Sustained CD4+ T cell-driven lymphopenia without a compensatory IL-7/IL-15 response among high-grade glioma patients treated with radiation and temozolomide

Abstract

Prolonged lymphopenia correlating with decreased survival commonly occurs among glioma patients undergoing radiation therapy (RT) and temozolomide (TMZ) treatment. To better understand the pathophysiology of this phenomenon, we prospectively monitored serum cytokine levels and lymphocyte subsets in 15 high-grade glioma patients undergoing combined radiation and TMZ (referred to as RT/TMZ) treatment. Sufficient data for analysis were acquired from 11 of the patients initially enrolled. Lymphocyte phenotyping data were obtained using cytofluorometric analysis and serum cytokine levels were measured using the a multiplex bead-based assays. Total lymphocyte counts (TLCs) were > 1000 cells per μL peripheral blood in 10/11 patients at baseline, but dropped significantly after treatment. Specifically, after RT/TMZ therapy, the TLCs were found to be < 500 cells/μL in 2/11 patients, 500-1000 cells/μL in 7/11 patients, and > 1000 cells/μL in the remaining 2 patients. Among residual mononuclear blood cells, we observed a proportional drop in B and CD4⁺ T cells but not in CD8⁺ T lymphocytes. Natural killer cells remained to near-to-baseline levels and there was a transient and slight (insignificant) increase in regulatory T cells (Tregs). The circulating levels of IL-7 and IL-15 remained low despite marked drops in both the total and CD4⁺ T lymphocyte counts. Thus, patients with malignant glioma undergoing RT/TMZ treatment exhibit a marked decline in TLCs, affecting both CD4⁺ T cells and B lymphocytes, in the absence of a compensatory increase in interleukin-7 levels. The failure to mount an appropriate homeostatic cytokine response may be responsible for the prolonged lymphopenia frequently observed in these patients.

Keywords: CD4+ lymphopenia; IL-7; glioblastoma; immunotherapy; lymphocytes; radiation therapy; radiotherapy.

Figure 1. Concurrent RT/TMZ effects on total peripheral lymphocyte count over time. (A and B) Total lymphocytes were counted in the peripheral blood of Grade III and IV glioma patients prior to (baseline) and following 6 wk of radiation therapy (RT) and temozolomide (TMZ) treatment, at the indicated time points. (A) Absolute total lymphocyte counts (TLCs) at baseline, week 10 (4 wk after completing RT/TMZ), and week 18 (12 wk after completing RT/TMZ). (B) Percent changes in TLC, plotted individually for each patient in the study.

Figure 2. Concurrent RT/TMZ adversely affects total T-cell counts by selectively depleting CD4⁺ T-cell populations. (A–F) The peripheral blood lymphocytes of Grade III and IV glioma patients were immunophenotyped with fluorophore-conjugated antibodies specific for the indicated marker prior to (baseline) and following 6 wk of radiation therapy (RT) and temozolomide (TMZ) treatment. Post-treatment time points analyzed were week 10 (4 wk after completing RT/TMZ) and week 18 (12 wk after completing RT/TMZ). Flow cytometry was used to calculate percentages of lymphocytes with the indicated marker profile. Absolute numbers were determined by calculating cell count per blood volume. (A–D) CD3⁺ cells (A), CD3⁺CD8⁺ cells (B), CD3⁺CD4⁺ cells (C), CD4⁺CD45RA⁺ cells (D). Left panel: representative flow cytometry results. Middle panel: change in mean (and range) % lymphocytes with indicated marker profile. Right panel: change in absolute cell counts. (E–F) Plot of ratio of cells with indicated marker profile: CD45RA^-/CD45RA⁺ T-cell ratio (E), CD4⁺/CD8⁺ T-cell ratio (F). Statistical analyses were performed using paired samples t-test; *P < 0.05.

Figure 3. Concurrent RT/TMZ depletes B cells but not NK cells. (A and B) The peripheral blood lymphocytes of Grade III and IV glioma patients were immunophenotyped with fluorophore-conjugated antibodies specific for the indicated markers prior to (baseline) and following 6 wk of radiation therapy (RT) and temozolomide (TMZ) treatment. Post-treatment time points analyzed were week 10 (4 wk after completing RT/TMZ) and week 18 (12 wk after completing RT/TMZ). Flow cytometry was used to calculate the percentage of (CD19⁺) B lymphocytes (A) and (CD56⁺) natural killer (NK) cells (B). Absolute numbers were determined by calculating cell count per blood volume. Left panel: representative flow cytometry results. Middle panel: change in mean (and range) % lymphocytes with indicated marker profile. Right panel: change in absolute cell count. Statistical analyses were performed using paired samples t-test; *P < 0.05.

Figure 4. Concurrent RT/TMZ treatment transiently expands Tregs despite overall depletion of CD4⁺ T cell counts. (A–C) The peripheral blood lymphocytes of Grade III and IV glioma patients were immunophenotyped with fluorophore-conjugated antibodies specific for the indicated markers prior to (baseline) and following 6 wk of radiation therapy (RT) and temozolomide (TMZ) treatment. Post-treatment time points analyzed were week 10 (4 wk after completing RT/TMZ) and week 18 (12 wk after completing RT/TMZ). Flow cytometry was used to calculate the percentage of T lymphocytes with the indicated profiles. Absolute numbers were determined by calculating cell count per blood volume. (A and B) CD4⁺/CD3⁺/FOXP3⁺ cells (A) and CD4/CD45RA⁺ cells (B). Left panel: representative flow cytometry results. Middle panel: change in mean (and range) % lymphocytes with indicated marker profile. Right panel: change in absolute cell count. (C) Ratio of antigen-experienced (CD45RA^-) to antigen-naïve (CD45RA⁺) T cells. Statistical analyses were performed using paired samples t-test; *P < 0.05.

Figure 5. Serum IL-7 levels after RT/TMZ treatment for malignant glioma. (A and B) The sera of Grade III and IV glioma patients were analyzed for cytokine levels via Bioplex200 multiplexed bead-based immunoassay prior to (baseline) and following 6 wk of radiation therapy (RT) and temozolomide (TMZ) treatment. (A) Serum interleukin (IL)-7 levels (pg/mL) at baseline, week 10 (4 wk after completing RT/TMZ) and week 18 (12 wk after completing RT/TMZ). (B) Scatterplot of serum IL-7 levels (y-axis; pg/mL) and absolute peripheral CD4⁺ T lymphocyte counts from Figure 2C (x-axis; cells/μL). Statistical analyses were performed using paired samples t-test; *P < 0.05.