TRANSPLANTATION
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Brief report
`Anti–IL6-receptor-alpha (tocilizumab) does not inhibit human monocyte-derived
`dendritic cell maturation or alloreactive T-cell responses
`Brian C. Betts,1-3 Erin T. St Angelo,1 Michael Kennedy,1 and James W. Young1-3
`
`1Laboratory of Cellular Immunobiology, Immunology Program, Sloan-Kettering Institute for Cancer Research, New York, NY; 2Adult Bone Marrow
`Transplantation Service, Division of Hematologic Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY; and
`3Weill Cornell Medical College, New York, NY
`
`Significant comorbidites and lethality com-
`plicate GVHD and its treatment. Targeting
`the cytokine milieu may improve GVHD con-
`trol; and IL6 is an attractive candidate, given
`its role in dendritic cell activation and T-cell
`differentiation. Tocilizumab is a humanized
`mAb to IL6-receptor-␣ (IL6R-␣), which is
`Food and DrugAdministration–approved for
`treatment of rheumatoid arthritis. Mouse
`Introduction
`
`transplant models have demonstrated that
`IL6 blockade also improves GVHD scores
`and survival. Definitive immunologic effects
`of IL6 inhibition have not emerged given
`inconsistent alterations in regulatory T cells
`(Tregs) and suppression of T-cell prolifera-
`tion. Despite on-target suppression of IL6R-␣
`signaling in human monocyte-derived den-
`dritic cells (moDCs) and T cells, our data
`
`show no effect on moDC maturation/
`activation, alloreactive T-cell proliferation,
`Treg expansion, or allogeneic Th1/Th17 re-
`sponses in vitro. These findings merit atten-
`tion in any clinical trials of tocilizumab for
`GVHD prevention or treatment and provide
`a rationale for evaluating more specific in-
`hibitors of downstream JAK2/STAT3 signal-
`ing as well. (Blood. 2011;118(19):5340-5343)
`
`Tocilizumab is a humanized mAb to IL6R-␣,
`inhibiting the
`JAK2/STAT3 signaling pathway.1 It is Food and Drug Administra-
`tion–approved for treatment of rheumatoid arthritis, with potential
`efficacy in other autoimmune diseases.2-4 Tocilizumab can cause
`significant adverse effects, including cytopenias, infections, and
`gastrointestinal perforation.2-4
`IL6 is a proinflammatory cytokine secreted by mature DCs and
`lymphocytes.5 IL6 is a constituent of monocyte-conditioned me-
`dium, and it enhances DC maturation and stimulatory potency.6
`Indeed, combinations of inflammatory cytokines that mature DCs
`include rhu-IL6.6 IL6 enhances the generation of CD8⫹ cytolytic T
`cells, supports the development of Th17 lymphocytes that are
`active in autoimmunity, and impairs Treg differentiation.7-13 IL6
`neutralization eliminates this suppressive influence over Tregs.12
`Two groups have investigated the efficacy of IL6 inhibition in
`treating GVHD in mice.14,15 Their data have shown that IL6
`inhibition results in decreased GVHD scores and improved sur-
`vival.14,15 The data are inconsistent, regarding Treg expansion or
`direct effects on alloreactive T-cell proliferation.14,15 Given the
`continued interest in IL6 inhibition in the management of GVHD
`and the paucity of human data, we investigated the immune
`mechanisms underlying tocilizumab’s effects on human DC-
`stimulated alloreactivity in vitro.
`
`Methods
`
`(Memorial Sloan-Kettering Cancer Center [MSKCC] Donor Room and
`Blood Bank; NY Blood Center, American Red Cross), in agreement with
`the Declaration of Helsinki and existing tissue procurement protocols
`approved by the Institutional Review and Privacy Board of Memorial
`Hospital, MSKCC. T cells and moDCs were obtained as published,16 with
`the exception of moDC maturation by exposure to LPS (10ng/mL;
`Sigma-Aldrich) whenever necessary to avoid IL6. Complete RPMI and
`IMDM (MSKCC Media Prep Core Facility) with heat-inactivated, pooled,
`human serum (PHS; Gemini Bioproducts) were supplemented as pub-
`lished.16 Tocilizumab (Actemra; Genen-tech) was purchased from MSKCC
`Pharmacy and used at 5 ug/mL final. Human immunoglobulin (Grifols)
`served as a negative control at 5ug/mL final.
