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Self-Organized Criticality Theory of Autoimmunity

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The cause of autoimmunity, which is unknown, is investigated from a different angle, i.e., the defect in immune 'system', to explain the cause of autoimmunity. Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4(+) T cells led to the development of autoantibody-inducing CD4(+) T (aiCD4(+) T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The aiCD4(+) T cell was induced by de novo TCR revision but not by cross-reaction, and subsequently overstimulated CD8(+) T cells, driving them to become antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE). Systemic autoimmunity appears to be the inevitable consequence of over-stimulating the host's immune 'system' by repeated immunization with antigen, to the levels that surpass system's self-organized criticality.
Expansion of CD8 + T cells and antigen cross-presentation. (A) Spleen cells stimulated with 50 ng/ml phorbol myristate acetate (PMA) and 500 ng/ml ionomycin for 4 h in the presence of brefeldin A (10 mg/ml) and stained for intracellular IFNc (upper). Subsets of CD8 + T cells categorized into naı¨venaı¨ve (CD44 low CD62L high ), effector (CD44 high CD62L low ), and memory (CD44 high CD62L high ) fractions (middle). Flow cytometry of IFNc + cells within naı¨venaı¨ve or effector/memory CD8 + T cell populations. Spleen cells were separated into naı¨venaı¨ve (CD44 low ) and effector/memory (CD44 high ) cells using CD44 MACS beads, and IFNc + cells within the CD8 + T population was evaluated (lower). (B) Adoptive transfer of splenocytes of OVA-immunized BALB/c mice into naı¨venaı¨ve recipients. The recipients were injected with 500 mg OVA 24 h after cell transfer, and proteinuria examined 2 weeks later. (C) Cross-presentation of OVA to CD8 + T cells. Splenic CD11c + DC from OVA-immunized or control mice were incubated in the presence (OVA(+)) or absence (OVA(2)) of 1 mg/ml OVA with or without chloroquine (CQ) (20 mg/ml) for 3 h, followed by a co-culture with KJ1-26 + CD8 + T cells of DO11.10 transgenic mice for 24 h to examine surface expression of CD69 (upper). Inhibition of cross-presentation in vivo by administration of 250 mg CQ per mouse 3 h prior to immunization with OVA or PBS. IFNc + CD8 + T cells (middle), autoantibodies and proteinuria (lower) after 126 immunization. (D) Requirement of autoantibody-inducing CD4 + T cells for CD8 + T cell-mediated autoimmune tissue injury. BALB/c mice were immunized 126with KLH, and CD4 + T cells were isolated using MACS beads. Cells were transferred into the anti-CD4 antibody-treated recipient mice immunized 86with OVA. Percent matured CTL, i.e., IFNc + CD8 + T cells, and proteinuria were measured 2 weeks after booster immunization 16 with KLH. doi:10.1371/journal.pone.0008382.g004
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Self-Organized Criticality Theory of Autoimmunity
Ken Tsumiyama
1
, Yumi Miyazaki
1
, Shunichi Shiozawa
1,2,3,4
*
1 Department of Biophysics, Kobe University Graduate School of Health Science, Kobe, Japan, 2 Department of Medicine, Kobe University Graduate School of Medicine,
Kobe, Japan, 3 The Center for Rheumatic Diseases, Kobe University Hospital, Kobe, Japan, 4 Global Center of Excellence (GCOE), Tokyo, Japan
Abstract
Background:
The cause of autoimmunity, which is unknown, is investigated from a different angle, i.e., the defect in
immune ‘system’, to explain the cause of autoimmunity.
Methodology/Principal Findings:
Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not
prone to spontaneous autoimmune diseases. Overstimulation of CD4
+
T cells led to the development of autoantibody-inducing
CD4
+
T(aiCD4
+
T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The
aiCD4
+
T cell was induced by de novo TCR revision but not by cross-reaction, and subsequently overstimulated CD8
+
T cells,
driving them to become antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-
presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE).
Conclusions/Significance:
Systemic autoimmunity appears to be the inevitable consequence of over-stimulating the host’s
immune ‘system’ by repeated immunization with antigen, to the levels that surpass system’s self-organized criticality.
Citation: Tsumiyama K, Miyazaki Y, Shiozawa S (2009) Self-Organized Criticality Theory of Autoimmunity. PLoS ONE 4(12): e8382. doi:10.1371/journal.
pone.0008382
Editor: Derya Unutmaz, New York University, United States of America
Received September 11, 2009; Accepted November 30, 2009; Published December 31, 2009
Copyright: ß 2009 Tsumiyama et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the Global Center of Excellence (GCOE) Program grant from the Ministry of Education, Culture, Sports, Science and
Technology of Japan, and the Japan Science and Technology Organization. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: shioz@med.kobe-u.ac.jp
Introduction
Since ‘clonal selection theory of immunity’ of F. Macfarlane Burnet
and subsequent molecular biological discoveries on V(D)J recombi-
nation and the diversity and individuality of immune response, how
autoimmunity arises remains unclear. Apart from the term ‘autoim-
munity’ which is now ready-made, in the present study, we tried to see
the pathogenesis of autoimmunity from different angle and test the
integrityofimmune‘system.Themethodwehavechosenwasto
stimulate the system maximally by antigen to the levels far beyond its
steady-state just like testing the capability of automobile. In a perfectly
reproducible experiments in which the mice not prone to autoimmune
diseases were immunized repeatedly with antigen, we have unexpect-
edly and surprisingly discovered that overstimulation of immune
system beyond its self-organized criticality inevitably leads to systemic
autoimmunity. Subsequent detailed molecular analyses revealed in the
first that autoantibodies are induced not by cross reaction to antigen
but by de novo T cell receptor (TCR) revision. Second, final maturation
of effector cytotoxic T lymphocyte (CTL) via antigen cross-presenta-
tion is sine qua non for generating autoimmune tissue injury. Most
importantly, we now show that autoimmunity arises not from ‘auto-
immunity’, but as a natural consequence of normal immune response
when stimulated maximally beyond system’s self-organized criticality.
