No abstract

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209–217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-γ expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-γ expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.

To detect possible differences in immunogenicity between tumors induced in T cell-deficient mice and phenotypically normal congenic mice, 16 sarcomas, 8 having developed in nude BALB/c mice and 8 having developed in congenic normal (nu/+) mice, were transplanted to normal BALB/c recipients and the rates of rejection or acceptance were registered. The 16 tumors were chosen randomly from a panel of 39 sarcomas induced with 0.5% or 0.1% 3-methylcholanthrene and maintained as cell lines in culture. Out of the tumors originating from nude mice, 66% were rejected by the normal BALB/c recipients, while only 30% of the tumors originating from normal mice were rejected. Tumors with short induction times from normal mice were more readily accepted than tumors with long induction times. Tumors originating from nude mice had significantly longer mean latency times after transplantation to both normal and nude recipients than tumors originating from normal mice. Contrary to what has been reported by others, there was no correlation between the rejection rates of the individual tumors and their Kd, Dd or Ld major histocompatibility complex (MHC) class I surface expression as measured by flow cytometric analysis of cultured tumor cells. The Kd, Dd and Ld proteins of the transplanted tumor lines were analyzed by isoelectric focusing for the occurrence of mutations resulting in altered charge of the MHC protein. No such mutations were found, ruling out MHC mutations of that kind as the source of immunogenicity in the cell lines used in these experiments. Our results suggest the existence of a T cell-mediated selection in the original tumor cell mass of tumors induced in normal mice, adapting the tumor to growth in a host with a functional T cell system, but apparently there is no connection between this loss of immunogenicity and loss of MHC class I expression.

Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209–217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient’s kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN-γ ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies.