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REDEFINING THE SELF
Paris, June 2014
António Coutinho
Instituto Gulbenkian de Ciência
“The heretic Paris School of the 1980’s” (M. Cohn)
PHYSIOLOGICAL AUTOREACTIVITY:
Autoreactive T and B cells exist in healthy
individuals and are naturally activated
Physiologic autoreactivity is necessary to
prevent pathologic autoimmunity
“Français encore un effort si vous voulez être républicains” (Sade)
Immunosomatics: the role of “homeostatic” immune
activity in other physiologic systems in the body
INFECTION: THE EVOLUTIONARY PROBLEM
•  Increases in complexity and size inexorably result
in prolonged developmental periods, slowing down
germ-line transmission of (selected) variation
•  Co-evolution of life forms with widely divergent
potential for transmissible genetic variation
(e.g., life cycles of 20 minutes vs 20 years)
THERE IS MORE THAN INFECTION IN “IMMUNITY”
When discussing “self integrity”, immunologists
concentrate on infections, but these are only a minor
fraction of all adverse “internal”, environmental or
metabolic conditions that decrease fitness of organisms
and to which they must, therefore, respond.
Evolutionary layers of protective mechanisms
Cytoprotection/stress responses
Innate immunity
Acute-phase resp
Phagocytosis
Inflammation
Adaptive immunity
[Commensals]
Respiration
Angiogenesis
Coagulation
Tissue remodeling
Neuro-endocrine
Homeostasis
Tissue conservation
(Pathogen elimination)
THE “PROMETHEIC” SOLUTION IN VERTEBRATES
• Transfer “VARIATION & SELECTION” to somatic time
• Generate very large Variable-region repertoires for
“COMPLETE” molecular identification: memory
[evolved twice independently in vertebrates]
• Re-deploy (older) activation mechanisms/ridding effector
functions by “coupling” them to (modern) V-region
repertoires for molecular identification
“Landsteiner’s Paradox” of Adaptive Immunity
Completeness of antibody repertoires
All adaptive IS identify every molecular shape whatsoever
irrespective of their total diversity (1012, 108, 105), and of the
presence of those molecules in evolution
DIVERSITY OF V-REGIONS
Immune systems with a very large diversity evolved twice
independently in vertebrates (>1014 in agnatha, > 1015 in jawed).
HIGH DEGENERACY OF V-REGION INTERACTIONS
Each V-region (Ab or TCR) reacts with millions of “antigens”;
cooperative specificity within a very large diversity.
D. Mason
D. Wilson: # of different peptides that are productively recognized by one TCR
Severe Combined Immunodeficiency (SCID)
Before treatment (bone marrow transplantation or somatic gene "
therapy), SCID patients succumb to infections and must be kept "
under specific pathogen free (SPF) conditions."
IPEX newborn: inflammatory bowel disease, diabetes mellitus, and thrombocytopenia
Maternidade São Luiz, SP
IPEX newborn: inflammatory bowel disease, diabetes mellitus, hypothyroidism, hemoly;c anemia UNIFESP-­‐EPM Pancreas from an IPEX infant (HE 100x)
Pancreas from an IPEX infant (IH cromogranina 40x)
Family 2
I
I
I
2
II
I
I
V
2
Male
P
2 hours, meconium in amnio;c fluid 51 days, P persistent diarrhea, meconium in amnio;c fluid males, 21 wk, hydrops 5
Males
female 9 wk Down Sd. female
fetus
Bloody diarrhea in the same IPEX newborn
Maternidade São Luiz, SP
Things we learned with IPEX patients and the Scurfy mouse
• 
Disease is truly autoimmune (adaptive IS):
no mature lymphocytes, no disease
independent of microbial colonization
independent of food antigens
• 
Disease may occur in the absence of B cells and MHC Class II
• 
Dominant tolerance (TREGS) is essential for natural
tolerance
• 
Recessive tolerance (clonal deletion) is not sufficient
(if at all necessary) to ensure natural tolerance
ADAPTIVE IMMUNITY HAS BECOME CRITICAL FOR
THE DEFENCE OF VERTEBRATES AGAINST INFECTIONS
Humans with selective defects in “adaptive immunity” (SCID
patients) are exquisitely sensitive to infections, and must be
kept under “specific pathogen free” conditions, before bone
marrow transplantation.
ADAPTIVE IMMUNITY IS ALSO EXTREMELY POWERFUL IN
SELF-DESTRUCTION
Humans and mice (vertebrates?) with null mutations in FOXP3
(IPEX, Scurfy) invariably succumb at very early ages (even
before birth) by destruction of normal, “self” tissues.
Regulatory T cells are essential for ensuring “self-tolerance”.
The Burnet-Lederberg proposal for a “negative definition” of
the immunological self is wrong because of:
(1)  size of V-region repertoires and cooperative “high degeneracy”
recognition;
(2)  the “time problem”: animals are tolerisable only in development,
(tolerant to antigens that are present in development) but
produce new lymphocytes throughout life;
(Obs: It was never shown that tolerance was due to clonal deletion in normal
animals)
In “adaptive immunity”, self is “defined positively” by selecting a
set of high affinity self-recognizing cells and differentiate
them to “regulatory” (non-ridding) functions that “dominate” over
ridding fucntions; “fail-safe”: “tolerise one, tolerize them all”
THE WINDOW OF “TOLERANCE” RELATES
TO THE AGE OF THE ORGANISM
THE “TIME PROBLEM”
• 
New lymphocytes are produced throughout life
but natural tolerance is exclusively established for
antigens that are present before or around birth.
• 
Newly formed lymphocytes must “discriminate”
antigens by presence in the body before they are
produced.
TOLERANCE IS A PROPERTY OF THE ORGANISM
[NOT OF SINGLE CELLS] BUILT ON A MEMORY OF THE
ANTIGENIC SELF IN DEVELOPMENT
In “adaptive immunity”, self is “defined positively” by
selecting a set of high affinity self-recognizing cells and
differentiate them to “regulatory” (non-ridding) functions that
“dominate” over ridding mechanisms;
“fail-safe”: “tolerise one, tolerize them all”
Positive “definition of self” by T cells:
Le Douarin’s phenomenon
The “immunological self” is not a biochemical listing, but the
developmentally constructed “point of view” of the IS, through
V-region interactions with themselves and with self ligands
(T cell – CD4, CD8 - and B cell “selves”). Limited “point of view”:
V-region repertoires early in development are very limited and
“germ-line encoded”.
Yet, the “immunological self” (for T cells) is not “what is there in
development” but what is in the thymus along development
Le Douarin’s Experiments
TCR-DEPENDENT REGULATORY T CELL SELECTION
Modigliani et al. model (1996)
•  TREG ARE A THYMICALLY COMMITTED T CELL LINEAGE
•  HIGH TCR SELF-AFFINITY LEADS TO TREG SELECTION
(thus, “isolating” positive and negative selection)
•  SELECTION (AND ACTIVATION) COMMITS TREG INTRATHYMICALLY
(in contrast to positively selected “naïve” T cells)
•  TREG ARE SELECTED ON TECs AND DELETED ON THCs
• 
THYMIC STROMA (TEC/THC) CHANGES IN DEVELOPMENT
(TEC predominance in embryonic/perinatal times)
•  PERIPHERY: TREG DOMINANTLY EDUCATE RTE AND INHIBIT PMR
(in contrast to “secondary responses” that are not suppressed by TREG)
ALL TCR SELF-AVIDITY
Coutinho et al. 1993
Modigliani et al. 1996
Dominant tolerance solves the classical “time problem”, as the
positive selection of auto-reactivities in the embryonic and perinatal period ensure the “learning” of “self” and a memory of the
“developmental self”.
A number of (genetic) developmental programs (both cellular
and systemic) have evolved to ensure that (1) TCR/BCR-depend.
positive selection of auto-reactivity is effective and occurs first,
(2) “non-ridding” effector functions are “coupled” to physiological
auto-reactivity; (3) these repertoires (maybe not the cells) persist
in the adult (memory of self composition in development).