`
`Fluorochrome-conjugated anti–human mAbs and flow
`cytometry
`
`MoDCs: FITC-, PE-, Alexa Fluor647 (AF647)–, APC-, and PE–cyanine-7
`(PE-Cy7)–conjugated mouse anti–human mAbs included anti-CD83, anti-
`CD86, anti–HLA-DR, and anti-pSTAT3 (pY705; BD Biosciences); and
`FITC-conjugated anti-CCR7 (R&D Systems).
`T cells: FITC-, PE-, AF647-, APC-, and PE-Cy7–conjugated mouse
`anti–human mAbs included anti-CD3, anti-CD8, anti-CD25, anti-pSTAT3
`(pY705), and anti–IFN-␥ (BD Biosciences); FITC-, AF647-, and APC-
`conjugated anti-CD3, anti-CD127, anti–human Foxp3, and anti-IL17a
`(eBioscience); and PE-Texas Red–conjugated anti-CD4 (Invitrogen).
`Corresponding fluorochrome-conjugated mouse immunoglobulins were
`used as isotype controls. Live events were acquired with a FC 500
`(Beckman Coulter) flow cytometer and analyzed using FlowJo Version
`8.8.7 software (TreeStar).
`
`Cells, media, and reagents
`
`STAT3 phosphorylation
`
`PBMCs were isolated over Ficoll-Paque Plus (GE Healthcare Biosciences)
`from leukocyte concentrates from healthy, consenting, volunteer donors
`
`Resting T cells or immature moDCs were starved in complete RPMI, with
`either tocilizumab or control Ig at 37°C for 3 hours. The cells were pulsed or
`
`Submitted June 27, 2011; accepted September 19, 2011. Prepublished online
`as Blood First Edition paper, September 22, 2011; DOI 10.1182/blood-2011-06-
`363390.
`
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
`
`The publication costs of this article were defrayed in part by page charge
`
`© 2011 by The American Society of Hematology
`
`5340
`
`BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
`
`

`

`BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
`
`ANTI–IL6R-␣ DOES NOT INHIBIT HUMAN DCs OR T CELLS
`
`5341
`
`Figure 1. IL6R-␣ neutralization with tocilizumab does
`not impair the phenotypic maturation of moDCs or
`alloreactive T-cell proliferation. (A-D) DCs were ma-
`tured for 48 hours with LPS, in the presence of tocili-
`zumab or control Ig, both at 5ug/mL final. Representative
`histograms depict selected markers of DC maturation.
`Bar graphs show the averaged MFI (mean fluorescent
`from 4 independent experiments, ⫾ SD;
`intensity)
`NS ⫽ not significant, paired t test. (E) Bar graphs show
`means from 4 independent experiments, ⫾ SD, evaluat-
`ing inhibition of IL6-induced pSTAT3 by tocilizumab in
`immature moDCs (HLA-DR⫹, top) and resting T cells
`(CD3⫹, bottom); *P ⬍ .05, paired t
`test. (F) A fixed
`number of T cells was cultured with variable doses of
`allogeneic DCs at DC:T ratios from 1:30 to 1:1000.
`Tocilizumab or control Ig (5ug/mL) was added once on
`d0. AlloMLRs were cultured for 5 days at 37°C. T-cell
`proliferation was assessed using a colorimetric assay.
`Graph shows the average of
`triplicate means from
`4 independent experiments, ⫾ SEM; NS ⫽ not signifi-
`cant, paired t test of area under the curve (AUC).
`
`ii: 4 511LNs
`:E 3
`r--
`~ 2
`0 1
`0
`
`E +
`M
`I- 15
`~
`<g_ 10
`♦-
`0::
`C 5
`ci:
`...I
`:I: 0
`
`~ .
`
`~ 10
`I-
`ct:
`8
`I-
`1/) 6
`Q.
`4
`♦-
`M
`C
`2
`0
`~
`
`ii: 8 10~ s
`:E 6
`M
`~ 4
`0 2
`
`0
`
`HLA-DR
`
`F
`E 2.0
`i 1.5
`
`C:
`
`0
`0
`QI g 1.0
`(I! .c ...
`0
`1: 0.5
`ct:
`~
`
`... Control
`o Tocilizumab
`-e PHA
`
`~'-..l
`~~~~-"-}s ..
