M' Julius IMM 3345 Lecture IV

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M. Julius IMM 334/5 Lecture IV
Addendum To Lecture III
Co-Stimulation in Antigen Presentation Successf
ul T cell activation requires the expression of
key co-stimulatory molecules on the APC in
addition to antigen/MHC complexes. While many
molecules expressed on the membrane of APC do
function as co-stimulators of T cell
activation, B7-1 (CD80) and B7-2 (CD86) play a
fundamental role as the physiological ligands of
CD28, expressed by most T cells. Professional
APC constitutively express members of the B-7
family, other cells, that can function as APC,
upregulate the expression of B7 in an activation
state dependent fashion. This is true of B cells
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• resting, high buoyant density B cells do not
  express B7
• activated, low buoyant density B cells do
  express B7
• As indicated above, the physiological ligand of
  B7 is CD28, that is expressed on most T cells.
  CD28 is a disulfide linked homodimer.
• CD28-B7 interaction provides an essential
  co-stimulatory signal required for T cell
  activation. In the absence of this CD28 mediated
  signaling..T cells are not activated, and
  moreover, are induced into a state called
  ANERGY.
• The induction of anergy is an active process and
  precludes subsequent activation by professional
  APC. i.e. anergized T cells will not respond to
  antigen presented by professional APC.

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Figure IV-A
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Lecture IV Notes At the end of Lecture III we
discussed the biochemical basis for the strict
association of CD4 with MHC Class II restricted
TcR, and of CD8 with MHC Class I restricted
TcR.providing the mechanism underlying the
absence of CD4 T cells expressing an MHC Class I
restricted TcR, and CD8 T cells expressing an
MHC Class II restricted TcR in the periphery of
an animal. First consider peripheral mature T
cells in the context of the paradigm we have
created Figure IV-1
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Given that we do not see mis-matched expression
of co-receptor molecules and TcR restriction
specificity in peripheral T cells ? the selection
processes occurring during T cell development in
the thymus must result in this phenotype of
mature T cells Intra-thymic T Cell Development
Two Models Instructional Model Figure IV-2
?
?
?
?
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• cannot know a priori whether TcR recognizes MHC
  Class I or II
• ? if the coaggregation of CD4 or CD8 with the TCR
  limits activation and subsequent development of
  DP cells? SP cells ? DP cells have optimized the
  possibility of ve and/or ve selection as they
  are equipped to support either MHC Class I or II
  recognition
• It follows
• if the TcR is MHC Class I restricted ? CD8-TcR
  coaggregation ?maturation ?CD8SP
• ? if the TcR is MHC Class II restricted ? CD4-TcR
  coaggregation ?maturation ?CD4SP
• The assumption is that
• TcR-CD8 coaggregation ? down regulated expression
  of CD4
• and
• TcR-CD4 coaggregaton ? down regulated expression
  of CD8

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Stochastic/Selection Model Figure IV-3 ?
random (stochastic) down regulation of either CD4
or CD8 expression ? 50 chance that the
specificity of the TcR will match that of the
co-receptor ? this stochastic process is then
followed by a selection check point ? only when
co-receptor and TcR specificities are matched
does maturation/selection proceed ? those cells
expressing mis-matched TcR and co-receptor die
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• Prediction in aid of distinguishing these models
• Stochastic/Selection model predicts the presence
  of immature T cells expressing mis-matched
  TcR/co-receptors, while the Instructive model
  predicts their absence
• ? mounting evidence supports a Blend of the two
  models
• T Cell Dependent Humoral Immune Responses
• We shall limit our discussion to in vitro
  experiments in which we are in control of all of
  the experimental elements that can be manipulated
• All of the properties of th are identical to
  those established for tk with exception that
  their usual ligand is MHC Class II
• selection of an MHC restricted repertoire
• selection within chimeras
• ? th?TH requires an MHC restricted interaction
  with an APC, as for tk?TK

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• What is T Cell Help?
• The simplest form of the generic experiment
• T cells B cells Ag ? Ab
• B cells Ag ? ? for so-called T
  dependent (TD)
• Agswe shall return
• The Components
• TH clone it is important that we know its allo
  specificity, it must NOT be alloreactive to the
  MHC expressed by B cells in the mixture
  experiments we will do
• We will use a CD4 H-2d th ? KLHH-2d clone
• ? KLH keyhole limpet hemocyanin
• ? KLH is the oxygen carrying protein of molluscs
• our clone recognizes a KLH derived peptide in
  conjunction with
• I-Ad (remember this is a CD4 clone)

