PCB 3233 Exam 3 Study GuideChapters 5 and 6

Chapter 5

1) BCR and TCR, how are they created and where in the body are they created?

BCR: @ Bone Marrowo Prior to Ag encounter:

Gene rearrangement of V-Region RNA splicing (IgM or IgD)

o Post Ag encounter: Ig is secreted! Isotype switching = C-Region rearranged Somatic Hypermutation= Ab of higher affinity

BCR Effector function depends on secreted Ab’s (…C-region isotype determines)

TCR: @Thymus

o Prior to Ag encounter: Gene Rearrangement of V-Region

o Post Ag encounter: Genes encoding TCR remain unchanged

TCR Effector Function no effector functions, that’s handled by other T-cells

2) Structure of Ig and the T cell receptor (TCR)

Igo 2 FAB armso C-region + V-regiono L-chain + H-chaino 2 Ag binding-siteo Membrane-bound or secreted

TCR o Resembles 1 FAB arm of Igo C-region + V-regiono and chains o Always transmembraneo 1 Ag binding-site (6 CDR’s)

3) The T cell receptor (TCR) type of antigen vs. Ig (BCR) type of antigen

Ig (BCR)o Variable binding site Wide range of Ag’so anything complementary + exposed.

“Soluble” molecules- proteins/carbs/lipids on pathogen

TCR o Variable binding site (cell binds to) 1 type of Ag (peptide)o Needs (Ag-presenting molecule) MHC + peptide to present so it

can bind

4) For expression of the TCR at the cell surface what is needed? (proteins + name given to proteins and TCR when on the surface).

CD3 complex (co-receptor) = 2 Epsilon, 1 Gamma, 1 Delta + 2 Zeta Chains

+ : TCR (total: 8 Polypeptides!) CD3 complex + TCR TCR complex Lack of functional CD3 delta & CD3 epsilon…

o low expression of TCR + impaired signaling immunodeficiency

5) Function of TCR; TCR complex

TCR function: recognize single Ag. (MHC + peptide)o Used only as cell-surface receptors o Multipoint attachment = multiple copies of TCR bind to multiple copies of

MHC+peptide

TCR Complex: 4 “invariant transmembrane” proteins aid : TCR with its expression on cell surface

o + Zeta chains aid in intracellular signaling

6) What is the CD3 complex?

CD3 Complex: Transmembrane proteins that aid : TCR with its expressiono Encoded by closely-linked genes on chromosome 11 o after Ag recognition by TCR!!!o 2 epsilons o 1 gamma o 1 delta o +2 Zeta Chains

7) T-cell receptor antigen-binding sites, how many per TCR and number of CDR’s per receptor; 1 binding site per TCR 6 CDR’s in 1 binding site (1 FAB arm) 3 CDR’s per chain

8) T cell receptor diversity and where are most of the differences concentrated?

TCR’s are all the same on a single cell!!! TCR diversity from cell-to-cell Most of the differences (variability/diversity) found in the Hypervariability region of the

V gene segments

9) (SCID); Omenn Syndrome; Bare lymphocyte syndrome, HIV (heterozygosity). SCID:

o Non-functional (non-enzymatic) RAG gene o both B-cells & T-cells absento no adaptive immune system!o Inherited (rare genetic defect)

Omen Syndrome:o “form of SCID”o Missense mutations produce some RAG proteins... partial enzymatic activityo Few T-cells made!o these T-cells are autoreactive (fight against ur own cells)o + immunodeficiency

Bare Lymphocyte Syndromeo “form of SCID”o Non-functional TAP MHC I not expressed on surface…NO peptides entering

the ER!!! deficient in CD8 T-cells poor response to viral infection

HIV heterozygosityo CCR5 = main co-receptor used by HIV to enter target cells during early

stages of HIV infectiono AIDS= CD4 T-cells drop below 200 per micro Litero Heterozygous individuals slower decrease in CD4 T-cell count per year

+ longer time of progression to AIDS

10) Organization + rearrangement of the : TCR genes

-chain gene segments are “within” the α-chain locus o between Vα and J α

-chain Chrom. 7 Rearrange β-chain first (DJ + V-DJ) … race against rearrangement of : β-chain wins! =95% of T-cells are α:β Adaptive T-cells Now rearrange α-chain (V,J)… race again.

o After 1 st α-chain rearrangement locus gets cut!!! -“Lose chance of that T-cell ever becoming a

: T-cell” During -gene rearrangement...

o D segments can be added into the final gene sequence increases variability (Junctional diversity) of the -chain

11) What is unique about : T-cells?

