695892

research-article2017
MSJ0010.1177/1352458517695892Multiple Sclerosis JournalE Giacomini, F Rizzo

MULTIPLE
SCLEROSIS MSJ
JOURNAL

Original Research Paper

Thymosin-α1 expands deficient IL-10- Multiple Sclerosis Journal

1­–13

producing regulatory B cell subsets in DOI: 10.1177/

https://doi.org/10.1177/1352458517695892
1352458517695892
https://doi.org/10.1177/1352458517695892

relapsing–remitting multiple sclerosis patients © The Author(s), 2017.

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Elena Giacomini, Fabiana Rizzo, Marilena P Etna, Melania Cruciani, Rosella Mechelli,
Maria Chiara Buscarinu, Francesca Pica, Cartesio D’Agostini, Marco Salvetti,
Eliana M Coccia and Martina Severa

Abstract Correspondence to:

EM Coccia
Background: B cells are key pathogenic effectors in multiple sclerosis (MS) and several therapies have Department of Infectious
Diseases, Istituto Superiore
been designed to restrain B cell abnormalities by directly targeting this lymphocyte population. di Sanità, Viale Regina Elena
Objectives: Moving from our data showing a Toll-like receptor (TLR)7-driven dysregulation of B 299, Rome 00161, Italy.
eliana.coccia@iss.it
cell response in relapsing–remitting multiple sclerosis (RRMS) and having found a low serum level Elena Giacomini
of Thymosin-α1 (Tα1) in patients, we investigated whether the addition of this molecule to peripheral Fabiana Rizzo
Marilena P Etna
blood mononuclear cells (PBMCs) would influence the expansion of regulatory B cell subsets, known to Melania Cruciani
dampen autoimmune inflammation. Eliana M Coccia
Martina Severa
Methods: Serum Tα1 level was measured by enzyme immunoassay. Cytokine expression was evaluated Department of Infectious
Diseases, Istituto Superiore di
by Cytometric Bead Array (CBA), enzyme-linked immunosorbent assay (ELISA), and real-time reverse Sanità, Rome, Italy
transcription polymerase chain reaction (RT-PCR). B cell subsets were analyzed by flow cytometry. Rosella Mechelli
Maria Chiara Buscarinu
Results: Tα1 pre-treatment induces an anti-inflammatory status in TLR7-stimulated RRMS PBMC Marco Salvetti
cultures, reducing the secretion of pro-inflammatory interleukin (IL)-6, IL-8, and IL-1β while sig- Centre for Experimental
Neurological Therapies
nificantly increasing the regulatory IL-10 and IL-35. Indeed, Tα1 treatment enhanced expansion of (CENTERS), Sapienza
CD19+CD24+CD38hi transitional-immature and CD24low/negCD38hi plasmablast-like regulatory B cell University of Rome, Rome,
Italy
subsets, which likely inhibit both interferon (IFN)-γ and IL-17 production. Francesca Pica
Conclusion:: Our study reveals a deficient ability of B cells from MS patients to differentiate into regula- Department of Experimental
Medicine and Surgery,
tory subsets and unveils a novel anti-inflammatory and repurposing potential for Tα1 in MS targeting B University of Rome Tor
Vergata, Rome, Italy
cell response.
Cartesio D’Agostini
Department of Experimental
Medicine and Surgery,
University of Rome Tor
Keywords: Multiple sclerosis, B-lymphocytes, regulatory, thymosin alpha1 Vergata, Rome, Italy/Clinical
Microbiology Laboratories,
Tor Vergata Hospital, Rome,
Date received: 4 November 2016; revised: 16 January 2017; accepted: 1 February 2017 Italy
E.G. and F.R. shared first
co-authorship. E.M.C. and
M.S. equally contributed to
this work.
Introduction pathology of MS has provided momentum to explore
Multiple sclerosis (MS) is a complex disease affect- more specific and hopefully more effective immune-
ing the central nervous system with both autoimmune based therapeutic strategies.
and neurodegenerative features, in which the strong
interaction between specific genetic components and In this study, for the first time we explore the potential
environmental factors leads to chronic activation of ability of Thymosin-α1 (Tα1) to modulate the inflam-
inflammatory processes. The complexity of the dis- matory milieu in MS.
ease and its diverse clinical course has lead over the
years to many therapeutic options. However, despite Tα1 is a 28-amino-acid peptide, mainly produced in
the enlargement of the drug armamentarium, approved the thymus gland, belonging to a family of hormone-
disease-modifying drugs display only partial efficacy. like molecules discovered back in the 1960s for their
Advances in understanding the pathophysiology and ability to stimulate the immune system.1 Considered a

