Lecture 1

How do we know antibodies come from B cells?Congenics. We know that a BALB/c mouse can only produce lymphocytes withallotype A. So we can say BALB/c are Iga. C57B/6 are Igb. We mate them and getheterozygous F1, then mate F1 to another BALB/c. We keep mating the F to theBALB/c, selecting for Igb at the IgH locus for 10 generations until all the other loci areIga. Then we mix T cells from thymocytes, B cells (bone marrow cells), and antigen.

Thymocyte donor BM donor Ag Ab Ab AllotypeBALB/c (H-2d, Iga) BAB/10 (H-2b, Igb) + + Igb

BAB/10 (H-2b, Igb) BALB/c (H-2d, Iga) + + Iga

Clonal selection vs. instructionalismInstructionalism: a) A cell must express a receptor in order to be selected.b) A cell expresses multiple receptors of different specificities.c) Binding of a receptor to an antigen results in no expression of other receptors on thesame celld) The molecule secreted closely resembles the receptor that was ligated.Clonal Selection: a) A cell must express a receptor in order to be selected.b) One cell expresses multiple receptors of the same specificity.c) The molecule secreted closely resembles the receptor that was ligated.

We go by clonal selection, because instructionalism implies that deletion of B cells specificfor a given Ag will affect responses to other Ags.

Affinity maturationGiving mouse with 2 types of B cells high [Ag] activates both types of B cells, low [Ag]activates only one type of B cells. BcR plays a central role in activation. Ag used plays arole in determining the affinity of the antibody response. Theres a threshold of B celloccupancy for B cell activation. BcR with high affinity need lower [Ag] than BcR withlow affinity. Thus there are a lot of B cells with low affinity and very few with highaffinity.

Affected by [Ag] and adjuvents; anything that affects the affinity of immune responses.

MHC restricted T cell functionTK from C57BK/6 kill BALB.B APC cells presenting antigen TNP. The only allotypethey have in common is MHC (H-2b).

Lecture 2 - Characterization of MHC restricted T cell function

1. CD4 and CD8 denote the restriction specificity of the T cell, not its function. CD4 canonly be activated by MHC II, CD8 can only be activated by MHC I.

2. MHC expression is not allelically excluded; one cell can express MHC class I and MHCclass II at the same time, and can express H-2b and H-2d at the same time.

3. T cell function is not limited to the MHC that they express.a. Pan experiment

b. TCR specificity is clonally distributed.In fact, T cell can recognize antigen + MHC if MHC matches at just one locus. Weonly care about 4 loci - K, I-A, I-E, and D. K and D are class I encoding loci, I-A andI-E are class II encoding loci. If a T cell is CD4 restricted, it would recognize antigen ifin context of correct haplotype of I-A or I-E.

4. Selection of T cell repertoirea. If you take stem cells from a BALB/c mouse (H-2d) and put it into a lethally

irradiated (H-2d/b) mouse, you can isolate T cells that kill H-2d+x and H-2b+x,but not H-2d or H-2b

b.

Lecture 3 - The role of MHC in shaping T cell repertoire

1. Proving that MHC on the TE is what restricts T cell repertoire

a. negative selection is mediated by MHC on TE and selfb. positive selection is mediated by MHC on TEc. so if MHC is on self but not TE, it does not appear in T cell repertoire

2. MHC on peripheral APC, which matches MHC of stem cell, is what restricts the T cellsthat get activated/expanded

Chimera H-2d H-2d+v H-2b H-2b+v

v->P->F - + - -H-2d/b APCv ->P->F - + - +v->F->P - + - -H-2d/b APCv ->F->P - + - -

3. Immune response genes; Holes in the repertoirea. If an MHC is really bad at binding to an antigen and no other allotype of MHC

is available, theres a chance that a T cell will never be raised to it4. The 5 big rules of Chimeras

a. MHC of stem cells used to reconstitute the host will be the MHC expressed bydaughter T cells, B cells, and APCs

b. MHC of intra-thymic stem cells and developing T cells will negatively selectfor those T cells expressing self-reactive TcR

c. MHC expressed on the thymic epithelium will negatively select those T cellsexpressing self-reactive TcR which are not deleted by donor stem cells

d. MHC expressed on the thymic epithelium will positively select those T cellsexpressing TcR able to recognize antigen in the context of self-MHC

e. MHC expressed by APC in the periphery of the animal will determine whether aT cell will be activated and expanded

