Determinants of Immunogenicity

and Tolerance

Abul K. Abbas, MD
Department of Pathology
University of California San Francisco

EIP Symposium Feb 2016

 Why do some people respond to
therapeutic proteins?
•  Characteristics of the protein
–  Conformation, post-translational modifications, etc

•  Features of the host

–  Genes that influence immune activation vs tolerance:
largely unknown
–  The immune “background” of the host
–  Defective control/tolerance mechanisms

•  Environmental factors
 Critical questions
•  Requirements for antibody response to a
protein

•  Why do some individuals produce antibodies

when exposed to a “self” protein?
–  Why does tolerance to the protein fail?

•  How can tolerance be restored?

•  What assays need to be developed to better

understand the loss and restoration of
tolerance?
 Antibody production: activation of B cells

Helper T cells,
other s#muli
Naive
IgG
B cell

Ac#vated B cells
Ac#vated differen#ate
Microbe B cell Prolifera#on
into an#body-secre#ng
plasma cells
 T cell responses
Effector T cells

CD4+ helper
T cells

Cytokine
Proliferation secretion
+ antigen

+ antigen
Differentiation
APC
Naïve T cells:
Can recognize Cell
antigen but killing
incapable of any CD8+ CTLs
functions
 6
Capture and presentation of antigens by dendritic cells

Sites of microbe entry:

skin, GI tract, airways
(organs with continuous
epithelia, populated
with dendritic cells).
Less often -- colonized
tissues, blood

Sites of lymphocyte
activation: peripheral
lymphoid organs (lymph
nodes, spleen), mucosal
and cutaneous lymphoid
tissues

Abbas, Lichtman and Pillai. Basic Immunology, 5th edition, 2016, Elsevier

Antigens and T cells come together in the same organs

 Schematic model of T cell recognition
of antigen: what antigens do T cells see

MHC molecules are the peptide display molecules of the immune system
Human MHC: HLA (human leukocyte antigens)
 HLA molecules
•  Most polymorphic genes in biology (many
variants – alleles – in the population)
–  Each of us inherits different combinations of
alleles
–  Each HLA variant can display many (but not all)
peptides; hence, individuals may display
different peptides and respond to different
antigens

•  Two major classes:

–  Class I MHC molecules bind to CD8
–  Class II MHC molecules bind to CD4
 Each HLA molecule presents a limited
number of peptides

Internalization Antigen Processing generates T cells

of antigen multiple peptides, respond to
processing one of which can bind immunodominant
into APC
to class II allele peptide epitope
Multiple possible
epitopes CD4+
T cell

Immunodominant
epitope
Costimulation: the second signal for T
lymphocyte activation
Costimulation: signal(s)
in addition to antigen
that are needed to
stimulate adaptive immune
responses

Costimulators are induced

on APCs by microbes and
by adjuvants administered
in vaccines

Self antigens and other

harmless substances fail
to induce costimulators,
do not elicit immune
reactions and may induce
tolerance
Role of costimulation in T cell activation

Abbas, Lichtman and Pillai. Basic Immunology, 5th edition, 2016 c Elsevier
 Costimulation determines the choice
of activation vs tolerance
Costimulation
(signal 2)

Immunogenic
antigen
(microbe, APC Naïve
TCR T cell
vaccine) Antigen (peptide + HLA):
signal 1
Effector and
memory cells

Tolerogenic
antigen (e.g.
self)
Tolerance: functional
inactivation or cell death,
or sensitive to suppression
 Naïve T cells are more dependent on B7
costimulation than are memory cells

Naïve CD4 T cells Memory CD4 T cells

500000 500000
Proliferation (CPM)

400000 400000

300000 300000

200000 200000

100000 100000

0 0
0 0.001 0.01 0.1 1 0 0.001 0.01 0.1 1

Antigen (µg/ml)
APCs wild type (normal; positive control)
B7.1/2-/-
None (negative control)
 Costimulation

•  Required for initiating T cell responses

(activation of naïve T cells)

•  Ensures that T cells respond to microbes

(the most potent inducers of costimulators)
and not to harmless antigens
–  Source of costimulation during responses to
tumors, transplants, proteins given without
adjuvants ?

•  Memory cells are less dependent on

costimulation than are naïve T cells
 Possible host determinants of antibody
responses to a biological
•  Size of antigen-specific T cell pool
–  Pre-existing pool of naïve T cells
–  High number of memory cells induced by cross-
reactive environmental antigens

•  Inheritance of HLA alleles that present the

antigen
–  Unlikely to be a key issue in an outbred population
(many different HLA alleles present)

•  High level of costimulation

–  Source? Unlikely to be provided by antigen itself
–  Activation state of dendritic cells?
 Exploiting absence of costimulation to
inhibit immune responses

•  Costimulatory blockade (blocking B7-CD28,

CD40 pathways)

•  Peptide administration (repeated low doses,

modeled after desensitization for allergies)
–  Peptide-coupled PBLs (spleen cells in mice)
The immunological equilibrium: balancing
lymphocyte activation and control

Tolerance,
Activation
regulation

Normal: reactions No response to self and

against pathogens other harmless antigens
 Where is tolerance induced?

Central tolerance Peripheral tolerance

 Best defined mechanisms of peripheral
T cell tolerance

•  Engagement of inhibitory receptors

•  Suppression by regulatory T cells (Tregs)

Inhibitory receptors of the immune system

•  One mechanism by which the system

maintains a balance between activation
and inhibition is to use different
receptors for different outcomes

•  Inhibitory receptors are present in NK

cells, T cells and B cells
–  Best defined activating and inhibitory
receptors of T cells are members of the
CD28 family
 21

The opposing functions of CD28 and CTLA-4

B7 CD28

B7-CD28
interaction APC Naïve
TCR T cell Proliferation,
differentiation
CTLA-4

B7-CTLA-4
interaction
Functional
inactivation

Knockout of CTLA-4 in mice and mutation in humans

results in immune dysregulation (lymphoproliferation,
multi-organ inflammation)
The landscape of T cell activating and
inhibitory receptors

TIGIT
 Engaging inhibitory receptors for
inducing tolerance

•  Can inhibitory immune receptors be

triggered to shut off immune responses?
–  Problem of reliably producing agonistic
antibodies against cellular receptors

•  Blocking inhibitory receptors has

revolutionized cancer immunotherapy
–  “Checkpoint blockade” stimulates anti-tumor
immune responses
 Regulatory T cells

CD4+, high
CD25 (IL-2
receptor),
Foxp3
transcription
factor
Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 8th edition, 2014, Elsevier
 The significance of Foxp3+ Tregs
•  Genetic evidence: Foxp3 mutations -->
autoimmune disease (IPEX); in mice,
disease can be corrected by providing
normal Foxp3+ cells

•  Do defects in Foxp3+ Tregs or

resistance to Treg-mediated suppression
contribute to common autoimmune
diseases?
–  Inconsistent and variable data
–  By definition, any abnormal immune response
reflects an imbalance of activation vs control
 The therapeutic potential of
regulatory T lymphocytes

•  Cell transfer of autologous Tregs to
suppress immune responses
–  Grow up patient’s Tregs ex vivo
–  Ongoing clinical trials in graft rejection, T1D
show it is safe
–  Very little efficacy data

 The therapeutic potential of
regulatory T lymphocytes
• 

Cell transfer of autologous Tregs to suppress immune
responses

•  Challenges:
–  Technically difficult, individualized

•  Administer antigen or antigen mimic in

ways that preferentially induce Tregs?
–  Weak stimulus (peptide antigen, anti-CD3); +
IL-2?
Functions of Interleukin-2: the dogma
 The unexpected biology of IL-2

•  Interleukin-2 is the prototypic T cell

growth factor (TCGF), required for
initiating clonal expansion of T cells in
response to antigen

•  BUT: knockout of IL-2 or the α or β

chain of the IL-2R results not in immune
deficiency but in systemic autoimmunity
and lymphoproliferation
 Dual roles of IL-2 in T cell responses

Surprising conclusion from knockout mice: the non-redundant

function of IL-2 is in controlling immune responses
 Therapeutic potential of IL-2: a
revision
•  IL-2 was originally used to boost immune
responses in cancer, HIV infection
(promoting effector and memory T cells)
•  Inconsistent clinical results

•  IL-2 treatment can increase number and

functional activity of Tregs
•  Low-dose IL-2 to treat steroid-resistant
chronic GVHD, vasculitis
•  More recent clinical trials ongoing in type 1
diabetes, SLE, graft rejection
 Antigen-specific lymphocytes
•  Are antigen-specific T (and B) cells present
before treatment? Are they altered by
treatment?
–  Frequency of naïve T cells predicts magnitude of
response following antigen exposure

•  What are the phenotypic and functional

characteristics of antigen-specific
lymphocytes?

Need for more sophisticated analyses of

antigen-specific lymphocytes
 Strategies for restoring tolerance

•  Administration of antigen in tolerogenic form

–  Repeated doses of peptides without adjuvants

•  Blocking costimulation

•  Engaging inhibitory receptors

•  Treg targeted therapies:

–  Treg cell transfer
–  IL-2