Activation of T Cells Specific to Endogenous Retroviral Peptides: Possible Association with Clinical Response to Azacitidine in Myeloid Malignancies

DNA methyltransferase inhibitors (DNMTi's), 5-azacytidine (5-aza-CR, azacitidine) and 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine), are drugs of choice for patients with higher-risk myelodysplastic syndromes (MDS). Although DNMTi's have shown clinical benefits, their mode of action is not completely understood. In vitro studies have shown that low-dose DNMTi causes up-regulation of transcripts derived from human endogenous retroviruses (HERVs) in target cells that may give rise to double and single stranded RNA formation that are sensed by the cells as foreign. This in turn induces the viral defense pathway that elicits an interferon response, which may play an important role in the efficacies of DNMTi's. In this study we wanted to explore whether DNMTi-treatment in patients causes up-regulation of HERVs and formation of HERV-derived peptides in vivo that can stimulate an adaptive immune response against the malignant cells.

We have developed a novel technology that allows multi-parallel assessment of T-cell reactivity for >1000 different peptide-MHC complexes in a single sample (Bentzen et al., Nat Biotechnol, 2016). To identify recognition of HERV-derived peptides by CD8⁺ T cells we created a library of 1174 such antigens coupled to the most frequent HLA-I types (HLA-A*01:01, HLA-A*02:01, HLA-B*07:02 and HLA-B*08:01). Peripheral blood mononuclear cells from 26 patients (17 MDS, 5 AML and 4 CMML) with matching HLA-I types and treated with azacitidine were sampled before and during treatment. Cells from onset of treatment and at the starting day of third and fifth treatment cycle were used in our multiplex technology to identify activated CD8⁺ T cells recognizing HERV-derived peptides embedded in their respective HLA class I molecules. For a comparative analysis we also included known exogenous viral antigens to assess whether the enhancement in CD8⁺ T-cell responses was a general phenomenon or restricted to the selected antigens. Moreover, CD8⁺ T cells from a number of healthy donors were investigated with the same peptide library for normalization. Sorted CD14⁺ and CD3⁺ cells from four CMML patients were analyzed at transcript-level by RNA sequencing for up-regulation of HERVs and viral defense genes. Besides, DNA from peripheral neutrophils or from mononuclear cells from 18 of the patients was sequenced with a targeted 20 gene panel, including genes frequently mutated in MDS. Clinical characteristics and treatment responses according to the IWG criteria were collected from patient files.

Nine of the 26 patients showed a clinical response to the treatment. Of these nine patients four had a significant induction of specific CD8⁺ T cells targeting HERV-derived peptides. For the non-responding patients the equivalent number was two of 17 patients (p<0.05); one of them with stable disease during treatment until undergoing allogeneic hematopoietic stem cell transplantation. When investigating individual HERV families across all patients we found a widespread induction of specific CD8⁺ T cells targeting HERV-derived peptides. The preliminary RNA sequencing data revealed that HERVs and downstream viral defense genes are indeed up-regulated in the patients during treatment and that more HERVs are up-regulated in the malignant cells compared with T cells. Conclusively, the up-regulation of HERVs and induction of immune responses, specifically adaptive immunity against HERV-derived peptides, may be essential for the patients' responses to this class of drugs. These findings point towards a rationale for combining DNMTi's with immunotherapy.

A.D.Ø, S.K.S. and A-M.B. as well as K.G. and S.R.H. contributed equally.