IMM 334 IMM 335

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IMM 334 / IMM 335 Lecture I M. Julius
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Lymphocyte Production Homeostasis
Primary lymphoid organs are sites of
lymphopoiesis T cells Thymus and intestinal
epithelium (although T cells generated at the
latter are restricted to that site, see
below) B cells Bone marrow Secondary
lymphoid organs are the sites of an immune
response involving interactions of cells within
the immune system Lymph nodes Filter
tissue-borne antigens Spleen Filter
blood-borne antigens - also consider Blood,
although not an organ
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Figure I-1
In the mouse B cell production ? 107 mature
naïve B cells produced daily...throughout the
life of the animal ? how many actually exit the
marrow and migrate to the secondary lymphoid
organs remains a question
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• T cell production
• bone marrow-derived precursors emigrate to
  thymus throughout
• gestation, undergo development and the selected
  population migrates to the periphery
• ? the thymus in the new born contains 1-2x108
  cells, and 5-10x106 mature thymocytes emigrate to
  the periphery per day
• at puberty - the thymus commences to involute
• ? post pubescent (4-6 weeks in mice) thymus
  contains 1-2 x107 cells.i.e. is now 10 of the
  size of the neonatal thymus,
• ? rate of cell emigration also decreases 10-fold
  i.e. 5-10 x 105 per day
• ? rate of production decreases but never stops

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• do T cells live longer than B cells?
• are there other sites of T lymphopoiesis? Do
  other sites take over? Yes, there are other
  sites of lymphoesis but they do not take over.
• ? another site of lymphopoiesis is the small
  intestine Intestinal Intra Epithelial
  Lymphocytes (iIEL) are T cellsBut iIEL do no
  access the inside of the animal.
• At any time during life, blood and secondary
  lymphoid organs, contain a roughly constant
  number of T and B cells notwithstanding
  alterations in the production rate of T cells!
• This process is termed lymphocyte homeostasis
  production and death are balanced

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Every day, lymphocytes with unique antigen
receptor specificities, are produced de novo, and
repopulate the periphery of the animal During
the course of immune responses ANTIGEN (Ag)
specific cells are expanding, but the available
lymphocyte repertoire remains heterogeneous
throughout the majority of life. It follows
that the majority of the cells involved in
specific immune responses must die after response
is over Memory Cells must be an exception to
this rule!we shall return
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• The Cellular Origin of Immunoglobulin (Antibody)
  
• Immunoglobulin Allotype Congenics
• The experimental system discussed below was
  developed before our understanding of
  immunoglobulin loci
• ? have antibodies that recognize markers that
  distinguish B cells and T cells
• T cells thy-1 or theta .thymocytes used as
  a source of T cells
• ? B cells membrane immunoglobulin (mIg).bone
  marrow (BM) used as a source of B cells

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• Experimental system
• ? Thymocytes isolated from a BALB/c mouse, are
  mixed with BM cells isolated from a BALB/c mouse,
  and injected with Ag, intravenously, into a
  lethally irradiated BALB/c recipient. The
  recipient is irradiated, resulting in the death
  of contained lymphocytes ? immune response of
  transferred cells is assessed, exclusively.
• must admix HISTOCOMPATIBLE cells (MHC matched)
  otherwise allogeneic responses ensue,
  confusing interpretation of the results.
• Can get positive allogeneic effects and
  negative allogeneic effects ..we will return

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But these results do not identify which of the
cells transferred are producing
Ab Immunoglobulin Allotypes discovered in the
mid 1960s Figure I-2
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• All of the Ig isotypes in a BALB/c are Iga
• i.e. IgMa IgG1a IgG2aa IgG2ba IgG3a IgAa
  IgEa
• ? All of the Ig isotypes in a C57BL/6 are Igb
• BALB/c Iga, H-2d H-2 is the notation used
  for MHC
• C57BL/6 Igb, H-2b
• Two points
• the amino acid sequence that characterizes Iga
  differs among isotypes of the BALB/c
• it follows that allotype specific Ab , say
  anti-IgG1a, will not recognize IgG1b, and hence
  will distinguish these isotypes derived from
  BALB/c and C57BL/6. This contrasts to isotype
  specific Ab, say anti-IgG1, which will not
  distinguish IgG1 derived from BALB/c and C57BL/6.