journals.sagepub.com/home/msj 1
Multiple Sclerosis Journal 
specific regulator of lymphocyte functions and intra-
cellular pathways, Tα1 has long been regarded as a
molecule retaining pleiotropic effects toward several
pathological conditions,2–4 especially acting as a mod-
ulator of immune responses and inflammation.5 In
this regard, effects of Tα1 were shown on different
immune cell subsets, most importantly on dendritic
cells (DC)6,7 and on subpopulations of T cells.8,9 In
particular, we recently demonstrated that Tα1, by act-
ing as a context-dependent molecule, could regulate
microbial-induced functional maturation in DC and
dampen pro-inflammatory cytokine milieu depending
on cell insult.7 Furthermore, Tα1 treatment was shown
to drive regulatory T cell differentiation and prolifera-
tion in different settings.8,10
Synthetic Tα1 has been used worldwide and is cur-
rently in advanced-stage clinical trials for the treat-
ment of many malignancies and infectious diseases as
well as an adjuvant in vaccine formulations display-
ing an excellent safety profile and tolerability and
increased effectiveness of chemotherapies or anti-
infection drugs in combination therapies.11
Tα1’s ability to establish a regulatory environment for
balance of inflammation and tolerance might be of
key importance in novel therapeutic applications
toward autoimmune diseases and MS.
In light of growing evidences on the disease-driving
relevance of B cells in MS12 and moving from our data
showing a Toll-like receptor (TLR)7-driven dysregu-
lation of B cell response in MS patients,13,14 in this in
vitro study we assessed the capacity of synthetic Tα1
to modulate the inflammatory milieu and the genera-
tion of regulatory B cell (B reg) subsets induced by
T cell-independent stimuli in MS-affected individuals,
interestingly showing a low serum level of endoge-
nous Tα1 as compared to matched healthy controls.
Materials and methods
Patients
A total of 20 untreated patients with definite relapsing–
remitting multiple sclerosis (RRMS) according to
McDonald’s criteria15 [median age ± standard devia-
tion (SD) = 39 ± 7 years (range = 20–51); female-to-
male ratio = 1:5] were enrolled at the S. Andrea
Hospital MS Center (Sapienza University, Rome,
Italy).
Median Expanded Disability Status Scale was 1
(range = 0–3), and median disease duration was
4 years (range = 1–9). Patients had not been taking
2 journals.sagepub.com/home/msj
steroids or any disease-modifying drugs for at least
three months prior to their enrollment in the study.
Additional exclusion criteria included presence of
other disorders that may be associated with abnormal
immune response and pregnancy. Demographic and
clinical data are detailed in Table 1.
Twenty age- and sex-matched healthy donors (HD)
were also enrolled (median age ± SD = 37.5 ± 7 years,
range = 27–53; female-to-male ratio = 1:5). Demo-
graphic data are listed in Table 2. The study was
approved by the Ethics Committee of S. Andrea
Hospital (CE 204/10), and all the subjects involved in
the study gave written informed consent.
Cell isolation and stimulation
Peripheral blood mononuclear cells (PBMCs) were
isolated from peripheral blood (~40 mL) withdrawn
from MS patients and paired HD by density gradient
centrifugation using Lympholyte-H (Cedarlane
Laboratories, Burlington, ON, Canada) and cultured
(1 × 106cells/mL) in Roswell Park Memorial Institute
(RPMI) 1640 (Lonza, BioWhittaker Europe, Verviers,
Belgium) supplemented with 2 mM L-glutamine,
100 U/mL penicillin, 100 µg/mL streptomycin (Gibco,
Thermo Fisher Scientific, Waltham, MA, USA), and
10% fetal bovine serum (FBS) (Lonza, BioWhittaker
Europe).
Whole PBMCs were then stimulated with optimal
dose of a specific TLR7 agonist (25 µM 3M001, a
kind gift of Dr Mark Tomai, 3M pharmaceuticals).13,16
Where indicated, 100 ng/mL Tα1 (Sigma–Tau,
Pomezia, Italy) was added 2 hours before the TLR7
agonist treatment as previously described.7
Supplementary Table 1 and Supplementary Figure 1
report preliminary dose-response experiments that
were conducted to choose dose to be used in the study
and evaluate impact of 10 or 100 ng/mL Tα1 on the
survival of CD19+ B cells, CD3+ T cells, and CD14+
monocytes (Supplementary Table 1), as well as Tα1
effect on Immunoglobulin (Ig) M or IgG production
(Supplementary Figure 1) upon 7 days of culture.
Flow cytometry analysis
Monoclonal antibodies (mAbs) for CD24 (clone #ML5,
R&D Systems), CD19 (clone #HIB19), CD38 (clone
#HIT2), CD86 (clone #FUN-1), CD27 (clone #M-T271),
CD138 (clone #M-T271), IgD (clone #IA6-2), and CD5
(clone #UCHT2) as well as IgG1, IgG2a isotype con-
trols (BD Pharmingen, San Diego, CA, USA), conju-
gated with different fluorochromes as needed, were used
to stain PBMCs. Briefly, cells (1 × 105) were collected
 E Giacomini, F Rizzo et al.

