Chemical castration of melanoma patients does not increase the frequency of tumor-specific CD4 and CD8 T cells after peptide vaccination

Abstract

Peptide vaccination against tumor-associated antigens remains one of the most common methods of immunization in cancer vaccine clinical trials. Although peptide vaccination has been reported to increase circulating antigen-specific T-cells, they have had limited clinical efficacy and there is a necessity to increase their capacity to generate strong antitumor responses. We sought to improve the clinical efficacy of peptide-based vaccines in cancer immunotherapy of metastatic melanoma using a LHRH agonist (leuprolide) as adjuvant. Seventy HLA-A*0201 stage IIb-IV melanoma patients were vaccinated with class I HLA-A*0201-restricted gp100209-2M peptide and stratified for HLA-DP4 restriction. HLA-DP4 patients were also vaccinated with class II HLA-DP4-restricted MAGE-3243-258 peptide. Patients from both groups were randomized to receive 2 doses of leuprolide or not. Here we report the increase in PBMC TREC levels at week 24 after peptide vaccination, which was independent of the leuprolide treatment. This change was mirrored by a small increase in the TREC-enriched CD8CD45RAROCD27CD103, but not the TREC-enriched CD4CD45RAROCD31 T-cell population. Serum concentration of 2 important factors for thymopoiesis was measured: insulin growth factor 1 (IGF-1) levels were not changed, whereas a moderate increase in IL-7 levels was noted in the sera of all patients 6 weeks after vaccination. Increased expression of CD127 (IL-7 receptor-α) at week 24, compared with baseline, was only seen in the CD8CD45RAROCD27CD103 T-cell population. Our results suggest that leuprolide has no effect on thymic output when used as peptide vaccine adjuvant, but IFA-based peptide vaccination may unexpectedly affect the thymus by increasing thymic output of new T cells.

FIGURE 1. Randomization of patients, treatment schema and time points for laboratory evaluations

All patients entering the trial were HLA-A*0201⁺. Patients were stratified by whether they were HLA-DP4⁺ or HLA-DP4⁻; Group 1 consisted of patients who were HLA-DP4⁻, while Group 2 consisted of patients who were HLA-DP4⁺. Within these groups, patients were randomized in a 1:1 ratio to receive Leuprolide or not (A). A schematic summary of treatments and time points for laboratory evaluations is also shown (B). The number in parenthesis is the total numbers of treatments and immunological evaluations for each patient.

FIGURE 2. Change in Frequency of gp100_209-2M –specific CD8⁺ and MAGE-3_243-258 –specific CD4⁺ T cells between baseline and weeks 12 or 24 after treatment, measured by tetramer (CD8) and multimer (CD4) staining assay

Frozen/thawed PBMC from patients at different time points were stained with class I HLA-A*0201-restricted gp100_209-2M tetramer (A) and class II HLA-DP4-restricted MAGE-3_243-258 multimer (not shown). How the net tetramer/multimer calculation is carried out is represented (A). Cells were stained with anti-CD11c/CD14/CD15/CD16/CD19 FITC (dump channel), MAGE3 Multimer PE, CD4 PerCP-Cy5.5, gp100 Tetramer-APC and CD8-APC/Cy7. The numbers in (B) represent the net change of gp100_209-2M –specific CD8⁺ and MAGE-3_243-258 –specific CD4⁺ T cell subsets within the CD8 and CD4 T cell population, respectively, at 12 and 24 weeks after vaccination. The numbers in (C) represent the net change of IFN-γ secretion after O/N stimulation of patients’ PBMC with peptides gp100_209-2M or MAGE-3_243-258 at 24 weeks after vaccination when compared to the Baseline (W24-BL).

FIGURE 3. Number of molecules of TRECs per million of PBMC detected in the blood of patients increase during the course of the intervention independently of Leuprolide treatment

Molecules of TREC in patients from age 23 through 85 yr (median = 52; n = 50) were analyzed by QC-PCR. The number of molecules of TRECs detected per million of PBMC in the blood of patients at baseline decreases with age (A). Analysis of blood testosterone levels in men (B) and estradiol levels in women (C) after Leuprolide treatment. Most men had testosterone levels compatible with clinical castration (50 ng/dL), whereas almost all women had postmenopausal estradiol levels (24 pg/ml), after 2 doses of Leuprolide. At 24 weeks post-vaccine, most patients showed an increase in total TREC cells/million PBMC compared with pretreatment, independently of Leuprolide treatment (D). In 18 of 24 patients for the Leuprolide-treated, and 15 of 25 for the non-Leuprolide group, changes were deemed substantial (>25% increase).

FIGURE 4. The Frequency of T regulatory cells does not change in patients after concomitant Leuprolide treatment and peptide vaccination

Multiparameter flow cytometric analysis of CD4⁺CD25^highFoxp3⁺ Tregs was performed using Frozen/thawed PBMC from 30 patients during peptide vaccination (12 treated with Leuprolide and 18 non treated) at Baseline, week 6, 12 and 24. Cells were stained with anti-CD4-PerCP-Cy5.5, Foxp3-Pacific Blue, CD25-FITC, CD3-PE and Aqua. Treg levels did not significantly change throughout the time course of the vaccination +/− Leuprolide (A). The mean frequency of CD4⁺CD25^highFoxp3⁺ Tregs was around 4% of the total CD4 T cells, independent from the Leuprolide treatment (B).

FIGURE 5. The TREC-enriched CD8⁺CD103⁺ naive CD8⁺ T cell subset, but not the TREC-enriched CD4⁺CD31⁺ naive CD4⁺ T cell subset, increased at week 24 after treatment in both Leuprolide treated and untreated patients

Multiparameter flow cytometric analysis of CD4⁺CD45RA⁺RO⁻CD31⁺ and CD8⁺CD45RA⁺RO⁻CD27⁺CD103⁺ subsets was performed using Frozen/thawed PBMC from 32 patients at different time points. Cells were stained with anti-CD4-AmCyan, CD8-Pacific Blue, CD45RA-FITC, CD103-PE, CD127-PerCP-Cy5.5, CD45RO-PE-Cy7, CD27-APC-H7 and CD31-Alexa Fluor 647. The gating strategy to study CD4⁺CD45RA⁺RO⁻CD31⁺ (A) and CD8⁺CD45RA⁺RO⁻CD27⁺CD103⁺ (B) subsets is shown. A comparison of CD4⁺CD45RA⁺RO⁻CD31⁺ (C) or CD8⁺CD45RA⁺RO⁻CD27⁺CD103⁺ (D) subpopulations between baseline and week 24 post-treatment of all patients shows that only an increase in the CD8⁺CD45RA⁺RO⁻CD27⁺CD103⁺ subpopulation correlates with the increase of TREC levels in PBMC.

FIGURE 6. Serum levels of IL-7, but not those of IGF-1, increase at week 6 after treatment

The levels of IL-7 and IGF-1 in the sera of the same thirty two patients used to identify the increase of the CD8⁺CD103⁺ naive subset were determined using ELISA (A). The percentage of expression of CD127 was determined by flow cytometry with the same gate strategy used in FIGURE 4B and using the quadrants depicted (B). The percentage of CD127⁺CD45RA⁺RO⁻CD27⁺CD103⁺ in CD8 T cells was calculated by multiplying the percentage of CD127⁺ cells in CD103⁺ (calculated as shown in B) by the percentage of CD8⁺CD45RA⁺CD27⁺ T cells. The graphic shows the result from these calculations for all 32 patients at baseline and at week 24 after treatment (C).