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Progress and prospects for the use and the understanding of the mode of action of autologous

hematopoietic stem cell transplantation in the treatment of multiple sclerosis

Fredrika Collins1, Majid Kazmi1,2 and Paolo A. Muraro*3

1 King’s College London Medical School, London UK; Division of Hematology, King's College Hospitals NHS Trust London, UK 2 Division of Brain Sciences, Imperial College, London *Corresponding Author. Address: Wolfson Neuroscience Laboratory Burlington Danes building, 4th floor Imperial College London 160 Du Cane Road London W12 0NN, United Kingdom Tel: +44 (0) 207 594 6670 Fax: +44 (0) 207 594 6548 E-mail: p.muraro@imperial.ac.uk

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Abstract

A substantial proportion of patients with multiple sclerosis do not respond to

pharmacological treatments and no currently approved therapy has been convincingly

demonstrated to prevent or stop disease progression. However, immunoablative therapy

followed by autologous haematopoietic stem cell transplantation (I/AHSCT), which has

been used to experimentally treat over 800 patients with multiple sclerosis, can induce

long term suppression of inflammatory disease activity and can halt or reverse

neurological deterioration, thus altering the fundamental disease course. Immunological

investigations of the reconstituting immune system have discovered that qualitative

changes take place at the cellular and molecular levels, which support the hypothesis of a

‘resetting’ of the immune system. With the results of phase III comparative trials only a

few years away, this article reviews the current landscape of I/AHSCT in the treatment of

MS.

Keywords

Multiple Sclerosis; Autologous haematopoietic stem cell transplantation; Immune

Ablation; Autoimmune disease; Immune reconstitution

Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease of the brain and spinal

cord affecting around 2.5 million people worldwide 1. It is amongst the leading cause of

neurological disability in young adults thus incurring massive social and economic

costs1,2. Whilst the precise pathophysiology of MS remains poorly defined, current

understanding is that an environmental trigger within a genetically susceptible individual

provokes a loss of self-tolerance with autoreactive lymphocytes infiltrating the central

nervous system (CNS) and mounting an inflammatory attack against CNS components,

most probably myelin sheath peptides1. Abnormalities of both adaptive and innate

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immune responses are also implicated, with inappropriate antigen presentation to T- and

B-cells and an ineffective regulatory T-cell (Treg) network fails to contain aberrant

responses of immune cells1. Clinically, a relapsing remitting (RR) form of the disease is

characterised by flares of inflammatory demyelination causing functional deficits which

resolve once inflammation subsides and neurons can remyelinate, a process that is

efficient in RRMS1. A progressive form of the disease, which usually develops 10 to 20

years after diagnosis but which is primary in 10% of cases, is characterised by persistent

demyelination, gliosis, irreversible axonal injury and loss, resulting in brain atrophy and

progressive accumulation of disability irrespective of inflammatory flares1.

Current disease modifying therapies (DMTs), which target various stages of the

inflammatory process, can be very effective at reducing the frequency and severity of

inflammatory flares in RRMS3. However, inflammatory activity persists in many patients

and there is no strong evidence that the rate of underlying disease progression is

affected3. No DMT is currently recommended for treating SPMS3.The shortfalls of

current treatment options alongside an inevitable neurological decline have galvanized

the MS patient population to seek out alternative therapies, accelerating clinical research

into experimental treatments.

Immunoablative therapy with autologous haematopoietic stem cell transplantation

(I/AHSCT) was first used to treat MS over 20 years age4 and over 800 patients have been

treated since5. Whilst early results were exciting but controversial, more recent trials have

produced compelling results and interest within the MS-affected population is

consequently growing rapidly. The rationale behind I/AHSCT is that immunoablative

therapy diminishes the self-reactive immune cell pool allowing re-engrafted stem cells to

generate a new, and potentially self-tolerant immune cell repertoire. Beyond

immunosuppression, I/AHSCT induces qualitative immunomodulatory changes which

are thought to tip the balance of the immune system from a pro-inflammatory to a

tolerant phenotype, effectively ‘resetting’ the immune system. Epigenetic changes, a

rebooted thymus and restored regulatory network and are all thought to contribute

towards these effects.

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This paper hopes to review the current progress of I/AHSCT in the treatment of

MS, exploring both clinical developments and advances in our understanding of the

immunological mechanisms of I/AHSCT.

Pre-Clinical Evidence for the use of I/AHSCT

Results from animal studies provided preclinical evidence for the feasibility of

I/AHSCT in the treatment of MS 6-13. In the 1990’s, an experimental allergic

encephalomyelitis (EAE) murine model demonstrated how total body irradiation (TBI)

followed by pseudo-autologous HSCT could cause a rapid regression of neurological

symptoms and suppression of spontaneous relapses in 70 % of cases 14. This result, whilst

promising, translated poorly to the clinical population who would be many years along

their disease pathway when receiving I/AHSCT.

Later studies addressed this and revealed that I/AHSCT was only effective when

administered during the acute, but not the chronic, phase of EAE 7,15. Whilst there will

always be limits to how well results from an animal model of MS will predict clinical

outcomes in humans, these studies provided proof of principle for the use of I/AHSCT in

the treatment of MS. In particular, they highlighted the superior efficacy of the therapy

during the early stages of disease.

Interestingly, a recent paper has presented a new potential avenue for therapeutic

intervention. Based on the premise that exposure to self-antigens is an important step in

the development of self-tolerant T-cells, Chan et al. 16 investigated whether the

therapeutic effect of I/AHSCT could be enhanced by pre-reinfusion genetic modification

of HSCs. The authors found that ex-vivo, retroviral transduction of myelin

oligodendrocyte glycoprotein (MOG) into HSCs induced total and sustained EAE

remission, even after MOG re-challenge. In contrast, 20% of the control group relapsed

spontaneously and 100% relapsed upon MOG re-challenge. These effects were long

lasting as disease resistance could be transferred with HSCs 7 months post-transplant. An

associated reduction in thymic CD4+ MOG-reactive cells was also seen. Modified HSCs

successfully repopulated the new immune system as molecular chimerism was evident in

T-cells, B-cells and dendritic cells (DC). DC’s are known to be mediators of central

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tolerance through their role in presenting self-antigens to developing T-cells in the

thymus, with any T-cells showing a high avidity for that self-antigen being deleted 17.

Therefore, increased MOG expression by DC’s could be another important driver of

tolerance post-transplant. Finally, the conditioning regimen only included corticosteroids,

not immunoablation. The potential prospect of creating a robust tolerant immune system

without the requirement of an immunoablative conditioning regimen is very appealing

considering the associated inherent risks. This potential therapeutic strategy should be

investigated further.

Clinical Evidence for the use of I/AHSCT

Whilst over 800 MS patients have been treated with I/AHSCT over the past 20

years 18, interpretation of the amassed clinical data is complex. Studies have employed

different conditioning regimens and there has been great heterogeneity within the patient

population studied, in terms of baseline disability, disease duration, age and MS disease

type. Furthermore, as of yet there are no published phase III clinical trials and only one

phase II randomised control trial (RCT) has been reported, therefore current evidence is

limited. Nonetheless, emerging patterns support the feasibility, tolerability and efficacy

of I/AHSCT in MS with recent studies consistently demonstrating that I/AHSCT can not

only arrest disease progression but can also reverse pre-existing disability 19.

Whilst early studies mainly included SPMS patients, an increasing proportion of

patient cohorts are now made up of RRMS patients 20. An increasing capacity to recruit

patients to trials at earlier stages of disease than before reflects increasingly convincing

results from phase I/II trials and a steadily declining toxicity profile. However, risks and

tolerability of I/AHSCT still remain the biggest deterrent to its use, especially when so

many pharmacological alternatives exist with more reassuring toxicity profiles20.

Three recent clinical trials have investigated I/AHSCT in a majority RR cohort: a

3 year interim analysis of high intensity I/AHSCT in 25 RRMS patients 21, a 4 year

retrospective nationwide survey of 41 patients (85% RRMS) treated with intermediate or

low intensity I/AHSCT 22 and a 5 year follow-up of a single-centre patient cohort of 145

patients (81% RRMS) treated with low intensity I/AHSCT 23. Treatment related mortality

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(TRM) was 0% in all these studies even though various intensities of conditioning

regimen were employed, which is reassuring considering that the main concern with

I/AHSCT is its toxicity. At last follow-up (3, 4 and 5 years respectively) these studies

found that disease-free survival (no relapses, no new MRI lesions and no EDSS

progression) was 78.4%, 68% and 68% respectively. EDSS progression free survival was

90%, 77% and 87% respectively. In addition, all three studies demonstrated that median

EDSS improved from baseline by 0.5, 0.75 and 1.5 points respectively. This

improvement was corroborated by two separate scoring systems for neurological function

in the studies by Nash et al. 21 and Burt et al. 23. Whilst valid comparisons between

distinct treatments requires a RCT, comparing these results with current best DMTs

reveals how I/AHSCT has a far superior efficacy, albeit with a less favourable toxicity

profile 24-26 (Fig 1).

The evidenced neurological recovery in these studies evolves the very nature of

I/AHSCT from one designed only to halt disease progression to one which can actually

reverse disability and return function, setting it apart from the capabilities of current

DMTs 3. Burman et al. 22 and Burt et al. 23 found that the majority of neurological

improvement was seen in RR, not SP, patients. This suggests that a significant proportion

of SP pathology is irreversible even in a post-inflammatory environment.

However, this is not to say that a certain degree of neurological recovery in SPMS

is not possible. Impressive results have recently been published by a multicentre, single-

arm, phase II trial 27 in which 12 RR and 12 SP patients were treated with high intensity

I/AHSCT. All patients had active inflammatory disease at baseline. A complete

suppression of all clinical and radiological relapses was observed up to 12.7 years

(median 6.7 years) post-transplant. In addition, the rate of brain atrophy slowed to control

levels after 2 years, similar to finding by Nash et al. 21. TRM was 4% which is high and

possibly a direct reflection of the high intensity conditioning regimen used, although

changes were made to the cytotoxic conditioning during the course of the trial in order to

improve safety. Impressively, by last follow-up 70% of patients were free from

neurological progression and 40% showed sustained improvement in EDSS. This extent

of disability reversal is remarkable considering half of the cohort were SP. I/AHSCT thus

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may have favourably impacted the progressive character of SPMS in these patients,

although a control group would be required for confirmation.

In 2015 the first RCT was published, The Autologous Haematopoietic Stem Cell

Transplantation trial in MS (ASTIMS). This multicentre phase II study found that

I/AHSCT (BEAM/rATG conditioning) was vastly superior to mitoxantrone (MTX) at

suppressing ongoing inflammatory activity in 21 patients with active inflammatory SP or

RRMS. Patients in the I/AHSCT arm (n=9) had 79% fewer new T2 lesions than in the

MTX arm (n=12), an effect which began in the first year and was sustained throughout

the entire follow-up. Furthermore, I/AHSCT-treated patients had significantly fewer

clinical relapses and experienced complete suppression of GD+ lesions, whilst Gd+

lesions were seen in over half of MTX-treated patients. Whilst no difference in EDSS

progression was found between the groups, the study was underpowered to find such a

difference. Tolerability profiles were comparable between the treatment groups. Whilst

this study had methodological limitations such as the small cohort size, it provides robust

evidence as to the superior action of I/AHSCT in suppressing CNS inflammation

compared with MTX 28.

It is important to consider which patient characteristics are associated with better

outcomes in order to inform more accurate patient selection in the future. Some studies

have found that disease type is important, with RR disease responding better than SP to

I/ASHCT 23,29,30. However other studies find that, rather than disease stage, the presence

of Gd+ lesions at baseline is important 22,29,31. This feature indicates three things that

could explain the enhanced therapeutic effect; inflammation is an active component of

the disease mechanism; the BBB is more permissive to drugs that enhance access to the

target CNS compartment; and immune cells within the CNS are proliferating rapidly and

so are more susceptible to lymphoablation. In addition, good outcomes have been

associated with a short disease duration 23,29-31, younger age 29-31 and absence of peri-

HSCT fever 23. Younger patients may respond better to treatment not only as a result of

the association of young age with relapsing inflammatory MS32, but also as they could

harbour a greater regenerative capacity for repair of pre-existing neuronal damage.

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Whilst the interpretation of results so far is that I/AHSCT can effectively suppress

ongoing CNS inflammation and in many cases halt or reverse recent neurological, we

await Phase III comparative trials for confirmation.

Implications of clinical findings on understanding MS pathophysiology

The fact that substantial neurological recovery can occur after I/AHSCT supports

the idea that the CNS harbours intrinsic repair mechanisms which must only be able to

function in a non-inflammatory environment. Recovery of neurological function

following an inflammatory flare in RRMS is another example of this 1. The majority of

neurological improvement following I/AHSCT occurs within the first year of transplant 22. This suggests that only recently accumulated disability is amenable to intrinsic repair

whilst more longstanding disability must be beyond the healing capacity of the CNS. This

is reflected in how short disease duration consistently correlates with therapeutic outcome 23,29-31 and how advanced disability in malignant MS patients can undergo astonishing

recovery after I/AHSCT 33. Due to highly aggressive disease, these patients rapidly

accumulate large amounts of disability in short periods of time, which must fortunately

mean the damage is still immune-dependent and therefore reversible

The proportion of disability that is irreversible following I/AHSCT must be due to

a distinct, immune-independent mechanism for which the body does not have the

capacity to heal, even in a post-inflammatory environment. Accordingly, patients can

undergo neurological progression after I/AHSCT despite complete suppression of CNS

inflammatory activity 27,34,35. This further demonstrates how a portion of MS pathology is

uncoupled from inflammatory mechanisms and progresses in its own autonomous,

neurodegenerative fashion. If I/AHSCT can successfully prevent RRMS from

progressing into SPMS it would support the theory that SP disease develops as a direct

consequence of RR disease rather than arising from a distinct pathophysiological

mechanism. The challenge would then be to treat MS early to prevent the SP phase from

developing. Alternative regenerative therapies 36 must be investigated to reverse immune-

independent neurological damage.

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The Conditioning Regimen

Resetting of the immune system is thought to be a prerequisite for durable

responses following I/AHSCT and the conditioning treatment plays an integral role in

this therapeutic success. However, owing to an absence of RCTs, there is a lack of

consensus amongst treatment centres over the optimum condition regimen. A positive

correlation exists between therapeutic efficacy and toxicity, whereby one can not be

improved without a concurrent compromise in the other (Fig 1), and the appropriate

balance of these two variable is hard to determine. This is complicated by the fact that

responses to the conditioning regimen may vary with patient age, disease duration and

phase of MS. In addition there may be a requirement for prolonged follow-up before any

benefit from a more intensive regimen becomes apparent.

The design of early clinical trials was based on results from animal studies 12,14,15

which advocated the use of high intensity immunoablation. Myeloablative conditioning

regimens were therefore employed, such as TBI or high dose busulfan, however TRM

and disease progression were consistently high throughout these early trials 30,37-39. TRM

in early studies may have been skewed by poor patient selection, with the inclusion of

large cohorts of SPMS patients with very advanced disease. In these cases, treatment

related neurotoxicity could have exacerbated underlying neuronal degeneration and

accelerated disease progression. More recent years have witnessed a shift towards the use

of intermediate and low intensity regimens. Intermediate intensity immunoablation is also

myelo-ablative and includes the most commonly used protocol in Europe: the BEAM

regimen combined with anti-thymocyte globulin (ATG) for enhanced immunoablation.

BEAM is composed of carmustine, etoposide, cytarabine and melphalan, all of which are

able to cross the blood-brain barrier and so can penetrate the auto-reactive compartment

in the CSF40. Low intensity immunoablation involves CY, melphalan or fludarabine-

based regimens and is lymphoablative but not myeloablative 5.

There is limited objective evidence to compare one regimen against another.

Hetereogeneity in trial design makes inter-trial comparisons difficult and many

immunological investigations have, as of yet, failed to analyse how the composition of

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the conditioning regimen affects immune reconstitution. Whilst several earlier studies 41,42 actually proposed that higher intensity immunoablation is associated with worse

therapeutic outcomes, these results have since been criticised due to methodological

flaws 20. Furthermore, recently published results from the Canadian MS experience 27

demonstrate impressive long term results with a very intensive regimen, consisting of

busulfan/Cy/rATG and ex-vivo CD34 graft selection, as described earlier in this paper.

They did report a 4% TRM, which is significantly higher than the 1.3% TRM reported by

the EBMT register between 2001 and 2007 40 and the 0.88% cumulative TRM reported

since 2007 (unpublished data, EBMT database). EBMT 2012 guidelines 5 acknowledge

that more profound therapeutic effects can be achieved with higher intensity regimens

however BEAM + ATG is currently recommended for MS, based on the more favourable

toxicity profile. With this recommendation in place, many of the larger clinical trials have

been, and are being, designed around BEAM + ATG 20,21.