Table 1. Main demographic and clinical characteristics of MS patients.

Sex Age (years) Disease duration EDSS Gd+ lesions on

(years) MRI
RRMS1 M 45 1 0 +
RRMS2 M 42 1 1 −
RRMS3 F 45 3 3 −
RRMS4 F 26 4 1 −
RRMS5 F 34 2 0 +
RRMS6 F 42 3 3 +
RRMS7 M 45 7 1 −
RRMS8 M 39 3 0 −
RRMS9 M 46 8 1 −
RRMS10 F 33 4 0 −
RRMS11 M 39 2 0 −
RRMS12 F 51 9 3 −
RRMS13 F 33 8 3 −
RRMS14 M 34 1 2 +
RRMS15 F 37 7 2 −
RRMS16 F 33 5 0 −
RRMS17 F 20 1 0 −
RRMS18 F 44 6 3 +
RRMS19 M 35 7 1 −
RRMS20 F 45 5 0 −
EDSS: expanded disability status scale; Gd: gadolinium; MRI: nuclear magnetic resonance imaging; RRMS: relapsing–remitting
multiple sclerosis.
The reported characteristics are referred to patients at the moment of blood withdrawals.

Table 2. Main demographic characteristics of healthy and washed once in phosphate-buffered saline (PBS)
donors.
containing 2% FBS, then incubated at 4°C for 30 min-
Sex Age (years) utes with Fixable Viability Dye (eBioscience, San Diego,
CA, USA) to exclude dead cells (see Supplementary
HD1 M 51
Figure 2 for live gating strategy) as well as mAbs. For
HD2 M 27
intracellular interleukin (IL)-10 production, PBMCs
HD3 M 34
from HD or MS patients at day 7 of culture were treated
HD4 M 35
HD5 F 39
for 6 hours with phorbol 12-myristate 13-acetate (40 ng/
HD6 M 30 mL) and Ionomycin (1 µg/mL) (Sigma–Aldrich, St.
HD7 F 39 Louis MO, USA). Then, upon surface labeling with B
HD8 F 47 cell lineage markers and dead cell exclusion, intracellu-
HD9 F 53 lar staining for IL-10 (anti-IL-10-APC mAb, clone
HD10 F 36 #JES3-19F1; BD Pharmingen) was performed using the
HD11 F 35 Intracellular Fixation & Permeabilization Buffer Set
HD12 F 35 (eBioscience).
HD13 M 39
HD14 F 36 After staining, cells were fixed with 2% paraformal-
HD15 F 27 dehyde and run on a Gallios Instrument (Beckman
HD16 M 44 Coulter, Brea, CA, USA). Data were analyzed by
HD17 F 39 Kaluza software (Beckman Coulter) and Flow Jo soft-
HD18 M 35 ware (Tree Star, Inc., Ashland, OR, USA).
HD19 F 43
HD20 F 44
HD: healthy donor. ELISpot assay
The reported characteristics are referred to donors at the PBMCs from MS patients were cultured for 7 days
moment of blood withdrawal.
with the specific TLR7 agonist 3M001 (25 µM) in the