: T-cells = small subset of T-cells (5%) Innate T-cells

No need for presentation via MHC + peptide Doesn’t have either co-receptor: CD4 or CD8

Co-receptor needed only for binding of : T-cells to MHC + peptide

Have fewer V gene segments

[Cell can express either : TCR or : TCR, never both!]

12) MHCI & MHC II molecules, what do they do? What do they activate? Which cells can present with these?

MHC Io Binds to intracellular pathogenic peptides

(VIRUS)o MHC + peptide is presented to CD8 “Killer

T-cells”o All nucleated cells have MHC I

MHC IIo Binds to extracellular pathogenic peptideso MHC + peptide presented to CD 4 “T-helper cells”

TH1activate macrophages by releasing cytokines {IFN-} TH2 activate B-cells (plasma) to produce

Ab’s {IgE}o Exclusive to Dendritic cells, Macrophages, & B-cells

To activate a naïve T-cell, a Dendritic cell is needed! @ secondary lymphoid tissue

13) Structure of MHC class I & II

MHC I o 1 chain ( 1, 2, 3) (“Like Heavy Chain”)o + -2 microglobulin

MHC II o 2 chains ( 1, 1, 2, 2) (“Heavy & Light Chain”)

[Simultaneous binding by MHC to TCR & Co-receptor!!]

14) Peptide (length) that can bind in MHC I & MHC II molecules?

MHC I = 8-10 Amino Acids MHC II = 13-25 Amino Acids

15) Peptide binding groove of MHC class I and MHC class II?

MHC I Peptide is grasped by pockets at each end of binding groove o Limited length!

MHC II Peptides are NOT pinned down, they extend out on each side of groove!o Can hold longer peptides!!!

16) What does the degenerate/promiscuous binding of MHC molecules allow?

Degenerate binding of MHC molecules allows it to bind to a variety of peptides (diff. AA) w/o having to change its structure

17) Which class of T cells will be activated for intracellular vs. extracellular pathogens

Intracellular MHC I present Ag to CD8 T-cells (cytotoxic) Extracellular MHC II presents Ag. to CD4 T-cells (TH1 phagocytose)

**Ag.’s can be intracellular or extracellular in 2 diff. intracellular compartmentsprocessed by 2 diff. intracellular pathways of degradation

18) Class of MHC molecules will be utilized for intracellular vs. extracellular pathogens

Intracellular MHC I

o Proteasome = breaks down protein (@cytosol) to peptide fragmentso TAP = transports peptides from cytosol to ER

dependent on binding of & hydrolysis of ATP! o Calnexin

Chaperone Ca 2+ dependent Lectin = retains partially folded MHC I in ER until it can fold

properly…-2 microglobulin comes!o Calreticulin & Tapasin = bind to TAP-1 to position the -2

macroglobulin to wait for suitable peptide coming from cytosol

Extracellular MHC II

o New MHC II & -chains move from ribosome ERo Invariant Chain (li) (Identical in all PPL) = blocks peptide binding-site

to prevent peptides in ER from binding + DIRECTS MHC II to endocytic vesicle (M2C)

o MII C (M2C) = degradative vesicle, has proteases that cleave the invariant chain leaving a small fragment…

o CLIP= small invariant chain fragment; blocks binding-site inside

vesicleo HLA-DM = catalyzes the release of CLIP + allows MHC II to sample

peptides to find one with highest affinity

19) Cytotoxic T cells & co-receptor CD8, do these stay in the secondary lymphoid tissue after activation of go to the site of infection?