As low intensity regimens consistently report 0% TRM follow I/AHSCT 23,43, it is

important to consider what level of risk is acceptable to expose patients to. It could be

argued that, despite reduced therapeutic efficacy, it is preferable to use a low intensity

regimen with a 0% TRM in order that I/AHSCT can be administered at an earlier stage of

the disease, where inflammatory mechanisms dominate pathogenesis 23,43. However, with

clinical evidence 23,43 suggesting that the effects of low intensity regimens are temporally

finite and immunological findings suggesting that myeloablation may be required for

certain reconstitution kinetics, such as thymus-dependent repopulation of the CD4+

population 44,45, many patients and doctors may feel that a higher TRM risk is justified.

An alternative approach to this problem is to focus on how the risk of TRM can

be reduced without having to compromise therapeutic efficacy by reducing the intensity

of immunoablation (see Fig 1 for the idealised ‘future of I/AHSCT’). For example, an

increasing number of studies employing intermediate intensity regimens are

demonstrating 0% TRM 22,29,30,46,47. Furthermore, a retrospective observational study of

12 years worth of patients treated for autoimmune diseases on the EBMT register found

that the transplant centres’ experience, not intensity of the conditioning regimen, was

significantly associated with 100 day mortality 48. In an attempt to maximise tolerability

of I/AHSCT, the EBMT has published recommendations aimed at minimising the risk of

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TRM and other adverse events 5. These include patients being treated exclusively within

specialist centres with JACIE accreditation, strict patient selection criteria and extensive

measures to prevent and manage infection and peri-HSCT fever.

Alongside TRM, it is important to acknowledge the risk of other adverse events

after I/AHSCT. These include neutropenic fever, infections, viral reactivations,

thromboses, gastrointestinal disturbances, infertility and even the development of

secondary autoimmune diseases and malignancies 19. Recent evidence also suggests

AHSCT, independent of immunoablation, can accelerate cellular ageing by the equivalent

of 30 years 49. This was shown by RNA transcriptional changes characteristic of aging,

telomere shortening and a significant increase in the T-cell expression of p16INK4a, a

cellular marker of senescence. This paper has several methodological limitations,

however it could help to explain why younger patients respond better to HSCT and is a

reminder of the potentially far reaching consequences of I/AHSCT.

Another area of uncertainty in the conditioning regime is whether or not to T-cell

deplete the graft before cryopreservation, either by positive selection of CD34+ cells or

negative depletion of T-cells. In principle, ex-vivo T-cell depletion should benefit

therapeutic outcome by removing any auto-reactive T-cells in the graft. However, a

retrospective analysis of all autoimmune patients in the EBMT database treated with

I/AHSCT before 2011 found that outcomes do not improve with ex-vivo depletion but

that the procedure increases cost 5. As a result, most studies do not integrate ex-vivo

depletion into their regimens. However, the publication of Atkins et al. 27 may renew

interest in ex-vivo graft manipulation after complete suppression of radiological and

clinical relapses was achieved. However, it is important to note that alongside the ex-vivo

graft manipulation this study administered high dose busulfan with Cy and in-vivo T-cell

depletion, so there is no way of knowing whether the role of ex-vivo depletion was

requisite for success. On top of this, Nash et al. 21 used ex-vivo depletion with the

intermediate BEAM + rATG regimen yet was unable to completely suppress

inflammatory activity, suggesting that the intensity of the conditioning regimen overall

rather than the inclusion of ex-vivo depletion is important. Instead, most trials integrate

in-vivo T-cell depletion, using alemtuzumab or more commonly ATG, without ex-vivo

graft manipulation. ATG is a polyclonal antibody with primary T-cell depleting, and to a

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lesser extent B-cell depleting, activity. It serves to deactivate lymphocytes that may have

either survived the conditioning regimen or been re-infused with the graft 40. Along with

its lymphoablative properties it has been suggested to have additional immunomodulatory

properties, with rapid and sustained expansion of CD4+CD25+ T-regs when cultured in

vitro with human peripheral blood lymphocytes. ATG is however a drug with significant

toxicity. In particular, infusion related events and delayed serum sickness can occur.

There is also a significant risk of EBV reactivation several weeks post-transplant.

In the long term, well designed RCT’s are needed to conclusively determine the

most appropriate conditioning regimen for I/AHSCT. In the short term there is a need for

more thorough and updated systematic reviews of the safety and efficacy of I/AHSCT for

MS. Currently there is no standardised assessment for the efficacy of I/AHSCT with

some studies reporting only on either radiological or clinical relapse suppression or EDSS

progression. It is clear that these parameters alone are not sensitive enough to allow

comparison between studies and, increasingly, composite end-points are being

incorporated in to trial design, such as no evidence of disease activity (NEDA). A

standardised follow-up assessment of disease activity must be agreed which integrates

clinical, radiological and immunological markers of disease activity and neurological

progression. This must then be adopted into the design of all future trials to allow ease of

inter-trial comparison.

Immunological Mechanisms underlying I/AHSCT

Clinical studies have clearly demonstrated that I/AHSCT can cause lasting

remission of MS disease activity and investigations into the reconstitution of the immune

system have revealed that qualitative immunodulatory changes occur alongside

lymphodepletion. Whilst a comprehensive understanding of these changes still eludes us,

work in recent years has identified thymic reactivation 44, restoration of the regulatory

network 22,45,50-53 and epigenetic changes 35,52 as key contributors to the formation of a

new and tolerant immune system. These qualitative changes set I/AHSCT apart from

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current DMT’s; where lymphodepletion is the sole mechanism of action and where

disease activity returns with lymphocyte count recovery upon cessation of treatment 3,5,22.

A New and Diverse T-cell Repertoire

The immune compartment reconstitutes quickly after I/AHSCT, with NK cells, B-

cells and monocytes returning to baseline frequencies by 6 months. T-cells undergo a

biphasic reconstitution, with CD8+ cell frequency recovering in 6 months whilst CD4+

cells take 2 years to recover, resulting in an inversion of CD4+/CD8+ ratio until this time

point 44. Further characterisation and analysis of T-cell subsets has revealed that this

occurs due to distinct mechanisms driving the recovery of CD8+ and CD4+ cell

populations, with late CD4+ pool repopulation by thymic production of naïve cells and

early CD8+ pool repopulation by peripheral expansion of pre-existing cells. The majority

of dominant CD8+ clones present at 2 months post-transplant were also detected in the

pre-transplant immune environment 54, suggesting that CD8+ clones survive

immunoablation and proliferate rapidly in the lymphopenic, post-transplant environment.

