The Development and Survival of Lymphocytes

1
Chapter 7 The Development and Survival of
Lymphocytes
Most of this chapter is about the
maturation/development of lymphocytes in the
absence of foreign antigen (from pre-lymphocyte
to mature naïve lymphocyte)

• Gene rearrangements
• Testing whether the rearrangements are productive
  and the receptor is functional (and replacing
  nonproductive rearrangements if possible)
• Testing the specificity of the receptor to
  provide assurance that it is not anti-self
  (tolerance) and get rid of cells with anti-self
  specificity
• T cells need to be MHC restricted (bind to self
  MHC) but not anti-self (bind to self molecules).
  This seems to be somewhat contradictory and
  introduces a technically difficult problem for T
  cells

Most lymphocyte die before solving all these
problems
2
Summary of the life of a B cell
Bone marrow
Periphery
With T help
Plasma cells mostly in bone marrow memory B
cells in lymphoid tissue and circulation
(Clonal
deletion)
In the bone marrow, B cell rearrange their Ig
genes in the absence of antigen (panel 1). Those
B cell with a BCR that bind antigen (self) in the
bone marrow die (panel 2). B cell that do not
bind antigen in the bone marrow can leave (mature
naïve). If they bind antigen in the periphery
(and get T help), they are activated (proliferate
and differentiate) (panel 3 and 4). Activated B
cell become plasma cells or memory cells (panel 4)
3
Lymphocytes develop from a hematopoietic stem
cells
Not exactly as shown in chapter 1
4
B cell development is dependent on bone marrow
stromal cells
5
Ig gene rearrangements occur in an orderly
fashion in developing B cells (every step in this
slide is antigen-independent)
periphery
bone marrow
6
When B cells rearrange their H chain genes they
make a pre-B cell receptor using a surrogate
light chain to test whether the rearrangment was
productive
If successful in making a functional H chain, the
cell stops rearrangement at the heavy chain locus
and divides several times before rearrangements
on the light chains
Here is whyCase 1 no division after making a
functional H chain. If 20 successful at H and
20 success at L then 4 (.2X.2) of the cells
will be successful (need 100 tries (cells) to get
4 successes). Case 2 division after making a
functional H chain. If 20 successful at H,
then the 1 in 5 (20) successful cells divide to
form 40 cells. Then 20 of the 40 (8) are
successful at L. So, 18 of the cells will be
successful at both L and H (lost 4 of 5 the first
round and 32 of 40 the second round so 8 of 45
(18) cells are successful)
7
Allelic exclusion.The expression of only one of
two co-dominant alleles in any given cell
(important for clonal selection) All the
antibodies made by one B cell (or TCR by T cell)
are identical even though each cell has the genes
to make up to 8 different antibodies In theory,
either of 2 H chains could pair with any of 2 k
and 2 l L chains. (2H X 4L 8 Ig binding sites)
a allotype
b allotype
Allelic and isotype exclusion provides that, in
any one B cell, only one heavy chain and one
light chain gene code for all the heavy and light
chains. Thus, one allotype and one specificity
per B cell (this is most efficient for clonal
selection) (same principle for the TCR)
Serum IgG1 is 50 a-allotype and 50 b-allotype
b allotype
a allotype
1 allotype per B cell
b allotype
a allotype
How is allelic/isotype exclusion achieved? By
stepwise, orderly gene rearrangements with
testing for success at each step (see slide 10)
Isotype exclusion (k vs l) and allelic exclusion
for each light chain and heavy chain locus
8
Because heavy (H) chains use up all their D gene
segments in an attempt to make an H chain, they
get only one chance per locus for a productive
rearrangement. However, in light chains multiple
attempts at gene rearrangements are possible.
delete
The maximum number of attempts in light chains is
equal to the number V gene segments or J gene
segments, which ever is fewer. Usually, less
than the maximum are possible because see
figure for why this cell had only three tries,
not five(even if n40 in Vkn and 5 Jk ).