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B cells derived from spleen cells ? contain T
cells, B cells, and M? (APC) ? remove T cells ? B
M?, which we will denote as B/APC ? this is a
B cell population ? contains a mixture of many
specificities ? if we immunize the B cell donor
with a specific Ag, we can increase the frequency
of B cells specific for that Ag, and hence will
subsequently be able to reveal them in
vitro..this is illustrated in the following
figure Figure IV-4
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• mice are immunized with a hapten-carrier i.e.
  dinitrophenyl(DNP)-Ferritin (F), that are
  covalently linked
• DNP is the hapten, a small molecule that will
  not elicit an Ab response if injected alone
• F is a large molecule, and functions as the
  carrier ? making the DNP moiety immunogenic
• ? we purposely chose F as it does not cross react
  with KLH, the specificity of the TH clone we are
  using
• We immunize with DNP-F to prime the animal. An
  immune response to both DNP and F will ensue ?
  the DNP specific B cells, denoted as B?DNP will
  be expanded. B?F will also be expanded, but were
  not interested. This scenario is illustrated in
  the following figure

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Figure IV-5 ? recall that the frequency of B
cells specific for any Ag x in a naïve animal
is roughly 10-5 ? subsequent to the immune
response to DNP-F, the frequency of B?DNP is
gtgtgt10-5 within the population ? as the immune
response wanes, the B?DNP will be resting, and no
longer secreting Ab ? we will denote these B
cells as long term primed (LTP) How is Ab
measured? We can assess serum Ab levels, specific
for DNP, however, this would be a mixture of Abs,
and as will become apparent, we must be able to
look at Ab production at the level of a single
cell.
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The Plaque Forming Cell assay This technique
enables the visualization of individual cells
secreting Ab. Its simplest from is illustrated in
the following figure Figure IV-6 ? mice
are immunized with sheep red blood cells (SRBC) ?
at various times post immunization, spleen cells
are isolated ? these splenocytes, which contain
Plaque Forming Cells (PFC) are mixed with an
excess number of SRBC (indicator cells), and
plated in agar, in a petri dish
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• ? each PFC is surrounded by SRBC
• anti-SRBC is secreted by the PFC, and binds to
  the surrounding SRBC
• ? complement (C) is added to the mixture this
  substance is a mixture of proteins that will be
  discussed later in the course. For the purposes
  of the present discussion, C binds to Ab
  molecules that are complexed with Ag. The
  interaction of C with AgAb complexes results in
  its activation ? punctures holes in the cell to
  which the AgAb complex is bound
• ? C will bind to anti-SRBCSRBC complexes, and
  lyse the SRBC
• ? it follows that SRBC surrounding an SRBC
  specific PFC will be lysed, creating a clearing
  or plaque
• The PFC assay can be tailored to detect Ab
  secreting cells of any specificity simply by
  coating the indicator cells with the appropriate
  Ag ? using SRBC coated with DNP will allow the
  detection of DNP specific PFC

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• Return to the Question What is T Cell Help?
• The first insight came with the deliberate
  separation of T and B cell specificities, through
  the use of the hapten-carrier system
• the T cell is specific for the carrier ? KLH
• the B cell is specific for the hapten ? DNP
• This system revealed the requirement for Linked
  Recognition (LR) Determinants recognized by T
  and B cells must be physically linked

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• Figure IV-7
• in the presence of DNP-KLH, covalently linked,
  LTP B?DNP are activated ? differentiate into DNP
  specific plaque forming cells (DNP-PFC)
• in the presence of DNP-F ? KLH specific T cells
  are not activated ? no help
• ? in the presence of DNP-F KLH ? as we shall
  see, the T cells ARE activated, but cannot help
  LTP B?DNP in the absence of LR

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• These results gave rise to the concept of an
  antigen bridge, which bridges the KLH specific
  TH with the DNP specific B cell.this is
  conceptually correct, but mechanistically
  incorrect.
• Consider
• T cells do not recognize native Ag, rather
  peptides derived from native Ag that are
  presented in the context of MHC
• thus, th ? TH is an MHC restricted process
• ? however the requirement for LR suggests that
  specific T cells must CONTACT specific B cells
• Does this mean that TH-B interaction is MHC
  restricted?
• This question is illustrated in the following
  figure

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• Figure IV-8
• this question can be addressed by mixing two
  sorts of B cells with the CD4 H-2d th ? KLHH-2d
  clone those that can be recognized by the TH
  clone in an MHC restricted fashion, and those
  that cannot.
• then we can determine which B cells are
  activated
• this can be assessed by allowing the culture to
  go to Ab production ? the T cells have done their
  job, and removing either the H-2d expressing B
  cells, or the H-2b expressing B cells using C
  mediated lysis in combination with antibodies
  specific for H-2d and H-2b
• ? the CD4 H-2d th ? KLHH-2d clone is NOT
  alloreactive to H-2b