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Multiple Sclerosis Journal 
Figure 1. Determination of endogenous Tα1 in sera of
RRMS patients and matched healthy controls. Sera from
20 therapy-free RRMS and 20 sex- and age-matched
healthy donors (HD) were collected and Tα1 levels
evaluated by EIA test. p-values for comparisons between
two groups were calculated by Mann–Whitney U-test.
***p = 0.0008.
presence or absence of 100 ng/mL Tα1. For ELISpot
assay, cells were recovered and incubated for 3 hours
at 37°C in IgM- or IgG-coated 96-wells flat-bottomed
microtiter plates. Wells were subsequently washed
and incubated overnight at 4°C with alkaline phos-
phatase-conjugated goat anti-human IgM or IgG
(Sigma, Ronkonkoma, NY, USA). After extensive
washings with PBS-Tween, the alkaline phosphatase
substrate 5-bromo-4-chloro-3-indolyl phosphate
(Sigma) was added to each well. After rinsing and
drying, the spots were enumerated under a stereomi-
croscope with 40-fold magnification.
Cytokine determination
Supernatants from PBMCs, cultured for 1 or 7 days as
indicated, were harvested and stored at −80°C.
Determination of IL-6, IL-1, IL-8, IL-10 was
performed by Cytometric Bead Array (CBA; BD
Biosciences, San Jose, USA). IL-17 and interferon
(IFN)-γ production was measured by specific enzyme-
linked immunosorbent assay (ELISA) kits (R&D
Systems). Shown values represent the means ± stand-
ard error of the means (SEM) of the cytokine concen-
trations detected in the supernatants of cultures
collected from independent individuals.
4 journals.sagepub.com/home/msj
RNA purification and real-time RT-PCR
DNase-I-treated total RNA was purified from MS- or
HD-derived PBMCs using the RNeasy Mini Kit
(Qiagen, Valencia, CA, USA) and reverse-transcribed
by the Murine Leukemia Virus Reverse Transcriptase
(Invitrogen Life Technologies, Carlsbad, CA, USA).
Quantitative RT-PCR for the housekeeping gene
TATA-box-binding protein (TBP) and for IL-10, IL-35/
p35, and IL-35/EBI3 transcripts were done in dupli-
cates using the LightCycler FastStart DNA SYBR
Green I Master Mix in the presence of 3 µM MgCl2 on
a LightCycler Instrument (Roche Diagnostics, Basel,
Switzerland) as previously described.14
Sample values of each messenger RNA (mRNA)
were normalized to TBP using the formula 2−ΔCt.
Primer pairs used in this study were as follows:
TBP forward: 5′-ATGTTGAGTTGCAGGGTGTG-′3;
TBP reverse: 5′-CCCAGATAGCAGCACGGTAT-′3;
IL-10 forward: 5′-TGAGAACAGCTGCACCCACT
T-′3;
IL-10 reverse: 5′-GCTGAAGGCATCTCGGAGATC-′3;
IL-35/p35 forward: 5′-TGCAGGCCCTGAATTTCA
AC-′3;
IL-35/p35 reverse: 5′-CAAGGGAGGATTTTTGTG
GC-′3;
IL-35/EBI3 forward: 5′-CGTGCCTTTCATAACAG
AGCA-′3;
IL-35/EBI3 reverse: 5′-GACGTAGTACCTGGCTC
GG-′3.
Tα1 serum level determination
Sera from 20 HD and 20 MS sex- and age-matched
individuals were collected and Tα1 levels were evalu-
ated by Tα1-specific enzyme immunoassay (EIA) test
(ALPCO Diagnostic, Salem, NH, USA), according to
manufacturers’ instruction.
Statistical analysis
Statistical significance of differences was determined
using Ministat 2.1 software. Student’s t-test was used
for paired data. For unpaired data, Mann–Whitney
U-test was applied for comparisons between two
groups, whereas two-way ANOVA was utilized for
comparisons between more than two groups using
two-tailed p-values.
Results were shown as mean values ± SEM. p ⩽ 0.05
was considered significant. In the figures, star scale