Dominant Tolerance built on self-reactive lymphocytes requires
that self-recognizing V-regions appear first in development
(and evolution?):
(1)  genetic developmental programs for V-region expression
(regulation of TdT expression, proximal VH-genes preference)
ensure direct reading of the (selected) germ-line and the
precedence of (multi)auto-reactivity;
Obs: multi-reactivity necessarily results in a V-region network
as experimentally demonstrated
(2) germ-line selection of V-region genes for auto-reactivity with
the “invariant” molecular self;
MULTIREACTIVITY (PROMISCUITY) IS A
CHARACTERISTIC OF NEWBORN TCR/ANTIBODY
• 
MECHANISMS
•  DEVELOPMENTALLY REGULATED TDT EXPRESSION
•  ORDERED REARRANGEMENT OF VH (TCR ?) GENES
•  “GERM-LINE” V-D-J JOINS
• 
METHODS
•  TDT KO AND TDT TRANSGENIC ANIMALS
•  VH7183.1 KO AND CHROMOSOMAL POSITION EXCHANGE
•  CLONAL ANALYSIS OF MULTIREACTIVITY
GENETIC PROGRAMS ENSURE DEVELOPMENTALLY
REGULATED MULTI/AUTOREACTIVITY:
1. Preferential VH-rearrangement
VH7183.1 (81X) IMPARTS MULTI/AUTO-REACTIVITY
DONOR
Wild type
81X KO
Fraction multireactive/positive clones
10/48 (440)
21%
7/84 (396)
8%
(François Huetz)
GENETIC PROGRAMS ENSURE DEVELOPMENTALLY
REGULATED MULTI/AUTOREACTIVITY:
2. TdT expression
ABSENCE OF TdT IMPARTS MULTI/AUTO-REACTIVITY
DONORS
# POS CLONES/TOT
% MULTIREACTIVE
NewBorn [TdT neg]
NewBorn [TdT pos (Tg)]
87/186
0/254
47
<0.5
Adult [TdT pos]
Adult [TdT neg (KO)]
0/246
40/160
<0.5
25
(François Huetz)
Holmberg et al. 1984, 1987; Kearney et al. 1987; Zoller et al 1991
Proposals on the evolution of protective mechanisms:
Cellular stress programs mediate “tissue damage control” allowing
“innate” inflammatory responses to evolve feed-forward
amplification;
The evolution of an “adaptive” system that further amplifies
inflammatory responses (e.g., pro-inflammatory cytokines from
CD4T cells, CD8 and antibody-dependent tissue destruction),
requires novel mechanisms now regulating the “immune”
inflammatory process itself (Tregs);
In turn, “adaptive” regulation of inflammation contributes to
“tolerance to infection”.
Proposals on the evolution of protective mechanisms:
“Primitive” regulatory (CD4T) cells were “connected” to cell stress
programs (e.g., HSPs activate Tregs; Class II-independent Tregs)
to install a new mechanism of tissue damage control;
Gain of MHC restriction, TCR diversity and selection on thymic
epithelial cells by “modern” Tregs then allowing for their co-option
to regulate all “adaptive” responses;
Evolution of “specific” Tregs and thymic selection solved the
problem of “specific tolerance” in the “adaptive” system, while
developmental programs of the thymus (MECs, AIRE, etc) and of
V-gene expression (“promiscuous”/multi-reactive V-regions)
established the developmental “time window” in self-tolerance;