Muraro et al. 54 suggests that peripheral expansion of CD8+ clones is driven by the

surrounding viral landscape, with viruses such as EBV and CMV activating and

expanding viral-specific CD8+ clonotypes. This causes an initial restriction in the TCR

repertoire with dominance of these viral-specific clones. However, the TCR repertoire

diversifies with the introduction of new CD4+ cells.

In contrast to the early reconstitution of the CD8+ pool, the CD4+ cell population

is regenerated through late thymic production of de novo naïve cells

(CD45RA+/CD45RO−/CD27+). Phenotypic markers of specific T-cell subtypes

characterised how the frequency of naïve CD4+ cells doubles from baseline by 2 years

whilst the frequency of central-memory CD4+ cells (CD45RA−/CD45RO+/CD27+)

halves (Fig 2). This means that one quarter of the CD4+ T-cell pool that had been

occupied by central-memory T-cells pre-transplantation is replaced with naive T-cells.

Naive T-cells are associated with immune tolerance whilst memory T-cells are mediators

of auto-immunity, so this shift in relative frequency could be a mechanism of immune

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tolerance post-transplant. In contrast, I/AHSCT has no effect on the relative proportions

of naive and central-memory cell types within the CD8+ T-cell pool 44.

Further, T-cell-receptor-excision-circle (TREC) analysis found that the newly

generated naive CD4+ T-cell cells were derived exclusively from the thymus (Fig 2). The

novel composition of the CD4+ compartment was confirmed in a later study by Muraro et

al. 54 where high-throughput deep TCRβ chain sequencing revealed that 82% of CD4+

clones present in the post-transplant environment were new. Taken together, these results

show that I/AHSCT stimulates thymic output to create a novel CD4+ T-cell population.

This could be viewed as a ‘resetting’ of at least the CD4+ cell compartment of the

immune system.

More work is required to further characterise the reconstitution of the other

immune cells. Whilst comparisons between distinct auto-immune diseases should only be

made with caution, owing to discrepancies in underlying pathophysiology, investigations

into immune reconstitution following I/AHSCT for systemic sclerosis55 and systemic

lupus eythematosus56 have revealed that the B-cell compartment also repopulates with

naïve, not memory, B-cells and these cells show enhanced tolerance towards self-

peptides57.

Reactivation of the thymus following I/AHSCT is an intriguing finding as thymic

output plays a critical role in immune tolerance through the production of Tregs and

diversification of the T-cell receptor (TCR) repertoire 1. Interestingly, MS patients have a

depressed thymic output with a corresponding reduction in the frequency of circulating

Tregs 58 and a restriction of their TCR repertoire 59. Revival of thymic output by

I/AHSCT therefore offers a convincing mechanism of immune tolerance. Indeed, along

with a normalisation of thymic output, levels of circulating Tregs increase after I/AHSCT 22,45,50-53 and the TCR repertoire diversifies to levels seen within healthy populations 54.

The importance of this mechanism in creating tolerance is reflected by the fact that early

post-transplant TCR diversity is a positive predictor of therapeutic outcome 54.

It is important to mention that clinical and radiological disease remission has been

achieved in a different cohort of patients without a biphasic T-cell compartment

reconstitution, when non-myelo- instead of myeloablative conditioning was used 45.

Instead, both the CD4+ and the CD8+ cell populations regenerated through peripheral

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expansion of pre-existing cells. Whilst thymic export did increase moderately, its action

was insufficient in increasing the frequency of naïve CD4+ cells. This either suggests that

several mechanisms contribute to disease suppression, not just thymic production of

naïve CD4+ cells, or that perhaps with longer term follow-up it will become evident that

thymic production of naïve cells is required for sustained remission.

Restoration of the regulatory T-cell network

Even after myeloablative I/AHSCT, myelin-reactive T-cells spontaneously re-

emerge and expand in-vivo despite sustained clinical and radiological remission 7,51,60.

This raises the question; what is preventing persisting auto-reactive T-cells from

reinitiating disease in the post-transplant environment? It could be that these auto-

reactive cells will drive relapse in the future or perhaps they never had a functional

pathogenic role in the first place. Alternatively, qualitative changes within the post-

transplant environment could restrain the activity of these myelin-reactive T-cells, in a

way that was previously impossible. With this school of thinking it then becomes

unnecessary to achieve a complete depletion of auto-reactive cells. Instead, the only

requirement is to tip the balance of the wider immune network from a pro- to an anti-

inflammatory phenotype (Fig 3).

One way in which I/AHSCT mediates this is by restoring the regulatory T-cell

network. Tregs are critical mediators of immune tolerance through their regulation and

suppression of T-cell activity 1. Evidence reveals how the frequency and suppressive

capacity of circulating Tregs is depressed in MS patients 58 due to inadequate thymic

production of naïve Tregs, impaired peripheral expansion of memory Tregs and skewing

of existing Tregs towards a pro-inflammatory phenotype 1. Clinical trials have

consistently demonstrated how both myelo- and non-myeloablative I/AHSCT induces a

significant and transient increase in levels of circulating CD4+/CD25high/FoxP3+ Tregs 22,45,50-53(Fig 2). Thymic renewal could be responsible for this phenomenon; natalizumab-

treated MS patients exhibit lower levels of CD4+/CD25high/FoxP3+ Tregs than both

healthy controls and I/AHSCT-treated patients and the discrepancy in Treg frequency

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was due to a paucity of thymus-derived, not peripherally expanded, Tregs 53.