Results
Induction of Autoantibodies
Consistent with the common observation that T cells become
anergic after strong stimulation with antigen [1], we observed
that 26 immunization with staphylococcus enterotoxin B
(SEB) caused SEB-reactive Vb8
+
CD4
+
T cells from BALB/c
mice to become anergized. However, these cells recovered
from anergy to divide and produce IL-2 after further immuni-
zation 86 with SEB (Figure S1A). This was accompanied by the
induction of autoantibodies, including IgG- and IgM-rheumatoid
factor (RF), anti-Sm antibody, and in particular, RF reactive
against galactose-deficient IgG, typically found in human
autoimmunity [2] (Figure 1A). Autoantibodies can also be
induced by other conventional antigens, including ovalbumin
(OVA) or keyhole limpet hemocyanin (KLH) (Figure S2) as long
as immunizing antigen is correctly presented to T cells (Figure
S1B). CD4
+
T cells of repeatedly-immunized mice become fully
matured, expressing CD45RB
lo
, CD27
lo
and CD122
hi
(data not
shown), and these primed CD4
+
T cells can confer RF generation
in naı
¨
ve recipients following adoptive transfer (Figure 1B). The
induction of autoantibodies is independent of CD8
+
T cells or
MHC class I-restricted antigen presentation for the following
reasons. First, both RF and anti-dsDNA antibody can be
consistently induced upon repeated immunization of b
2
-micro-
globulin (b
2
m)-deficient BALB/c mice with OVA. b
2
m-deficient
mice are deficient in CD8
+
T cells, which are reduced to
,0.8% of splenic T cells [3] (Figure S3). Second, the ability to
induce autoantibodies was transferable from OVA-immunized
BALB/c mice to b
2
m-deficient mice solely via CD4
+
T cells
(Figure 1C). Thus, CD4
+
T cells from repeatedly-immunized
mice acquire the ability to induce autoantibodies. We refer to
these as autoantibody-inducing CD4
+
T(aiCD4
+
T) cells in this
communication.
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Figure 1. Induction of autoantibodies and proteinuria. BALB/c mice were repeatedly injected i.p. with 25 mg SEB, 500 mg OVA or PBS every 5 d.
(A) Serum IgG- and IgM-RFs, anti-galactose-deficient IgG and anti-Sm antibodies were measured using ELISA. The arbitrary unit (AU) of 1.0 is equivalent
to the titer obtained from sera of prototypic autoimmune MRL/lpr mice. Data from each mouse are connected by dotted lines. (B) Adoptive transfer of
splenic B, T, CD4
+
TorCD8
+
T cells of SEB-, OVA- or PBS-immunized BALB/c mice into naı
¨
ve BALB/c mice. The recipient mice were given single i.p.
injection of 25
mg SEB or 500 mg OVA 24 h after cell transfer, and autoantibodies were measured 2 weeks later. (C) Adoptive transfer of cells from
OVA-immunized BALB/c mice into b
2
m-deficient mice.
doi:10.1371/journal.pone.0008382.g001
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Mechanism of Autoantibody Induction
To further clarify the characteristics of aiCD4
+
T cells, we
examined their TCR repertoire by spectratyping of their
complementarity determining region 3 (CDR3) [4]. Combinato-
rial assessment of Vb and Jb showed that the CDR3 length profiles
of CD4
+
splenocytes in mice immunized either 86 with PBS or 26
with SEB fit a normal Gaussian curve, typical of a diverse and
unbiased TCR repertoire (Figure 2A). However, splenocytes, but
not thymocytes, from mice immunized 86 with SEB showed
skewed length profiles, suggesting that TCR revision was in
progress at periphery of the spleen. Genes encoding components
of the V(D)J recombinase complex were specifically re-expressed
in mice immunized 86 with SEB, including the recombination-
activating genes 1 and 2 (RAG1/2), terminal deoxynucleotidyl
transferase (TdT) and surrogate TCRa chain (pTa) [5] (Figure 2B).
The RAG1 gene is expressed in vivo after immunization 86 with
Figure 2. TCR revision upon repeated immunization with antigen. (A) TCR CDR3 length profiles of mice immunized 86 with PBS, 26 or 86
with SEB. TCR repertoire of splenic CD4
+
T cell was skewed only after immunization 86 with SEB. (B) Expression of V(D)J recombinase complex and
related molecules in the spleen of PBS- or SEB-injected BALB/c mice. (C) GFP
+
cells in the Vb8
+
CD4
+
T population of rag1/gfp knock-in mice. IgG-RF as
induced in rag1/gfp knock-in mice after immunization 86 with SEB (lower left). The GFP
+
T cell fraction was also increased among Vb8
+
CD4
+
T cells
(mean 6 SD, 4–5 mice/group). (D) TCRa chain revision in the spleen of mice immunized 86 with SEB was determined by LM-PCR detection of dsDNA
breaks at the RSS flanking the TCRAV2, with PCR-amplified TCRa constant region (TCRAC) as a DNA quality control.
doi:10.1371/journal.pone.0008382.g002
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SEB in rag1/gfp knock-in mice [6] (Figure 2C). In these mice,
serum RF was increased in conjunction with an increase of GFP-
expressing Vb8
+
CD4
+
T cells in the spleen. To directly prove that
V(D)J recombination took place at the periphery in spleen, we
used ligation-mediated PCR (LM-PCR) to detect blunt-end DNA
fragments harboring a rearranged coding V region flanked by
recombination signal sequences (RSS) [7,8]. We identified
rearranged intermediates corresponding to the TCRa variable
region 2 (TCRAV2) in the splenocytes of mice immunized 86 with
SEB (Figure 2D). These findings indicate that repeated immuni-
zation with conventional antigen can induce the generation of
aiCD4
+
T cells which have undergone TCR revision and are
capable of stimulating B cells [9]. This observation is in line with
previous findings showing that such somatic mutations occur often
in lymphocytes, a process which is considered to be a major
stochastic element in the pathogenesis of autoimmunity [10,11].
Thus, overstimulation of CD4
+
T cells by repeated immunization
with antigen and induction of full maturation inevitably leads to
the generation of aiCD4
+
T cells which have undergone TCR
revision and are capable of inducing autoantibodies. Importantly,
the present study shows that such aiCD4
+
T cells are induced by de
novo TCR revision but not by cross-reaction to antigen.
Induction of Autoimmune Tissue Injury
Repeated immunization with OVA can also lead to autoim-
mune tissue injury and the production of autoantibodies reactive
against IgG, Sm and dsDNA (Figure 3 and Figure S2A). Serum
immune complex (IC), proteinuria, and the deposition of IC and
OVA in the kidney were noted in mice immunized 126 with OVA
(Figure 3A). Typical diffuse proliferative glomerular lesions were
seen in the kidney, and these glomeruli were infiltrated with CD8
+
T cells. These observations resemble the clinical features observed
in lupus patients, who typically exhibit an increase in CD8
+
T
cells in the peripheral blood and infiltration of CD8
+
T cells in
kidney [12,13]. Immunization of mice 126 with OVA led to re-
expression of the V(D)J recombinase complex and enlargement of
the spleen (Figure S4A), and an increase in anti-dsDNA antibody,
which is uniquely linked to autoimmune tissue injury in lupus
nephritis [14] (Figure S2A). Pathological findings included diffuse
membranous (wire-loop) and/or proliferative glomerulonephritis
in the kidney (Figure 3A), infiltration of plasma cells around
hepatic bile ducts (Figure S4B), enlarged lymphoid follicles with
marked germinal center in spleen (Figure S4B), occasional
lymphocyte infiltration into the salivary glands (data not shown),
lymphoid cell infiltration into the thyroid, and perivascular
infiltration of neutrophils and macrophages into the skin dermis
of the auricle (Figure S4B). The lupus band test, diagnostic of SLE,
was positive in the skin at the epidermal-dermal junction
(Figure 3B).
