Monocyte-derived dendritic cells reflect the immune functional status of a chromophobe renal cell carcinoma patient: could it be a general phenomenon?

Abstract

Purpose: The chromophobe renal cell carcinoma (ChRCC), though associated with a hereditary cancer syndrome, has a good prognosis after tumor removal. The lack of recurrence could be related to the absence of immune system compromise in patients or to an effective functional recovery of immune functions after tumor removal. Thus, we evaluated monocyte-derived dendritic cells (Mo-DCs) in a 34-year-old male who had a ChRCC, before and after tumor removal.

Methods: CD14(+) monocytes from the patient's peripheral blood, 1 week before and 3 months after partial nephrectomy, were differentiated in vitro into immature and mature Mo-DCs. These were harvested, analyzed by flow cytometry and used as stimulators of allogeneic T cells. Supernatants from cultures were collected for cytokine analysis.

Results: Tumor removal was associated with decreased expression of PD-L1, but also, surprisingly, of CD205, HLA-DR, CD80 and CD86 by Mo-DCs. Also, Mo-DC's ability to stimulate T cell proliferation increased, along with IL-2Rα expression and IFN-γ production. Simultaneously, the patients' Mo-DCs ability to induce Foxp3(+) T cells decreased after surgery. One-year postoperative follow-up shows no tumor recurrence.

Conclusion: The presence of a ChRCC affected Mo-DCs generated in vitro, which recovered their function after tumor removal. This indicates that the favorable outcome observed after ChRCC resection may be due to the restoration of immunocompetence. Furthermore, since functional alterations described for DCs within tumors may be also found in Mo-DCs, their accurate functional analysis-not restricted to the determination of their surface immunophenotype-may provide an indirect "window" to the tumor microenvironment.

Fig. 1

Surface phenotype of Mo-DCs derived from cells obtained before and after tumor removal surgery. a MRI obtained one month before (left) and 1 year after (right) partial nephrectomy due to chromophobe renal cell carcinoma localized at the inferior pole of the left kidney. b Mo-DCs were gated within SSC × FSC parameters, and then, histograms for each molecule were obtained to determine the molecule expression in Mo-iDCs (top) and Mo-mDCs (bottom), 1 week before (dashed line) and 3 months (black line) after surgery. Gray histograms represent unlabeled cells obtained before, which overlapped to histograms of unlabeled cells obtained after surgery

Fig. 2

Function of Mo-DCs derived from cells obtained before and after tumor removal surgery. a T cells were gated within SSC × FSC parameters (I) and then selected as CD3⁺ (II). Histograms in (III) represents the percentage of proliferation, determined as the frequency of CD3⁺CFSE^lo T cells stimulated by Mo-iDCs and Mo-mDCs derived from the patient, before (dashed line), after surgery (black line) and derived from a healthy donor (HD # 1—dotted line). Gray histograms in (II) represent unlabeled cells and in (III) represent the frequency of CD3⁺CFSE^lo in non-stimulated T cells. b Histograms for frequency of CD25 (IL-2Rα) frequency were obtained within CD3⁺CFSE^lo, CD3⁺CFSE^loCD4⁺ and CD3⁺CFSE^loCD8⁺ T cells, stimulated by Mo-iDCs (top) and Mo-mDCs (bottom), before (left) and after surgery (right). Gray histograms represent CD25 expression by CD3⁺ non-stimulated T cells. c Frequency of CD4 and CD8 proliferating T cells were obtained from CD3 × CD4 parameters. Plots were gated within non-stimulated CD3⁺ T cells, Mo-DCs-stimulated CD3⁺CFSE^lo T cells (upper plots) and Mo-DCs-stimulated CD3⁺ T cells (lower plots) for groups stimulated by Mo-iDCs and Mo-mDCs obtained before (left) and after surgery (right). CD8 T cells were considered as CD3⁺CD4^neg T cells since purity of T cells was determined as of ≥97 %

Fig. 3

Frequency of CD4⁺CD25⁺Foxp3⁺ T cells induced by Mo-DCs derived from cells obtained before and after tumor removal surgery.a CD4⁺CD25⁺Foxp3⁺ T cells were gated within SSC × FSC parameters, b selected as CD3⁺, then, c selected as CD25⁺CD4⁺ T cells and finally, d selected as Foxp3⁺ T cells, stimulated by Mo-iDCs (left) and Mo-DCs (right) derived from the patient, before and after surgery and from a healthy donor (HD # 1). Gray histograms in (b) represent unlabeled cells. e Presented the absolute T cell numbers of CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25⁺Foxp3^neg for non-stimulated control T cells (left), Mo-iDCs-stimulated T cells (middle) and Mo-mDCs-stimulated T cells (right), before and after surgery

Fig. 4

Phenotypical and functional changes of Mo-DCs derived from cells obtained before and after tumor removal surgery Before tumor removal, Mo-iDCs and Mo-mDCs derived from the patient’s blood monocytes expressed higher levels of molecules related to antigen uptake (CD205), antigen presentation (HLA-DR) and co-stimulation (CD80 and CD86) but were poor T cell stimulators and favored the proliferation of CD8⁺ T cells. Both CD4⁺ and CD8⁺ T cells had low levels of CD25 (IL-2Rα) expression, and consequently, higher levels of free IL-2 were found in supernatants. Moreover, Mo-DCs obtained before the surgery expressed higher levels of PD-L1 and induced higher frequencies of CD4 and CD8 CD25⁺Foxp3⁺ T cells. Remarkably, after tumor resection, Mo-iDCs downregulated the expression of CD205, HLA-DR, CD80 and CD86, molecules that were upregulated consistently with the maturation stimulus. Mo-iDCs and Mo-mDCs also displayed an enhanced ability to stimulate T cell proliferation, along with IL-2Rα expression, IL-2 consumption and IFN-γ production. Additionally, Mo-DCs obtained after the surgery expressed lower levels of PD-L1 and decreased their ability to induce CD4 and CD8 CD25⁺Foxp3⁺ T cells