`
`.......... .
`
`not with rhu-IL6 (105 IU/mL; CellGenix) for 10 minutes. The cells were
`then fixed (Cytofix; BD Biosciences); permeabilized (cold methanol, 90%
`vol/vol); and stained with anti-CD3 (T cells) or anti–HLA-DR (moDCs),
`together with anti-pSTAT3.
`
`Allogeneic mixed leukocyte reactions (alloMLR)
`
`AlloMLRs comprised 105 T cells stimulated by moDCs at DC:T ratios of
`1:30 to 1:1000. Tocilizumab or control Ig was added once on d0 of the
`5-6 days alloMLR. T-cell proliferation was determined by a colorimetric
`assay (Promega).
`
`Tregs and Th1/Th17 staining
`
`Cytokine matured moDCs were cultured with allogeneic T cells at a DC:T
`ratio of 1:30, to which tocilizumab or control Ig was added on d0. After
`5 days, Tregs were identified by gating on the live CD3⫹, CD4⫹, CD25bright
`cells, then assessing for Foxp3 expression and absence of CD127.17,18
`To assess Th1 and Th17 responses, CD4⫹ T cells were negatively
`selected from PBMCs (EasySep; Stemcell Technologies) and stimulated by
`cytokine-matured moDCs at a DC:T ratio of 1:30 with tocilizumab or
`control Ig. CD4⫹ T cells were harvested after 6 days and stimulated with
`PMA/ionomycin in IMDM-10% PHS for 6 hours. Monensin (eBioscience)
`stimulation. CD4⫹ T cells
`was added after
`the first hour of
`were surface-stained for CD3, CD4, and CD25, followed by fixation
`and permeabilization (BD Biosciences) for intracellular staining of IFN-␥
`and IL17.
`
`Statistical analysis
`
`Statistical comparisons used the paired, 2-tailed, Student t test (GraphPad/
`Prism Version 5 software). Statistical significance required P value ⬍ .05.
`
`Results and discussion
`
`IL6R-␣ blockade does not impair moDC maturation or suppress
`alloreactive T-cell proliferation
`
`The addition of tocilizumab to LPS-matured moDCs did not
`diminish expression of CCR7,19 CD83,20 CD86,21 or HLA-DR21
`(Figure 1A-D). We verified that tocilizumab (5ug/mL) blocked IL6
`signaling by confirming the absence of IL6 induced pSTAT3 in
`tocilizumab-treated moDCs and T cells (Figure 1E).1 While IL6
`enhances moDC potency, the addition of tocilizumab at the outset
`of the alloMLR had no effect on T-cell proliferation at any DC:T
`ratio (Figure 1F). This finding is similar to mouse data, where
`blocking the IL6R could not inhibit donor T-cell proliferation in
`alloMLRs.15
`
`IL6R-␣ neutralization does not alter the Treg/Th17 axis
`
`While IL6 antagonizes Treg development, data from mouse GVHD
`models regarding donor Treg expansion are inconsistent.14,15 We
`
`

`

`5342
`
`BETTS et al
`
`BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
`
`A
`
`3.83 6.1
`
`Tocilizumab
`2.97
`
`...
`0
`ti
`C1I
`in
`+LI) 0.20
`N
`C 0.15
`()
`+00 0.10
`C
`~ 0.05
`
`NS
`
`C
`
`NS
`
`15
`
`10
`
`Cl
`.,
`E
`C:
`Cl
`.......
`'i:
`.o N
`LI)
`....
`NC
`C U
`() +
`+
`C')
`~ C.
`C X
`
`5
`() &
`
`D
`
`20.3
`
`·
`
`Tocilizumab
`0.53 21.7 .·
`·
`0.51
`
`F
`
`NS
`
`E +
`Ill
`E 30
`E
`Ill
`"t' 20
`z
`!:!::
`+- 10
`~
`C
`()
`~
`0
`
`0
`
`21- ~
`~ ~
`o(:' v
`c;
`,,...
`.~"v
`~o
`
`0.95
`
`NS
`
`+ ..... 2.5
`.... 2.0
`I :::!