Mechanism of Autoimmune Tissue Injury
It has been shown previously that IFNc is increased in
association with autoimmune tissue injury [15–17]. Consistent
with this, we found that the number of IFNc
+
CD8
+
T cells, but
not regulatory T or T helper 17 cells, was increased following
immunization 126 with OVA (Figure 4A and data not shown).
We also observed an expansion of IFNc-producing effector/
memory CD8
+
T cells, which are necessary for adaptive immunity
[18] (Figure 4A). These IFNc-producing CD8
+
T cells were
observed to have infiltrated into OVA-deposited glomeruli of
OVA-immunized mice (Figure 3A). CD8
+
T cells are required for
tissue injury based on the following observations. First, the transfer
of CD8
+
T cells can induce renal lesions in mice (Figure 4B), as
well as the generation of new IFNc
+
CD8
+
T cells in the spleens of
recipient mice following cell transfer (Figure S5). Second,
autoimmune tissue injury is not induced by the transfer of CD8
+
T cells from OVA-immunized wild-type mice into b
2
m-deficient
mice (Figure 1C). And finally, CD8
+
T cell transfer must be
accompanied by at least a 16 booster immunization with OVA to
induce autoimmune tissue injury in the recipient mice (Figure S6).
The findings indicate that full-matured, IFNc-producing effector
CD8
+
T cells are required for the induction of autoimmune tissue
injury, provided that the relevant antigen is correctly presented on
the target organs. These are well-established characteristic of CTL
and not novel. We show, however, that (i) CTL is induced through
an immune, but not ‘autoimmune’, process, and that (ii)
autoimmune tissue injury inevitably occurs when CD8
+
T cells
are overstimulated to become matured effector CTLs. The latter
means that regardless of how CTL is induced, the consequence of
CTL over-induction is immune tissue injury.
Antigen Cross-Presentation
We next show that antigen cross-presentation is required for the
induction of CTL and tissue injury. To test this, we co-cultured
OVA-pulsed dendritic cells (DC) from mice immunized 126 with
OVA together with T cells from OVA-TCR transgenic DO11.10
mice exclusively expressing OVA-reactive TCR [19]. We show
that OVA-reactive DO11.10 CD8
+
T cells are activated upon co-
culture with OVA-pulsed DCs (Figure 4C and Figure S7). Further,
autoimmune tissue injury and the increase in IFNc
+
CD8
+
T cells,
but not of autoantibody generation, were both abrogated by
adding chloroquine (CQ), an inhibitor of antigen cross-presenta-
tion (Figure 4C). This indicates that antigen cross-presentation is
required for the expansion of IFNc-producing CD8
+
T cells and
autoimmune tissue injury.
aiCD4
+
T Cell Helps CD8
+
T Cell to Induce Tissue Injury
Since CTL appear to play a rather passive role in autoimmu-
nity, we next studied whether or not aiCD4
+
T cell help is required
for the induction of autoimmune tissue injury. Since anti-CD4
treatment almost abrogates generation of IFNc-producing CD8
+
T cell and autoimmune tissue injury in OVA-immunized BALB/c
mice (Figure S8), to test whether this CD4
+
T cell-mediated help is
mediated by aiT cells or antigen-specific T cells, we have
transferred CD4
+
T cells from mice immunized 126 with KLH
into CD4
+
T-depleted BALB/c mice immunized 86 with OVA
(Figure 4D). Because full-matured IFNc
+
CTLs do not develop
with less than 86 immunization with OVA (Figure S9), this
experiment can test the ability of aiCD4
+
T cells that have
undergone TCR revision to promote the maturation of OVA-
specific CTL. The result showed that both autoimmune tissue
injury and OVA-specific IFNc
+
CD8
+
T cells arose in these mice
after transfer, indicating that aiCD4
+
T cells with de novo TCR
revision are required for the maturation of CD8
+
T cell and
autoimmune tissue injury (Figure 4D).
Discussion
The present findings are consistent with the current consensus
that CD4
+
T cells normally die via activation-induced cell death
(AICD) after repeated exposure to a single antigen, while naı
¨
ve
CD4
+
T cells having a ‘cross-reactive’ TCR with lower affinity can
be activated through repeated exposure to the same antigen and
survive due to weak TCR signaling, ultimately acquiring
autoreactivity [20]. We show here, however, that aiCD4
+
T cells
are induced not by cross-reaction, but by de novo TCR revision.
The aiCD4
+
T cells thus generated induce not only autoantibodies
but also full-maturation of CD8
+
T cells leading to autoimmune
Cause of Autoimmunity
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Figure 3. Induction of autoimmune tissue injury. BALB/c mice were injected i.p. with 500 mg OVA every 5 d. (A) Serum IC measured 2 d after
final immunization, expressed as AU. Proteinuria assessed 9 d after final immunization: grades 1, 2 and 3 represent 30–100 mg/dl, 100–300 mg/dl and
300–1000 mg/dl of urinary protein, respectively (upper left). Representative histopathology of kidneys from mice immunized 126 with PBS or OVA
(lower left) (H&E staining, bar = 20
mm; original magnification 6400): glomerular expansion with cellular infiltration including eosinophils seen under
the same magnification. Immunohistochemistry for deposited IC, IgG, C3 and OVA (upper right) (bar = 50
mm; original magnification 6200), and cells
infiltrated into glomeruli (bar = 20
mm; original magnification 6300), stained in serial tissue sections using anti-CD8a (53–6.7) and anti-IFNc (R4-6A2)
monoclonal antibodies, in the specimens of mice immunized 126 with OVA (lower right). (B) Lupus band test stained with anti-IgG and anti-C3
antibodies (bar = 20
mm; original magnification 6400).
doi:10.1371/journal.pone.0008382.g003
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Figure 4. Expansion of CD8
+
T cells and antigen cross-presentation. (A) Spleen cells stimulated with 50 ng/ml phorbol myristate acetate
(PMA) and 500 ng/ml ionomycin for 4 h in the presence of brefeldin A (10
mg/ml) and stained for intracellular IFNc (upper). Subsets of CD8
+
T cells
categorized into naı
¨
ve (CD44
low
CD62L
high
), effector (CD44
high
CD62L
low
), and memory (CD44
high
CD62L
high
) fractions (middle). Flow cytometry of IFNc
+
cells within naı
¨
ve or effector/memory CD8
+
T cell populations. Spleen cells were separated into naı
¨
ve (CD44
low
) and effector/memory (CD44
high
) cells
using CD44 MACS beads, and IFNc
+
cells within the CD8
+
T population was evaluated (lower). (B) Adoptive transfer of splenocytes of OVA-immunized
BALB/c mice into naı
¨
ve recipients. The recipients were injected with 500
mg OVA 24 h after cell transfer, and proteinuria examined 2 weeks later. (C)
Cross-presentation of OVA to CD8
+
T cells. Splenic CD11c
+
DC from OVA-immunized or control mice were incubated in the presence (OVA(+)) or
absence (OVA(2)) of 1 mg/ml OVA with or without chloroquine (CQ) (20
mg/ml) for 3 h, followed by a co-culture with KJ1-26
+
CD8
+
T cells of DO11.10
transgenic mice for 24 h to examine surface expression of CD69 (upper). Inhibition of cross-presentation in vivo by administration of 250
mg CQ per
mouse 3 h prior to immunization with OVA or PBS. IFNc
+
CD8
+
T cells (middle), autoantibodies and proteinuria (lower) after 126 immunization. (D)
Requirement of autoantibody-inducing CD4
+
T cells for CD8
+
T cell-mediated autoimmune tissue injury. BALB/c mice were immunized 126 with KLH,
and CD4
+
T cells were isolated using MACS beads. Cells were transferred into the anti-CD4 antibody-treated recipient mice immunized 86 with OVA.