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Preparation of allotype congenic strain
BAB/10 HISTOCOMPATIBLE animals that differ
only in their Ig allotype expression. In this
case, a mouse that is genetically identical to a
BALB/c mouse, with the exception of the IgH
locus, that is derived from C57BL/6 (Igb). ?
mouse has 20 chromosomes in its genome, one of
which contains the IgH locus
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Figure I-3
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• Backcross this F1 to BALB/c
• What Ig allotypes can be donated in the form of
  gametes
• 50 of the pups will contain an Igb allele.
  These pups are chosen for further backcross to
  BALB/c
• the pups of every subsequent backcross will have
  identical distribution of Ig genotypes as the
  pups derived in the first backcross, i.e. 11
  distribution of pups expressing Iga/aIga/b
• BUT

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• due to Crossing Over, all loci, with the
  exception of the Ig locus (because we are
  selecting for it) will become increasingly of
  BALB/c origin. Thus, each backcross to BALB/c
  results in pups with an increased amount of DNA
  of BALB/c origin.
• ? the frequency of crossing over between two
  loci is directly related to the distance between
  them ? the further apart the loci are, the
  likelihood of cross overs between the two of them
  increases

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• the proportion of the desired genome is
  calculated according to the following formula
• 1-(1/2)n where n number of crosses
• for example after 10 backcrosses
• 1-(1/2)10 1-1/1024
• 100-0.1
• 99.9
• thus, progeny of the 10th backcross will contain
  DNA which is 99.9 of BALB/c origin, but 50 of
  these progeny will be heterozygous Iga/b because
  we have been selecting for it.

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• for congenic strains, must have the animals
  homozygous (in this case Igb/b). This is achieved
  through intercrossing, i.e. brother x sister
  matings.
• set up mating between Iga/b pups derived from
  10th backcross
• The pups of this intercross will express aaabbb
  in a 121ratio. Thus, 25 of the offspring of
  this intercross will be homozygous Igb/b and
  their genome will be 99.9 of BALB/c origin.
• This strain, called BAB/10, is an allotype
  congenic with BALB/c, and is fully
  HISTOCOMPATIBLE. No allogeneic effects will be
  observed when cells from these two strains are
  mixed!

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• Using cells derived from these immunoglobulin
  allotype congenic strains, we can now determine
  whether Ig is of T cell or B cell origin.
• As described above thymocytes and bone marrow
  cells as a source of T and B cells, respectively,
  are transferred along with Ag into lethally
  irradiated recipients, and the allotype of the Ab
  found in the serum was assessed
• Conclusion Antibody (Ig) is produced by bone
  marrow derived B cells!

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• Clonal Selection vs. Instructionalism
• immune system is able to recognize and respond
  to a huge (gt107) variety of Ags, and these immune
  responses are specific!
• the human genome is comprised of roughly 105
  genes, not all of which are structural
• we now appreciate the organization of Ig loci,
  and the mechanism through which a limited number
  of genes can create, through random
  recombinatorial processes, an infinite variety of
  immunoglobulin molecules, in terms of specificity
• But how is the selection of lymphocytes
  expressing the appropriate specificity achieved?
• Two theories have been proposed

