Response: validity of evidence demonstrating efficacy of extracorporeal photochemotherapy

We appreciate the opportunity that the letter of Drs George and Roederer offers us to clarify and highlight our findings relevant to the mechanism(s) underlying the immunotherapeutic efficacy of extracorporeal photochemotherapy (ECP). Specifically, we reported several lines of convergent evidence that collectively demonstrate that ECP efficiently and rapidly induces processed monocytes to activate genes intricately involved in monocyte-to-dendritic cell (DC) differentiation, leading to expression of phenotypic and functional properties of these important antigen-presenting cells.¹  Because DC can be either selectively immunostimulatory or immunosuppressive, we suggested that ECP's large-scale conversion of monocytes to DCs contributes to ECP's paradoxical, but established, ability to either immunize against cancer antigens in cutaneous T-cell lymphoma patients or immunosuppress in the transplantation setting.

Because it is standard practice, we did not detail in the “Methods” section the care that we took in performing our phenotyping studies to ensure compensation for fluorescence spectral overlap in those cytofluorographic experiments involving 2-color analyses. In particular, Drs George and Roederer express concern that, under circumstances involving simultaneous measurement of class II major histocompatibility antigen expression and the DC marker CD83, the relatively high level expression of the former might have caused us to inadvertently overstate the positivity of the latter, had we not adequately compensated the samples for fluorescence spillover. In view of their comments, we have reviewed our data and remain confident of the validity of our reported findings for several reasons.

First, we did, in fact, use single-color staining controls to set compensation levels. These single color controls, in combination with negative, isotype-matched antibody controls, were then used to carefully set gates and permit subtraction of background fluorescence in our 2-color cytofluorographic determinations. Second, we separately confirmed our 2-color results by showing comparable CD83 positivity in single-color analyses, in which fluorescence compensation is, of course, not a concern. Third, our phenotypic findings were supported by parallel selective expression of multiple genes contributing heavily to DC maturation. Fourth, dose response curves clearly revealed that ECP induces monocytes to develop into quite functional DCs supportive of both CD4- and CD8-specific responses.

We agree that flow cytometry results are prone to qualitative interpretations and, by themselves, would have provided inconclusive results even in the context of our fluorescence-compensated analyses. It is for that reason that we have confirmed our observations by molecular methods (microarray expression analysis, quantitative reverse transcription–polymerase chain reaction) and functional studies (recall antigen presentation, cytotoxicity assays). Yet we are grateful for this chance to clarify our methodology. In full context, our results provide complementary sets of evidence revealing that ECP produces highly functional DCs, meeting established criteria for identification of these important cells and suggesting that this method is a potentially valuable source of DCs for cellular immunotherapy.