Prospective Study of the Evolution of Blood Lymphoid Immune Parameters during Dacarbazine Chemotherapy in Metastatic and Locally Advanced Melanoma Patients

<div><p>Background</p><p>The importance of immune responses in the control of melanoma growth is well known. However, the implication of these antitumor immune responses in the efficacy of dacarbazine, a cytotoxic drug classically used in the treatment of melanoma, remains poorly understood in humans.</p><p>Methods</p><p>In this prospective observational study, we performed an immunomonitoring of eleven metastatic or locally advanced patients treated with dacarbazine as a first line of treatment. We assessed by flow cytometry lymphoid populations and their activation state; we also isolated NK cells to perform in vitro cytotoxicity tests.</p><p>Results</p><p>We found that chemotherapy induces lymphopenia and that a significantly higher numbers of naïve CD4⁺ T cells and lower proportion of Treg before chemotherapy are associated with disease control after dacarbazine treatment. Interestingly, NK cell cytotoxicity against dacarbazine-pretreated melanoma cells is only observed in NK cells from patients who achieved disease control.</p><p>Conclusion</p><p>Together, our data pinpoint that some immune factors could help to predict the response of melanoma patients to dacarbazine. Future larger scale studies are warranted to test their validity as prediction markers.</p></div

Correlation between T cell transcription factors and survival.

<p>We performed RT-qPCR on RNA from PBMC. Figure depicts relation between survival (in months) and expression level of indicated genes: <i>EOMES</i> (A), <i>TBX21</i> (B), <i>RORC</i> (C).</p

Antibodies for flow cytometry analysis.

<p>PerCP: Peridinin chlorophyll; PE: Phycoerythrin; Cy7: Cyanine-7.</p><p>Antibodies for flow cytometry analysis.</p

Patients' clinical characteristics.

<p>PD: progressive disease.</p><p>PR: partial response.</p><p>SD: stable disease.</p>†<p>: dead.</p><p>(*): Patient n°2 was inoperable, due to initial extension of its nasal tumor.</p><p>Patients' clinical characteristics.</p

NK cell activity is related to chemotherapy response.

<p>A: CD56^dimCD16⁺ (left panel) cells and CD56^hiCD16⁻ (right panel) NK cells counts. Black filled diamonds represent data from patients with progressive disease and white squares represent data from patients with disease control. B: CD69 expression by CD56^dimCD16⁺ (left panel) cells and CD56^hiCD16⁻ NK cells (right panel). C: Death of MelC cells pretreated with PBS or DTIC, and cocultured with NK cells isolated from patients.</p

Treatment impacts T cell sub-populations.

<p>Panel A shows CD8⁺ (left panel) and CD4⁺ (right panel) T cell counts before and after chemotherapy. B and C: Naïve CD4⁺ T cell counts before and after chemotherapy (B) and between patient groups (C). Black filled diamonds represent data from patients with progressive disease and white squares represent data from patients with disease control.</p

Chemotherapy causes changes in blood cells.

<p>We retrieved clinical data about leukocytes (A), lymphocytes (B), monocytes (C) and granulocytes (D) before the first (“Before”) and second (“After”) DTIC treatment. Black filled diamonds represent data from patients with progressive disease and white squares represent data from patients with disease control. For panel E, we compared granulocytes counts between patient groups before the first and second treatment (left and right panel, respectively).</p

BLM induces in vivo Treg accumulation throught TGFb production.

<p>A: Spleens from CT26 tumor-bearing mice, treated with PBS or BLM, were harvested and the cells were analyzed through flow cytometry for Foxp3⁺CD4⁺ Treg detection. B: Treg from PBS- or BLM- treated CT26-bearing mice were isolated from spleen and co-cultured with OT-I in presence of SIINFEKL (SII). After 3 days of culture, IFNγ was titrated in the supernatant using ELISA method. C: Schematic representation for D and E panel experiments. D: GFP⁺ CD4⁺ cells were sorted from CD45.2 FOXP3-EGFP mice, then injected i.v. in CD45.1 mice bearing CT26 tumor. The mice then received PBS, IL-2 or BLM treatment. All mice received EdU injection. One day after treatment, spleens and tumors were collected and the proliferation status of transferred cells was assessed by revealing EdU by flow cytometry. E: CD4⁺ CD62L⁺ GFP⁻ naive T cells were sorted from CD45.2 FOXP3-EGFP mice, and injected i.v. in CD45.1 mice bearing CT26 tumor. The mice then received PBS or BLM treatment. Spleens were harvested and the cells were analyzed through flow cytometry for Treg detection. F: Mice were injected with 1.10⁶ CT26 cells i.p. Ten days later mice received PBS or BLM injection. The following day ascites were collected, and TGFβ was assessed using ELISA method. G: CT26 cells were treated with PBS or BLM <i>in vitro</i> for 24 h, then the supernatant was collected and assessed for TGFβ using ELISA method. H: Mice bearing CT26 tumor cells were treated with PBS or BLM. The day after, CT26 tumors were collected and tumor cells were separated from the tumor infiltrating lymphocytes. Tumor cells were stained for LAP and analyzed by flow cytometry. I: GFP⁺ CD4⁺ cells were sorted from CD45.2 FOXP3-EGFP mice, then cultured <i>in vitro</i> under TCR-stimulating conditions. We added culture supernatant of CT26 treated for 24 h with PBS of BLM. In some wells, blocking anti-TGFβ antibody was added. After three days of culture, the cells were incubated with BrdU for 3 h, and then proceeded to BrdU detection by flow cytometry. J: Same as A, and mice received injection of blocking anti-TGFβ antibody. All data presented are representative of one out of two (panels B, D, E, G and I) or three experiments (panels A, F, H and J). Graphs show mean +/− SEM.</p

BLM in vivo antitumor effect through immune-based mechanisms.

<p>A: Footpad injection of different vaccines was performed: PBS-, DOX- or BLM-treated B16-OVA, or ovalbumin protein. 5 days later, the popliteal lymph nodes were harvested and cells were rechallenged with OVA peptide SIINFEKL. After 3 days of culture, IFNγ was titrated in the supernatant using ELISA method. B: shControl CT26 (left panel) or shCRT CT26 (right panel) were injected in the flank of mice. When the tumor reached about 25 mm², mice were treated with PBS or BLM and tumor growth was monitored with a caliper over time. C and D: CT26 cells were injected in the flank of mice. When the tumor reached about 25 mm², mice were treated with PBS or BLM. Some of the mice received injection of isotype control or depleting anti-CD8 (C) or anti-IFNγ (D). We monitored tumor growth with a caliper over time. All data presented are representative of one out of two experiments. Graphs show mean +/− SEM.</p

BLM anti-tumor effect is enhanced by Treg or TGFβ depletion.

<p>Five hundred thousand CT26 cells were injected in the flank of mice. When the tumor reached about 25 mm², mice were treated with PBS or BLM. Some of the mice received injections of isotype control antibody or depleting anti-CD4 (A), anti-CD25 (B) or blocking anti-TFGβ (C) antibodies. Tumor growth was monitored with a caliper over time. All data presented are representative of one out of two experiments. Graphs show mean +/− SEM.</p