may provide a molecular basis for some autoimmune diseases in humans.
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We describe a family with 12 members carrying a heterozygous germline FAS c.3G>T start codon mutation leading to FAS haploinsufficiency. One patient had autoimmune lymphoproliferative syndrome (ALPS), one had recovered from ALPS, and ten mutation-positive relatives (MPRs) were healthy. FAS-mediated apoptosis and surface expression of FAS in single-positive T cells were lower for MPRs but did not discriminate between them and the ALPS patient. However, double-negative (DN) T cells of the ALPS patient had no FAS expression due to somatic loss of heterozygosity. Our results in this kindred suggest that FAS haploinsufficiency does not cause ALPS-FAS, but that modifying genetic events are crucial for its pathogenesis. FAS surface expression on DN T cells should be assessed routinely and FAS haploinsufficient patients should be followed as its potential for lymphomagenesis is not well defined and a second hit might occur later on.
Article
The autoimmune lymphoproliferative syndrome (ALPS) is characterized by non-malignant lymphoproliferation and signs of autoimmunity. A hallmark of ALPS are high amounts of circulating CD3+/CD4 − /CD8 − double negative T-lymphocytes (DN T cells). The origin of these cells remains elusive. To investigate the relationships of DN T cells and the single positive T cell populations (CD4+ and CD8+), we analyzed by spectratyping the complementarity determining regions 3 (CDR3) of the T cell receptors in sorted “single positive” (CD4+, CD8+) and DN T cells in a patient with ALPS type 1a. We observed signs for clonal expansion in all three T cell subpopulations. Strong and weak clonal expansions were to be seen in 16 and 14 for DN, 6 and 12 for CD8+, and 1 and 5 for CD4 + T cells, respectively. Most importantly, 24 out of 30 aberrant peaks in the spectratype histograms of the DN T cells where unique for this population and were not to be detected in the histograms of the single positive T cells. In contrast to published data, we conclude that expanded DN T cell populations in ALPS are not generally derived from expanded CD3+/CD4+ or CD3+/CD8+ populations.