Percent matured CTL, i.e., IFNc
+
CD8
+
T cells, and proteinuria were measured 2 weeks after booster immunization 16 with KLH.
doi:10.1371/journal.pone.0008382.g004
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tissue injury akin to human SLE. Thus, induction of aiCD4
+
T
cells is a critical step, and subsequent induction of effector CTL is
a critical next step in the development of autoimmunity [21,22].
The question of how autoimmunity is triggered can therefore be
deduced to the quantitative response of host against immunizing
antigen, i.e., the ability of host to present and/or cross-present
antigen. It then follows that the ability of certain antigens such as
measles virus to cause autoimmunity may be due to their ability, in
conjunction with its ability to present antigen, to overstimulate
CD4
+
and/or CD8
+
T cells of certain hosts beyond integrity of
their immune system. Living organisms are constantly exposed to
a broad range of environmental antigens, as exemplified by the
recent re-emergence of measles virus infection among a subpop-
ulation of Japanese young adults who were not vaccinated against
the virus. We therefore conclude that systemic autoimmunity
necessarily takes place when host’s immune ‘system’ is overstim-
ulated by external disturbance, i.e., repeated exposure to antigen,
to the levels that surpass system’s self-organized criticality, and
propose here ‘self-organized criticality theory’ explaining the cause
of autoimmunity.
Materials and Methods
Ethics Statement
This study was approved by the Institutional Animal Care and
Use Committee and carried out according to the Kobe University
Animal Experimental Regulations.
Reagents
APC (allophycocyanin)-conjugated antibody against CD4
(RM4-5), and PE-conjugated antibodies against CD62L (MEL-
14), CD69 (H1.2F3) and were purchased from BioLegend (San
Diego, CA); FITC-conjugated antibodies against CD44 (IM7.8.1)
and DO 11.10 clonotypic TCR (KJ1-26) and PE-conjugated rat
IgG1 isotype control from CALTAG Laboratories (Burlingame,
CA); PE-Cy5 (phycoerythrin-cyanin 5)-conjugated antibody
against CD8a (53-6.7), PE-conjugated antibodies against Vb8
TCR (F23.1) and IFNc (XMG1.2) from BD PharMingen (San
Diego, CA).
Animal Studies
Animal studies with BALB/c female mice (Japan SLC, Inc.,
Hamamatsu, Japan) and DO11.10 TCR transgenic mice [19]
(Jackson Laboratory, Bar Harbor, ME), b
2
m-deficient mice [3]
and rag1/gfp knock-in mice [6] of BALB/c background were
performed with the approval of the Institutional Review Board.
Mice (8 weeks-old) were immunized with 25
mg SEB (Toxin
Technologies, Sarasota, FL), 500
mg OVA (grade V; Sigma, St.
Louis, MO), 100
mg KLH (Sigma) or PBS by means of i.p.
injection every 5 d.
Frozen sections of kidney and dermis were stained for C3, IgG
or OVA using goat anti-C3 (Bethyl laboratories, Inc., Montgom-
ery, TX) and Alexa Fluor 488-conjugated anti-goat IgG antibodies
(Molecular Probes, Eugene, OR), Alexa Fluor 594-conjugated
anti-mouse IgG antibody (Molecular Probes), or rabbit anti-OVA
antibody (Sigma). For CD8 or IFNc staining, paraffin-embedded
sections of kidney were stained with rat antibodies against CD8a
(53-6.7; BD PharMingen) or IFNc (R4-6A2; BD PharMingen),
followed by reaction with VECTASTAIN Elite ABC rat IgG kit
(Vector, Burlingame, CA).
To detect intracellular IFNc, cells (1610
6
/ml) were stimulated
with 50 ng/ml phorbol myristate acetate (PMA; Sigma) and
500 ng/ml ionomycin (Sigma) in the presence of brefeldin A
(10
mg/ml; Sigma). After 4 h, cells were stained with anti-CD8
antibody, followed by fixation with 2% formaldehyde, permeabi-
lization with 0.5% saponin (Sigma) and stained for IFNc.
For adoptive cell transfer, B, T, CD4
+
T and CD8
+
T cells were
isolated from spleens to .90% purity using MACS beads
(Miltenyi Biotec, Germany). The cells were transferred into naı
¨
ve
BALB/c or b
2
m-deficient mice via i.p. (5610
6
/mouse) or i.v.
(2.5610
7
/mouse) injection. The recipients received a single i.p.
injection of 25
mg SEB or 500 mg OVA 24 h after cell transfer,
and sera, urine and organ of recipients were studied 2 weeks
afterwards.
BALB/c mice were injected i.p. with 200
mg anti-CD4 antibody
(GK1.5; BioLegend) to deplete CD4
+
T cell 24 h after
immunization 86 with OVA. Four days later, CD4
+
T cells from
mice immunized 126 with KLH were transferred to the CD4
+
T-depleted mice. The recipient mice received a single i.p. injection
of 100
mg KLH 24 h after the cell transfer.
Assay for Mediators
Sera were assayed for anti-Sm antibody using Sm antigen
(ImmunoVision, Springdale, AR), RF (Shibayagi Co., Gunma,
Japan), RF for galactose-deficient IgG (Eisai Co., Ltd., Tokyo,
Japan) and anti-dsDNA antibody using dsDNA (Worthington
Biochemical Co., Lakewood, NJ) after digestion by S1 nuclease
(Promega, Madison, WI). Serum IC was detected using goat anti-
C3 antibody (Bethyl Lab.).
CDR3 Length Spectratyping
cDNAs from thymocytes and CD4
+
splenocytes were subjected
to PCR amplification using Cb- and Vb8-specific primers.
Amplified products were subjected to run-off reactions using three
fluorophore-labeled Jb primers, Jb1.1, Jb1.3 and Jb2.4, and
analyzed by GeneScan software (Perkin-Elmer Applied Biosys-
tems, Emeryville, CA) [4].
