Heat-shock proteins (HSPs), in addition to their known housekeeping roles as chaperones, can stimulate an immune response to bound antigenic cellular peptides. This response is thought to have two components: stimulation of an innate response through pro-inflammatory, adjuvant effects on dendritic cells (DCs), and the delivery of antigen for crosspresentation on DC MHC class I molecules to induce an adaptive CD8+ cytotoxic T lymphocyte (CTL) response. How efficient is the response and what is the relationship between these two components?

Using an assay that measures the ability of antigen-pulsed DCs to generate an effector CTL response, Paul Lehner and colleagues showed that only a small number of human DCs pulsed with influenza-A-derived peptides bound to mycobacterial HSP70 are necessary to induce an efficient antigen-specific CTL response (T-cell:DC ratio of 5000:1). Pulsing DCs with the same peptides in the absence of HSP70, even in the presence of a known DC activator such as lipopolysaccharide, was not effective, and CD8+ T cells could not be stimulated by peptide–HSP70 complexes alone. Therefore, both DCs and HSPs are crucial for the response.

Next, they measured the affinity of peptide binding to HSP70 and used this to calculate that 120 pM HSP70-bound peptide can generate a CTL response in vitro, whereas a 2000-fold higher concentration of free peptide was unable to do so. The low concentration of mycobacterial HSP-bound peptide and the small number of stimulated DCs required for a CTL response without additional adjuvants in this human system requires further investigation as a potential vaccination method.

To determine the minimal HSP70 requirement for generation of a peptide-specific CTL response, the authors created truncated HSP70 proteins containing the known carboxy-terminal peptide-binding domain only. After taking slight differences in binding affinity into account, such mutant forms of HSP70 were as efficient as full-length HSP70 at generating CTLs, indicating that they were able to both stimulate and deliver peptide to DCs. By contrast, an HSP70 mutant with markedly decreased peptide-binding affinity, owing to a point mutation in the peptide-binding domain, could still induce the production of pro-inflammatory cytokines by DCs but did not lead to CTL generation, and therefore the delivery of antigen can be separated from DC stimulation. This mutant HSP70 was unable to generate CTLs even in the presence of excess free peptide, which shows that to be crosspresented, the peptide must be bound to HSP. However, when the concentration of wild-type peptide–HSP70 was limiting, addition of excess mutant HSP70 that cannot bind peptide did enhance the CTL response. So, the two components of the response can be delivered by separate HSP molecules, indicating that other HSPs might be able to enhance the response to a low concentration of peptide-bound HSP in physiological situations.