was assigned as follows: *p ⩽ 0.05; **p ⩽ 0.01;
***p ⩽ 0.001.
Results
Tα1 serum level is halved in sera of MS patients
as compared to healthy individuals
MS patients show a highly inflammatory condition
and dysregulated immune responses. Because of that
we wanted to evaluate whether the endogenous Tα1
serum levels would differ in matched healthy and
MS-affected individuals. Quantitative determination
in sera collected from 20 HD and 20 RRMS subjects
clearly showed that endogenous Tα1 was signifi-
cantly lower in patients than in controls (Figure 1).
Providing that Tα1 molecule is considered as an
important player in controlling central and peripheral
immune tolerance and inflammatory status,5,7 this
result may suggest an association of the deficient
endogenous production of Tα1 with the altered
inflammatory condition observed in MS.
Tα1 promotes a regulatory milieu in cells of MS
patients upon TLR7 stimulation
Since we recently demonstrated that TLR7 pathway is
dysregulated in MS patients affecting B cell-mono-
cyte crosstalk,13,16 next we investigated whether the
exogenous addition of synthetic Tα1 to MS cells
would modulate TLR7-induced responses.
In this study, we analyzed the effect of an in vitro
exposure to Tα1 on PBMCs derived from therapy-
free RRMS patients treated for 1 and 7 days with a
specific TLR7 agonist (3M001). Culture superna-
tants were then harvested and assayed for level of the
pro-inflammatory cytokines IL-1β, IL-8, and IL-6 by
CBA (Figure 2(a)). Interestingly, the increasing
TLR7-induced production of these inflammatory
factors in culture was drastically reduced at both
analyzed time points when PBMCs were pre-treated
with Tα1. Moreover, Tα1 drove an enhancement of
IL-10 production in TLR7-treated PBMCs of MS
patients (Figure 2(b)). In particular, the already
higher induction of IL-10 observed in cells stimu-
lated for 1 day was even more pronounced at day 7 of
culture in the presence of Tα1 and TLR7 agonist as
compared to TLR7 stimulation alone. IL-10 is a
potent anti-inflammatory and immunosuppressive
cytokine that blocks immune responses by acting
directly and indirectly at different levels inhibiting
production of pro-inflammatory factors, antigen
presentation, and cell proliferation.17
journals.sagepub.com/home/msj
E Giacomini, F Rizzo et al.
Consistently with a promotion of an anti-inflamma-
tory milieu by Tα1 treatment, together with an
increased IL-10 gene expression upon 1 week of cul-
ture, we also found an induced transcription of the
two subunits p35 and EBI3 composing IL-35, another
immune-suppressive cytokine produced by regulatory
cells17 (Figure 2(c)).
Tα1 in vitro affects the differentiation of
plasmablasts in TLR7-treated PBMCs from
therapy-free RRMS patients
Having found that Tα1 impacts cytokine production
in TLR7-stimulated PBMCs derived from therapy-
free RRMS patients, we sought to investigate whether
this molecule could exert any immune-modulatory
effect on TLR-induced B cell responses.
Together with TLR9 ligands, TLR7-specific agonists
were shown to sustain B cell differentiation and matu-
ration status by driving in vitro cytokine production
and proliferation into Ig secreting cells.18
In our experimental setting, we found that a 7-day
stimulation with TLR7 agonist in the presence of Tα1
significantly increased the differentiation of circulat-
ing CD19+ B cells derived from RRMS patients into
CD38+CD138+ plasmablasts (Figure 3(a)) to a level
closer to that found in TLR7-treated cells derived
from HD (Figure 3(b)). Conversely, Tα1 displayed no
effect in HD (Figure 3(b)). However, this enhanced
differentiation into plasmablasts did not correlate
with an increased maturation status. Indeed, CD86
expression, whose signaling normally enhances Ig
secretion,19 was significantly reduced (Figure 3(c)
and (d)). Furthermore, when Tα1 was added to TLR7-
treated RRMS PBMC cultures, the capacity of B cells
to differentiate into IgM- or IgG-secreting cells was
also lowered (Figure 3(e)). This scenario is in line
with a more regulatory phenotype of these cells.20
Tα1 treatment expands IL-10-producing
regulatory B cell subsets in TLR7-treated PBMC
cultures of RRMS patients
Over the last decade, many studies have identified
distinct human B reg subsets.21 In particular, two pop-
ulations have been characterized in health and dis-
ease, CD19+CD24+CD38hi transitional-immature B
reg arising from CD27− B cells22 and CD24low/neg
CD38hi plasmablast-like B reg cells differentiating
from CD27+ memory B cell subset.23
Our in vitro studies show that upon TLR7 stimula-
tion in PBMC cultures of RRMS patients B
5
Multiple Sclerosis Journal 

Figure 2. Recombinant Tα1 treatment skews the TLR7-induced pro-inflammatory status in RRMS patients toward a
more anti-inflammatory and regulatory environment. PBMCs from therapy-free RRMS patients were stimulated for 1
or 7 days as specified with either 100 ng/mL synthetic Tα1, 25 µM 3M001 (a specific TLR7 agonist) or both of them.
(a) Levels of IL-1β, IL-8, and IL-6 or (b) IL-10 were measured in culture supernatants by Cytometric Bead Array.
*p ⩽ 0.03. (c) Transcription level of IL-10 gene and genes encoding the subunits IL-35/p35 and IL-35/EBI3 were
evaluated by quantitative real-time RT-PCR in total RNA of PBMCs cultured for 7 days as described above. Tata-binding
protein (TBP) was used as housekeeping gene and Ct values normalized by 2−ΔCt formula. **p = 0.01. Data are shown as
mean ± SEM obtained from six MS patients. p-values for paired data were calculated by Student’s t-test.

cells differentiate mainly in CD19+CD24intCD38int
(primarily mature) and CD19+CD24hiCD38− (primar-
ily memory) B cells (Figure 4(a)). However, Tα1 treat-
ment expands both transitional-immature and
plasmablast-like B reg subsets, which were almost
completely absent in RRMS PBMCs treated only with
TLR7 agonist (Figure 4(a)), to the levels found in HD
(Figure 4(b) and (c)). Interestingly, both populations
differentiated at a much higher level in TLR7-treated
HD cells than that found in RRMS, and Tα1 did not
change their frequency (Figure 4(b) and (c)).
In accordance, the combination of synthetic Tα1 and
TLR7 ligation strongly enhanced the intracellular
IL-10 production in CD19+-gated B cells (Figure 5(a))
to a level similar to TLR7-treated HD cells (Figure
5(b)). Interestingly, IL-10 expression was confined to
both transitional-immature and plasmablast-like B reg

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 E Giacomini, F Rizzo et al.

