Resetting the with immunoablation and autologous haematopoietic stem cell transplantation in autoimmune diseases T. Alexander1,2, R. Arnold3, F. Hiepe1,2, A. Radbruch4

1Department of Rheumatology and ABSTRACT Current immunosuppressive and novel Clinical , Charité - University Over the past 20 years, immunoablation biologic treatments may suppress or at- Medicine Berlin; followed by transplantation of autolo- tenuate autoimmune responses as long 2German Rheumatism Research Centre gous haematopoietic stem cells (ASCT) as they are applied, but do not target the (DRFZ) Berlin, a Leibniz Institute, Autoimmunology Group, Berlin; has emerged as a promising treatment autoreactive immunologic memory and 3Department of Haematology, option for patients with severe forms of cannot switch off the underlying mech- Oncology and Tumorimmunology, autoimmune diseases (ADs) that insuf- anisms to induce therapy-free remis- Charité - University Medicine Berlin; ficiently respond to standard immuno- sion, i.e. cure. In contrast, the general 4German Rheumatism Research Centre suppressive or novel biologic treatment. concept of autologous haematopoietic (DRFZ) Berlin, a Leibniz Institute, Cell Meanwhile, mechanistic studies have stem cell transplantation (ASCT) is Biology Group, Berlin, Germany. provided the proof-of-concept that the that an extensive immune re-education Tobias Alexander, MD long-term, treatment-free remissions after eradicating the autoreactive im- Renate Arnold, MD achieved by ASCT are associated with munologic memory with high-dose Falk Hiepe, MD Andreas Radbruch, PhD the eradication of the autoreactive im- chemotherapy, using agents such as munologic memory and a fundamental Please address correspondence to: anti- globulin (ATG), would Tobias Alexander, MD, reconfiguration of the immune system. restore immunologic self-tolerance to Department of Rheumatology The latter comprises regeneration of induce sustained remissions (5, 6). and Clinical Immunology, naive B cells and a stable thymic reac- Based on experimental data from Charité - University Medicine Berlin, tivation with re-emergence of thymic- animal models (7, 8), immunoabla- Charitéplatz 1, derived naive T cells, including Foxp3+ tion followed by ASCT is performed 10117 Berlin, Germany. regulatory T cells, with new re- since 1996 and was shown to induce E-mail: [email protected] ceptors, i.e. immune reset. In this arti- long-term, treatment-free remissions Received and accepted on June 29, 2016. cle, we discuss mechanistic studies that for several ADs (9-12). To date, more Clin Exp Rheumatol 2016; 34 (Suppl. 98): investigated how such immune renewal than 2100 cases of ASCT for ADs S53-S57. after ASCT may rewire a faulty immune have been reported to the European © Copyright Clinical and system in ADs into a self-tolerant state, Group for Blood and Marrow Trans- Experimental Rheumatology 2016. to induce long-term remissions. plantation (EBMT) Working Party for Autoimmune Diseases (ADWP), and Key words: stem cell transplantation, Introduction controlled randomised trials recently autoimmune diseases, immune reset, Antigen-specific T cells and long-lived demonstrated superiority to standard immune reconstitution, autologous plasma cells (PCs) that persist long treatment in the major indications, such haematopoietic stem cells after pathogen clearance are responsi- as systemic sclerosis (SSc) (13) and transplantation (ASCT) ble for immunologic memory, provid- Crohn’s disease (14). ing protective to subsequent The present review focuses on the re- challenge to the original microbe. In constitution of various components of systemic autoimmune diseases, how- the immune system following immu- ever, such immunologic memory could noablation and ASCT and how such potentially contribute to the chronicity immune reconfiguration may rewire a of and/or flare induction chronic autoimmune system into a self- (1). In fact, we could recently demon- tolerant state, to mediate long-term re- strate that long-lived PCs, unlike short- missions in ADs. lived PCs, are refractory to cyclophos- phamide or depleting therapies Immunoablation largely depletes the (2, 3), and their adoptive transfer from (autoreactive) immunologic memory Funding: the research was supported by -prone mice (NZB/W) into im- Reduction of and the Deutsche Forschungsgemeinschaft, munodeficient RAG1-/- mice could in- vaccine-induced . project no. SFB 650. duce chronic lupus manifestations, in- Depletion of the autoreactive immu- Competing interests: none declared. cluding immune complex nephritis (4). nologic memory after ASCT is best

S-53 Resetting the immune system in autoimmune diseases / T. Alexander et al. reflected by the reduction of serum data from clinical trials indicated that row or secondary lymphoid organs autoantibodies in systemic autoim- lymphodepletion alone, as performed (23), which are hardly to access for im- mune diseases such as systemic lupus e.g. for (18), and munologic analyses, their fate after im- erythematosus (SLE) and SSc. In con- use of unmanipulated stem cell grafts munoablation may only be determined trast to B cell depletion therapies with after in vivo depleting conditioning indirectly. With this regard, lessons rituximab, where titres regimens for SLE were associated with from vaccination studies in paediatric mostly persist after treatment (15), im- a higher relapse incidence post-trans- patients with juvenile arthritis and SLE munoablation has been demonstrated plantation (10), indicating that a more revealed that the immunological mem- to significantly reduce or even normal- stringent immune depletion is associ- ory to a neoantigen is lost in most pa- ise autoantibody titres in such