Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

An Erratum to this article was published on 03 June 2015


Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient’s tumour with vaccines produced ‘just in time’.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cancer‐associated mutations are frequently immunogenic and pre‐dominantly recognized by CD4+ T cells.
Figure 2: Efficient tumour control and survival benefit in B16F10 melanoma with an RNA vaccine encoding a single mutated CD4+ T‐cell epitope.
Figure 3: RNA pentatope immunization confers disease control and survival benefit in murine tumours.
Figure 4: RNA pentatope vaccines with mutations selected for in silico predicted favourable MHC class II binding and abundant expression confer potent antitumour control.


  1. Castle, J. C. et al. Exploiting the mutanome for tumor vaccination. Cancer Res. 72, 1081–1091 (2012).

    Article  CAS  Google Scholar 

  2. Castle, J. C. et al. Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma. BMC Genomics 15, 190 (2014).

    Article  Google Scholar 

  3. Holtkamp, S. et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009–4017 (2006).

    Article  CAS  Google Scholar 

  4. Kreiter, S. et al. Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals. J. Immunol. 180, 309–318 (2008).

    Article  CAS  Google Scholar 

  5. Kuhn, A. N. et al. Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther. 17, 961–971 (2010).

    Article  CAS  Google Scholar 

  6. Bloom, M. B. et al. Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma. J. Exp. Med. 185, 453–459 (1997).

    Article  CAS  Google Scholar 

  7. Schumacher, T. et al. A vaccine targeting mutant IDH1 induces antitumour immunity. Nature 512, 324–327 (2014).

    Article  ADS  CAS  Google Scholar 

  8. Tran, E. et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344, 641–645 (2014).

    Article  ADS  CAS  Google Scholar 

  9. Britten, C. M. et al. The regulatory landscape for actively personalized cancer immunotherapies. Nature Biotechnol. 31, 880–882 (2013).

    Article  CAS  Google Scholar 

  10. Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883–892 (2012).

    Article  CAS  Google Scholar 

  11. Koebel, C. M. et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903–907 (2007).

    Article  ADS  CAS  Google Scholar 

  12. Matsushita, H. et al. Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting. Nature 482, 400–404 (2012).

    Article  ADS  CAS  Google Scholar 

  13. Robbins, P. F. et al. Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nature Med. 19, 747–752 (2013).

    Article  CAS  Google Scholar 

  14. van Rooij, N. et al. Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma. J. Clin. Oncol. 31, e439–e442 (2013).

    Article  Google Scholar 

  15. Shen, Z. et al. Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J. Immunol. 158, 2723–2720 (1997).

    CAS  PubMed  Google Scholar 

  16. Schoenberger, S. P. et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature 393, 480–483 (1998).

    Article  ADS  CAS  Google Scholar 

  17. Linnemann, C. et al. High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma. Nature Med. 21, 81–85 (2015).

    Article  CAS  Google Scholar 

  18. Arnold, P. Y. et al. The majority of immunogenic epitopes generate CD4+ T cells that are dependent on MHC class II-bound peptide-flanking residues. J. Immunol. 169, 739–749 (2002).

    Article  CAS  Google Scholar 

  19. Wolfel, T. et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281–1284 (1995).

    Article  ADS  CAS  Google Scholar 

  20. Wang, R. F. et al. Cloning genes encoding MHC class II-restricted antigens: mutated CDC27 as a tumor antigen. Science 284, 1351–1354 (1999).

    Article  ADS  CAS  Google Scholar 

  21. Lu, Y. C. et al. Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression. J. Immunol. 190, 6034–6042 (2013).

    Article  CAS  Google Scholar 

  22. Gubin, M. M. et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 515, 577–581 (2014).

    Article  ADS  CAS  Google Scholar 

  23. Snyder, A. et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N. Engl. J. Med. 371, 2189–2199 (2014).

    Article  Google Scholar 

  24. Herbst, R. S. et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515, 563–567 (2014).

    Article  ADS  CAS  Google Scholar 

  25. Tumeh, P. C. et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568–571 (2014).

    Article  ADS  CAS  Google Scholar 

  26. Rizvi, N. A. et al. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348, 124–128 (2015).

    Article  ADS  CAS  Google Scholar 

  27. Castle, J. C. et al. Mutated tumor alleles are expressed according to their DNA frequency. Sci. Rep. 4, 4743 (2014).

    Article  Google Scholar 

  28. Löwer, M. et al. Confidence-based somatic mutation evaluation and prioritization. PLOS Comput. Biol. 8, e1002714 (2012).

    Article  Google Scholar 

  29. Boegel, S. et al. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines. OncoImmunology 3, e954893 (2014).

    Article  Google Scholar 

  30. Kreiter, S. et al. Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity. Cancer Res. 70, 9031–9040 (2010).

    Article  CAS  Google Scholar 

Download references


We thank M. Holzmann, A. König, U. Schmitt, R. Roth, C. Worm and N. Krause for technical assistance; L. Ralla, J. Groß, A. Spruß, M. Erdeljan, S. Wöll and C. Rohde for immunohistochemical staining and analysis; C. Paret for sequence validation of mutations; M. Brkic for immunofluorescence staining; S. Witzel and Bodo Tillmann, S. Wurzel and Z. Yildiz for cloning of constructs; S. Kind, M. Mechler, F. Wille, B. Otte and S. Petri for RNA production as well as L. Kranz and colleagues involved in RNA formulation development. We are grateful to B. Kloke, S. Heesch, A. Kuhn, J. Buck, C. Britten and H. Haas for conceptual and technical discussions. Moreover, we would like to thank V. Bukur, J. de Graf and C. Albrecht who supported the next-generation sequencing of samples. Furthermore we like to acknowledge A. Kong for critical reading and A. Orlandini for help with graphic design. The results shown here are in part based on data generated by the TCGA Research Network The study was supported by the CI3 excellence cluster program of the Federal Ministry of Education and Research (BMBF).