Figure 3. In vitro Tα1 stimulation of PBMCs from RRMS patients enhances TLR7-induced CD38+CD138+ plasmablast
differentiation but reduces their maturation status and Immunoglobulin (Ig) production. PBMCs from therapy-free RRMS
patients or healthy donors (HD) were stimulated for 7 days with 25 µM of specific TLR7 agonist 3M001 in presence or
absence of 100 ng/mL synthetic Tα1. B cell phenotype was assessed by staining with CD19, CD27, CD138, and CD38
surface antigens and analyzed by flow cytometry in gated Fixable Viability dye negative live cells. The percentage (%)
of CD38+CD138+ plasmablasts was evaluated in CD19+CD27+ B cell gate. (a) Representative dot plots derived from one
RRMS patient are depicted. (b) Mean ± SEM of results derived from six MS and six matched HD are shown. p-values
for paired data were calculated by Student’s t-test; *p = 0.022. p-values for unpaired data were calculated by two-way
ANOVA test; **p = 0.0015. CD86 expression was evaluated in CD19+ total B cell gate. (c) Representative overlays on
untreated cells derived from one patient. (d) Mean ± SEM of results derived from six MS and six HD. (e) In the same
culturing conditions, IgM- and IgG-secreting cells (SC) were detected and enumerated by isotype-specific ELISpot assay.
Values represent mean ± SEM of results obtained from six MS patients. p-values were calculated by Student’s t-test. For
IgM: **p = 0.01, for IgG: *p = 0.036.

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Multiple Sclerosis Journal 

Figure 4. Tα1 treatment drives proliferation of regulatory B cell subsets in PBMC cultures of RRMS patients. PBMCs
from therapy-free RRMS patients were stimulated for 7 days with 25 µM of specific TLR7 agonist 3M001 in presence or
absence of 100 ng/mL synthetic Tα1. B cell subsets were characterized by staining PBMCs with CD19, CD27, CD24, and
CD38 surface antigens and analyzed by flow cytometry in gated Fixable Viability dye negative live cells. The percentage
(%) of CD24hiCD38hi transitional-immature B reg and CD24low/negCD38hi regulatory plasmablasts was evaluated in
CD19+ total B cell gate. (a) Representative dot plots derived from one patient are depicted. (b) and (c) Mean ± SEM of
percentage (%) of subsets in CD19+ B cells derived from six MS and six matched healthy donors (HD) are shown. p-
values for paired data were calculated by Student’s t-test; whereas p-values for unpaired data were calculated by two-way
ANOVA test; **p ⩽ 0.01.

subsets and in our settings, it was mainly a preroga- Discussion

tive of CD24low/negCD38hi regulatory plasmablasts
(Figure 5(c)).
In inflammatory conditions, the regulatory activity of
IL-10-producing B reg subsets is exerted by function-
ally suppressing IFN-γ+ Th1/IL-17+ Th17 CD4+ T cell
responses.23–25 Taking advantage of our experimental
setting based on the mixed cell population of MS
patient–derived PBMCs, we evaluated whether the
expansion of both Tα1-induced transitional-immature
and plasmablast-like B reg subsets correlated with the
reduction of pathogenetic Th1 and Th17 populations,
as evaluated by the release of IFN-γ and IL-17.
Interestingly, the addition of Tα1 to TLR7 agonist-
treated PBMC cultures partially decreased the release
of IFN-γ and dramatically suppressed the production
of IL-17 suggesting the involvement of IL-10-
producing regulatory B cell populations in this inhibi-
tory effect (Figure 6).
All together, these findings highlight the therapeutic
potential of Tα1 in MS as well as in other autoim-
mune conditions that show reduced differentiation of
B reg subsets and chronic immune activation.
Accumulating evidence has brought B cells into focus
as critical players in MS immune-pathogenesis.12,26
However, even if historically B cell implication in MS
was linked to abnormally increased Ig levels in the
cerebro-spinal fluid of patients as well as Ab-deposition
in brain lesions,27 important Ab-independent patho-
genic roles of B cells are emerging, also in light of
successful results of B cell-depleting therapies in
MS.28
In particular, B cells can efficiently present antigens
to T cells and modulate local immune responses by
secreting soluble factors. Distinct B cell subsets with
different functional properties have been identified in
humans based on their ability to express on one hand
IL-10 and IL-35 (B reg cells) and on the other hand
Tumor necrosis factor (TNF)-α, lymphotoxin (LT),
IL-6, or granulocyte-macrophage colony stimulating
factor (the so-called pro-inflammatory B cells).17 This
cytokine network was found dysregulated in patients
with MS, whose B cells exhibit a decreased average
production of the anti-inflammatory cytokine IL-10.29
Furthermore, B cells of MS patients exhibit aberrant
pro-inflammatory responses, with increased LT:IL-10

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 E Giacomini, F Rizzo et al.

