To the Editor.
The beneficial effects of Allergen Specific Immunotherapy (AIT) relies
on the induction of allergen-specific Regulatory T-cells (Tregs) (1).
Tregs, a subpopulation of CD4+CD25+T-cells expressing the specific transcription factor Foxp3, are not
functionally homogeneous and their detection is complex and uncertain
due to FoxP3 intracellular localization. Furthermore,
FoxP3+ Tregs might become unstable and halt the
production of their functional suppressive cytokines in inflammatory
conditions (2) (1). In its place, the surface antigen CD127, whose
expression inversely correlates with FoxP3, conveniently identifies
Tregs as
CD4+CD25+CD127negcells (3) (2), so surmounting the problems of FoxP3 stability and
intracellular detection. Tregs also constitutively express the
inhibitory antigen CD39, enhanced in highly suppressive memory Tregs (4)
(3). Furthermore, HLA-DR expression is a monitor of Treg differentiation
status and identifies a functionally and greatly suppressive population
(1,5) (1,5). Lack of CD45RA characterizes memory T cells enabled to
survive for long periods, even in absence of specific antigen, showing
increased activity upon re-exposure and able to induce apoptosis in
target cells (6) (3).
CD4+CD25highCD39+CD127negcells are subtyped as Resting
(CD45RA+/HLA-DRneg: rTreg),
Activated (CD45RAnegHLA-DRneg:
aTreg) and Effector
(CD45RAnegHLA-DRlow/high: eTreg)
Tregs (6) (6). This latter subtype includes terminally differentiated
Tregs, the most highly suppressive (5) (7) (Supplementary Figure 1).
They are different from secreting or type III Tregs expressing CD127
that represent a short-lived terminally differentiated population
(5,6,8) . In order verify possible correlations between specific subsets
of Treg and the effectiveness of AIT, we applied this analytical
approach to study Treg profile in adolescents suffering from mite
allergic rhinitis, pre and 12 months post Sublingual Immunotherapy
(SLIT) with mite monomeric allergoid, an acid-resistant allergen known
to elicit early T reg-activation (7,8). The study was approved by the
Ethical Committee of University “G. d’Annunzio”, Chieti-Pescara. All
patients and parents signed a written informed consent after having been
informed about the procedures of the study.
Twenty patients diagnosed with mite-allergic persistent rhinitis with or
without asthma were enrolled. Allergic rhinitis (AR) was graded
according to ARIA guidelines in 1) intermittent mild, 2) intermittent
moderate/severe, 3) persistent mild and 4) persistent moderate/severe.
At the enrollment, each patient marked in a 100 mm visual analogic scale
(VAS) the level of its health status related to allergy with 0 the best
status and 100 the worst.
All patients were treated by SLIT with mite monomeric allergoid (LAIS -
Lofarma, Milan, Italy) at 1000 UA four times/week every other day, for
12-months. No adverse local and systemic reactions were detected. The
effectiveness of SLIT was established comparing VAS, ARIA grading and
ACT questionnaire performed after 12-months of treatment with their
basal values. Two blood samples were drawn pre/post SLIT to be analyzed
for Regulatory T-cells. Clinical and demographic details of the studied
population, analytical methods, statistical approach and the outline of
the study are detailed in the online supplementary material .
Rhinitis scores VAS and ARIA significantly decreased after SLIT (Table
1), with the same statistical significance (Wilcoxon z -3.7236; p =
0.0002). Improvement was evidenced also in the subgroup of asthmatic
patients (n=7) since ACT scores significantly increased from the
baseline value of 18 (16-19) up to 24 (20-25) after 12 months of
treatment (the low number of patients does not allow application of
efficient statistics).
Tregs were analyzed as frequency of total Treg cells and their three
subsets, namely Resting (rTregs), Activated (aTregs) and Effector
(eTregs), within the parental population of CD4+cells. Total Tregs did not change significantly; rTreg significantly
decreased (Wilcoxon z-3.6214,
p<0.0003), while, the abundance of aTregs and eTregs
significantly incremented (Wilcoxon z-2.9011, p<0.05 and
z-3.077, p=0.002, respectively) (Table 1). A significant negative
correlation has been observed between the decrease in rTreg and the
increase in aTreg (Spearman’s ρ-0.69391, p<0.02) and increase
in eTreg cells (Spearman’s ρ-0.56845, p<0.02) (Figure 3 in
supplementary material).
HLA-DR resulted significantly up-regulated in all Tregs from 4.93±3.1 to
6.92±5.1 MFI (Wilcoxon z-4.2026, p <0.00001). HLA-DR increased
on aTregs from 3.4±3.03 to 4.91±3.2 MFI (Wilcoxon z-3.2479, p=0.001) and
on eTregs from 1.54±0.66 to 2.0±1.45 MFI (Wilcoxon z-2.9664, p=0.005).
CD39 was found differently expressed in the three subsets of Tregs at
baseline, with Resting<Activated<Effector. After 12
months of SLIT, CD39 surface expression was found significantly
increased in all Tregs from 6.9±4 to 8.02±5 MFI (Wilcoxon z-3.1049,
p=0.001) (HLA-DR and CD39 changes are reported in Table 1). We found
some interesting correlations between laboratory data and clinical
parameters. Changes in eTregs significantly correlated with both ARIA
(Spearman’s ρ=0.58728, p=0.013) (Figure 1A) and VAS (Spearman’s
ρ=0.49172, p=0.044) (Figure 1B) variations after SLIT. While a
significant negative correlation was found between rTregs and clinical
parameter changes after treatment (Spearman’s ρ-0.48482, p=0.0491).
Changes in HLA-DR expression on all Treg cells significantly correlated
with variation in VAS pre-/post-SLIT (Spearman’s ρ=0.54104, p= 0.01376)
(Figure 1C). No other correlations were found except for the lowest
increase (< 8%) of memory Tregs (CD45RAneg)
detected in patients with the lowest levels of mite-specific serum IgE
(not shown).
To our knowledge this is the first report on successful SLIT being
associated with re-patterning of the differentiation status of Tregs,
with high rates of the most suppressive Treg subtypes: activated and
effector, characterized by higher expression of HLA-DR and CD39 both
playing inhibitory function in Tregs. Moreover, effective SLIT seems to
be associated with the generation of cells lacking CD45RA that
characterizes memory T cells with increased activity upon re-exposure to
the antigen. Our results suggest that SLIT also induced empowerment of
Treg inhibitory function, likely compensating the under-representation
of Tregs observed in allergic patients (9) (9). In AR children, there
are evidences that Tregs have defect in suppressing IgE production and
that they can be incremented by mite SLIT.
Next step of our study will be to evidence if such relationship between
effective SLIT and Treg re-patterning is present in the first months of
SLIT, with a view to profiling Tregs for the early identification of
SLIT responders/non-responders by mean of a straightforward and
non-invasive blood test.