disease. ated with more favourable outcomes tients after immunoablative pre-treat- For example, single-centre phase II after ASCT. ment, yet memory to a recall antigen clinical trials for SLE from the North- The degree of immune depletion fol- boosted before bone marrow harvest is western University Chicago and our lowing immunoablation may also vary only lost in part of the patients (24). group demonstrated that serum anti- depending on the type of the underly- double-stranded (ds)DNA antibodies ing AD. According to previous data Immune reconstitution after completely normalised in responding (19) and our own observations, the immunoablation and ASCT patients whereas they were persistently reduction of antinuclear and vaccine- for present before ASCT under chronic induced titres is less signifi- Reconstitution of the innate immunosuppression, and serum ti- cant in SSc compared to SLE patients immune system tres for antinuclear antibodies (ANA) and a recent study demonstrated a per- Following engraftment, the first phase were significantly reduced or became sistence of pulmonary hypertension of immune reconstitution is character- negative (12, 16). Notably, depletion of (PH)-related specific antibody titres, ised by a significant though transient the immunologic memory was not re- such as anti-endothelin-receptor anti- increase in the proportion of CD56high stricted to the autoreactive memory. In bodies, in SSc patients after immune natural killer (NK) cell subsets in vari- addition, vaccine-induced antibody ti- depletion (20), suggesting a resistance ous autoimmune diseases (19, 22) with tres were largely extinguished in serum of bone marrow PCs to ATG for so far a similar kinetic observed in studies after immune reset (16). This drastic unknown reasons. investigating immune reconstitution in ablation of humoral memory suggests patients receiving ASCT for malignant that the ATG used for immunoablation Depletion of (autoreactive) memory diseases (25). Data on the recovery directly targets the PCs secreting such T cells following ASCT of , and in- antibodies. In line with this notion, In autoimmune diseases, pathogenic T nate lymphoid cell (ILC) subsets after previous in vitro experiments indicat- cell functions are mediated by autore- ASCT for ADs are scarce and merit ed that polyclonal rabbit ATG directly active memory effector T cells; their further investigations. Here, especially targets PCs via complement-mediated elimination is therefore presumed to ILC could be of particular relevance lysis and (17) and were able ameliorate autoimmune inflamma- in the immune reconstitution for ADs, to stain autoreactive PCs with ATG ex tion. In line with this notion, Sun and given their regulatory functions as in- vivo and demonstrate that PCs disap- colleagues (21) demonstrated largely dicated by their ability to prevent graft peared from the bone marrow after im- reduced T-cell responses to myelin ba- -versus-host disease (GvHD) after al- mune reset (16). sic protein (MBP) in the reconstituted logeneic stem cell transplantation for Different transplantation protocols are immune system in MS patients after haematologic malignancies (26). being employed in the treatment of ASCT compared to pre-treatment. Fur- ADs, varying from lymphodepletion thermore, recent data demonstrated that reconstitution alone, e.g. with cyclophosphamide, a CD161high pro-inflammatory CD8+ Recurring T cells after immunoablation followed by ASCT or with additional T cell mucosal-associated invariant T may theoretically derive from four dif- in vivo T cell depletion using ATG or cell (MAIT) subset, which was recent- ferent sources: (1) from expansion of monoclonal antibodies, such as Cam- ly connected to MS immunopathol- residual T cells that either survived the path, or ex vivo graft manipulation ogy, could be depleted from peripheral conditioning or (2) are reinfused along using CD34-selection to minimise the blood following immunoablation and with the stem cells, (3) from haemat- risk of re-infusing autoreactive lym- ASCT, while they were maintained at opoietic stem cells residing after the phocytes. Depending on the transplan- similar frequencies under treatment conditioning or (4) from transfused tation regimens used, immunologic with interferon β (22). CD34-selected stem cells through a memory may either reside, especially Even with the most intensive nonmy- process of thymic education. Although if no in vivo depletion is performed, or eloablative-conditioning regimen used dissecting the role of residual vs. trans- re-infused with the stem cell graft in for ASCT, a complete eradication of planted stem cells as source for T cell case of transplantation of unmanipulat- the existing immunologic repertoire regeneration is only feasible in allo- ed stem cell grafts with potential impli- may not be feasible. Since experienced geneic stem cell transplantation us- cations on clinical outcomes. Indeed, memory T cells reside in the bone mar- ing chimerism analyses, immunologic