Figure 5. In vitro Tα1 stimulation expands IL-10-producing regulatory B cell subsets in TLR7-treated PBMCs of
RRMS patients. PBMCs from therapy-free RRMS patients were stimulated for 1 or 7 days as specified with either
100 ng/mL synthetic Tα1 or 25 µM 3M001 (a specific TLR7 agonist) or both of them. A total of 40 ng/mL PMA and
1 µg/mL Ionomycin were added for the last 6 hours of culture. CD19+IL-10+ B cells were measured by intracellular
cytokine staining. IL-10 expression in CD19+ live B cells was assessed relative to an isotype matched control mAb. (a)
Representative dot plots derived from one patient are depicted. (b) Mean ± SEM of percentage (%) of CD19+IL-10+ B
cells derived from six MS and six matched healthy donors (HD) are shown. p-values for paired data were calculated by
Student’s t-test; **p = 0.01. p-values for unpaired data were calculated by two-way ANOVA test; ***p = 0.0002. (c) IL-
10-producing B cell subsets were characterized by staining PBMCs with CD19, CD27, CD24, and CD38 surface antigens
and analyzed by flow cytometry in CD19+ live B cells. Representative dot plots from one patient are shown.

Figure 6. Tα1-expanded IL-10-producing regulatory B cell subsets are functionally suppressive. PBMCs from therapy-
free RRMS patients were stimulated for 7 days with synthetic Tα1 (100 ng/mL) in presence or absence of the specific
TLR7 agonist 3M001 (25 µM). IFN-γ and IL-17 production were assayed by specific ELISA kits in culture supernatants.
Shown values represent mean ± SEM obtained from experimental data of four independently processed RRMS patients.
p-values were calculated by Student’s t-test; *p < 0.05.

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Multiple Sclerosis Journal 
ratio and exaggerated LT and TNF-α secretion, that
may mediate “bystander activation” of disease-rele-
vant pro-inflammatory T cells, resulting in new
relapsing MS disease activity.30
Thus, research in MS field is seeking for strategies to
target or modulate the pro-inflammatory responses into
a more anti-inflammatory scenario and, in this regard,
manipulating B reg subsets might be successful.
Wolf et al. first observed that B10.PL mice lacking B
cells suffered an unusually severe and chronic form of
experimental autoimmune encephalomyelitis, the MS
mouse model, suggesting that anti-inflammatory B
cells in charge of negatively regulating inflammatory
reactions might possibly be depleted.31 However, the
term “regulatory B cells” was first introduced by
Mizoguchi and Bhan32 10 years later. Since then, the
family of B reg is expanding with many subsets iden-
tified and shown to arise at different stages of B cell
differentiation in a context-dependent manner.21 In
particular, among many others, two main B reg popu-
lations have been deeply characterized: CD24+CD38hi
transitional-immature B reg arising from CD27− B
cell compartment22 and CD24low/negCD38hi plasmab-
last-like B reg cells differentiating from CD27+ mem-
ory B cells.23
While many studies showed that B cell-derived IL-10
production is strongly down-regulated in MS
patients,29,30 only few studies have characterized so far
B reg populations in MS using different Ab cocktails,
thus having contrasting results and showing either no
difference or reduction in IL-10-producing B reg sub-
sets in MS as compared to healthy individuals.33–36
Here, we set up an experimental procedure to study
differentiation of B reg subsets not in cultures of puri-
fied B cells but in the context of the mixed cell popu-
lation of PBMCs resembling the peculiar in vivo
scenario found in either patients or HD. In particular,
upon stimulation with TLR7 agonist, PBMCs could
account for cytokine production as well as triggering
of CD40 signaling generating an in vitro B cell prolif-
erating milieu unique for either healthy individuals or
patients.
Our interest on TLR7 pathways in MS is long-lasting.
Besides the knowledge that dysregulation in TLR7
gene expression was implicated at different levels in
autoimmunity as well as specifically in MS disease
progression,37 we also recently demonstrated that
TLR7 responses of MS-derived plasmacytoid DC and
B cells are dysregulated.13,16 Thus, besides TLR9
stimulation (the most used stimulus in B reg inducing
10
in vitro settings22,23), understanding TLR7-mediated
skewing of MS B cells into regulatory cells might
reveal important insights of this disease.
Our study demonstrated a striking difference in the abil-
ity of B cells of RRMS patients, as compared to matched
controls, to differentiate into both CD24+CD38hi transi-
tional-immature and CD24low/negCD38hi plasmablast-
like B reg subsets upon TLR7 stimulation.
Interestingly, in the same patients we found, for the
first time, a deficient endogenous production of Tα1.
Concentration of Tα1 in human serum is very high in
fetuses and newborns, when the immune system is
first developing but rapidly drops in early childhood
coincident with the maturation of T cells in the body
remaining to a steady-state level through adulthood.
However, deregulation in Tα1 serum concentrations
were found in different types of cancers or infectious
diseases and, more recently, in patients affected with
chronic inflammatory autoimmune diseases (includ-
ing psoriatic arthritis, rheumatoid arthritis, and sys-
temic lupus erythematosus), which have a much
lower level of serum Tα1 as compared to healthy
controls.38
We hypothesized that the deficient endogenous Tα1
level found in sera of MS patients could be related to
MS-associated altered inflammatory status. Thus, we
investigated whether the exogenous addition of
recombinant Tα1 to MS cells would regulate TLR7-
induced responses of peripheral blood cells from
RRMS patients. In vitro exposure to Tα1 drastically
reduced the TLR7-induced production of the pro-
inflammatory cytokines, IL-1β, IL-8, and IL-6, while
increasing expression of the anti-inflammatory medi-
ators IL-10, and IL-35. Accordingly, Tα1 treatment
restored the ability of RRMS B cells to differentiate
into IL-10-producing transitional-immature B reg and
regulatory plasmablasts to the level found in HD.
B reg cells suppress proliferation of inflammatory
CD4+ T cells as well as the release of IFN-γ, TNF-α
and IL-17; this mechanism is partially mediated
directly through the production of IL-10.22,25 In this
study, the B reg subsets expanded in Tα1 plus TLR7
agonist-treated MS patient–derived PBMC cultures
are likely to be involved in the reduction of IFN-γ and
IL-17 production, thus suggesting a suppressive activ-
ity of these cells.
However, further studies are needed to identify in a
larger cohort of MS patients and at different stages of
disease if the positive effects described in this study
are ascribable to a specific group of patients or
journals.sagepub.com/home/msj