5. Activation of T cells requires coaggregation of TcR and CD4 or CD8a. CD4 is MHC II restricted because it binds to beta 2 domain of Class II

alpha-beta heterodimersb. CD8 is MHC I restricted because it binds to alpha 3 domainc. TcR and CD4 or CD8 could interact with the same MHC moleculed. used tridiated thymidine and saw that DNA synthesis didnt occur in plate

coated with anti-TcR or with anti-CD4, but it did in plate coated with anti-TcRand anti-CD4

i. could be homodimers causing activation tested this using beads, andanti-TcR and anti-CD4 on different beads didnt trigger activation buton same bead did, thus it must be bringing together 1 TcR and one CD4that does the trick

e. mechanism: CD4 and CD8 tend to drag along lck, which has an innatephosphorylation activity. Every TcR/CD3 complex contains 10 ITAMs.Phosphorylation by lck brought by CD4/CD8 allows SH-2 to dock to ITAMs,more stuff docks, signalling cascade

Lecture 4 - Co-stimulation in antigen presentation

1. Resting B cells do not express B7, but activated B cells doa. physiological ligand of B7 is CD28b. CD28-B7 interaction is required to activate T cell, or it goes into anergyc. selection processes during T cell development in the thymus must result in

phenotype of mature T cells where theres no mismatched expression ofco-receptor molecules and TcR restriction specificity

2. Two models for intra-thymic T cell dvelopmenta. Instructional model: at DP, co-aggregation of Tcr/CD4 decreases CD8

expression, or co-aggregation of TcR/CD8 decreaes CD4 expressionb. Stochastic/selection model:

c. Difference: Stochastic predicts presence of immature T cells with mismatchedTcR/co-receptors, Instructive predicts their absence

d. Its probably a blend of these two models3. T cell dependent humoral immune responses

a. making LTP B cellsi. inject hapten-carrier into mouse, careful that carrier doesnt cross react

with specificity of Th cloneii. come back in 6 weeks and harvest B cells

b. measuring antibodies - plaque forming cell assayi. immunize mouse with SRBC

ii. isolate spleen cells at various timepoints after immunizationiii. mix isolated cells with SRBC and plate in agariv. add complement which binds to Ag:Ab complexes and activates, lysing

the SRBC, forming a plaquec. what is t cell help?

i. T cell is specific for carrier, B cell for haptenii. Linked recognition: determinants recognized by T and B cells must be

physically linked

iii.iv. implies that theres an antigen bridge, but T cell must recognize

antigen in context of restricted MHCd. Is TH-B interaction MHC restricted?

i. CD4 H-2dthCKLHH-2D + H-2dB vs CD4 H-2dthCKLHH-2D + H-2bB, allow cultures togo into Ab production, add C and observe lysis

ii. th not alloreactive to H-2biii. TH-B interaction is MHC restricted

e. Requirement for LR for TH dependent activation of LTP BCDNPi. BcR binds to DNP. BcR mediates internalization of DNP and KLH.

DNP-KLH is processed. B cell presents KLH in context of H-2d4. Non-Specific Ig: The Bystander B Cell response

a. we dont know what the Ags are, so we call them NSIgs.b. quantitate using a plaque assay. coat indicator cells with Ab specific to Ig.c. in an non-immunized animal, there are B cells secreting Anti-DNP and NSIgsd. in an immunized animal, theres a 100-fold increase in anti-DNP secreted and

10-fold increase in NSIge. how are they made?