`1.5
`+-
`~ 1.0
`C
`() 0.5
`~ 0
`0.0
`
`4-6' ~
`o~ v~
`c;
`-~~
`~o"
`
`Figure 2. IL6R-␣ neutralization with tocilizumab does not alter the Treg/Th17
`axis. (A) Representative contour plots of allogeneic Treg populations. Tregs were
`identified by gating on the moDC-stimulated allogeneic CD3⫹, CD4⫹, CD25bright
`T cells, and then assessing for expression of Foxp3 and lack of CD127. (B) Bar
`graph depicts the means from 4 independent experiments (paired t test), ⫾ SD,
`for the percentages of Tregs in alloMLRs treated with tocilizumab or control Ig
`(5ug/mL). (C) Effect of IL6R ␣ inhibition on the ratio of CD4⫹ Tregs to CD8⫹CD25⫹
`effector T cells in alloMLRs, compared with control. Bar graph shows means from
`3 independent experiments, ⫾ SD; NS ⫽ not significant, paired t
`test.
`(D) Representative contour plots of moDC-stimulated allogeneic Th1 and Th17
`cells. Th1 and Th17 cells were identified by gating on the CD3⫹ CD4⫹ population
`and then evaluating for intracellular IFN-␥ and IL17, respectively. (E) Bar graph
`shows means from 3 independent experiments, ⫾ SD (paired t test, NS ⫽ not
`significant), representing the percentages of CD3⫹, CD4⫹, IFN-␥⫹ cells in the
`alloMLRs treated with tocilizumab (5 ug/mL) or control. (F) Bar graph shows
`means from 3 independent experiments, ⫾ SD (paired t test, NS ⫽ not signifi-
`cant) for the percentages of CD3⫹, CD4⫹, IL17⫹ cells in the alloMLRs treated with
`tocilizumab (5 ug/mL final) or control.
`
`identified Tregs in human alloMLRs by gating on CD3⫹, CD4⫹,
`CD25bright T cells, followed by confirmation of Foxp3 expression
`and absence of CD127 (Figure 2A).17,18 The addition of tocili-
`zumab neither increased the percentage of alloreactive human
`Tregs nor the ratio of CD4⫹ Tregs to effector CD8⫹ CD25⫹ T cells
`(Figure 2B-C).
`IL6 induces naive CD4⫹ T cells to differentiate into Th17 cells,
`suggesting that tocilizumab should impair Th17 expansion.9,13
`Mouse studies have shown that IL6 blockade does suppress Th1
`responses in GVHD.14 We identified moDC-stimulated Th1 and
`Th17 responder lymphocytes by gating on CD3⫹ CD4⫹ cells,
`followed by assessment of intracellular IFN-␥ or IL17, respectively
`(Figure 2D). The addition of tocilizumab did not diminish the
`Th1 or Th17 response in human moDC-stimulated alloMLRs
`(Figure 2E-F).
`Apart from the amelioration of mouse GVHD by blockade of
`IL6 signaling,14,15 there is limited information regarding details of
`the relevant allogeneic interactions. In fact, while the observed
`effects of IL6 inhibition have not been in complete accord, the
`benefit occurs primarily through protection of the host gastrointes-
`tinal epithelium.14,15 Because of the additional absence of published
`information regarding human allogeneic interactions after IL6/
`IL6R blockade, we have examined the immune mechanisms of
`tocilizumab on human moDC-stimulated alloreactivity in vitro. We
`have identified potential limitations of IL6 blockade as a strategy to
`control clinical GVHD. Tocilizumab failed to suppress moDC
`maturation, alloreactive T-cell proliferation, or alter the Treg/Th17
`axis in human alloMLRs. We have not yet evaluated the effects of
`tocilizumab on other conventional human DC subsets. Their
`similar potency to stimulate alloreactive T cells is well-
`established,22 so any differences with tocilizumab should be
`negligible.
`Clinical case reports and a small clinical trial have ascribed
`some benefit
`to tocilizumab therapy in steroid-refractory
`GVHD,23,24 although prior and ongoing treatment with other
`immunosuppressants can always have confounding contribu-
`tions to outcomes. While these reports have been intriguing, the
`findings presented herein merit careful attention in any clinical
`trials of tocilizumab for GVHD prevention or treatment. Even if
`tocilizumab were to prove effective in clinical GVHD,
`the
`inconsistent immune effects would be difficult to correlate with
`disease response and clinical outcome. Finally, as
`in
`most biologic systems, there may be sufficient redundancy for
`other unrecognized factors to signal
`through shared distal
`pathways, thus circumventing IL6R-␣ blockade by tocilizumab.