After activation, cytotoxic T-cells (+CD8) go to site of infection

20) Process for generating peptides from intracellular vs. extracellular pathogens (video)

21) Proteasome; TAP-1 and TAP-2; Calnexin; Invariant chain; HLA-DM; CLIP; Chaperones molecules

Proteasome breaks down proteins (cytosol) to peptide fragments TAP-1 & TAP-2 TAP transports peptides (8-10) from cytosol to ER

o TAP-2 = what tapasin & calreticulin bind too Dependent on binding and hydrolysis of ATP

Calnexin retains the partly-folded -chain ‘til -2 microglobulin comes (ER)

o Chaperoneo Ca2+ dependent lection (carb-binding proteins that

retain TCRs & Igs) Calreticulin + Tapasin bind to TAP-2 to stabilize the -2 microglobulin

and position it better ‘til it finds suitable peptide

Chaperones = helps with assembly & peptide-loading of MHC I

Invariant Chain Block peptide binding-site on MHC II so peptides in ER don’t bind

o Directs MHC II to endocytic vesicle M2Co Identical in all ppl

CLIP small fragment of invariant chain on MHC II (clipped by proteases) HLA-DM catalyzes the release of CLIP + allows MHCII to sample peptide

to find the one with highest affinity

22) MHC genetic complex; chromosomes; genes; region I, II and III of MHC

MHC genes do NOT undergo gene rearrangement MHC I & II genes occupy diff. regions on Chromosome 6 MHC II: upstream

o HLA-DP, DQ, DR human-presentingo HLA-DO (DOA & DOB) separated by HLA-DMo moleculeso 5 MHC II isotypes

MHCIII irrelevant

MHC I: downstreamo HLA-A,B,C human-presenting molecules o HLA-D,F

HLA-E = normal MHC inhibitory molecule on surface for NK cells … w/o it NK cells activate and tells cell to die

o 6 MHC I isotypes

23) How do MHC I & MHC II molecules get their diversity? Where is that diversity located in the molecule?

MHC diversity comes from parents

o 12 MHC molecules heterozygouso 6 MHC molecules homozygous

3 MHCI + 3MHC II Multiple similar gene families

o Encoding MHC I (heavy) -chains o Encoding MHCII and chains

Polymorphism many alternative forms (alleles) of MHC I & II across the human pop. Majority of AA sequence difference from allotype to allotype in the…

o Binding Groove + shoulder of MHC

24) The reason for graft rejection? MHC molecule different from one individual to another. The host recognizes the grafted tissue as “foreign” (graft vs host) T-cells will not recognize the MHC, won’t bind, instead it will attack it! Polymorphism!

25) Types of human MHC class I molecules present to which T-cells?

HLA-A,B,C MHC 1 present to CD8 T-cells

26) Types of human MHC class II molecules present to which T-cells?

HLA-DP,DQ,DR MHC II present to CD4 T-cells

27) Chromosomes; genes that encode MHC I & II

MHC I & II genes on different regions on Chromosome 6

MHC IIo HLA-DO (DOA & DOB) separated by HLA-DMo HLA-DP, DQ, DR human-presenting molecules o *Class II region of the MHC dedicated to genes involved in Ag. presentation

α:β chains of 5 HLA isotypes TAP Proteasome subunits LMP2 + LMP7

MHC Io HLA-A,B,C human-presenting molecules o HLA-D,F HLA-E

-are the 2-microglobulin and the invariant chain genes in the MHC region?

NO. 2-microglobulin Chromosome 15 NO. Invariant chain Chromosome 5 NEITHER POLYMORPHIC

28) Genes that encode protein products 2-microglobulin + invariant chain & what do these proteins do? 2-microglobulin Chromosome 15

o Serves as support for the MHC I (heavy) -chains

o Helps MHC I bind to TAP

Invariant Chain Chromosome 5

o Blocks the peptide binding-site of MHC II… prevents peptides floating in ER from binding

o Directs MHC II towards the degradative endocytic vesicle M2C (MIIC)

29) What does MHC restriction mean and what cells does it refer to?

MHC restriction T-cell/TCR needs to bind to the appropriate MHC + appropriate peptide (Ag)

TCR recognition of peptide (Ag) is MHC restricted Refers to : TCR

30) MHC polymorphism, what does this mean? What does it mean for us as a population and what does it mean for you as an individual?

Polymorphism = large # of alleles across the population at each locus Population numerous alt. forms (Alleles) of the MHC gene Individual Heterozygous for MHC : 12 MHC molecules

3 MHC I + 3 MHC II (MOM) + 3 MHC I + 3 MHC II (DAD) Homozygous = six

3MHC I (mom/dad same) + 3MHC II (mom/dad same)

31) How are the MHC genes expressed? Can you potentially express more than just one DRB chain?

Genes for the MHC loci lie close together = LINKED genes Individuals inherit the alleles as two sets, one from each parent – haplotype Alleles are Co-Dominantly expressed Everyone gets one DRB1 + one DRA

o But…. you can inherit DRB 3, 4, or 5

32) What are anchor residues and what do they do?

Anchor residues AA residues that allow the binding of peptides to MHCo Need specific AA in specific places to bind polypeptide to MHC

binding grooveo Interact with the pockets of the peptide binding-groove

33) TH1 and TH2 cells; roles; B cells; macrophages. Th1 vs. Th2, does one of these stay in the secondary lymphoid tissue after activation? Does one of them go to the site of infection after activation?

TH1 CD4 Effector T-cell o Exits secondary lymphoid tissue after activationo Activate macrophages to phagocytose o Produces IFN-γ to stimulate macrophage

TH2 CD4 Effector T-cello Remains in secondary lymphoid tissue to stimulate B-cellso Now B-cells can proliferate & make Ab’s

Chapter 6

34) Pre-B cell receptor, there are several things taking place in the B cell at this point, what are they? What makes up the pre-B cell receptor?

Pre-BCR @ Large Pro-B Cell Pre-BCR = μ chain + “surrogate” light chain “Surrogate” L-chain = VpreB (sub. for V-region) + 5 (sub. for C-region)

At this point, H-chain is rearranged & “surrogate” L-chain allows for testing of appropriate μ chain

Allelic Exclusion = only 1 μ chain is expressed

Ig and Ig expressed on surface to help Pre-BCR with intracellular signaling

35) What do stromal cells provide for developing B-cell? What does IL-7 do and when/where is it needed in B-cell development?

Stromal Cells provide specialized microenv. for B-cell to mature

o Cell-Surface contact VLA-4 integrin binds to selectinV-CAM1 (stromal cell)o Produce growth factors

Early Pro-B cell Kit receptor on B-cell binds to SCF…promotes proliferation!

Late Pro-B Cell IL-7 produced by stromal cells binds to IL-7 receptor & stimulates growth/proliferation

36) How many chances does a cell have to make a functional heavy chain rearrangement? Does the light chain have more chances than the heavy chain?

H-chain = 2 chances (one on each chrom.) L-chain = 10 chances ( or κ) (five on each chrom.)

o Unproductive L-chain rearrangement can be replaced! loci organization allows further arrangement…

Ex: Vκ -Jκ fails first time… try Vκ2 with any other Jκ downstream 5 J gene segments (much more V seg.) … 5 successive attempts at a productive rearrangement of L-chain on single chromosome

37) What might happen if there wasn’t allelic exclusion?

Allelic Exclusion give homogenous BCR’s w/high avidity (single Ag. specificity) Without Allelic Exclusion

o RAG-1 & 2 aren’t shutdown after productive rearrangemento get heterozygous BCR’s w/low avidity…can’t bind properly!

38) What does allelic exclusion mean and how is it achieved? Are certain genes turned off? If so, which genes are not expressed to help provide allelic exclusion? Are there any enzymes tagged for destruction to help provide allelic exclusion?

Allelic Exclusion= ensures B-cell gets only one H-chain & one L-chain RAG transcription stops (RAG-2 tagged for destruction) Achieved by successful gene rearrangement signaled by protein product on the surface by

Ig and Ig! (“hey this is good, stop re-arranging!)

39) Which chain rearranges VDJ and which chain rearrange VJ only? Which are rearranged first, heavy, light, D-J or V-D? There are quite a few ways I could ask these questions, look at these topics from different angles and make sure you know it well.