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Figure IV-9 We conclude that TH-B
interaction is MHC restricted. Specifically, only
those B cells that can present Ag to the TH clone
are activated ? the requirement for LR reflects
the requirement for an MHC restricted TH-B
interaction Now we can reassess the requirement
for LR for TH dependent activation of LTP B?DNP
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Figure IV-10 ? the B cell is specific for
DNP ? both H-2d and H-2b expressing LTP B?DNP
will bind DNP through their BcR ? since KLH is
physically linked to DNP (LR), the B?DNP will
indirectly bind KLH ? ? receptor (BcR) mediated
internalization of DNP-KLH ? the DNP-KLH will be
processed as it is in other APC ? ? only the
H-2d expressing B cell will express determinants
derived from KLH in the context of I-Ad, i.e.
creating the Ag that can be recognized by the
I-Ad restricted TH clone
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One aspect of T cell help involves TH-B contact,
mediated through an MHC restricted recognition of
the B cell by the TH cell Non Specific Ig
The Bystander B Cell Response The term
non-specific is a misnomer ? the Ig is actually
not nonspecific, rather, we dont know what its
specificity is! Consider that a mouse reared in
an open environment is under constant
stimulation by environmental Ags ? Abs. As we
dont know what the Ags are, we cannot determine
the specificity of the Abs ? we call them
non-specific Ig (NSIg) to distinguish them from
the specific Ab we are analysing during the
course of an immune response that we deliberately
induce e.g. a response to DNP-KLH
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We can quantitate NSIg using a plaque assay, in
which the indicator cells are coated with
antibody specific to Ig, independent of its
specificity, as illustrated in the following
figure Figure IV-11 If we assess the
number of DNP PFC and NSIg PFC in a
non-deliberately immunized animal, as well as in
an animal immunized with DNP-carrier, we observe
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1 there are a few B cells secreting anti-DNP in
non-immunized mice ? likely reflects stimulation
with environmental Ag that looks like DNP 2
reflects the level of B cell stimulation by
environmental antigen 3100-fold increase in the
anti-DNP response upon immunization 4 there is
a concomitant 10-fold increase in NSIg secreting
cells in deliberately immunized animals As
these NSIg secreting cells are unrelated
(specificity) to the antigen specific response,
how are they generated?
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• TH Dependence of NSIg Production
• Increases in NSIg only accompany immune responses
  to T-dependent Ags
• T-dependent Ags (TD) are those that require the
  presence of TH cells
• T-independent Ags (TI) are those that induce
  quantitatively similar responses in the presence
  and absence of TH cells e.g. in an ATX BM
  chimera, or B animals
• a classical example of a TI Ag is bacterial
  flagellin, and indeed bacteria themselves e.g.
  Brucella abortus (BA)
• ? that BA is a TI Ag is demonstrated, as
  illustrated in the following table

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1 demonstrates that BA is a TI Ag 2
demonstrates that DNP-KLH is a TD Ag Can use TI
Ags as carriers for haptens and thus convert the
anti - hapten response from TD ? TI
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Now consider the NSIg response associated with
immunizing a conventional mouse (euthymic) with a
TI or a TD form of DNP 1 this is
considered the bystander B cell response 2 no
increment over background in NSIg PFC Conclude
Induction of the Bystander B cell Response is T
cell dependent, i.e. requires the activation of T
cells
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Differential Requirements for LR in Ag Specific
and NSIg Responses Using the CD4 H-2d th ?
KLHH-2d clone, in combination with B cells
derived from an animal LTP with DNP-F, and
therefore containing an expanded population of
B?DNP as well as all of the other B cells of
unknown specificity, consider requirements for LR
of the DNP and NSIg responses
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Figure IV-12 ? in the presence of
DNP-KLH we observe both a DNP and a NSIg
response ? in the presence of DNP-F, we observe
neither response, as the th are not activated ?
in the presence of DNP-F KLH, we observe a NSIg
response, exclusively
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• requirement for th activation and LR for LTP
  B?DNP
• ? requirement for th activation BUT NOT LR for
  the NSIg response
• Does the lack of requirement for LR correlate
  with the lack of requirement for an MHC
  restricted TH-B interaction ? NSIg PFC ?
• Repeat the B cell mixing experiment that we used
  to demonstrate the requirement for an MHC
  restricted TH-B interaction for the activation of
  LTP B?DNP but consider both the DNP as well as
  the NSIg PFC responses, as illustrated in the
  following figure

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Figure IV-13 Correlation between the
requirements for LR and MHC restricted TH-B
interaction ? an animal LTP with DNP-F contains a
population of B cells that gives rise to NSIg
with activation requirements distinct from those
of the B?DNP , requiring neither LR, nor an MHC
restricted interaction with TH