Development of Immune Cells; B and T Lymphocytes and Immune Receptor Diversity

Question 1

• Hematopoietic stem cells (HSCs) can be identified as CD[] cells that lack any mature lymphocyte phenotypic markers (e.g. CD3 and CD4/CD8 for T cells, CD19 or CD20 for B cells, etc.)

Answer 1

• Hematopoietic stem cells (HSCs) can be identified as CD34+ cells that lack any mature lymphocyte phenotypic markers (e.g. CD3 and CD4/CD8 for T cells, CD19 or CD20 for B cells, etc.)

Question 2

Cytokines that induce hematopoietic activity in the Myeloid lineage?

Answer 2

IL-3, IL-5 and GM-CSF: Induce hematopoietic activity in the Myeloid lineage..

Question 3

Cytokines used to increase neutrophils, platelets and monocytes in patients receiving drugs that kill progenitor cells?

Answer 3

G-CSF, GM-CSF (and IL-3)

Question 4

Cytokines that induce hematopoietic activity in the Lymphoid lineage?

Answer 4

IL-3 and IL-7: Mediate hematopoietic activity in the Lymphoid lineage. IL-3 for B cell and IL-7 for B, T and NK cells development/survival.

Question 5

B cell development takes place in the [], while T cell development takes place in the [].

Answer 5

B cell development takes place in the bone marrow, while T cell development takes place in the thymus.

Question 6

For the heavy chain of the BCR/Ig and the TCR b chain, 3 segments must be successfully recombined: [].

Answer 6

For the heavy chain of the BCR/Ig and the TCR b chain, 3 segments must be successfully recombined: variable (V), diversity (D) and joining (J) segments.

Question 7

For the light chain of the BCR/Ig and the TCR a chain, 2 segments must be successfully recombined: [].

Answer 7

For the light chain of the BCR/Ig and the TCR a chain, 2 segments must be successfully recombined:variable (V) and joining (J) segments (no D gene segments).

Question 8

Name of the enzyme that combine different V-D-J segemnts?

Answer 8

V-D-J recombinase

Question 9

V-D-J Recombinase Components

Answer 9

• RAG-1 and RAG-2 proteins (Recombinase-Activating Gene) are lymphoid-specific, and work like scissors to cut the DNA open, and initiate the recombination of V-D-J gene segment.
• DNA repair enzymes come in next, to join the ends and repair the double stranded DNA breaks.

* The convention is to refer to this process as V-D-J rearrangement, even though the D gene segments are not present in Ig light chain or TCRa chain rearrangement.

Question 10

V-J recombination is for…

Answer 10

…Ig light chain or TCRa chain.

Question 11

V-J-D recombination is for…

Answer 11

…heavy chain of the BCR/Ig and the TCR b chain.

Question 12

junctional diversity

Answer 12

There is a second, very important strategy for creating receptor diversity also occurring during this somatic recombination process, termed junctional diversity - removal and/or additiona of nucleotides.

Question 13

exonucleases

Answer 13

Exonucleases may non-specifically remove nucleotides from the ends of the VD-J segments during the recombination process.

Question 14

terminal deoxyribonucleotidyl transferase (TdT)

Answer 14

The terminal deoxyribonucleotidyl transferase (TdT) enzyme can randomly insert nucleotides into the sites where the VD-J segments are rejoined (called “Ninsertions” for nontemplate encoded insertions).

Question 15

Although all three CDRs that make up the regions that recognize antigen (CDR1, CDR2, and CDR3) are encoded within the V-D-J recombined segment, [] are entirely contributed by the V segment.

Answer 15

Although all three CDRs that make up the regions that recognize antigen (CDR1, CDR2, and CDR3) are encoded within the V-D-J recombined segment, CDR1 and CDR2 are entirely contributed by the V segment.

Question 16

. It is the [] that spans the V- (D)-J joins, which means that most of the antigen specificity and variability of immune receptors is attributed to [].

Answer 16

. It is the CDR3 that spans the V- (D)-J joins, which means that most of the antigen specificity and variability of immune receptors is attributed to CDR3.

Question 17

In addition to the V-D-J rearrangement (involved in the variable region of the antigen receptors), choices are made at this time about the [] regions of the Ig/BCR and TCR as well.

Answer 17

In addition to the V-D-J rearrangement (involved in the variable region of the antigen receptors), choices are made at this time about the constant regions of the Ig/BCR and TCR as well.

*This is particularly important for B cells, in which the constant region determines the isotype of the resulting antibody.

*For B cells, the Heavy chain is responsible for providing the constant region. During B cell development, the µ Constant Heavy chain is always utilized – it is the closest to the V-D-J segments. That chain gets replaced by other chains only after naïve B cells have been activated by antigen.

Question 18

Answer 18

Question 19

Answer 19

•After the common lymphocyte progenitor commits to the B cell lineage, the cell undergoes proliferation in response to the cytokines IL-3 and IL-7 to generate B cell precursors called pro-B cells. These cells have not yet begun to recombine and generate their antigen receptors.

Question 20

Answer 20

• Pro-B cells then undergo somatic gene recombination in the Heavy chain. All B cells first rearrange their heavy chain locus to express µ. If this is successful, these cells are allowed to continue their development as pre-B cells. Some of the heavy µ chain is expressed on the cell surface with surrogate light chains as a pre-BCR.
• The expression of the pre-BCR is a survival checkpoint for the pre-B cell. If the pre-BCR can successfully make it to the surface and form a complex with the Iga and Igb signaling components, the cell is allowed to undergo another round of proliferation. This also signals the cell to shut down all concurrent rearrangement at the second heavy chain locus on the other chromosome – a process called allelic exclusion – to make sure that each cell only expresses a single antigen receptor

Question 21

Answer 21

•Pre-B cells next rearrange their light chains, which are then paired with µ and delivered to the cell surface for a second survival checkpoint - if the cell has generated a functional IgM on the surface, the cell is allowed to live as an immature B cell.

Question 22

Answer 22

•The final maturation step for B cells is the co-expression of IgD along with IgM on the B cell surface. Note that these receptors still express the same Variable domains, thus are still specific for the same antigens. Once the cell is IgD+ IgM+ it is considered a mature naïve B cell, and is now able to respond to antigen in the secondary lymphoid organs.

Question 23

What is the final step that the B cell has to go through before leaving the bone marrow?

Answer 23

•Prior to leaving the bone marrow, the negative selection process tests B cells for their capability to bind self-antigens. This mechanism works to eliminate B cells that might trigger autoimmune responses to normal tissue antigens. Any B cell whose BCR binds strongly to self-antigens found in the bone marrow dies by activationinduced cell death.

Question 24

AT what point in development does the B cell have its first checkpoint?

Answer 24

• The expression of the pre-BCR is a survival checkpoint for the pre-B cell. If the pre-BCR can successfully make it to the surface and form a complex with the Iga and Igb signaling components, the cell is allowed to undergo another round of proliferation.
• This also signals the cell to shut down all concurrent rearrangement at the second heavy chain locus on the other chromosome – a process called allelic exclusion – to make sure that each cell only expresses a single antigen receptor

Question 25

What is allelic exclusion?

Answer 25

•At the pre-BCR checkpoint, signals to the B cell shut down all concurrent rearrangement at the second heavy chain locus on the other chromosome – a process called allelic exclusion – to make sure that each cell only expresses a single antigen receptor.

Question 26

Where does T cell maturation take place?

Answer 26

•T cell maturation takes place exclusively in the thymus, after the T cell progenitor has left the bone marrow and has migrated into the thymus in response to chemokines

Question 27

The most immature progenitors arriving from the bone marrow are called []. They do not express signature molecules that mark a T cell: the CD4 or CD8 T cell coreceptors or the CD3/TCR complex – thus they are often called []. Like their pro-B cell counterparts, pro-T cells are highly dependent on the cytokine [] for their proliferation and maintenance.

Answer 27

The most immature progenitors arriving from the bone marrow are called **pro-T cells**. They do not express signature molecules that mark a T cell: the CD4 or CD8 T cell coreceptors or the CD3/TCR complex – thus they are often called **double (or triple) negative (DN) thymocytes**. Like their pro-B cell counterparts, pro-T cells are highly dependent on the cytokine **IL-7** for their proliferation and maintenance.

Question 28

After initial proliferation, DN thymocytes first rearrange their TCRb chain using the V-D-J recombinase machinery described above. Similar to B cells, the DN T cell will then attempt to express the rearranged TCRb chain on the cell surface with a surrogate a chain in a complex known as the [].

Answer 28

After initial proliferation, DN thymocytes first rearrange their TCRb chain using the V-D-J recombinase machinery described above. Similar to B cells, the DN T cell will then attempt to express the rearranged TCRb chain on the cell surface with a surrogate a chain in a complex known as the **pre-TCR.**

Question 29

Where is the first developmental checkpint for T cell maturation?

Answer 29

•Expression of the pre-TCR is a developmental checkpoint, ensuring that the TCRb chain V-D-J recombination resulted in a functional protein. If the pre-TCR can signal (indicating a “good” TCRb chain), then the cell undergoes several rounds of proliferation. Just like in B cells, this checkpoint closes the b chain locus on the other chromosome (allelic exclusion). If the pre-TCR is no good, the cell will try again with the other locus. Continued failure to pass this checkpoint leads to death by apoptosis.

Question 30

After passing this checkpoint, each cell expresses both CD4 and CD8, becoming []. These cells now independently rearrange their TCRa chain – potentially pairing the good TCRb chain with lots of different TCRa chains, in order to diversify the population.

Answer 30

After passing this checkpoint, each cell expresses both CD4 and CD8, becoming **double positive (DP) thymocytes**. These cells now independently rearrange their TCRa chain – potentially pairing the good TCRb chain with lots of different TCRa chains, in order to diversify the population.

Question 31

Where is the second developmental checkpoint in T cell maturation?

Answer 31

* POSITIVE SELECTION * After the cells independently rearrange their TCRa chain – potentially pairing the good TCRb chain with lots of different TCRa chains, in order to diversify the population and and transport to the cell surface for a second developmental checkpoint. * If the TCR is able to assemble and loosely bind to self-peptide: MHC (pMHC) complexes in the thymic cortex (Goldilock’s “not too cold”), the DP thymocyte is allowed to proceed; if not, the cellwill try to generate a new TCRa chain over the next 2-3 days. If this fails, or if the newly generated TCRa pairs with the existing TCRb and still does not recognize self pMHC, the cell dies by apoptosis.

Question 32

The positive selection step tests TCR to ensure they can mildly recognize [] in the context of either self MHC-I or MHC-II.

Answer 32

The positive selection step tests TCR to ensure they can mildly recognize **self-peptides** in the context of either self MHC-I or MHC-II. ## Footnote •This ensures that the selected TCR will be able to work with the set of MHC molecules available in the organism. Both the TCR and correct CD4 or CD8 coreceptor must recognize a selfpeptide/MHC complex to pass this second developmental checkpoint

Question 33

Answer 33

* This ensures that the selected TCR will be able to work with the set of MHC molecules available in the organism. * Both the TCR and correct CD4 or CD8 coreceptor must recognize a selfpeptide/MHC complex to pass this second developmental checkpoint

Question 34

Positive selection of T cells occurs in the [].

Answer 34

Positive selection of T cells occurs in the **thymic cortex.**

Question 35

At the DP stage, cells also undergo [] selection, where T cells that are highly responsive to self-peptides are eliminated, to prevent autoimmunity.

Answer 35

Question 36

Answer 36