Heavy chain rearranged 1st L-chain rearranged 2nd Heavy Chain (μ chain) rearranges D-J first, then V- DJ Light Chain ( or κ) rearranges V-J

40) Surrogate light chain, 5 and VpreB. What each subunit substitutes for, when and where is this happening? Why do you need this subunit produced? Is this one of the checkpoints during maturation?

“Surrogate” L-Chain: 5 sub. for C-region VpreB sub. for V-region @ Large Pro-B Cell @ ER (mostly, but also on surface) μ chain + “surrogate” L-chain + Ig and Ig = Pre-BCR You need this subunit cuz it’s a checkpoint!

o Pre-BCR tests diff. H-chain until functional one has been made!... then signals w/ Ig and Ig help to stop further rearrangement :]

o Pre-BCR (+) signal that prevents apoptosis & allows B-cell to proliferate (small pre-B cells <3)

41) Ig and Ig, what do these do and when would you expect to see them expressed?

1st expressed in Large Pro-B Cell along with Pre-BCR Anytime there is any BCR on surface, they’re expressed Help B-cell with intracellular signaling signals to stop further gene rearrangement

42) Immature B cell vs. mature B cell, are there differences?

Immature B-cell IgM on surface Mature B-cell IgM + IgD on surface

43) Large Pre-B cell ---> Small Pre-B cell, what significant happens or is formed at this point in B cell maturation? Large Pre-B Cell H-chain + “surrogate” L-chain = Pre-BCR + Ig and Ig Small Pre-B Cell L-chain rearrangement (H-chain in ER)

*Large Pre-B Cells have exact same CDR’s in H-chain…

o after proliferation, Small Pre-B Cells have same H-chain, but they are free to make their own L-chain rearrangement

44) Anergy vs. clonal deletion

Clonal Deletion if BCR binds to multivalent self-antigen it will undergo apoptosis Anergy if BCR binds to soluble monovalent self-antigen it will express low levels of

IgM and enter peripheral circulation…will express IgD but will never be activated

45) Development of B cells, where and steps and names.

Stem Cell Ig genes in germline configuration Early Pro-B Cell H-Chain rearrangement of D-J Late Pro-B Cell H-Chain rearrangement of V-DJ Large Pre-B Cell μ chain + “Surrogate” light chain = testing to find appropriate

μ chain… Pre-BCR on surface, mostly in ER + Ig and Ig expressed on surface Small Pre-B Cell L-Chain rearrangement ( or κ) of V-J (μ chain @ ER chillin) Immature B-Cell H-chain (μ) & L-chain ( or κ) rearranged = Expression of IgM

(NEW BCR ) Mature B-cell IgM + IgD on surface (RNA splicing)

46) Signal

for

successful gene rearrangement (checkpoints), what happens if the cell fails the checkpoint and what would cause it to fail at the checkpoint

Checkpoints:I. Pre-BCR Large Pre-B Cells

Checks for functional H-chain rearrangement (+) signal when done promotes proliferation & prevents apoptosis

II. (new!) BCR Immature B-Cells Checks for functional L-chain rearrangement L-Chain + H-Chain = IgM Presence of IgM Ig and Ig signal to prevent further rearrangement

If cell fails at checkpoint it will undergo apoptosis It could fail due to unproductive rearrangement of H-chain or L-chain (used up all

chances on each chromosome)

47) What stage of B cell development is the first check point and what stage of B cell development is the second check point?

Checkpoints:I. Pre-BCR Large Pro-B Cells

II. BCR Immature B-Cells

48) When would you expect to find Ig and Ig turned on and are they ever turned off?

Ig and Ig turned on in Large Pre-B Cell stage Never turned off so long as a type of BCR is on the surface

49) What is Pax-5 and what does it do? = B-cell specific transcription factor

o 1 st synthesized in early Pro-B cell. .. continues to be synthesized throughout B-cell development + lifetime of mature B cell.

50) Somatic recombination, somatic hypermutation, mRNA alternative splicing, N and P addition of nucleotides.

Somatic Recombination = V(D)J recombination that takes place in BCR & TCR Somatic Hypermutation = creates higher Ab specificity. (BCR)

o Point mutation of variable regionso Affinity Maturation

N and P addition of nucleotideso part of somatic recombination by TDT

Junctional Diversityo Random N & P nucleotide addition after RAG-mediated cleavage.

