Common mutations in the tumor suppressor p53 & the oncogene Kras as targets for long peptide anti-cancer vaccination

Tumor-associated antigens (TAAs) are promising targets for immunological therapeutic intervention in cancer therapy. Particularly, mutated proteins are a source for true tumor-specific antigens (TSAs) because they are exclusively expressed in the tumor and are not shared with normal tissue. Moreover, TSAs reduce the risk of autoimmunity and increase the chance to overcome tolerance compared to non-mutated protein sequences. In this study we sought to show for the first time, that a mutation-specific multi-peptide vaccine, which targets simultaneously common mutations in gastrointestinal tumors in a human HLA context, is capable to induce multifunctional and polyvalent CD4+ and CD8+ effector T cell responses with a tumor-protective capacity. Furthermore, we aimed to investigate whether this strategy results in immune-suppressive counter-reactions, like the induction of regulatory T cells (Treg). As target-antigens, we created a panel of peptides with sequences derived from the most frequently mutated variants of the tumor suppressor p53 and the oncogenes Kras and Braf described for colorectal (CRC) and pancreatic carcinomas. More precisely, the peptides represent wild-type (wt) or mutated sequences and have a length of 28-35 amino acids to facilitate a presentation of MHC I and II epitopes. In the presented study we analyzed the potency of the long peptide panel for active vaccination and its tumor-protective capacity in a murine cancer model system. For this purpose we utilized C57BL/6J mice, as well as an HLA-class I/II humanized mouse strain (HLA.A2/HLADR1 transgenic), in a multi-peptide vaccination setting. T cell responses of immunized mice were monitored by flow cytometry measuring cytokine secretion after antigen-specific in vitro re-stimulation. Thereby, we observed simultaneous, polyvalent CD8+ cytotoxic and CD4+ helper T cell responses against the majority of the peptides. Moreover, the peptide-specific T cells possessed a multifunctional cytokine-secretion profile and CD4+ T cells displayed a TH1 like phenotype. Notably, two of the mutation-comprising long peptides (Kras G12V and p53 R248W) induced a significantly higher secretion of cytokines than the corresponding wt sequences in both CD4+ as well as CD8+ T cells, which implied mutation-specificity. For these two peptides we were able to revalidate (Kras G12V) and identify (p53 R248W) HLA.A2 and HLA.DR1 restricted mutation-comprising epitopes. To investigate the tumor-protective capacity of the vaccination approach syngenic fibrosarcoma cell lines were generated in the HLA-class I/II transgenic mouse model by carcinogen-induced tumorigenesis. In tumor challenge experiments we employed cell lines carrying intrinsic Kras/Tp53 mutations and cell lines which were engineered to express the most immunogenic mutations found in our vaccination studies. Vaccination with mutated long peptides resulted in delayed tumor outgrowth compared to vaccination with wt counterparts regarding tumors with intrinsic mutations. However, animals vaccinated with highly immunogenic wt and mutated peptides showed a strong increase of immunosuppressive, peripheral Treg numbers correlating with an accelerated outgrowth of transgene-expressing tumors. In conclusion, we showed that long peptide vaccination targeting multiple mutated oncogenes and tumor suppressor genes is capable of eliciting polyvalent, multifunctional, and mutated antigen-specific effector T cells responses, which have the potential to eradicate tumor cells. Furthermore, we suggest the induction of immune-suppressive, possibly mutation-specific regulatory T cells as a critical issue for the success of this therapeutic approach

Octamer-dependent transcription in T cells is mediated by NFAT and NF−κBNF-\kappa B

The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and $NF-\kappa B$-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/$NF-\kappa B$ sites. An array of genetic and biochemical analyses illustrates the involvement of the $Ca^{2+}$/calmodulin-dependent phosphatase calcineurin as well as NFAT and $NF-\kappa B$ transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or $NF-\kappa B$ transcription factors