`Hence there is a strong rationale to evaluate more specific
`inhibitors of downstream JAK2/STAT3 signaling to control
`alloreactivity.25
`
`Acknowledgments
`
`This work was supported by a Mortimer J. Lacher Fellowship from
`the Lymphoma Foundation (B.C.B.), T32 CA009207 (B.C.B.);
`R01 CA83070 (J.W.Y.), P01 CA23766 (J.W.Y.) from the National
`Cancer Institute, National Institutes of Health; William H. Good-
`win and Alice Goodwin of the Commonwealth Foundation for
`Cancer Research through The Experimental Therapeutics Center of
`MSKCC (J.W.Y.); and Swim Across America (J.W.Y.).
`
`

`

`BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
`
`ANTI–IL6R-␣ DOES NOT INHIBIT HUMAN DCs OR T CELLS
`
`5343
`
`Authorship
`
`Contribution: B.C.B. designed the overall study, performed experi-
`ments, analyzed and interpreted data, and wrote the manuscript;
`E.T.S.A. and M.K. assisted with performance of experiments; and
`J.W.Y. designed the overall study, supervised performance of
`experiments, supervised analysis and interpretation of data, and
`cowrote and edited the manuscript.
`
`Conflict-of-interest disclosure: The authors declare no compet-
`ing financial interests.
`The current affiliation for B.C.B. is Moffitt Cancer Center,
`University of South Florida, Tampa, FL. The current affiliation
`for M.K.
`is School of Medicine, Wayne State University,
`Detroit, MI.
`Correspondence: James W. Young, MD, Memorial Sloan-
`Kettering Cancer Center, 1275 York Ave, New York, NY 10065;
`e-mail: youngjw@mskcc.org.
`
`References
`
`4.
`
`1. Atreya R, Mudter J, Finotto S, et al. Blockade of
`interleukin 6 trans signaling suppresses T-cell
`resistance against apoptosis in chronic intestinal
`inflammation: evidence in crohn disease and ex-
`perimental colitis in vivo. Nat Med. 2000;6(5):583-
`588.
`2. Genovese MC, McKay JD, Nasonov EL, et al.
`Interleukin-6 receptor inhibition with tocilizumab
`reduces disease activity in rheumatoid arthritis
`with inadequate response to disease-modifying
`antirheumatic drugs: the tocilizumab in combina-
`tion with traditional disease-modifying antirheu-
`matic drug therapy study. Arthritis Rheum. 2008;
`58(10):2968-2980.
`3. Jones G, Sebba A, Gu J, et al. Comparison of
`tocilizumab monotherapy versus methotrexate
`monotherapy in patients with moderate to severe
`rheumatoid arthritis: the AMBITION study. Ann
`Rheum Dis. 2010;69(1):88-96.
`Illei GG, Shirota Y, Yarboro CH, et al. Tocilizumab
`in systemic lupus erythematosus: data on safety,
`preliminary efficacy, and impact on circulating
`plasma cells from an open-label phase I dosage-
`escalation study. Arthritis Rheum. 2010;62(2):
`542-552.
`5. Blanco P, Palucka AK, Pascual V, Banchereau J.
`Dendritic cells and cytokines in human inflamma-
`tory and autoimmune diseases. Cytokine Growth
`Factor Rev. 2008;19(1):41-52.
`6. Jonuleit H, Kuhn U, Muller G, et al. Pro-inflammatory
`cytokines and prostaglandins induce maturation of
`potent immunostimulatory dendritic cells under fetal
`calf serum-free conditions. Eur J Immunol. 1997;
`27(12):3135-3142.
`7. Ming JE, Steinman RM, Granelli-Piperno A. IL-6
`enhances the generation of cytolytic T lympho-
`cytes in the allogeneic mixed leucocyte reaction.
`Clin Exp Immunol. 1992;89(1):148-153.
`8. Chen W, Jin W, Hardegen N, et al. Conversion of
`peripheral CD4⫹CD25- naive T cells to
`CD4⫹CD25⫹ regulatory T cells by TGF-beta in-
`
`duction of transcription factor Foxp3. J Exp Med.
`2003;198(12):1875-1886.
`9. Littman DR, Rudensky AY. Th17 and regulatory T
`cells in mediating and restraining inflammation.