Author information

Authors and Affiliations



U.S. is principal investigator, conceptualized the study and experimental strategy. S.K., M.V., N.vdR., M.D., J.D., F.V. and U.S. planned and analysed experiments. M.V. and N.vdR. performed experiments. S.K., M.V., M.D., S.P.S., C.H., Ö.T. and U.S. interpreted the data and wrote the manuscript. M.L., S.B., A.D.T. and J.C.C. processed next-generation sequencing data and identified mutations. M.V. and B.S. analysed murine MHC II binding predictions. S.B. analysed TCGA data and human MHC II binding predictions.

Corresponding author

Correspondence to Ugur Sahin.

Extended data figures and tables

Extended Data Figure 1 Non synonymous cancer‐associated mutations are frequently immunogenic and pre‐dominantly recognized by CD4+ T cells.

T‐cell responses obtained by vaccinating C57BL/6 mice with antigen‐encoding RNA in the B16F10 tumour model (n = 5). Left, prevalence of non‐immunogenic, MHC-class-I- or class-II-restricted mutated epitopes. Right, detection and typing of mutation‐specific T cells (individual epitopes shown in Extended Data Table 1).

Extended Data Figure 2 Mutant epitope-specific T cells induced by RNA vaccination control tumour growth.

a, Splenocytes of mice (n = 5) vaccinated with B16‐M30 RNA were tested by ELISpot for recognition of mutated peptides as compared to the corresponding wild‐type (B16‐WT30) sequence. Right, testing of truncated variants of B16‐M30 (mean + s.e.m.). b, Mean ± s.e.m. tumour growth (left) and survival (right) of C57BL/6 mice (n = 10) inoculated s.c. with B16F10 and left untreated (control) or injected i.v. with irrelevant RNA. c, Lungs of B16F10‐Luc tumour bearing mice shown in Fig. 2b (day 27 after tumour inoculation). d, Therapeutic antitumour activity against B16F10 tumours in mice (B16‐M27, Trp2 n = 8; B16‐M30 n = 7; others n = 10) conferred by immunization with epitopes encoding immunogenic B16F10 mutations or an immunodominant wild type Trp2 epitope6. The area under the tumour growth curve at day 30 after tumour inoculation was normalized to untreated control mice and depicted as mean ± s.e.m. Red and black columns represent mutations recognized by CD8+ or CD4+ T cells, respectively. e, Spontaneous immune responses in splenocytes of irrelevant RNA treated B16F10 tumour bearing C57BL/6 mice (n = 3) were tested by ELISpot for recognition of peptides (mean + s.e.m.).

Extended Data Figure 3 Mechanism of antitumour activity of mutation specific poly‐epitope vaccines in CT26 tumour‐bearing mice.

a, BALB/c mice (n = 5) were vaccinated either with pentatope (35 µg) or the corresponding mixture of five RNA monotopes (7 µg each). T‐cell responses in peptide‐stimulated splenocytes of mice were measured ex vivo via ELISpot (medium control subtracted mean ± s.e.m.). b, c, BALB/c mice (n = 10) were inoculated i.v. with CT26 tumour cells and left untreated or injected with irrelevant, CT26‐M19 or pentatope1 or 2 RNA in absence (b) or presence of a CD8 T cell depleting antibody or a CD40L blocking antibody (c). Mean ± s.e.m. of tumour nodules per lung are shown. d, Immunofluorescence analyses of tumour‐infiltrating lymphocytes in pentatope2‐vaccinated mice. Upper panel, lung tumour tissue stained for CD4 and CD8 or CD4 and FoxP3. Scale bar, 50 µm. Lower panel left, proportion of infiltrating cells in sections of irrelevant (CD4: n = 13; CD8 = 9; FoxP3: n = 13) or pentatope (CD4: n = 17; CD8: n = 6; FoxP3: n = 10) RNA‐treated animals. Lower panel right, tumour area in sections of control (n = 22) and pentatope2‐treated (n = 20) animals (mean ± s.e.m.).

Extended Data Figure 4 Immunogenicity testing of PME pentatope‐encoded mutations.

Splenocytes of PME RNA vaccinated BALB/c mice (n = 6) were tested ex vivo for recognition of peptides representing the mutated 27mer sequences represented in PME pentatopes with or without addition of an MHC class II‐blocking antibody. Mean + s.e.m. of background (medium control) subtracted responses are shown.

Extended Data Table 1 Immunogenic B16F10 mutations
Extended Data Table 2 Immunogenic CT26 mutations
Extended Data Table 3 Immunogenic 4T1 mutations
Extended Data Table 4 CT26 mutated epitopes encoded in pentatope 1+2
Extended Data Table 5 In silico prediction of CT26 mutations with abundant expression and favourable MHC class II binding properties

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kreiter, S., Vormehr, M., van de Roemer, N. et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692–696 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing