International Symposium on Lysosomal Storage Diseases

1
Overview of immune mechanisms perspectives from
B cell immunology

• International Symposium on Lysosomal Storage
  Diseases
• ISLSD, Paris, April 2008. This talk is available
  from http//tinyurl.com/yq72ks
• Simon Hunt
• Sir William Dunn School of Pathology
• simon.hunt_at_path.ox.ac.uk
• You are welcome to use the material in this
  lecture for your own private study or for
  non-profit educational use provided you cite the
  link to this page. Further distribution is not
  allowed.
• Travel and accommodation expenses and honorarium
  kindly provided by Shire Pharmaceuticals

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Anaphylaxis in Man
Effraction (Fr.) breaking and entering, burglary
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Serum sickness in Man
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Anti-aGalA antibodies during ERT in Fabry
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In this talk Ill cover

• Antigen-antibody interactions and effector
  mechanisms
• Immunogenicity
• the strength of an antigenic stimulus
• how to minimise it
• Strategies for avoidance
• dose and form of enzyme
• induction of immunological tolerance
• cellular mechanisms Regulatory T lymphocytes T
  regs
• patient selection
• A new technology platform, CPAI
• to measure calcium signals
• potential diagnostic, monitoring, research
  applications?

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The antibody molecule IgG
Fc
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Two LSD enzymes antigens
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Each IgG can bind two antigens - divalent
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At certain ratios, Enzyme-Ab complexes form
Multivalent binding -gt high overall strength of
binding avidity
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Effects of antibody fluid phase
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Anaphylactic and anaphylactoid reactions
anaphylactic
ERT enzymes?
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Effects of immune complexes cellular regulation
FcReceptor subtypes can be (i) activating(ii)
inhibitory
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Immunogenicity

• Antigen-antibody interactions and effector
  mechanisms
• Immunogenicity
• the strength of an antigenic stimulus
• how to minimise it

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Nature of antigens

• Minimum size (MW) to induce immunity
• 3000 to 5000 Daltons
• much smaller molecules (Haptens) become
  immunogenic if covalently coupled to larger
  Carrier
• Chemical nature practically unlimited
• e.g. proteins, nucleic acids, synthetic chemicals
  (especially drugs)
• And very importantly, carbohydrates
• often thymus-independent (TI) trigger only
  IgM
• T cells can see proteins and peptides, not
  carbohydrates
• Antigenic determinants epitopes
• Conformational like proteins in their native
  shape B cell antigens
• Linear denatured short peptides T cell
  antigens

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T and B cells see fundamentally different kinds
of epitopes
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How to tame a vigorous immunogen

• An immunogen is an antigen in its role as inducer
  of an immune response. Tends to provoke weaker
  responses when
• its dose is low
• low zone tolerance
• it can be sneaked in initially at low
  concentration
• there is little or co-stimulus (lack of help)
• from immune lymphocytes (T lt-gt T Tlt-gt B)
• from innate immune cells
• Macrophages, Natural Killers, dendritic cells
• the immune system is already somewhat
  immunosuppressed
• e.g. by drugs, monoclonal Ab preconditioning
• because of disease
• there are no adjuvants
• adjuvants provide very strong co-stimulatory
  signals
• inflammation generates adjuvant-like molecules

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Low zone tolerance
?Flagellin challenge in adjuvant
Low zone tolerance
High zone tolerance
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Synergistic signalling

• Bretscher and Cohn (1970) Hypothesis suggested

? Anergy
? Activation
Their theory originally applied to B cells but
is now extended to T cells
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Two signals for B cells
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Blockade of co-receptor on T cells
Bluestone et al (2006) Immunity 24 233
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Predisposition to altered immune system
reactivity in some LSDs
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Strategies for avoidance

• Antigen-antibody interactions and effector
  mechanisms
• Immunogenicity
• the strength of an antigenic stimulus
• how to minimise it
• Strategies for avoidance
• dose and form of enzyme
• induction of immunological tolerance
• cellular mechanisms Regulatory T lymphocytes T
  regs
• patient selection

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Antibody tolerance in gene-treated Fabry mice
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Tolerance transfer by CD4ve spleen cells
28 days
?
?rhaGalA
?
35 days
?
49 days
? Spleen CD4
?
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New technology platform

• Antigen-antibody interactions and effector
  mechanisms
• Immunogenicity
• the strength of an antigenic stimulus
• how to minimise it
• Strategies for avoidance
• dose and form of enzyme
• induction of immunological tolerance
• cellular mechanisms Regulatory T lymphocytes T
  regs
• patient selection
• A new technology platform, CPAI
• to measure calcium signals
• potential diagnostic, monitoring, research
  applications?

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Cell Population Array Imager, CPAI

• a very low-power wide-field microscope
• for measuring dynamic light changes in living,
  single cells in suspension
• cells settle into wells in a close-packed
  ultra-microarray on an optical fibre conduit
• mounted in micro-chamber through which reagents
  flow
• capture images for up to a few hours

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CPAI schematic
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The apparatus
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Calcium responses in anti-CD3CD28 triggered
Jurkats
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Selected Oscillators
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Finding similar shapes - 1
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Finding similar shapes - 2
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Identifying the wells
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Ca2 signal patterns in primary T cells
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Loss of Ca2 buffering in lysosomal storage
diseases
Inhibition of Ca-dependent processes
Activation of Ca-dependent proteases
Kiselyov Muallem Cell Calcium 2008
10.1016/j.ceca.2007.12.005 
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In this talk we have visited

• Antigen-antibody interactions and effector
  mechanisms
• Immunogenicity
• the strength of an antigenic stimulus
• how to minimise it
• Strategies for avoidance
• dose and form of enzyme
• induction of immunological tolerance
• cellular mechanisms Regulatory T lymphocytes T
  regs
• patient selection
• A new technology platform, CPAI
• to measure calcium signals
• potential diagnostic, monitoring, research
  applications?

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B cell immunologists love to be

• where self molecules get treated as though
  they are non-self
• where it matters basic immunology concepts
  translate into practice
• where things are described in hard-to-decipher
  hieroglyphs
• where theres no protection
• in Paris!

Image of Alain Robert, French spiderman, climbing
the obelisk, Place de la Concorde http//www.franc
etoday.com/thismonth/frenchspiderman.php
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Dr Steve Young Infection Immunity, Birmingham
Co-investigator
Pete Stroud, Martin Osgathorpe Dunn School
Workshop
Wendy Brownsill Research Technician
Thanks too to Dr Nick White and the Bioimaging
suite at the Dunn School Funded by a Wellcome
Trust Showcase Award