RT-PCR
Total RNA was reversely transcribed to cDNA and amplified by
PCR [23]. The products were fractionated by electrophoresis and
transferred to nylon membranes (Roche Diagnostics, Mannheim,
Germany). The membranes were hybridized to fluorescein end-
labeled probes and visualized by alkaline phosphatase (ALP)-labeled
anti-fluorescein antibody and Gene Images CDP-Star chemilumi-
nescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ).
The primers and probes were: 59-CCAAGCTGCAGACATTC-
TAGCACTC-39 (forward), 59-CAACATCTGCCTTCACGTCG-
ATCC-39 (reverse) and 59-AACATGGCTGCCTCCTTGCCG-
TCTACCCT-39 (probe) for RAG1 [24]; 59-CACATCCACAAG-
CAGGAAGTACAC-39 (forward), 59-GGTTCAGGGACATCT-
CCTACTAAG-39 (reverse) and 59-GCAATCTTCTCTAAAGA-
TTCCTGCTACCT-39 (probe) for RAG2 [24]; 59-GAACAAC-
TCGAAGAGCCTTCC-39 (forward), 59-CAAGGGCATCCGT-
GAATAGTTG-39 (reverse) and 59-ATTCGGTCACCCACATT-
GTGGCAGAGAAC-39 (probe) for TdT; 59-CAACTGGGTCAT-
GCTTCTCC-39 (forward), 59-TGGCTGTCGAAGATTCCC-39
(reverse) and 59-CCGTCTCTGGCTCCACCCATCACACTG-
CT-39 (probe) for pTa.
LM-PCR
DNA (1 mg) was ligated to 20 mM BW linker using T4 ligase
(Takara Bio Inc., Shiga, Japan) [25]. Primary PCR was performed
using 200 ng ligated DNA, BW-1HR primer (59-CCGGGA-
GATCTGAATTCGTGT-39) [24], primer specific for 39 flanking
sequence of TCRAV2 (59-AGATGATACAGAGACAAAATGT-
GAGC-39) and 2 U of AmpliTaq Gold DNA polymerase (Applied
Cause of Autoimmunity
PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8382
Biosystems, Foster City, CA). A second PCR was performed using
1
ml of the first PCR product (diluted 1/100), BW-1HR, and nested
primer specific for 39 flanking sequence of TCRAV2 (59-TATTGTG-
GATGCTAACAAGTGCTTTC-39). Amplified DNA was trans-
ferred to membranes and visualized using fluorescein end-labeled
probe specific for TCRAV2 (59-TAACATAAGAATGCACCGCT-
TACACC-39) and ALP-labeled anti-fluorescein antibody. Primers
for control TCRAC region were amplified using the primers 59-
CAGAACCCAGAACCTGCTGTG-39 and 59-ACGTGGCAT-
CACAGGGAA-39.NomenclatureoftheTCRA gene segments
was according to the ImMunoGeneTics (IMGT) database (http://
imgt.cines.fr).
Antigen Cross-Presentation
OVA-reactive CD8
+
T cells were isolated from spleens of DO
11.10 mice using MACS beads (Miltenyi Biotec). CD11c
+
DCs
(4610
5
/well) were isolated using MACS beads (Miltenyi Biotec)
and incubated with 1 mg/ml OVA for 3 h, then co-cultured with
DO11.10 CD8
+
T (KJ1-26
+
CD8
+
) cells (2610
5
/well) for 24 h,
and the expression of CD69 on DO11.10 CD8
+
T cells was
examined. IL-2 and IFNc in culture supernatants were measured
by ELISA (Biosource, Camarillo, CA).
To inhibit cross-presentation, mice were immunized in vivo with
250
mg of chloroquine (Sigma) 3 h prior to immunization with
500
mg OVA or PBS every 5 d. Presence of autoantibodies was
analyzed 2 d after each immunization, and proteinuria and
IFNc
+
CD8
+
T cells were examined 9 d after the final
immunization.
Statistical Analysis
Statistical analyses were performed using Student’s t test, and
the data are expressed as the mean 6 SD.
Supporting Information
Figure S1 Induction of autoantibodies depends on correct
presentation of antigen to T cells. (A) BALB/c mice were
repeatedly injected i.p. with 25
mg of SEB or PBS every 5 d.
Sorted Vb8
+
CD4
+
splenocytes obtained 9 d after the final
immunization were stimulated in vitro with plate-bound 2
mg/ml
anti-CD3 (145-2C11; Cederlane, Ontario, Canada) and 5
mg/ml
anti-CD28 (37.51; BD PharMingen) antibodies for 24 h. Culture
supernatant assayed for IL-2 (mean 6 SD, 5 mice/group), or the
cells were labeled with carboxyfluorescein diacetate succinimidyl
ester (CFSE; Molecular Probes) and further cultured for 72 h
followed by flow cytometry. (B) Requirement of correct antigen
presentation for induction of RF. Induction of RF after
immunization 86 with SEB in B10.D2 and BALB/c mice
(efficient in presenting SEB) and in C57BL/6 (B6) mice (inefficient
in presenting SEB).
Found at: doi:10.1371/journal.pone.0008382.s001 (1.17 MB TIF)
Figure S2 Generation of autoantibodies after repeated immu-
nization with antigen. (A) The 8 week-old BALB/c mice were
injected i.p. with 500
mg OVA every 5 d, and serum RF and anti-
Sm, and anti-dsDNA antibodies (upper), and serum IgG and anti-
OVA antibodies (lower) were quantified by ELISA 2 d after
respective immunization. An arbitrary unit (AU) of 1.0 is the
equivalent titer in sera of MRL/lpr mice. Serum IgG was
quantified by ELISA (Bethyl Laboratories), and anti-OVA
antibody was quantified using mouse anti-OVA monoclonal
antibody (OVA-14; Sigma) as reference. (B) BALB/c mice were
immunized i.p. with 100
mg KLH every 5 d. Serum RF and anti-
Sm antibodies were measured by ELISA 2 d after respective
immunization, AU 1.0 = equivalent detected in sera of MRL/lpr
mice.
Found at: doi:10.1371/journal.pone.0008382.s002 (1.00 MB TIF)
Figure S3 Induction of autoantibodies in CD8
+
T cell-deficient
mice. b
2
m-deficient mice were immunized with 500 mg OVA via
i.p. injection every 5 d, and IgG-RF, anti-dsDNA antibody, and
proteinuria were measured.
