1. T/B Collaboration Associative recognition/carrier effect Hapten cannot elicit an immune response without a carrier Hapten must be physically linked to carrier to generate response Secondary immunization must be with the same hapten-carrier conjugate for a memory response to occur. *Carrier effect can be circumvented by priming separately with The unrelated carrier used in the 2 o immunization

2. In vivo Injection of Hapten/Carriers Hapten and carrier need to be physically Linked to allow T/B interactions and observe responses Interpretation: Ag “bridges” T and B cells

3. T/B Collaboration in vitro Cells added to upper or lower chamber In the presence of APCs This is accomplished by soluble cytokines Close proximity of cells eliminates need for Ag “bridge” T and B cells can interact at a distance in vitro

4. MHC Restriction at the Ab production Level Bone Marrow Chimeras B B A Make BM chimera, wait 3 months, remove splenocytes Incubate in vitro with TNP-KLH and look for primary response T cell help for B cells is restricted MHC II on APCs must be compatible

5.  -KLH CD4  -TNP B Help provided X  -KLH CD4 A strain T cells Restricted To B haplotype B strain T cells Restricted To B haplotype BM from BB mouse BM from A B mouse MM MHC II TNP-KLH Bone Marrow Chimeras  -TNP B MHC II MM MHC II

6. MHC Restriction CTL will Only kill Infected Cells of The correct (self) haplotype Specificity Defined by Both peptide And MHC

7. MHC I Variability

8. Dual-Reactive Hybridoma Reacts w/ 2 different Cyt C molecules Same MHC restriction (IE k ) Make point mutants in MHC II (APC) AA of MHC II bind TCR in a Different pattern if peptide different Look for IL-2 production by T cell Similar changes if mutate one AA in TCR V  or V  TCR/MHC-peptide interactions Are fluid. Different conformations May influence affinity of binding.

9. Alloreactivity 1 - 10% of T cells will react against an allogeneic stimulus Explained by Cross-reactivity 1. Peptides bound by allo APC (but not self) Form complexes self T cells not tolerized to, self T cells will react Allo peptide strongly binds TCR that normally binds self MHC + different peptide 2. Direct binding by self T cells to distinct features on allo MHC Independent of peptide (although peptide must be present in Ag cleft) High concentration of foreign MHC creates a strong signal