`Cell. 2010;140(6):845-858.
`10. Marie JC, Letterio JJ, Gavin M, Rudensky AY.
`TGF-beta1 maintains suppressor function and
`Foxp3 expression in CD4⫹CD25⫹ regulatory T
`cells. J Exp Med. 2005;201(7):1061-1067.
`11. Zheng Y, Rudensky AY. Foxp3 in control of the
`regulatory T cell lineage. Nat Immunol. 2007;8(5):
`457-462.
`12. Pasare C, Medzhitov R. Toll pathway-dependent
`blockade of CD4⫹CD25⫹ T cell-mediated sup-
`pression by dendritic cells. Science. 2003;
`299(5609):1033-1036.
`13. Zhou L, Lopes JE, Chong MM, et al. TGF-beta-
`induced Foxp3 inhibits T(H)17 cell differentiation
`by antagonizing RORgammat function. Nature.
`2008;453(7192):236-240.
`14. Chen X, Das R, Komorowski R, et al. Blockade of
`interleukin-6 signaling augments regulatory T-cell
`reconstitution and attenuates the severity of graft-
`versus-host disease. Blood. 2009;114(4):891-
`900.
`15. Tawara I, Koyama M, Liu C, et al. Interleukin-6
`modulates graft-versus-host responses after ex-
`perimental allogeneic bone marrow transplanta-
`tion. Clin Cancer Res. 2011;17(1):77-88.
`16. Ratzinger G, Reagan JL, Heller G, Busam KJ,
`Young JW. Differential CD52 expression by dis-
`tinct myeloid dendritic cell subsets: implications
`for alemtuzumab activity at the level of antigen
`presentation in allogeneic graft-host interactions
`in transplantation. Blood. 2003;101(4):1422-
`1429.
`17. Seddiki N, Santner-Nanan B, Martinson J, et al.
`Expression of interleukin (IL)-2 and IL-7 receptors
`discriminates between human regulatory and ac-
`tivated T cells. J Exp Med. 2006;203(7):1693-
`1700.
`
`18. Liu W, Putnam AL, Xu-Yu Z, et al. CD127 expres-
`sion inversely correlates with FoxP3 and sup-
`pressive function of human CD4⫹ T reg cells.
`J Exp Med. 2006;203(7):1701-1711.
`19. Dieu MC, Vanbervliet B, Vicari A, et al. Selective
`recruitment of immature and mature dendritic
`cells by distinct chemokines expressed in differ-
`ent anatomic sites. J Exp Med. 1998;188(2):373-
`386.
`20. Zhou LJ, Tedder TF. Human blood dendritic cells
`selectively express CD83, a member of the im-
`munoglobulin superfamily. J Immunol. 1995;
`154(8):3821-3835.
`21. Rossi M, Young JW. Human dendritic cells: po-
`tent antigen-presenting cells at the crossroads of
`innate and adaptive immunity. J Immunol. 2005;
`175(3):1373-1381.
`22. Ratzinger G, Baggers J, de Cos MA, et al. Mature
`human Langerhans cells derived from CD34⫹
`hematopoietic progenitors stimulate greater cyto-
`lytic T lymphocyte activity in the absence of bio-
`active IL-12p70, by either single peptide presen-
`tation or cross-priming, than do dermal-interstitial
`or monocyte-derived dendritic cells. J Immunol.
`2004;173:2780-2791.
`23. Drobyski WR, Pasquini M, Kovatovic K, et al. To-
`cilizumab for the treatment of steroid refractory
`graft-versus-host disease. [published online
`ahead of print July 13, 2011]. Biol Blood Marrow
`Transplant. doi:10.1016/j.bbmt.2011.07.001.
`24. Gergis U, Arnason J, Yantiss R, et al. Effectiveness
`and safety of tocilizumab, an anti-interleukin-6 recep-
`tor monoclonal antibody, in a patient with refractory
`GI graft-versus-host disease. J Clin Oncol. 2010;28:
`e602-604.
`25. Betts BC, Abdel-Wahab O, Curran SA, St.
`Angelo ET, Koppikar P, Heller G, Levine R,
`Young JW. Janus kinase-2 inhibition induces du-
`rable tolerance to alloantigen by dendritic cell-
`stimulated human T cells, yet preserves immunity
`to recall antigens. Blood. 2011;118(19):5328-
`5337.
`
`