Found at: doi:10.1371/journal.pone.0008382.s003 (0.69 MB TIF)
Figure S4 Expression of V(D)J recombinase complex and
histopathology of OVA-immunized BALB/c mice. (A) Expression
of V(D)J recombinase complex after immunization 126 with OVA
as detected using RT-PCR (upper left). GFP
+
cells in the CD4
+
T
cell of rag1/gfp knock-in mice after immunization 126 with OVA
(lower left). Appearance and weights of spleens and a represen-
tative low-magnification view of the spleens from PBS- and OVA-
immunized mice (right, mean 6 SD, 9 mice/group). Enlarged
lymphoid follicles with marked germinal centers were seen in mice
immunized with OVA (H&E staining, bar = 200
mm; original
magnification 620). (B) Representative renal and extra-renal
histopathology in the mice immunized 126 with OVA. A wire-
loop-like massive membranous glomerulonephritis in the kidney
(upper left) (PAS staining, bar = 20
mm; original magnification
6400), plasma cell infiltrates around bile ducts (upper middle) (bar
=20
mm; original magnification 6400), expansion of lymphoid
follicle in the white pulp of spleen (upper right) (bar = 200
mm;
original magnification 640), focal infiltrates of mononuclear cells
to thyroid (lower left) (bar = 50
mm; original magnification 6100),
and diffuse infiltration of inflammatory cells into auricular
subcutaneous tissue (upper right) (bar = 50
mm; original magni-
fication 6200).
Found at: doi:10.1371/journal.pone.0008382.s004 (6.01 MB TIF)
Figure S5 The de novo generation of IFNc-producing CD8
+
T
cells in recipient mice after cell transfer. Percentage of IFNc
+
cells
within the CD8
+
T population of the recipient mice was examined
2 weeks after cell transfer (mean 6 SD, 5 mice/group).
Found at: doi:10.1371/journal.pone.0008382.s005 (0.73 MB TIF)
Figure S6 Transfer of the ability to induce anti-ds DNA
antibody or tissue injury by transfer of CD4
+
or CD8
+
T cells,
respectively. Adoptive transfer of cells from OVA-immunized mice
into naı
¨
ve BALB/c mice, with or without 16 booster injection of
OVA (500
mg, 24 h post-transfer). Autoantibodies and proteinuria
measured 2 weeks later.
Found at: doi:10.1371/journal.pone.0008382.s006 (0.70 MB TIF)
Figure S7 Antigen-specific activation of T cells and the
expression of MHC class I on DC. (A) Spleen cells were cultured
with or without 1 mg/ml of OVA for 24 h, and the expression of
CD69 on CD4
+
T or CD8
+
T cells was examined by flow
cytometry. (B) DC from PBS- or OVA-immunized mice (PBS DC
or OVA DC) were incubated in the presence or absence of
chloroquine (CQ) (20
mg/ml) for 2 h and OVA (1 mg/ml) for 3h.
OVA- and/or CQ-pulsed DCs were stained with biotin-
conjugated anti-H-2k
d
antibody (SF1-1.1; BD PharMingen) and
PE-conjugated streptavidin (BioLegend).
Found at: doi:10.1371/journal.pone.0008382.s007 (1.62 MB TIF)
Figure S8 Requirement of CD4
+
T cell help for inducing
autoimmune tissue injury. The mice were depleted of CD4
+
T
cells by treatment with 200
mg anti-CD4 antibody (Ab) (GK1.5;
BioLegend) 24 h prior to 66,96 and 126 immunization with
OVA. Control mice were injected with 200
mg rat IgG (CALTAG
Lab.). (A) A representative flow cytometry plot showing that CD4+
T cells were depleted to 5.5662.30% in the spleen and
Cause of Autoimmunity
PLoS ONE | www.plosone.org 8 December 2009 | Volume 4 | Issue 12 | e8382
3.4261.02% in peripheral blood mononuclear cells (PBMC) 9 d
after 3rd treatment with anti-CD4 Ab. (B) Mice were immunized
126 with OVA with or without adding anti-CD4 antibodies, and
the number of IFNc
+
cells within the CD8
+
T population (upper
and lower left) (mean 6 SD, 5 mice/group) and proteinuria (lower
right) were evaluated.
Found at: doi:10.1371/journal.pone.0008382.s008 (2.02 MB TIF)
Figure S9 Study on the requirement of autoantibody-inducing
CD4
+
T cells for autoimmune tissue injury. Neither OVA-specific
matured IFNc
+
CD8
+
T cells or autoimmune tissue injury were
observed until BALB/c mice were immunized at least 106 with
OVA. The percent splenic IFNc
+
CD8
+
T cells (left, mean 6 SD, 4
or 5 mice/group) and proteinuria (right) were examined after
immunization 66,86,106 and 126 with OVA.
Found at: doi:10.1371/journal.pone.0008382.s009 (0.67 MB TIF)
Acknowledgments
We dedicate this work to the Late Professor Emeritus Atsushi Okabayashi,
a mentor to SS, who introduced us to this area of study. We thank Prof.
Masaaki Miyazawa, Deartment of Immunology, Kinki University School
of Medicine, and Prof. Nobuo Saaguchi, Department of Immunology,
Kumamoto University Graduate School of Meicine, for useful advice,
authorization of results and providing mice. We also thank Dr.Sachiyo
Tsuji-Kawahara, Kinki University, for kindly providing b
2
m-deficient mice
and Dr. Hideya Igarashi, Kumamoto University Graduate School of
Medicine, for kindly providing rag1/gfp knock-in mice. We thank Mai
Takimoto and Toshie Nakashima, graduate students of our department,
for studies of rag1/gfp knock-in mice, CDR3 spectratyping and LM-PCR,
Dr. Akira Hashiramoto, Division of Rheumatology, Kobe University, for
helpful discussions, and Dr. Marc Lamphier for reviewing the manuscript.
Author Contributions
Conceived and designed the experiments: SS. Performed the experiments:
KT YM. Analyzed the data: KT YM SS. Wrote the paper: KT SS.
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PLoS ONE | www.plosone.org 9 December 2009 | Volume 4 | Issue 12 | e8382
... During the course of our investigation, we cloned the SH2D1A gene that is critically involved in immune protection against EBV and applied humanized mice to generate a mouse model of EBV-induced RA-like arthritis ( Figure 1). As reported by Tsumiyama et al. (self-organized criticality theory) [103], an autoimmune phenomenon is not always essential for the onset of autoimmune diseases. We sincerely hope that the knowledge accumulated so far will be useful in developing new treatments for rheumatic diseases. ...
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Objectives To evaluate the safety and immunogenicity of third and fourth BNT162b2 boosters in patients with SLE and rheumatoid arthritis (RA). Methods Patients with SLE and RA aged 18–65 years who completed a series of inactivated, adenoviral vector, or heterogenous adenoviral vector/mRNA vaccines for at least 28 days were enrolled. Immunogenicity assessment was done before and day 15 after each booster vaccination. The third BNT162b2 booster was administered on day 1. Patients with suboptimal humoral response to the third booster dose (antireceptor-binding domain (RBD) IgG on day 15 <2360 BAU/mL) were given a fourth BNT162b2 booster on day 22. Results Seventy-one patients with SLE and 29 patients with RA were enrolled. The third booster raised anti-RBD IgG by 15-fold, and patients with positive neutralising activity against the Omicron variant increased from 0% to 42%. Patients with positive cellular immune response also increased from 55% to 94%. High immunosuppressive load and initial inactivated vaccine were associated with lower anti-RBD IgG titre. Fifty-four patients had suboptimal humoral responses to the third booster and 28 received a fourth booster dose. Although anti-RBD IgG increased further by sevenfold, no significant change in neutralising activity against the Omicron variant was observed. There were two severe SLE flares that occurred shortly after the fourth booster dose. Conclusions The third BNT162b2 booster significantly improved humoral and cellular immunogenicity in patients with SLE and RA. The benefit of a short-interval fourth booster in patients with suboptimal humoral response was unclear. Trial registration number TCTR20211220004.