Nucleotide additions done by TdT. Adds Ig variability.

51) Understand what receptor editing/rescue means

BCR that binds to multivalent self-Ag. will undergo developmental arrest!o IgM expressed is reducedo RAG genes stay on to allow cell to continue L-chain rearrangement

new functional rearrangement “Rescued B-Cell”

52) Burkitt’s lymphoma. What causes it? What does the MYC gene normally do? Where does it end up in the lymphoma? What is Bcl-2 and if the gene is mutated what can it lead to?

Proto-oncogenes = genes that cause cancer when function/expression is perturbed Burkitt’s Lympoma = translocation of parts on chromosome 8 & 14

Chromosome 8 MYC gene (proto-oncogenes) Chromosome 14 Ig H-chain gene (or L-chain 22 or κ 2) MYC gene normally regulates cell division…translocation causes

increased growth BCL-2 = proto-oncogene; regulates apoptosis, if muted cancer cells will

have growth advantage

53) B-1 cells vs. B-2 cells (know the differences). Which is also called a CD-5 B cell?

B-1 o minority (Innate)o prenatalo IgM on surface primarily (lil-0 IgD)o distinctive repertoire of Ag. receptors o “CD5 B-cells”o V-region repertoire limited o lack N-nucleotides (no TDT) less diverse VDJ junction low affinity

but, each bind to many diff. Ag.’s = Polyspecificity (no T-cell aid!)o Postnatal after repertoire is selected, no more will be made

B-2o majority (Adaptive) o WideV,DJ segments + diverse V-repertoire

memory, somatic hypermutation, Isotype switching (after activation)o T-cell help! helps activate it and go thru ^^^^o IgG>IgMo High specificity = High Affinity o Made all da time (bone marrow!)

54) When would you expect to find RAG 1 and 2 being expressed and not being expressed?

RAG genes expressed during gene rearrangement RAG genes OFF during proliferation

55) CCL-19, CCl-21 and CXCL-13, what are they, where are they with respect to secondary lymphoid tissues and what effects do they have on B-cells?

Chemokines!o CCL-21 attracts B-cells to HEV(high endothelial venules)o CCL-19 attract B-cells into Lymph node to T-cell area o CXCL-13 attracts B-cells to primary follicle

Interaction with follicular dendritic cells & chemokines drives immature B-cell to mature

56) What is BAFF and what role does is play in a B-cells life? What cell secretes BAFF?

BAFF signal = final maturation/survival signal from follicular Dendritic cells

57) Where does Central tolerance vs. peripheral tolerance take place? What is a Centrocyte and where would you find it? What is a Centroblasts and where would you find it?

Central Tolerance @Bone Marrow (B-cells) @Thymus (T-cells)

Peripheral Tolerance 2° Lymphoid organs

Centroblasts = Large proliferating B-cells o @ Dark Zoneo Undergoing somatic hypermutation, isotype switching = Affinity

Maturation Centrocytes = what Centroblasts mature into; slow/non-dividing B-Cells

o @ Light Zoneo New BCR on surface

58) What are the light and dark zones of a germinal center and what is found in each?

Dark Zone whereo Centroblasts are undergoing somatic hypermutation + isotype switching and turn

into Centrocytes Light Zone

o Centrocytes are located o Follicular Dendritic Cells harbor Ag.’s that new BCR can test (if good its

activated by T-cell!)

59) What can a plasma cell differentiate from? (what type of B-cell, there are 3 cell types)

Activated Mature B-Cell (secreting high affinity Ab with Ig switched) Centrocytes (undergone somatic h. + isotype s.) Memory B-cell (high affinity Ab when re-exposed to same Ag)

60) What are the isotypes of the light chains and which is usually rearranged first?

L-chain (22) or κ (2) κ is rearranged first

61) Define naïve and mature in relationship to B-cells.

Naïve B-Cell B-cell that has not encountered Ag. Mature B-Cell IgM + IgD on surface

62) What are the lymphocytes?

T-Cells B-Cells NK cells