Immune Monitoring

Previous reports have indicated upregulation of the cancer-testis antigens (CTAs) by treatment with de-methylating drugs and in parallel other studies have suggested increased immune competence following administration of such drugs. Thus, if patients’ immune reactivity would be partly responsible for the clinical benefit it would possibly be beneficial to combine demethylating treatment with more directed immune boosting such as a vaccine or indirect immune treatments such as an immune checkpoint inhibitor blocker.

In the academic setting we sat out to investigate this effect further by obtaining blood samples prior to treatment and twice every fourth week from a cohort of 17 patients diagnosed with myeloid malignancies and treated with the de-methylating agent 5-Azacytidine (Gang & Frøsig et al., Blood Cancer Journal, 2014).

We investigated three hypotheses on the effect of 5-Azacytidine treatment:

• Hypothesis I: The established increase in expression of CTAs by 5-Azacytidine treatment results in a heightened T-cell recognition of T-cell epitopes from such antigens.


• Yes. By use of combinatorial encoded MHC multimers and a panel of 43 CTA-related T cell epitopes we showed a significant increase in the sum of T cell specifically recognizing these in complex with relevant MHC molecules.

Methods available: combinatorial encoding of MHC multimers, Hadrup & Bakker et al., Nature Methods, 2009; Andersen et al., Nature Protocols, 2012 or DNA-barcoding of MHC multimers, Bentzen et al., Nature Biotechnology, 2016.

• Hypothesis II: 5-Azacytidine treatment leads to enhanced anti-tumor reactivity


• Yes, by affecting the tumor cells and not the T cells. By cell sorting with magnetic beads and following 5 hours co-culture of myeloid blasts and T cells across time points, we were able to investigate separately the effect of treatment on the myeloid blasts and on the T cells. Staining with T cell markers, CD34 (a surrogate marker for the myeloid blasts) and CD107a an enhanced recognition of the myeloid blasts affected by the demethylating treatment compared to those obtained before treatment.

Methods available: all kinds of magnetic cell separation available from Miltenyi Biotec with following: VITAL-FR cytotoxicity assay, Stanke et al., Journal of Immunological Methods, 2010; extracellular staining of activation marker CD107a/b Betts et al., Journal of Immunological Methods 2003 and CD137 (Wolfl et al., Blood 2007).

• Hypothesis III: 5-Azacytidine treatment affects the general effector and suppressor immune populations


• No. We measured the absolute counts of CD4 and CD8 T cells, Natural Killer (NK) cells, myeloid-derived suppressor cells and regulatory CD4 T cells and found no significant effect of treatment as measured direct ex vivo. For the NK cells we did observe a minor change in the frequency of cells with an inhibitory phenotype. In vitro we were able at repeating findings from previous studies suggesting an inhibitory effect on the general NK cell population. The latter pointed to the caution needed when extrapolating from in vitro data to the in vivo situation, as further elaborated on in (Frøsig & Hadrup, Mediators of Inflammation, 2015).

Methods available: established SOPs for measuring several immune effector and suppressor cell populations.

Note, several of the methods mentioned above are patented and licenses are required in order to use these.

ImmuMap Services offer to perform immune monitoring in our laboratory or to train or advice you on how to perform these studies yourself.