Rearranges until there is a functional Ig (heavy
and light) or until it runs out of V or J
segments and dies
9
Bone Marrow
Periphery
For simplicity, we will refer to all these stages
(before the cell has a functional BCR) as Pre-B
cells
Pre-B cells Immature B cells
Mature naïve B cells
Nomenclature for this classPre-B cell all cells
in the B cell lineage before they have a
functional BCR Immature B cell B cell in the
bone marrow with a functional BCR and which, upon
binding antigen, will be deleted (killed) Mature
naïve B cell B cell in the periphery that have
never bound antigen and, upon binding of antigen
can be activated (or deleted) (in later chapters,
additional names for activated B cell)
10
Gene rearrangements in B cells is an orderly(?)
process (remember, these are diploid cells with
two heavy chain loci, two k loci and two l loci)
What is the test for a productive heavy chain
rearrangement?
What is the test for a productive light chain
rearrangement?
Q Why an orderly process such as this? A So
only one heavy and one light chain per B cells
(i.e. one antigen specificity per B cell). To
achieve this, cells need allelic exclusion (heavy
and light) and light chain isotype exclusion (see
slide 7). This a achieved by the orderly
rearrangements and testing for functionality
after each rearrangement.
11
After gene rearrangements and production of a
functional Ig molecule, the cell tests whether
its specificity is anti-self
Maintenance of tolerance requires the persistence
of antigen
Bone marrow
because self-antigens are always present but
foreign antigens are transient
or
periphery
Next slide
Clonal deletion(death) (most important)
Alive but not functional
Ag binding is too weakly to get a response (least
important)
These are the cells that leave the bone marrow
and constitute a normal immune response
12
Before clonal deletion of an anti-self B cell,
the cell can attempt receptor editing of the
light chain
So, light chain can use repeated rearrangements
(figure 7.9) and can receptor edit. Repeated
rearrangements are to make a functional molecule
whereas receptor editing is to avoid clonal
deletion of anti-self specific B cells.
Immature B cell edits light chain if it binds
antigen (gets negative selection signal). This
could rescue the cell from negative selection
(i.e., death). (needs signal to edit)
Later we will see that a pre-T cells continues to
rearrange a chain if it does not gets positive
selection signal. (will eventually die if it does
not receive positive selection). (needs signal to
stop editing figure 7.26)
Recall, pre B cells can also undergo repeated
light chain locus rearrangements until a function
Ig light chain is produced (slide 10, fig 7-9).
13
Summary of the life of a T cell
T cells in the thymus are called thymocytes
(negative selection is clonal deletion positive
selection is to select for MHC restriction
specificity)
In older individuals there is little thymus
function and most T cells come from division of
mature T cells in the periphery (new B cell are
continually generated in the bone marrow)
14
Organization of the human thymus
15
Nude mice and DiGeorges syndrome humans are
thymic deficient and have no T cells. Neonatally
thymectomzed mice are T cell deficient, too.
The thymus is required for T cell development
SCID mice have a functional thymus but cannot
rearrange Ig and TCR genes (RAG-/-) so they
cannot make Ig or TCR and thus have no B or T
cells(but they have a normal thymus)
Lymphocytes in nude mice have everything needed
to rearrange Ig and TCR gene but do not have a
functional thymus for T cell development
T cells in the thymus are called thymocytes
severe combined immune deficiency
CD3- CD3
CD3- CD3
16
Double negative refers to the absence of CD4 and
CD8 on the cell surface
TCR?
Thymocyte differentiation correlates with
expression of surface markers
pTa surrogate TCRa chain comparable in function
to the surrogate Ig light chain
Less than 5 of the double positives survive to
become single positive.
ab abTCR
Do these single positive cell express CD3 on
their surface? How about a TCR?
Single positive refers to the presence of either
CD4 or CD8, but not both, on the cell surface
17
The fate of thymocytes in a young mouse. 100x106
- 200x106 total thymocytes. 50x106 new thymocytes
each day (immigration and division). Therefore
50x106 cells must be removed each day. 1x106 -
2x106 mature T cells leave the thymus each
day. 48x106 - 49x106 thymocytes die each
day. That is, 96-98 of the thymocytes never
become fully functional and die in the thymus (by
apoptosis).