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Intructionalism Paul Ehrlich circa 1895 Figure
I-4 Tenets (i) a cell must express a
receptor in order to be selected (ii) each cell
expresses multiple receptors of different
specificities (iii) once a given receptor is
ligated, expression of all other receptors
ceases (iv) there is a precursor?product
relationship i.e. the molecule ultimately
secreted is closely related to that which
functioned as the receptor. Problem If we
remove cells that can react to, Ag3, then the
responses to other Ags for which these cells also
express receptors should be compromised!
Empirically, this is not seen to occur.
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Clonal Selection Theory Joshua Lederberg,
Macfarlane Burnet, Neils Jerne c.1959 Tenets
(i) a cell must express a receptor in order to
be selected (ii) one cell expresses multiple
copies of ONE receptor specificity (iii) there is
a precursor?product relationship i.e. Ab is a
secreted form of the membrane associated Ig
molecule (mIg), and expresses the same VHVL as
the mIg which functioned as the receptor. NB In
the case of B cells, tenet (ii) refers to the
VHVL combination, and NOT the isotype, as resting
B cells express two different mIg isotypes, IgM
and IgD simultaneously, both of which express the
identical combining site.
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Figure I-5 Clonal Selection Theory ?
unlike Instructionalism, deletion of B cells
specific for a given Ag will not affect the
responses to other Ags. ? the clonal selection
theory is our current paradigm Antigen does not
affect the specificity of a cell, rather, it
selects those cells expressing receptors of the
the appropriate specificity
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Affinity Maturation of Immune Responses ? the
initial Ab produced during an immune response has
a lower affinity for Ag than that Ab produced
later in the immune response ? must understand
cross reactivity to understand this
phenomenon Consider a mouse with only two sorts
of B cells Figure I-6 ? if the mouse is
immunized with a high dose of Ag1 ?Ag1 both
B1 and B2 cells are selected and activatedgiving
rise to both Ab1 and Ab2. ? if the mouse is
immunized with ?Ag1 B1 cells are
preferentially selected and activated .giving
rise to Ab1 and not Ab2 ? the reverse would be
true for Ag2
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• Therefore
• B cell receptor for Ag, BcR, plays a central role
  in B cell activation
• (ii) the Ag used to immunize plays a central
  role in determining the affinity of the ensuing
  Ab response
• (iii) taken together, these results imply a
  threshold of BcR occupancy is required to
  achieve B cell activation
• (iv) B cells with high affinity BcR require less
  Ag to be activated than B cells with low affinity
  BcR
• NB one other empirical fact the frequency of B
  cell specific for ANY Ag x is 10-5
• We can now expand our 2 B cell mouse to
  consider a conventional mouse that contains 20-50
  x 106 B cells applying the above principles

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Figure I-7 ? number of B cells that
recognize Ag x with low affinity is high ?
number of B cells that recognize Ag x with high
affinity is low ? how low can affinity be and Ig
still be considered an Ab? 105 L/mole ?
although can measure such low affinities
experimentally, will not find Ab with such low
affinities.consistent with the notion of a
threshold of activation, that in turn is
related to the intrinsic affinity of the BcR,
that in turn defines the concentration of Ag
required to activate it!
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Now we can consider Affinity Maturation Figure
I-8 ? each activated B cell divides creating
daughter cells that differentiate into cells
that secrete Ab that has an affinity for Ag which
is related to that of the BcR on the mother B
cell. ? most of the Ab will be low affinity, as
most of the Ag specific B cells activated using
?Ag will express low affinity receptors for
Ag ? Ab binds and clears Ag from the system? Ag
falls ? ?Ag favours the continued
activation/differentiation of B cells expressing
high affinity BcR ? subsequent Ab produced will
be on average of higher affinity
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Anything affecting the affinity of immune
responses, will therefore affect the process of
affinity maturation. I Ag ? the average
affinity of Ab is inversely related to the Ag
used for immunization II Adjuvents ?
Non-specific immune stimulators that help
increase the immunogenicity of substances i.e.
non-specifically activates T and B cells
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• Freunds Adjuvent (i) Complete mineral oil
  containing mycobacteria (ii) Incomplete
  mineral oil
• create a water (Ag) in oil emulsion
• ? keeps Ag localized (doesnt go systemic)
• ? slow release from bolus? maintenance of
  relatively ?Ag in the system
• ? Maintain a high concentration of Ag, therefore
  average affinity of response is low but the
  response is larger (since more B cells of low
  affinity respond)