Alternatively, the expansion of the CD4+/CD25high/FoxP3+ T-reg pool could be a non-

specific response to lymphodepletion as alemtuzumab monotherapy can also increase its

frequency and ATG can cause rapid and sustained in vitro expansion of

CD4+/CD25high/FoxP3 Tregs 61.

I/AHSCT also enhances the functional immunosuppressive capacity of Tregs

alongside an increased frequency, as up-regulation of Treg surface immunoregulatory

molecules, CTLA-4 and GITR, occurs 52. Tolerance mechanisms are up-regulated within

the innate as well as adaptive immune system as a transient increase in regulatory natural

killer cells is also seen after I/AHSCT 45. Furthermore, the chemokine network is

modified in the post-transplant environment51. This could affect interactions between

immune cells and recruitment of inflammatory cells to the CNS, however further studies

are required to elucidate the significance of these changes and to determine whether they

contribute to, or are a consequence of, treatment effect 51.

An increase is also seen in circulating levels of CD8+/CD28-/CD57+ T-cells 44,45,50(Fig 2), an effector-memory T-cell subtype which is thought to exert an

immunosuppressive effect on CD8+ cells 62-64. This change occurs following both myelo-

and non-myeloablative I/AHSCT and is sustained for up to 4 years50. An expanded

population of these cells could enhance the regulatory network by working

synergistically with Tregs to maintain self-reactivity of T-cells within the post-transplant

environment

It is interesting that most of the evidenced modifications in the immunoregulatory

network are transient yet lasting clinical remission can be seen. Perhaps enhanced

immunosuppressive qualities of regulatory cells persist even after kinetics have

normalised or perhaps re-emerging effector T-cells are more easily suppressed.

Alternatively, an early transient augmentation in the regulatory network could be

sufficient for creating a new and tolerant T-cell repertoire by regulating antigen priming

and education of re-emerging and naive T-cells in the early post-transplant environment 65.

Reduced pro-inflammatory IL-17 response

PAGE17

Whilst reactivity to myelin self-peptides re-emerges in the post-transplant

environment 7,51,60 the balance between pro- and anti-inflammatory T-cell responses

evolves to a more tolerant pattern (Fig 3). Flow cytometry assays reveal that peripheral

CD8+ (Tc17) and CD4+ (Th17, Th1/Th17) T-cells implicated in MS pathogenesis1

mount a greatly diminished pro-inflammatory interleukin-17 (IL-17) response after

myeloablative I/AHSCT, and this is corroborated by parallel changes in RNA expression 51. IL-17 promotes an inflammatory CNS environment by increasing permeability of the

blood brain barrier (BBB) and recruiting lymphocytes into the CNS 66. The observed

diminution in the IL-17 response should therefore promote an anti-inflammatory CNS

environment. This same study demonstrated reductions in levels of polarising cytokines,

leading the authors to hypothesise that altered interactions between antigen presenting

cells (APCs) and T-cells could contribute towards the formation of a more anti-

inflammatory T-cell phenotype51.

Likewise, a different study demonstrated that myeloablative I/AHSCT, but not

natalizumab, completely suppressed the pro-inflammatory IL-17 response of Th17 CD4+

cells when activated by MOG 53. Unlike Darlington et al. 51 they also demonstrated a

suppression of the pro-inflammatory interferon-gamma response by Th1 cells, another

CD4+ subtype implicated in MS pathogenesis. The authors propose that, within the new

and tolerant immune environment, an enhanced Treg secretion of transforming growth

factor-b-1 (TGF-b1) suppresses the pro-inflammatory IL-17 response towards MOG as

significantly higher levels of TGF-b1 were produced by T-cells from healthy controls and

I/AHSCT-treated patients than from natalizumab-treated patients 53.

In addition, non-myeloablative I/AHSCT has been found to deplete to almost

undetectable levels an IL-17 producing CD8+ T-cell subtype called the mucosal-

associated invariant T (MAIT) cell45 (Fig 2). MAIT cells have convincingly been

implicated in MS pathogenesis; they are pro-inflammatory, producing IFN-gamma, TNF-

alpha and the highest levels of IL-17 of all the CD8+ cells 45 and whilst they originate in

the gut they express CCR6, a CNS homing receptor, which facilitates transmigration

across the BBB 67. Accordingly, MAIT-cells are found within the immunological cell

infiltrate in MS lesions at autopsy and circulating levels are increased in the peripheral

PAGE18

blood of MS patients compared to controls 45,68. It is therefore of significant interest that

even non-myeloablative I/AHSCT can induce sustained suppression of this cell type. The

treatment of MS with alemtuzumab and cyclophosphamide alone also depletes MAIT-

cell levels but not to the same extent. This suggests that lymphodepletion, even without

myeloablation, is an important component of therapeutic success.

Normalisation of microRNA expression

Intriguing evidence from a paper by Arruda et al. 52 proposes that epigenetic

changes could underlie many of the immunological modifications which occur in the

post-transplant environment. MicroRNAs (miRNAs) are small non-coding RNA

molecules which contribute to immune regulation through post-transcriptional

modulation of protein-encoding genes 69. Up-regulation of three miRNAs; miR-15570,71

miR-142-3p 72 and miR-16 73, has been described in MS. I/AHSCT results in down-

regulation of all three miRNAs and a concurrent increase in the expression of their

normally silenced target genes, FOXP3, FOX01 and IRF2BP2 52. These genes are

implicated in the formation of Tregs and the maintenance of anti-inflammatory cytokine

networks 74-76. Re-activation of these genes could therefore underlie subsequent down-

stream restoration of the regulatory T-cell network, as was described earlier in this paper 22,45,50-53. These epigenetic changes were sustained throughout the entire 2 year follow-up,

although longer term investigations are required to elucidate whether these changes are

transient, as is seen with increases in Treg frequency.