Why the carnage? Because most thymocytes either
(1) fail to make functional TCR or, if they make
a functional TCR, they are (2) anti-self
(negative selection) or (3) not self
MHC-restricted (no positive selection).
18
positive selection here
negative selection here
Single positive
Positive selection for self MHC restriction
Negative selection against cells that are
anti-self
19
The sequential rearrangements at the TCR a and b
loci are similar to the Ig rearrangements
pTa is a surrogate a chain
Double positive
20
In a TCR a locus there are about 70 V gene
segments and 60 J segments. This provides for
many attempts at a productive rearrangement.
Rearrangements stop when there is positive
selection.
This is selection for self MHC restriction
Q what stops light chain rearrangements? A
productive rearrangement at a light chain locus
and subsequent signaling from surface Ig (HL)(no
antigen or test for specificity) (see figure 7-9
or slide 8)
Stated differently, TCRa rearranges until there
is positive selection whereas Ig light chains
rearrange until there is a productive
rearrangement (and no more light chain receptor
editing, figure 7.13, slide 12)1. stop TCRa
rearrangements by positive selection for
restriction to self MHC2. stop light chain by
productive rearrangement3. restart light chain
rearrangement if BCR is anti-self (receptor
editing)for light chains, 2 and 3 until
successful (productive and NOT anti-self) or out
of light chain gene segments (unsuccesful) and
dies
21
MHC restriction is learned (in the thymus)
All cells, including bone marrow-derived cells
(e.g., T cells) express both MHCa and MHCb on
their surfaces
(MHCa/b)
Irradiation kills bone marrow stem cells and all
lymphocytes (wipes out the immune system)
MHCaMHCa/a and MHCbMHCb/b
Thymus epithelial cells (and all other tissues)
express MHCb
Thymus epithelial cells (and all other tissues)
express MHCa
MHCa/a
MHCb/b
These T cells have MHCa and MHCb on their surface
but are restricted to MHCb
These T cells have MHCa and MHCb on their surface
but are restricted to MHCa
Conclusion T cells that mature in an MHCa
environment are restricted to MHCa. T cells that
mature in an MHCb environment are restricted to
MHCb. The T cells MHC genotype does not affect
its restriction specificity. T cells learn their
restriction specificity, they are not born
knowing it.
22
The environment that determines the restriction
specificity is the thymus
Neonatal thymectomy results in no mature T cells
The thymic epithelium is MHCa. All other the
tissue in this chimera, including T cell, is MHCa
and MHCb
Inject Virus-x
Results CTLs kill MHCaxb infected cellsCTLs kill
MHCa infected cellsCTLs cannot kill MHCb
infected cells
Conclusion The CTLs express MHCa and MHCb but
their TCRs are restricted to MHCa only. Thus, the
thymus determines restriction specificity
23
Fully allogeneic chimera
MHCa?MHCb radiation chimera cannot function
without MHCb APCs
MHCa bone marrow into irradiated MHCb mouse
(thymusT cell education)
(irradiated)
APCs from the bone marrow
Figure 7-28 7th edition
This fully allogeneic chimeras is immune
incompetent because the thymus is MHCb so T cells
are restricted to MHCb. However the professional
APCs (B cell, dendritic cells and macrophages)
are MHCa (bone marrow derived) so APCs present
antigens in association with MHCa. The MHCb
restricted T cell cannot interact with the MHCa
APCs and thus there are no adaptive immune
responses. See next slide for an explanation of
this phenomenon.