NATURAL ANTIBODIES (NA) Some 10 20 anKbody molecules: Largest pool of V-­‐regions (> 1,000 fold the number of BCR Igs); Unknown how many plasma cell clones are represented (108-­‐109 ?); “Homeostasis”: Kept in lymphopenia and a]er lymphoid reconsKtuKon or in Rag+ BCR Tgs (new B cell input <1:10,000); Physiology: AnK-­‐infecKous protecKon (bacteria and virus); “House-­‐keeping” and homeostasis of other physiological systems (hormonal); AnK-­‐inflammatory, “Kssue-­‐protecKve” (e.g., atheroprotecKve, neuroprotecKve); “AnK-­‐autoimmune” (?); [TherapeuKc effects of IvIG] NATURAL ANTIBODIES (NA) InducKon: IgM is at control levels in T cell-­‐deprived and “an;gen-­‐free” animals; IgG and IgA require both T cells and bacterial flora colonizaKon; Repertoires are strongly selected: Specificity and “mulK-­‐reacKvity”; Predominant auto-­‐reacKvity to a sub-­‐set of self an;gens; Liile variaKon in newborns, and with exposure to external anKgens; T cell-­‐dependent; Varies with “self-­‐composiKon” (single proteins, MHC, etc.); Frequency of some reacKviKes increases 40,000 fold in with age; MulK-­‐parametric tesKng (immunoblots, anKgen arrays): “paierns of reacKvity”, measure increases/decreases reacKvity; May offer valuable “biomarkers” in diagnosKc/therapeuKc; Hypothesis on positive self-definition by antibody repertoires
•  The “invariant molecular self” includes a set of ligands for TLRs
•  Germ-line (primordial ?) V-region repertoires are “TLR-like”
ü  mechanism for “autonomous activation” of B cells
ü  homeostasis of natural antibody production (as NatAbs
inhibit internal activation by removing TLR ligands)
Göran Moller
Niels K. Jerne
Luciana Forni, Dan Holmberg
Francisco Varela, Nelson M. Vaz, Alberto Nóbrega,
António Bandeira, François Huetz, Yves Modigliani,
S. Avrameas, P.-A, Cazenave, M. Kazatchkine
Nicole Le Douarin, Josselyne Salaün
Jocelyne Demengeot, Shoei Hori, Santiago Zelenay
Coutinho & Möller, 1974:
“One-nonspecific signal”
Caramalho et al, 2003;
Demengeot & Coutinho, submitted
T REG
B CELL
APC
TLR
TLR-LIGAND
BCR/TCR
HAPTEN/pepMHC
ADAPTIVE IMMUNITY ADDS “MEMORY” TO INNATE
(P. chabaudi PARASITEMIA IN C57BL/6 MICE)
B cell competent; Ig deficient mice
µS KO
AID KO
AIDµS KO
AIDµS double KO mice [B cell competent; sIg null]
accumulate activated B cells in the periphery
AIDµS
µS
AID
WT AID
µS
AIDµS
WT
Circulating Igs regulate numbers of naturally activated B cells
(15x increase in Ig-null mice); no change in the resting B cell pool
J. Demengeot et al, 2013
OTHER FUNCTIONS OF THE ADAPTIVE IMMUNE SYSTEM ?
A general homeostatic role for the molecular composition
of the body (systemic, but microbiota) established in development
(fetal and early post-natal life) defining the range of tolerance ?
Regulating itself ?
The Immune System is not a bag of cells and molecules
LE DOUARIN’S TOLERANCE EXPERIMENTS:
• 
EMBRYONIC (PRE-LYMPHOID STAGES) TISSUE GRAFTS
DO NOT INDUCE TOLERANCE TO THEMSELVES
• 
PURE THYMIC
EPITHELIUM (TE) INDUCES TOLERANCE
TO (ALL ?) OTHER TISSUES IN THE BODY
MIXED (CHIMERIC THYMUSES) INDUCE FULL TOLERANCE
TO BOTH DONOR AND RECIPIENT
HENCE
TISSUE-SPECIFIC (PERIPHERAL) TOLERANCE IS
(CENTRALLY) INDUCED IN THE THYMUS AND MEDIATED BY
DOMINANT MECHANISMS
Three evolutionary layers of protective mechanisms
Angiogenesis
Coagulation
Tissue Remodeling
Respiratory
1.  Cytoprotection/stress responses
Inflammation
Acute Phase Responses
Phagocytosis
2.  Innate immunity
Tissue conservation
Pathogen elimination
3.
Adaptive immunity
THE “OLDEST SOLUTION”
CELLULAR STRESS PROGRAM
CYTOPROTECTIVE RESPONSES
•  Extremely conserved from unicellulars
•  Heat shock, protein folding stress, oxidative and
metabolic stress, etc.
