Optimized combinatorial pMHC class II multimer labeling for precision immune monitoring of tumor-specific CD4 T cells in patients

With immunotherapy gaining increasing approval for treatment of different tumor types, scientists rely on cutting edge

This new optimized staining procedure represents an advance toward better detection and analysis of antigen-specific CD4 T cells. It should facilitate state-of-the art precision monitoring of tumor-specific CD4 T cells and contribute to accelerate the use and the targeting of these cells in cancer immunotherapy.

We have used combinatorial staining strategies with peptide major histocompatibility complex class II (pMHCII) multimer constructs of different alleles to establish an optimized staining procedure for in vitro and direct ex-vivo visualization of tumor-specific CD4 T cells, in patient samples. Furthermore, we have generated reversible multimers to achieve optimal cell staining and yet disassemble prior to in vitro cell expansion, thus preventing activation induced cell death.

We observed a vastly improved detection of tumor-specific, viral-specific and bacterial-specific cells with our optimization methods compared with the non-optimized staining procedure. By increasing the variety of fluorochromes used to label the pMHCII multimers, we were also able to increase the parallel detection of different specificities within one sample, including antigen-specific CD8 T cells. A decrease in cell viability was observed when using the full optimization method, but this was mitigated by the removal of neuraminidase and the use of reversible multimers.