Another target gene of miR-16, PDCD1, is of particular interest because it

encodes for the production of a cell surface protein, Programmed cell death protein 1

(PD-1). PD-1 is an inhibitory receptor expressed on T-cells, B-cells, natural killer cells,

dendritic cells and monocytes 50 which plays a critical role in the maintenance of self-

tolerance through regulation of T-cell proliferation and reactivity. Interaction with the co-

stimulatory molecule, PD-L1, results in an increased secretion of the potent anti-

inflammatory cytokine, IL-10, and suppression of T-cell proliferation 77,78. PD-1 is also

implicated in polarising peripheral Tregs 79 and in mediating apoptosis of self-reactive T-

cells 78,80. Animal and clinical results have convincingly implicated the protective role of

PAGE19

PD-1 in MS development and progression. Animal studies have shown how blockade or

genetic deletion of PD-1 results in an exacerbation and acceleration of EAE acquisition 81-83 and clinical studies have demonstrated how PD-1 expression is tightly correlated

with patterns of MS disease activity 78 and how a PD-1 polymorphism is associated with

MS progression 84.

It is therefore of considerable interest that Arruda et al. 52 demonstrates how a

down-regulation of miR-16 following I/AHSCT is associated with a corresponding

increase in PDCD1 expression and a transient rise in PD-1 receptor expression on the

surface of both B- and T-cells. A transient up-regulation of PD-1, like the transient

expansion of the Treg pool 22,45,50-53, could be sufficient for the establishment of long-

lasting tolerance through the cultivation of an anti-inflammatory environment into which

emerging T-cells are shifted towards a more self-tolerant, anti-inflammatory phenotype.

Interestingly, a later study by the same group found that the only immunological

correlate of long term neurological outcome following non-myeloablative I/AHSCT was

the early up-regulation of PD-1 on B- and T-cells 50. Likewise CD8+ T-cell exhaustion,

as exhibited by high expression of PD-1, acts as a positive predictor of recovery in

autoimmune, but not viral, disease 85. More targeted manipulation of PD-1 without the

need for I/AHSCT could offer a new therapeutic target in the future.

Normalisation of gene expression profiles

I/AHSCT induces immune reconstitution at the molecular level with potent

reprogramming of transcriptional expression within peripheral CD4+ and CD8+ cells,

which could contribute towards the formation of a more tolerant immune environment.

Sousa et al. 35 investigated with microarray DNA-chip technology the gene expression

profiles of peripheral CD8+ and CD4+ cells from 16 patients with MS, before and after

non-myeloablative AHSCT, and compared them to healthy controls. All patients

underwent successful radiological stabilization supporting a complete abrogation of CNS

inflammatory activity. Before I/AHSCT, expression profiles from MS patients were

distinct to that of controls with over 2000 differentially expressed genes (DEG) - genes

that are expressed differently between MS patients and controls - in both CD4+ and

PAGE20

CD8+ cells. Of note, many of the DEG’s are implicated in the regulation of immune

tolerance and inflammatory responses, which supports a strong genetic contribution

towards the dysfunctional nature of T-cells.

I/AHSCT induced extensive transcriptional changes in the expression profiles of

both CD4+ and CD8+ cells. Following I/AHSCT, the majority of DEGs in CD4+ and

CD8+ cells were down-regulated, 70% and 77 % respectively 35. It could therefore be

reasoned that DEGs are pathogenic and their subsequent down-regulation drives clinical

remission. Remarkably, I/AHSCT normalised gene RNA expression within CD8+ cells;

at 2 years post-transplant the expression profile of CD8+ cells was similar to controls.

Whilst significant modifications also took place within CD4+ cells, their expression

profiles remained distinct from that of healthy controls at 2 years. Longer follow-up is

required to determine whether normalisation within CD4+ cells is delayed.

More detailed analysis into the function of genes which underwent robust changes

revealed that they regulate T-cell activation, migration and effector function. Modulation

of these genes could therefore contribute towards the observed shift towards an anti-

inflammatory T-cell phenotype following I/AHSCT 22,45,50-53. It is important to note that

myeloablation is not required for these potent transcriptional changes.

Interestingly, AHSCT in the treatment of malignant disorders has also been

shown to induce transcriptional changes associated with immune tolerance, with an

increase in the expression Treg-associated transcripts 49. The authors of this study

propose that these transcriptional changes are characteristic of cellular ageing, a process

which is accelerated due to damaging effects of forced bone marrow expansion. Whilst

accelerated cellular senescence is disadvantageous in the context of malignant disorders

where the integrity of the immune system is important, these changes are more

favourable in the context of autoimmunity where a depressed immune system acts as a

mechanism of fighting disease.

In the future larger studies will be required to corroborate the results of small

phase I/II trials and work is required to determine how immune reconstitution is affected

by the intensity of the conditioning regimen. A comprehensive understanding of how

I/AHSCT affects MS will also require investigations into the qualitative reconstitution of

PAGE21

B-cells and cells of the innate immune system. Of particular interest is microglia, which

are thought play an increasingly important role in MS pathogenesis 1.

It will also be important to elucidate how differences in immune reconstitution are

associated with treatment response, to identify biomarkers of response which will allow

for more careful patient selection in the future. Even if I/AHSCT never becomes a

mainstream treatment, immunological investigations of I/AHSCT will evolve our

understanding of MS pathophysiology and may help to identify new more selective

therapeutic targets for the future.