... Autoimmune diseases such as Guillain-Barré Syndrome (Shao et al., 2021), autoimmune induced hepatitis (Avci &Abasiyanik, 2021), and acute autoimmune transverse myelitis (Hirosae et al., 2021) along with a wider spectrum of autoimmune phenomena have been attributed to the novel DiCoReTh-injections. Evidence is even mounting that repeated vaccinations with the same antigen at short intervals ("booster shots") destabilise the immune system so that autoimmune processes are triggered (Tsumiyama et al., 2009) and in some cases are lethal (Shimoyama et al., 2021). ...
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The engineered spike protein of SARS-COV-2, and the corresponding infectious disease COVID-19 attributed to it, hold in their grip a large portion of humanity. The global race for a counter strategy quickly turned into a search for a vaccine as the preferred means to contain the virus. An unusually rapid development of different and completely new classes of experimental therapies that would widely be referred to as “vaccines” raised questions about safety, especially with regard to emergency use approval (EUA) being granted with unprecedented urgency and hardly any critical scrutiny. At present, independent researchers, even some former proponents and insiders, of the currently ongoing global experiment represented as a “vaccination” campaign point primarily to the lack of public safety studies based on empirical datasets that should be obtainable for the tens of millions, even hundreds of millions, of doses of mRNA and DNA vector therapeutics being distributed as “vaccines”. Studies regarding efficacy and “side effects” (sometimes fatalities or permanent iatrogenic injuries) of these experimental therapies have been by-passed in favor of short-term field data from real patients which inevitably raises scientific and ethical questions particularly in view of the fact that the persons and entities responsible for public safety hold deep financial and other vested interests in speeding along the distribution of the experimental pharmaceutical products. The lack of an open discussion about the experimental therapies for COVID-19 now being applied across all age groups, even children hardly impacted by COVID-19, is worrying. The core principle of open debate without pre-conceptions or vested interests in outcomes has been and continues to be utterly ignored. We hope to engage scientific discussion that will help decision-makers, the general public, and the media alike to consider the subject-matter of what is at stake in a context of reason rather than panic.
... Sono state attribuite alle nuove iniezioni di TeReCoMa: malattie autoimmuni come la sindrome di Guillain-Barré (Shao et al., 2021), l'epatite autoimmune indotta (Avci & Abasiyanik, 2021), la mielite trasversa autoimmune acuta (Hirosae et al., 2021) assieme a un ampio spettro di fenomeni autoimmuni . Stanno anche aumentando le prove che vaccinazioni ripetute con lo stesso antigene a brevi intervalli ("colpi di richiamo") destabilizzano il sistema immunitario in modo che siano innescati processi autoimmuni (Tsumiyama et al., 2009) in alcuni casi letali (Shimoyama et al., 2021). Nessuna delle citate evidenze dovrebbe sorprendere data la tumorigenicità dei substrati cellulari utilizzati nella produzione dei costituenti delle iniezioni di TeReCoMa che possono includere l'uso di allotrapianti tumorali, il trasferimento di virus noti o sconosciuti e l'incorporazione intenzionale di agenti oncogeni o componenti cellulari che possono produrre "de novo" o riaccendere cellule cancerose esistenti (Aubrit 2015;Arvay, 2020;Sumi et al., 2021;Goldman et al., 2021;VAERS, 2021) 3 . ...
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La proteina ingegnerizzata spike della SARS-COV-2 e la corrispondente malattia infettiva COVID-19 attribuita ad essa tengono in pugno una gran parte dell'umanità. La corsa globale per una strategia di contrasto si è rapidamente trasformata nella ricerca di un vaccino come mezzo preferenziale per contenere il virus. Uno sviluppo insolitamente rapido di diverse classi completamente nuove di terapie sperimentali diffuse come "vaccini", ha sollevato interrogativi sulla sicurezza, in particolare per quanto riguarda l'approvazione dell'uso di emergenza (EUA) che è stata concessa con un'urgenza senza precedenti e priva di qualsiasi esame critico contrario. Attualmente, ricercatori indipendenti, come anche alcuni ex proponenti e addetti ai lavori dell'esperimento globale attualmente in corso e rappresentato come una campagna di "vaccinazione", sottolineano soprattutto la mancanza di studi sulla sicurezza della campagna vaccinale che ha finito invece per strutturarsi su set di dati empirici che verranno ottenuti attraverso decine di milioni, anche centinaia di milioni, di dosi di mRNA e terapie vettoriali del DNA distribuite col nome di "vaccini". Gli studi riguardanti l'efficacia e gli "effetti collaterali" (talvolta fatalità o lesioni iatrogene permanenti) di queste terapie sperimentali sono stati omessi a favore di dati a breve termine presi sul campo su pazienti reali. Questa evidenza solleva inevitabilmente questioni scientifiche ed etiche, in particolare in considerazione del fatto che le persone e gli enti responsabili per la sicurezza pubblica hanno vasti interessi finanziari e di altro tipo che li portano ad accelerare la distribuzione di questi prodotti farmaceutici sperimentali. La mancanza di una discussione aperta sulle terapie sperimentali per il COVID-19 ora applicate su tutte le fasce di età, anche i bambini, che difficilmente sono colpiti dal COVID-19, è preoccupante. Il principio fondamentale del dibattito aperto senza preconcetti o sugli interessi nei risultati è stato e continua ad essere completamente ignorato. Speriamo di impegnare una discussione scientifica al fine di aiutare chi deve decidere, l'opinione pubblica e i media a considerare l'oggetto di ciò che è in gioco in un contesto di ragione piuttosto che di panico.
... Multiple studies have reported acceptable safety profiles in SLE patients after initial vaccine series [11][12] . However, in an animal model, repeated exposure to the same antigen was shown to induce autoimmunity 13 . Given this uncertainty, the benefit of administering a short interval fourth booster dose should be balanced against the risk of disease activation. ...