•  Gene expression programs (HSPs, GRPs, HO-1):
cell survival and performance (apoptosis)
•  Involve other physiological/organ systems
•  Intracellular pathways, but cell-surface display
and intercellular communication
•  “Connected” to both innate and specific immunity
(e.g.,TLR-ligands, peptide transporters/presenters)
THE “ANIMAL SOLUTION”
(INNATE IMMUNITY)
•  Phagocytic cells that are mobile
•  Inflammation and other “ridding” mechanisms
•  Germ-line encoded receptors for “invariant” microbial
molecules (DNA, ss/dsRNA, phosphated glycolipids)
[Little diversity; no discrimination of “target” molecular
structures by effector mechanisms]
REGULATION OF INNATE RESPONSES
INNATE IMMUNITY
FEEDFORWARD
MECHANISMS
INNATE RESPONSE
TISSUE DAMAGE
MICROBES
REGULATION OF INNATE RESPONSES
INNATE IMMUNITY
MICROBES
STRESS
FEEDFORWARD
MECHANISMS
INNATE RESPONSE
TISSUE DAMAGE
TISSUE PROTECTIVE
GENES
REGULATION OF INNATE RESPONSES
INNATE IMMUNITY
FEEDFORWARD
MECHANISMS
MICROBES
STRESS
REGULATORY
T CELLS
INNATE RESPONSE
TISSUE DAMAGE
TISSUE PROTECTIVE
GENES
TREGS SUPPRESS INFECTION-ASSOCIATED
CD4 T CELL-DEPENDENT IMMUNOPATHOLOGY
CD4 cells into Rag-/- mice:
CD25-neg
CD25-neg+CD25-pos
LUNG
(P. carinii)
COLON
(H. hepaticus)
TREGS SUPPRESS BOTH IMMUNOPATHOLGY AND
THE RESOLUTION OF INFECTION
Microbial load
Hori, Carvalho & Demengeot, 2002
GENETIC BASES OF “MULTIREACTIVITY”:
A DEVELOPMENTAL PROGRAM
Most NatAbs (all in the newborn) are encoded by:
•  germ-line sequences
•  D-proximal VH-genes
•  no TdT activity
There is a genetic developmental program ensuring that
the normal Immune System always starts with a BCR/NatAb
repertoire that is highly multi(auto)reactive and “connected”;
many such Abs also react (and are protective) with conserved
bacterial antigens (PC/OxLDLs, carbohydrates/cellular ags...)
PHYSIOLOGIC SIGNIFICANCE OF A V-REGION NETWORK ?
(many types of experiments did not address the question)
Working postulate
FUNCTIONAL SIGNIFICANCE = CELL ACTIVATION
Experiment
Deprive the system of external stimulation (Ag-free mice)
Result
SIGNIFICANT “INTERNAL ACTIVITY” BUT ONLY
IN A PART OF THE SYSTEM
Hypothesis
PERIPHERAL IMMUNE SYSTEM (PIS)
•  A “burnetian” immune system of resting cells, deprived of
self-reactive and connected specificities, ensuring clonal
responses to non-self; Ag-dependent and -specific regulation;
[Efficient BCR/TCR-dependent deletion of conventional
B and T cells in BM and thymus.]
CENTRAL IMMUNE SYSTEM (CIS)
•  A developmentally programmed “jerneian” immune system
of connected self-reactivities, ensuring natural tolerance; nonridding effector functions; network regulation;
[BCR/TCR engagement leads to positive selection and
activation: TREGs and MZ B cells.]
Coutinho, A. Beyond clonal selection and network. Immunol. Rev. 110 : 63-87 (1989)
PRIMORDIAL “STRATEGY” TO DEAL WITH THE PROBLEM:
1.  “COUPLE” INNATE/V-REGION “RECOGNITION”:
LYMPHOCYTE ACTIVATION REQUIRES CONSERVED
GERM-LINE RECEPTORS (e.g.TLR) FOR MICROBIAL
PRODUCTS (“INFECTION CORRELATES”)
(Coutinho & Möller 1974)
2. ESTABLISH MECHANISMS OF DOMINANT TOLERANCE:
DEVELOPMENTAL SELECTION FOR SELF-REACTIVITIES
“COUPLED” TO SUPPRESSIVE FUNCTIONS: TREGS
(Modigliani, Bandeira & Coutinho 1996)
The repertoire of Natural Antibodies in healthy individuals
(man and mouse)
1. Is predominantly self-reactive (IgM and IgG)
2. Defines a conserved set of reactivities
3. Is independent of environmental stimulation
4. Is T cell- (and MHC haplotype-) dependent
5. Varies with the “protein self”
6. Typical pathological deviations in reactivity patterns
7. Multireactivity (auto) is abundant in new-borns
T REG SELECTION “ISOLATES”
POSITIVE vs NEGATIVE SELECTION
T REG SELECTION
PROBAB
SELECT.
POSITIVE SELECTION
(NAÏVE CELLS)
NEGLECT
DELETION
TCR-DEPENDENT AVIDITY
T REG SELECTION “ISOLATES”
POSITIVE vs NEGATIVE SELECTION
T REG SELECTION
PROBAB
SELECT
&
CELL
FATE
POSITIVE
SELECTION
(NAÏVE CELLS)
NEGLECT
DELETION
TCR-DEPENDENT AVIDITY