Expert Commentary

I/AHSCT is a multi-step procedure aimed at inducing fundamental “immune

resetting” changes that promote suppression of disease activity in autoimmune disease

including MS. Evidence from clinical trials demonstrates that I/AHSCT can have a

powerful effect on inflammatory disease activity in both RR and SPMS and can even

cause clinical improvements in patients with reversible immune-dependent neurological

damage. I/AHSCT can induce sustained effects however longer follow-ups will be

required before it can be offered as a potential ‘cure’. Whilst there is no doubt about the

efficacy of I/AHSCT, toxicity of the therapy remains a barrier to patient recruitment into

phase III trials although safety profiles have been improving. For this reason I/AHSCT

must be restricted to JACIE accredited specialist centres and all efforts taken to improve

transplant tolerability. There is a sub-set of patients who do not respond to treatment in

both the RR and SPMS population which raises the question whether distinct

pathophysiological etiologies exist for the same clinical phenotype. RCTs are required

not only to demonstrate superior efficacy against current DMTs but also to determine the

most appropriate conditioning regimen.

Immunological investigations are beginning to unravel the mechanisms

underlying I/AHSCT, revealing how the immune system is modulated at the cellular and

molecular level and is tipped into a more tolerant and anti-inflammatory state. This sets

I/AHSCT apart from current DMTs and offers an explanation for its apparently superior

PAGE22

and longer-lasting action.

Five-Year View

We await the completion of phase III clinical trials. The MIST trial, currently

recruiting, is comparing current best DMT to low intensity I/AHSCT for RRMS

(clinicaltrials.gov identifier: NCT00273364) and a European trial is currently being

designed to compare current best DMT to intermediate intensity I/AHSCT 20. A trial

should also be considered to compare I/AHSCT against SPMS in patients with active

inflammatory disease, for whom no therapy is currently recommended. If phase III

comparative trials demonstrate that I/AHSCT is more effective than current best

treatment, and with a justifiable toxicity profile, the landscape for treating MS could be

dramatically altered. I/AHSCT could be offered as a first line treatment in order to target

MS when the pathogenesis is dominated by inflammatory rather than neurodegenerative

mechanisms. Rather than being on life-long, expensive pharmaceutical therapy patients

could undergo an early I/AHSCT which could dramatically alter the course of their

disease and leave them drug free for years. Already, the Swedish national health

authorities have approved I/AHSCT for highly active refractory MS and more countries

may follow this lead.

Progress in mechanistic understanding of I/AHSCT will accelerate due to

advancing methodologies such as mass cytometry and RNA sequencing as well as due to

immunological analysis being incorporated into larger phase III trials. Even if I/AHSCT

is never introduced into mainstream MS treatment, the immunological investigations into

its effects on the immune system will advance our understanding of MS pathophysiology

and may reveal new drug targets for the future

Key Issues

• A cohort of RRMS patients with aggressive disease do not respond to current

treatments and no current therapies are recommended for SPMS

PAGE23

• I/AHSCT can induce a sustained suppression of clinical and radiological

inflammatory activity in both RR and SPMS

• Stabilisation and even reversal of neurological disability is possible in both RR

and SPMS patients with active inflammatory disease, although this effect is more

profound in SPMS

• Preliminary evidence, including from a phase II randomised controlled trial,

suggest that I/AHSCT has superior efficacy over current pharmacological

therapies although phase III trials will be required to confirm this

• Tolerability of I/AHSCT remains a pertinent issue although safety profiles are

improving

• No clear consensus exists over the appropriate conditioning regimen for immune

ablation

• Immunological investigations have revealed that qualitative changes take place at

the cellular and molecular level which could explain sustained clinical remission

despite return of auto-reactive T-cells

• I/AHSCT activates thymic production of naïve CD4+ cells, restores the regulatory

T-cell network, diminishes pro-inflammatory T-cell responses, normalises gene

expression and down-regulates microRNAs

• In the future there is a need for phase III trials to validate the superiority of

I/AHSCT against current best therapies and to determine the optimal conditioning

regimen.

• Further mechanistic understanding of I/AHSCT is required including how

immune ablation intensity affects immune reconstitution and how the innate

immune system, including microglia, reconstitute

Conflict of interest statement

FC and MK have no relevant conflicts of interests to report. PAM declares honoraria for

speaking and travel support from Merck Serono, Biogen, Bayer, and Novartis.

PAGE24

Reference Annotations

** Atkins et al. 2016

Phase II clinical trial demonstrating complete suppression of inflammatory activity in RR

and SPMS patients up to 12.7 years after I/AHSCT.

* Nash et al. 2015

3 year interim analysis of HALT-MS trial investigating I/AHSCT in RRMS patients.

Results shows disease stabilisation and neurological improvement.

** Mancardi et al. 2015

Phase II comparative trial demonstrating superior action of I/AHSCT over mitoxantrone

at suppressing inflammatory activity in RR and SPMS patients.

** Burt et al. 2015

Non-myeloablative I/AHSCT can induce significant neurological improvement in RRMS

patients

* Muraro et al. 2005

First demonstration that immunomodulatory changes take place alongside

lymphodepletion after I/AHSCT. A new and diverse T-cell repertoire is formed due to

thymus activation

** Muraro et al. 2014

In the post-transplant environment, T cell receptor diversity increases, the majority of

CD4+ cell clones are new and the majority of CD8+ cell clones were also present in the

pre-transplant environment

* Sousa et al. 2015

I/AHSCT causes a renewal and relative normalization of gene expression profiles in

CD8+ and CD4+ cells

PAGE25

* Arruda et al. 2014

I/AHSCT results in a down-regulation of microRNAs implicated in MS pathophysiology

with a subsequent up-regulation of their target genes and downstream proteins

** Darlington et al. 2013

Auto-reactive T-cells have a reduced pro-inflammatory interleukin-17 response post-

transplant

* Abrahamsson et al. 2013

Non-myeloablative I/AHSCT, including alemtuzumab in the conditioning regimen, causes

a sustained depletion in MAIT cells, whose implication in MS pathophysiology is

demonstrated by their presence in MS post-mortem CNS lesions

PAGE26

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Pre-transplant 6mo 12mo 24mo18mo

TimePost-Transplant

Freq

uency

CMCD4+

NaïveCD4+

Thymicoutput

MAIT

Tregs

EMCD4+

CD8+/CD28-/CD57+