Tcf-1 promotes genomic instability and T cell transformation in response to aberrant β-catenin activation

Understanding the mechanisms promoting chromosomal translocations of the rearranging receptor loci in leukemia and lymphoma remains incomplete. Here we show that leukemias induced by aberrant activation of β-catenin in thymocytes, which bear recurrent Tcra/Myc-Pvt1 translocations, depend on Tcf-1. The DNA double strand breaks (DSBs) in the Tcra site of the translocation are Rag-generated, whereas the Myc-Pvt1 DSBs are not. Aberrantly activated β-catenin redirects Tcf-1 binding to novel DNA sites to alter chromatin accessibility and down-regulate genome-stability pathways. Impaired homologous recombination (HR) DNA repair and replication checkpoints lead to retention of DSBs that promote translocations and transformation of double-positive (DP) thymocytes. The resulting lymphomas, which resemble human T cell acute lymphoblastic leukemia (T-ALL), are sensitive to PARP inhibitors (PARPis). Our findings indicate that aberrant β-catenin signaling contributes to translocations in thymocytes by guiding Tcf-1 to promote the generation and retention of replication-induced DSBs allowing their coexistence with Rag-generated DSBs. Thus, PARPis could offer therapeutic options in hematologic malignancies with active Wnt/β-catenin signaling

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

Mutated proteins arising from somatic mutations in tumors are promising targets for cancer immunotherapy. They represent true tumor-specific antigens (TSAs) as they are exclusively expressed in tumors, reduce the risk of autoimmunity and are more likely to overcome tolerance compared to wild-type (wt) sequences. Hence, we designed a panel of long peptides (LPs, 28–35 aa) comprising driver gene mutations in TP35 and KRAS frequently found in gastrointestinal tumors to test their combined immunotherapeutic potential. We found increased numbers of T cells responsive against respective mutated and wt peptides in colorectal cancer patients that carry the tested mutations in their tumors than patients with other mutations. Further, active immunization of HLA(-A2/DR1)-humanized mice with mixes of the same mutated LPs yielded simultaneous, polyvalent CD8+/CD4+ T cell responses against the majority of peptides. Peptide-specific T cells possessed a multifunctional cytokine profile with CD4+ T cells showing a TH1-like phenotype. Two mutated peptides (Kras[G12V], p53[R248W]) induced significantly higher T cell responses than corresponding wt sequences and comprised HLA-A2/DR1-restricted mutated epitopes. However, vaccination with the same highly immunogenic LPs strongly increased systemic regulatory T cells (Treg) numbers in a syngeneic sarcoma model over-expressing these mutated protein variants and resulted in accelerated tumor outgrowth. In contrast, tumor outgrowth was delayed when vaccination was directed against tumor-intrinsic Kras/Tp53 mutations of lower immunogenicity. Conclusively, we show that LP vaccination targeting multiple mutated TSAs elicits polyvalent, multifunctional, and mutation-specific effector T cells capable of targeting tumors. However, the success of this therapeutic approach can be hampered by vaccination-induced, TSA-specific Tregs

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling

Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism

Omicron BA.1 breakthrough infection drives cross-variant neutralization and memory B cell formation against conserved epitopes

Omicron is the evolutionarily most distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) to date. We report that Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individuals resulted in strong neutralizing activity against Omicron BA.1, BA.2, and previous SARS-CoV-2 VOCs but not against the Omicron sublineages BA.4 and BA.5. BA.1 breakthrough infection induced a robust recall response, primarily expanding memory B (BMEM) cells against epitopes shared broadly among variants, rather than inducing BA.1-specific B cells. The vaccination-imprinted BMEM cell pool had sufficient plasticity to be remodeled by heterologous SARS-CoV-2 spike glycoprotein exposure. Whereas selective amplification of BMEM cells recognizing shared epitopes allows for effective neutralization of most variants that evade previously established immunity, susceptibility to escape by variants that acquire alterations at hitherto conserved sites may be heightened