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Objectives To evaluate the safety and immunogenicity of third and fourth BNT162b2 boosters in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients. Methods SLE and RA patients aged 18-65 years who completed a series of inactivated, adenoviral vector, or heterogenous adenoviral vector/mRNA vaccines for at least 28 days were enrolled. Immunogenicity assessment was done before and day 15 after each booster vaccination. The third BNT162b2 booster was administered on day 1. Patients with suboptimal humoral response to the third booster dose (anti-receptor binding domain (RBD) IgG on day 15 < 2,360 BAU/mL) were given a fourth BNT162b2 booster on day 22. Results Seventy one SLE and 29 RA patients were enrolled. The third booster raised anti-RBD IgG by 15 fold and patients with positive neutralizing activity against the Omicron variant increased from 0% to 42%. Patients with positive cellular immune response also increased from 55% to 94%. High immunosuppressive load and initial inactivated vaccine were associated with lower anti-RBD IgG titer. Fifty four patients had suboptimal humoral responses to the third booster and 28 received a fourth booster dose. Although anti-RBD IgG increased further by 7 fold, no significant change in neutralizing activity against the Omicron variant was observed. There were 2 severe SLE flares that occurred shortly after the fourth booster dose. Conclusions The third BNT162b2 booster significantly improved humoral and cellular immunogenicity in SLE and RA patients. The benefit of a short interval fourth booster in patients with suboptimal humoral response was unclear. Key messages What is already known about this subject? - The SARS-CoV-2 omicron variant (B.1.1.159) has multiple mutations that have resulted in greater escape from immune protection elicited by COVID-19 vaccines. - More attenuated immune response to SARS-CoV-2 vaccination has been observed in patients with autoimmune rheumatic diseases. The additional third dose of SARS-CoV-2 vaccine has been recommended in immunocompromised populations. - Some immunocompromised patients have a suboptimal humoral response to a third booster dose. Factors associated with poor immune response have not been adequately studied. - Administration of more than 3 doses has been shown to enhance immune response in some severely immunocompromised patients. What does this study add? - The third BNT162b2 booster was well tolerated, and significantly improved both humoral and cellular immunogenicity in SLE and RA patients previously vaccinated with either inactivated, adenoviral vector, or heterogenous adenoviral vector/mRNA vaccines. - High intensity of immunosuppressive therapy and initial inactivated vaccine were associated with lower humoral immune response to the third BNT162b2 booster. - Administration of a fourth BNT162b2 booster in poor humoral immune responders may not offer additional protection against the omicron variant, and flares were observed in SLE patients. How might this impact on clinical practice or future developments? - This study supported a third BNT162b2 booster dose administration in SLE and RA patients to enhance immune protection against the Omicron variant. - Patients who receive a high dose of immunosuppressive therapy or initial inactivated vaccine could be unprotected from SARS-CoV-2 infection. Benefits and risks of additional boosters or second generation of SARS-CoV-2 vaccine should be further studied.
... Pour ce qui concerne les lymphocytes CD8 + Treg, il y a moins de travaux publiés dans la littérature chez l'homme. Ils ont été étudiés en particulier chez les patients lupiques, pour lesquels un déficit quantitatif et/ou fonctionnel des CD8 + Treg a été rapporté (Filaci et al. 2001;Tulunay et al. 2008 V. La théorie de la criticité auto-organisée de l'auto-immunité de Tsumiyama (Tsumiyama, Miyazaki, and Shiozawa 2009) selon laquelle le défaut du système immunitaire pourrait expliquer la cause de l'auto-immunité. L'auto-immunité systémique semble être la conséquence inévitable de la stimulation excessive du système immunitaire par une immunisation répétée avec un antigène, à des niveaux qui dépassent la criticité auto-organisée du système. ...
Thesis
Les maladies auto-immunes et auto-inflammatoires (MAI) représentent un problème scientifique extraordinaire. Étant donné la complexité des MAI et la nécessité de disposer de marqueurs de diagnostic, de pronostic, d'activité de la maladie et de réponse à la thérapie, il est nécessaire d'avoir une approche holistique qui prend en compte tous les acteurs de ce processus. Nous avons appliqué cette approche holistique à une étude observationnelle (TRANSIMMUNOM), ainsi qu’à des études interventionnelles (DF-IL2 et TRANSREG). L'objectif principal du projet TRANSIMMUNOM est de revisiter la nosologie de plusieurs MAI avec une approche « atlas » incluant 1000 participants avec différentes MAI et des volontaires sains. Un effort important a été apporté pour concevoir un Case Report Form approprié. Nous avons sélectionné, organisé et harmonisé plus de 5000 variables multiparamétriques codées. L'approche holistique appliqué à des études interventionnelles nous a permis: i) de déterminer la dose et modéliser le schéma optimal d'IL-2 (DF-IL2), ii) de constater que les fables doses d'IL-2 (fd d’IL-2) induisent une activation accrue de cellules Treg dans différentes MAI, iii) les fd d’IL-2 sont bien tolérées quelle que soit la MAI et la thérapie de fond et iv) les fd d’IL-2 ont une efficacité clinique dans un groupe de patients très hétérogènes.Nous avons démontré que l'intégration de ce type d'approche est très utile dans des essais de phase précoce. La méthodologie de nos essais peut être appliquée en pratique clinique courante pour étudier la population des patients « tout-venant » et pour tester de nombreux médicaments en cours de développement dans les MAI.
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Pathogens including autoantigens all failed to induce systemic lupus erythematosus (SLE). We, instead, studied the integrity of host’s immune response that recognized pathogen. By stimulating TCR with an antigen repeatedly to levels that surpass host’s steady-state response, self-organized criticality, SLE was induced in mice normally not prone to autoimmunity, wherein T follicular helper (Tfh) cells expressing guanine nucleotide exchange factor DOCK8 on the cell surface were newly generated. DOCK8⁺Tfh cells passed through TCR re-revision and induced varieties of autoantibody and lupus lesions. They existed in splenic red pulp and peripheral blood of active lupus patients, which subsequently declined after therapy. Autoantibodies and disease were healed by anti-DOCK8 antibody in the mice including SLE-model (NZBxNZW)F1 mice. Thus, DOCK8⁺Tfh cells, generated after repeated TCR stimulation by immunogenic form of pathogen, either exogenous or endogenous, in combination with HLA to levels that surpass system’s self-organized criticality, cause SLE.
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The somatic diversity immunoglobulin and T-cell receptor diversity is largely provided by the junctional variation created during site-specific rearrangement of separately encoded gene segments. Using a transient transfection assay, we demonstrate that the recombination activating genes Rag1 and Rag2 direct site-specific rearrangement on an artificial substrate in poorly differentiated as well as in differentiated nonlymphoid cell lines. In addition to a high frequency of precise recombination events, coding joints show deletions and more rarely P-nucleotide insertions, reminiscent of immunoglobulin and T-cell receptor junctions found in fetal tissues. N-region insertions, which are characteristic of adult junctional diversity, are obtained at high frequency upon transfection of a terminal deoxynucleotidyltransferase expression vector together with Rag1 and Rag2. These results show that only three lymphoid-specific factors are needed to generate all types of junctional diversity observed during lymphoid development.
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