24
The Fully Allogeneic Bone Marrow ChimeraMHCa
bone marrow into a MHCb recipient (previous slide)
MHCa?MHCb
Thymus
MHCb
MHCa
Bone marrow
TCR restricted to MHCb
Periphery
Note on this slide, we are examining the
restriction specificity of the T cells, not the
antigen specificity
25
T cells learn their restriction specificity by
positive selection
Positive selection also determines CD4 or CD8
expression and T cell function CTL or TH (not
TH1 or TH2, that choice comes later)
Positive selection1. lets cells live (is
required for survival)2. affects CD4 and CD8
expression3. affects function
The requirement to undergo positive selection
biases the repertoire toward MHC of the species
and partly explains high alloreactivity
26
Thymic epithelium mediates positive selection
(see previous slides) but
bone marrow-derived cells mediate negative
selection in the thymus
Figure 7.35
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The Semi Allogeneic Bone Marrow Chimera (from
previous slide)
(MHCa x MHCb)F1 MHCa chimera
MHCa Thymus
MHCa/b Bone marrow
Skip this slide
No anti MHCa or MHCb reactivity (tolerant to MHCa
an MHCb skin graft in previous slide)
Dendritic cell
Periphery
TCR specific for MHCa or MHCb means
alloreactive (MHC is the target antigen) and can
mediate graft rejection here, it does NOT refer
to MHC restriction specificity
Note on this slide, we are examining the antigen
specificity of the T cells, not the restriction
specificity
28
MHCa (MHCa x MHCb)F1 radiation
chimera
bone marrow recipient
donor
What is the restriction specificity of the T
cells in this semi-allogeneic chimera?
T and B cells express MHCa haplotype (but this is
not important for the question at hand) Since the
thymus is MHCa and MHCb, some T cells will be
restricted to MHCa and others to MHCb (any one T
cell can have only one restriction specificity so
any one T cell will be restricted to MHCa or MHCb
haplotype but not both)
What is the MHC-type of B cells and macrophages
that will mediate immune responses?
29
One specificity per T cell (one antigen and one
restriction specificity combination).For
example, lets look at MHC class I restricted T
cells
6 mutually exclusive restriction specificities
per individual
X represents an antigen (e.g., a virus) however,
it is not likely that all the T cells recognize
the same peptide derived from X
haplotype
haplotype
Within an individual there will be many different
T cell clones, each with one of the six
restriction specificities, responding to various
peptides derived from the same antigen (or
pathogen).
The more common HLA nomenclature is B0702
Cw0203 A0209
30
The example on the previous slide is for MHC
class I. However, the principle of one
restriction specificity and one antigen
specificity per T cell is exactly the same for
MHC class II restricted T cells.
Also, recall the T cells probably do not
recognize the antigen and the allele-specific
determinants of MHC as separate entities but
recognizes a new antigenic determinant formed by
the combination of antigen (peptide) and MHC.
31
How can cells be positively selected for MHC
restriction and not killed by negative selection
that removes anti-self reactive T cells?
Avidity hypothesis
Not differential signaling
T cells with low avidity for self-MHC plus self
peptides survive (positive selection without
negative selection)
nothing here so this cannot be correct
Figure 7-36 7th edition
32
Survival of B and T cells requires that they
circulate through lymphoid organs.
33
Organization of a lymphoid organ
Stromal cells (Interdigitating) dendritic
cells T cells B cells Follicular dendritic cells
High endothelial venules (HEV)
34
Survival of B and T cells requires that they
circulate through lymphoid organs.
B cell survival signal is probably not received
via the antigen receptor. Without antigenic
stimulation, most B cells die shortly after
leaving the bone marrow some will survive
several weeks (3-8 weeks). B cell will not
divide in the periphery without antigenic
stimulation. Memory B cells can survive for many
years but must circulate through lymphoid organ
and receive survival signals.
Mature naïve T cells can divide in the periphery
(probably get stimulation via weak binding to
self peptide and MHC, similar to positive
selection in the thymus). Memory T cells can
divide without MHC peptide stimulation.
35
B and T cell tumors have many normal cell
phenotypes and have been instrumental in
understanding lymphocyte development. They often
mimic a stage in lymphocyte development that is
normally transient but is permanent in the tumor
cell lineage.
36
Normal
Burkitts lymphoma
Productive rearrangement at IgH locus
37
Promoters and enhancers regulate the expression
of heavy chains
A promoter is a site on DNA where RNA polymerase
binds when initiating transcription. An enhancer
is a DNA sequence containing a binding site for a
transcription factor (protein) that works to
enhance RNA transcription from a nearby promoter
SKIP THIS SLIDE
Prototype V gene segment in the germline
configuration