Nuclear factor kappaB is required for peptide antigen-induced differentiation of a CD4+CD8+ thymocyte line.

NF-kappaB transcription factors are known to regulate the expression of a number of genes involved in T cell activation and function. Some evidence has suggested that they also play a role in T cell development. However, the role of NF-kappaB in Ag-induced thymocyte differentiation has not been directly addressed to date. Here we critically examine this role by employing DPK, a CD4+CD8+ thymocyte line that undergoes differentiation upon TCR engagement in a process that closely mimics positive selection. Expression of a degradation-resistant form of IkappaBalpha in DPK cells results in constitutive inhibition of NF-kappaB activity. We find that in the absence of NF-kappaB activity, MHC-peptide-induced differentiation of DPK is blocked. Furthermore, differentiation induced by a nonphysiologic stimulus, anti-TCR Ab, is greatly reduced. Altogether, our data indicate a requirement for NF-kappaB in the developmental changes associated with positive selection.

The ability of peptides to induce cytotoxic T cells in vitro does not strongly correlate with their affinity for the H-2Ld molecule: implications for vaccine design and immunotherapy.

The hypothesis that the ability of a peptide to bind to a class I molecule correlates with its immunogenicity is controversial. In this paper we have measured the affinity constants of nine synthetic peptides, which have been previously identified as binding to H-2L(d) molecules, and have determined their immunogenicity in an in vitro cytotoxic T lymphocyte (CTL) induction assay. We find that six peptides bind with high affinity (K(a) > 10(7)/M); of these, four are of viral origin but only two elicit potent CTLs, one is a self peptide which is not immunogenic, while the sixth is of bacterial origin and also does not generate effective CTLs. Two peptides bind with intermediate affinity (K(a) > 10(6)/M); one of these elicits a moderate CTL response, while the other, a tumor-derived epitope, is highly immunogenic. Intriguingly, the peptide with lowest affinity (p2Ca) is exceedingly effective at eliciting CTLs. The efficacy of peptides with modest affinity for their restriction elements appears to correlate well with the CTL precursor frequency. We have also examined intrinsic parameters of some of the peptides such as solubility and stability. Taken together, our results underscore the relevance of factors other than affinity which affect immunogenicity and which may be critical in the design of peptide-based vaccines as well as tumor immunotherapy approaches.

H-2Ld-alloreactive T cell hybridomas utilize diverse V alpha and V beta T cell receptor chains.

We have sequenced the TCRs from Ld-specific alloreactive T cell hybridomas, whose reactivities we have found to be quite representative of those of a primary dm2 anti-BALB/cJ mixed lymphocyte reaction. We find V beta 6, V beta 7, V beta 8 and V beta 10 gene segments. V alpha usage is diverse, although closely related to that from peptide-specific Ld-restricted CTLs. V alpha-V beta selection provides evidence of preferential pairing. Amino acid frequency analysis shows that the alpha CDR2 region is rich in charged amino acids, in contrast to the beta CDR2 region. Our data suggests the beta chain may be more immunoglobulin-like than the alpha chain, and that charge complementarity may be important in TCR-MHC interactions. We do not consider our results to be contradictory to those previously reported but rather they may represent an early, more diverse response.