5. TH Dependence of NSIg productiona. T-dependent Ags require presence of TH cells to induce NSIg production

i. DNP-KLH is TD; DNP PFC is only in animals with thymusb. T-independent Ags induce similar responses in presence and absence of TH cells

i. BA is TI; induces BA PFC in animals both with and without thymusc. Using TI Ags as carriers would convert anti-hapten response from TD->TId. th activation and LR required for LTP BCDNPe. th activation required for NSIg response

Lecture 5 - Requirement for MHC restricted TH-B interaction: Dependence on state of B cellActivation

1. Recap:a. Linked recognition and MHC-restricted TH-B interaction correlateb. Theres a subset of B cells giving rise to NSIg that doesnt require LR or MHC

restricted interaction with TH2. Generation of LTP B cells

a. immunize donor with DNPb. helper T cells with specificity for DNP become activated by APC presenting

DNPc. at the same time, DNP specific B cells pick up DNP and carrier, metabolizes

them, presents them on MHC, receive MHC restricted interaction with THthrough LR

d. activation: blastogenesis->division->concomitant differentiation->some cellsbecome Ab secreting cells, others become memory cells that go back to restingstate

3. Separating resting b cells from activated b cellsa. activated B cells have low buoyant density and float upb. resting B cells have high buoyant density and float downc. can use Percoll to separate them by density; dilute Percoll to different densities,

and the B cells, when centrifuged, will migrate to the interface thats closest toits density

d. helper T cell can help activated allogeneic B cells

e. Conclusion: TH can secrete soluble mediators that activate and differentiate Bcells that are not MHC restricted, and those soluble mediators effectiveness isdependent on state of B cell activation

4. MHC restricted interaction puts B cell in state susceptible to cytokine/lymphokinemediated growth and differentiation

a. should be able to transform activation requirement of LTP BCDNP that requiresneither LR nor MHC restricted interaction

b. boost LTP with same Agc. harvest B cell before 2o responsed. mix with resting T helper cells, APCs, and antigen, look for PFCse. Results

f. Conclusion: Boosting LTP B cells removes requirement for LR5. Rules when thinking about TH-B collaboration

a. Can th be activated?b. Activated T cells produce non-MHC restricted lymphokinesc. Check the state of activation of the B cellsd. Activated low p B cells dont require LR and MHC restricted interaction, resting

high p B cells do6. IL-2 loop

a. MHC-restricted th-APC interaction causes APC to secrete IL-1 and th to expressIL-2R (high affinity)

b. IL-2R and IL-2 ligate; high enough affinity of interaction between th and APCcauses th to express IL-1R

c. IL-1 is utilized to make more IL-2d. IL-2 is utilized for clonal expansion

7. Cytokines that affect unboosted B cellsa. IL-5 drives B cell differentiation to antibody production but no growth (unless

supplemented with IL-2)b. IL-4 increases the chances of B cell getting the MHC restricted interaction it

needs by inducing de novo transcription and translation of MHC Class II8. MHC Class II interaction is not what causes B cell activation

a. Take H-2dtH-2d and H-2bBLTPDNPb. Throw in antibody specific for the TcR decorated with DNP to block TcR-MHC

interaction and allow DNP ligationc. Find production of anti-DNP antibodies. Because T cells and B cells are

allogeneic, this couldnt possibly be due to MHC restricted interaction.9. B cell activation is actually done by CD40 ligation

a. MHC specific interaction between B cell and T cell just bring them closertogether

b. B cell expresses CD40 (induced by membrane Ig processing), T cell expressesCD40L (induced by interaction with professional APC)

c. So can we just induce CD40 expression, ligate it with CD40L, throw cytokines

at the B cell, and activate the B cell?i. Yes we can.

10. Making monoclonal antibodies (mAbs)a. allows standardization of antibodies used in labs; before, no one used the same

antibodies because B cells cant be cloned and they produce antibodiespolyclonally

b. first, hyperimmunize the animal with antigen xc. isolate anti-x secreting cells (HAT resistant) as well as tumor cells (HAT

sensitive)d. glue cells together using PEG; eventually cells will fusee. throw in HAT

i. only anti-x secreting cell-tumor fusions will be able to growf. screen for the hybridoma (fusion) that makes the desired antibodyg. harvest antibodies, which will all be identical given that they came from the

same hybridoma