S-54 Resetting the immune system in autoimmune diseases / T. Alexander et al. studies investigating the T cell recon- (34, 35). Depending on the condition- IgD- CD27+ memory B cells and expan- stitution after autologous transplanta- ing regimen used and the amount of sion of CD27high CD20neg plasmablasts tion provided important insights into CD34+ stem cells transplanted, RTEs in peripheral blood (40). These find- T cell regeneration. After the initial usually recover to normal levels at 2 ings support the notion that the B cell lymphopenia following immunoabla- years post-transplant as demonstrated compartment predominantly regener- tion, T cell recovery occurs at different in studies for MS (29), RA (18), SLE ated from the haematopoietic stem cells paces in different lineages and subsets (16) and SSc (19) and mostly exceed rather than from residual B cell clones. (27). During the first months of im- pre-transplant levels during the fol- Here, quantification of kappa-deleting mune reconstitution, numeric recovery lowing years even in older adults, in- recombination excision circle (KREC) after ASCT for ADs is predominated dicating a stable thymic reactivation. could - in analogy to TREC assess- by CD45RO+ CD27- effector memory As expected, such rejuvenation of the ment - provide a practicable method to CD4+ and CD8+ T cells with increased CD4+ T cell compartment is associated investigate the bone marrow output for proliferative history and activation sta- with the vast diversification of the TCR B cells, as recently demonstrated in pa- tus (22), and highly restricted T cell re- repertoire in MS (28, 29), SSc (19) and tients with acute leukaemia after allo- ceptor (TCR) repertoire usage (16, 19, SLE (16). Among recurring T cells, geneic transplantation (41). Apparent- 28, 29), while the size of the CD4+ naïve both CD4+ CD25high Foxp3+ (22, 32, 36, ly, recurring B cells after immune reset subset was consistently found dimin- 37) and an unusual CD8+ Foxp3+ T reg- for ADs are tolerant to self-, ished for at least 1 year after transplan- ulatory (Treg) subset (32) returned to either resulting from a renewed B cell tation (16, 19, 29). The initial expan- levels seen in healthy individuals; they receptor repertoire and/or facilitated by sion of mature T cells could be induced expressed markers of RTEs and He- recurring IL-10 producing B regulatory by homeostatic peripheral proliferation lios (37), a marker of thymic-derived (Breg) cells, although available data driven by low-affinity antigens during Tregs (38), and displayed a diverse are scare to prove this assumption. lymphopenia (30), which may pose TCR repertoire (37, 39), confirming the risk that such lymphopenia-driven a reset of the Treg compartment fol- Conclusions proliferation might induce autoimmun- lowing ASCT. Of note, although Tregs There is now accumulating evidence ity, as demonstrated in animal models are thought to play a key role in main- that immunoablation followed by (31). However, we and others found no taining self-tolerance, their adoptive ASCT not solely exerts prolonged im- evidence of clonal expansion of autore- transfer after immunoablation together munosuppressive effects as initially active T cells early after immune reset with the stem cell graft did not elicit proposed, but rather provides funda- for SLE upon in vitro stimulation with additional clinical improvement in a mental changes of the immune system SLE-associated autoantigens, such as murine model of ASCT, but conversely that may rewire a chronic autoimmune nucleosomes or SmD1, (16, 32). Rath- resulted in a delayed reconstitution of system into a naïve and self-tolerant er, we showed that expanded memory the graft-derived T cell compartment. state, which was already assumed by T cells reacted to virus-specific anti- Therefore, Treg therapy should be ap- the pioneering research of van Bek- gens during infections that are com- plied with caution as it may hamper kum’s group (7). Such re-induction of monly observed in the early phase of immune renewal (39). self-tolerance is achieved by two major immune reconstitution. Expansion of principles of ASCT, that is eradication virus-specific T cells was recently con- B cell reconstitution of the pathogenic autoreactive immune firmed by next generation sequencing Analogous to T cells, repopulating B repertoire and profound immunologic (NGS) analyses after immune reset for cells could either derive from residual renewal. In analogy to stem cell trans- MS, predominantly within the CD8+ T B cell clones or originate from hae- plantation for haematologic malignan- cell compartment (28). matopoietic progenitor cells following cies that yields at the eradication of The second phase of T cell reconstitu- ASCT. Unlike T cell reconstitution, malignant clones, immunoablation for tion is characterised by recurrence of which is characterised by an initial ex- ADs is performed with the premise thymic-derived T cells. As opposed to pansion of memory cells, recurring B to deplete immunologic memory cells peripheral expansion, thymic education cells after ASCT predominantly dis- that are refractory to conventional im- is the prerequisite for immune renewal play a naïve phenotype including im- munosuppressive therapies. This no- and could provide a pool of naïve T mature transitional B cells. Naïve B tion is supported by serologic data and cells with new antigen receptor specifi- cells returned to normal levels within TCR repertoire analyses that demon- cities. Recent studies support a role for 12 months post-transplantation as dem- strated disappearance of pre-existing de novo regeneration of naïve T cells onstrated in immunologic studies for autoantibodies (16) and prominent T from the thymus (16, 19, 29, 33), in- SLE and SSc (16, 19), whereas the lev- cell clones (28) post-transplantation, dicated by the co-expression of CD31 el of memory B cells was diminished respectively. and CD45RA in combination with for up to three years. This is remark- In addition, immunological studies in high levels of T cell receptor excision ably in view of the disturbed B cell AD patients treated with ASCT have circles (TRECs) that are characteristic homeostasis characteristic for SLE, demonstrated substantial post-trans- for recent thymic emigrants (RTEs) including a relative predominance of plant modifications of the adaptive

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