Abstract B062: Differences of transcriptional profiles between long-term tolerized and memory CD4 T cells
Authors: Imai Naoko , Tsuji Takemasa , Altorki Nasser K. , Kuo Pei-Yu , Perumal Deepak , Parekh Samir , Gnjatic Sacha 
Published: May 2016

Abstract
It is well known that immunological adjuvants improve antigen-specific immune responses to cancer vaccines. We previously reported a clinical trial in non-small cell lung cancer where half of the patients received a cycle of 4 vaccines every 3 weeks with recombinant MAGEA3 protein alone in the absence of adjuvant, while the other half were vaccinated with MAGEA3 protein administered in adjuvant AS02B containing saponin and TLR4-ligand Monophosphoryl Lipid A. Results showed that vaccination without adjuvant led to very limited or no humoral and T cell immune responses to MAGEA3, while vaccine with adjuvant elicited robust humoral and CD4 T cell responses. Two to three years later, patients received a second cycle of vaccination, but this time with AS02B adjuvant for everyone. Remarkably, patients initially vaccinated without adjuvant still failed to mount immune responses even after 4 vaccines with adjuvant, exhibiting a tolerized phenotype, while remaining patients had a typical recall memory response reaching a plateau of high antibody and CD4 T cell levels after a single injection. This data suggested that the initial environment during priming dictates subsequent long-term capacity of CD4 T cells to expand and produce cytokines. While long-term tolerized CD4 T cells were induced in patients primed without adjuvant, in contrast to long-term memory CD4 T cells in patients vaccinated with adjuvant throughout, markers associated with T cell responsiveness vs. tolerance remain unclear. Here, we assessed antigen-specific cells using RNASeq analyses to elucidate at the transcriptional level mechanisms associated with qualitative differences in the CD4 T cell repertoire
We applied a sensitive method based on CD154 upregulation to detect and sort MAGEA3-specific CD4 T cells throughout the study, including even rare precursors present at baseline, from two patients representative of tolerized vs. memory profile respectively. Unexpectedly, we found that the number of CD154-expressing cells after MAGEA3 in vitro restimulation were highly increased in both patients after the first vaccine injection, but durably reduced thereafter in the tolerized patient, while still increasing in the responder patient. To characterize differences in transcriptional profiles, MAGEA3 specific CD4 T cells were subjected to RNASeq at baseline and after the 1st and 2nd immunization. Sorted MAGEA3 specific CD4 T cells were non-specifically expanded, and restimulated with MAGEA3, followed by RNA isolation and deep sequencing by RNASeq. A total of ~16,000 individual genes were found transcribed in all samples, and many were related to expected CD4 T cell characteristics. When expression levels of all genes were considered together in unsupervised principal component analysis, there was a remarkable difference in profiles between the two patients. While the responder had a consistent shift in gene transcript post-vaccine compared to baseline, the tolerized patient had a transient shift in genes expressed by MAGEA3-specific CD4 T cells after one vaccine, which was abolished and returned to a profile closer to baseline after the 2nd vaccine. We identified a set of 413 genes with differential expression profiles between tolerized and responder patients over early vaccination time points. These genes can be further classified according to whether they increase or decrease specifically in the tolerized or in the responder patient, when comparing baseline to 1st vaccine to 2nd vaccine, and by their constitutive vs. inducible nature following antigen stimulation. Immune-related genes were heavily predominant within this subset, and we found an array of cytokines and checkpoint molecules that we propose as candidates for a tolerance signature. These results could be useful for early prediction of vaccine efficacy and patient benefit in future immunotherapy trials.
Citation Format: Naoko Imai, Takemasa Tsuji, Nasser K. Altorki, Pei-Yu Kuo, Deepak Perumal, Samir Parekh, Sacha Gnjatic. Differences of transcriptional profiles between long-term tolerized and memory CD4 T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B062.