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III Somatic mutation ? mutations occur within
the VH and VL regions of Ig during clonal
expansion ? these mutations are localized in
hotspots called Complementarity Determining
Regions or CDR ? these mutations can alter the
specificity and affinity of the Ig for Ag x ?
these mutations are NOT directed i.e. they may
result in either an increase or decrease in Ig
affinity for Ag in a non-predictable fashion ? if
as a consequence of somatic mutation the affinity
of the BcR for Ag x is increased, then the B
cell will move to the right on the curve (Fig
I-8), and will be selected in circumstances of
?Ag ? if as a consequence of somatic mutation
the affinity of the BcR for Ag x is decreased,
then the B cell will move to the left on the
curve (Fig. I-8), and will be excluded from the
immune response as the Ag falls
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The clonal selection theory applies to the
selection of both B and T cells during the course
of an immune response. But, as you understand, T
cells are somewhat more complicated to deal with
as they recognize antigen in the context of
molecules encoded by the Major Histocompatibility
Complex (MHC). We must therefore consider the
role of the Antigen Presenting Cell (APC)
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• Antigen Recognition by T Cells
• central role for APC
• ? many different cell types function as APCs,
  each of which have specialized function and
  tissue distribution monocytes/macrophages (M?)
  marginal zone M? dendritic cells (DC)
  follicular dendritic cells (FDC) and B cellsin
  certain states of activationwill return
• Consider the generic APC in the context of
  creating a notation for T cell activation we
  will first deal with the generation of cytotoxic
  T cells, or killer T cells

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Figure I-9 Two things are
happening (i) clonal expansion (ii) differentiati
on into effector T cells Now we need a method
for quantitating these events
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An Assay for T cell killer function Figure
I-10 ?perforin induced target cell
death is one mechanism associated with TK
function ? creation of holes in the plasma
membrane of target cells ? loss of
semi-permeability ? leads to cell death, which in
tun leads to the release of 51Cr into the culture
supernatant that can be quantitated ? there is a
direct relationship between the extent of killing
and the amount of 51Cr in the supernatant
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MHC Restricted T Cell Function The Generic
Experiment Ag splenocytes from a BALB/c (H-2d)
a mixture of T cells, B cells, and M???which have
been covalently modified with trinitrophenyl
(TNP), using a reactive form of this hapten,
trinitrobenzenesulfonic acid (TNBS). Figure I-11
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TARGETS killing of targets - no killing
of targets Only targets expressing Ag and
appropriate MHC are killed, ? MHC restricted
function of Tk ..NB there is a white lie
in this statement, we shall return! But there
are many molecules on the surface of these APC,
how was it determined that MHC is restricting TK
function?
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Another Congenic Experiment Using BALB/c (Iga,
H-2d) and C57BL/6 (Igb, H-2b) prepare a congenic
strain in which H-2b derived from C57BL/6 is back
crossed onto a BALB/c background. ? prepare an
F1, and then back cross these pups to BALB/c,
selecting for those expressing H-2d/b at each
cross ? pups of the 10th backcross that express
H-2d/b are then intercrossed ? 25 of the pups
from this mating will express H-2b/b with 99.9
of the remainder of the genome being of BALB/c
origin ? this strain is called BALB.B TK are
then prepared in BALB/c and in C57BL/6 mice using
TNP-modified syngeneic (genetically identical)
splenocytes as Ag, followed by activation/expansio
n of these cells in vitro using the same source
of Ag, as described above.
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• Prepare TNP-coated targets from BALB/c, C57BL/6
  and BALB.B, and assess the specificity of TK
  derived from each of BALB/c and C57BL/6
• TARGETS
• TK derived from C57BL/6 kill BALB.B targets
  modified with TNP, all other gene products in
  BALB.B are of BALB/c origin, thus TK must be MHC
  restricted, all other self molecules are
  irrelevant