clinical stage or rather generalized to individuals
affected by MS disease.
The results reported in this study are interesting in
view of the impressive number of data supporting the
potential clinical benefits and tolerability of synthetic
Tα1 produced after 1985, when this molecule was first
used in a clinical trial. Since then, Tα1 was tested in
combination with chemotherapeutic agents or antiviral
drugs for the treatment of cancer or chronic infectious
diseases, as well as an adjuvant for influenza and hep-
atitis B antiviral vaccines in subjects immunocompro-
mised due to age or hemodialysis.11 This clinical usage
in cancer or chronic infections, in which a pro-inflam-
matory activity and an immune potentiation is needed,
might seem difficult to reconcile with its use in auto-
immune conditions where an anti-inflammatory action
is desirable. However, Tα1 was characterized as a
pleiotropic molecule acting as a context-dependent
immune-modulator able to shift an inflammatory
milieu toward a more anti-inflammatory one or vice-
versa, depending on the stimuli or pathological condi-
tion.7,39 In accordance with this view, it has been
already demonstrated that Tα1 treatment may induce
regulatory T cell generation and IL-10 production,6 a
beneficial and advisable function for a drug to be used
in autoimmune diseases. Furthermore, another mem-
ber of the thymosin family, thymosin-β4, was shown
to promote neurological functional recovery in murine
model of MS40 as well as oligodendrogenesis in the
demyelinating central nervous system41 suggesting
that Tα1 may exert a similar action, even if further
studies are needed to investigate this aspect.
In light of the growing interest in the MS field for
repurposing already licensed molecules, due to sky-
rocketing costs and never-ending process for develop-
ing new therapeutics, Tα1 could be taken into account,
alone or in combination with other approved drugs,
for treatment of MS or other autoimmune conditions
that display reduced differentiation of B reg subsets
and chronic immune activation.
Acknowledgements
The authors acknowledge Dr Silvia Romano and Dr
Arianna Fornasiero, who took care of patients and
helped with sampling. They are thankful to Eugenio
Morassi (Division Service for Data Management,
Documentation, Library, and Publishing Activities,
Istituto Superiore di Sanità, Rome, Italy) for prepar-
ing drawings and to Prof. Enrico Garaci (IRCCS San
Raffaele Pisana and San Raffaele University (Rome,
Italy) for helpful discussion. E.G. and F.R. shared first
co-authorship. E.M.C. and M.S. equally contributed
to this work.
journals.sagepub.com/home/msj
E Giacomini, F Rizzo et al.
Declaration of Conflicting Interests
The author(s) declared the following potential
conflicts of interest with respect to the research,
authorship, and/or publication of this article: Marco
Salvetti received lecture fees from Biogen–Dompé,
research support from Bayer–Schering, Biogen–
Dompé, Merck Serono, and Sanofi–Aventis. Enrico
Garaci is a thymosin patent holder.
Funding
The author(s) disclosed receipt of the following
financial support for the research, authorship, and/or
publication of this article: This work was supported
by the Italian Multiple Sclerosis Foundation projects
#2013/R/9 (to E.M.C.) and #2014/R/19 (to M.S.) and
from Istituto Superiore di Sanità (to E.M.C.).
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