Activation, dysfunction and retention of T cells in vaccine sites after injection of incomplete Freund's adjuvant, with or without peptide

Abstract

We conducted a randomized clinical trial in 45 patients with resected AJCC stage IIB-IV melanoma to characterize cellular and molecular events at sites of immunization with incomplete Freund's adjuvant (IFA) alone, or a melanoma vaccine in IFA. At a primary vaccine site, all patients received a multi-peptide melanoma vaccine in IFA. At a replicate vaccine site, which was biopsied, group 1 received IFA only; group 2 received vaccine in IFA. Lymphocytes isolated from replicate vaccine site microenvironments (VSME) were compared to time-matched peripheral blood mononuclear cells (PBMC) in ELISpot and flow cytometry assays. Compared to PBMC, the VSME had fewer naïve and greater proportions of effector memory CD8(+) T cells (TCD8). The vast majority of TCD8 within the VSME were activated (CD69(+)), with a concentration of antigen-specific (tetramer(pos)) cells in the VSME, particularly in vaccine sites with peptide (group 2). CXCR3(+) lymphocytes were concentrated in the VSME of all patients, suggesting IFA-induced chemokine recruitment. TCD8 expression of retention integrins αEβ7 and α1β1 was elevated in VSME, with the highest levels observed in antigen-specific cells in VSME containing peptide (group 2). TCD8 retained in the VSME of both groups were strikingly dysfunctional, with minimal IFN-γ production in response to peptide stimulation and few tetramer(pos) cells producing IFN-γ. These data suggest that vaccine-induced selective retention and dysfunction of antigen-specific TCD8 within VSME may represent a significant mechanism underlying transient immune responses and low clinical response rates to peptide vaccines administered in IFA.

Fig. 1

Mel48: A multi-peptide vaccine in melanoma patients with evaluation of the injection site microenvironment. Patients with resected AJCC stage IIB-IV melanoma underwent cutaneous vaccination with 12-melanoma peptide vaccine and tetanus helper peptide in IFA. At a replicate vaccine site, Group 1 received IFA alone, Group 2 received full vaccination. Replicate vaccine sites were biopsied and peripheral blood collected as shown

Fig. 2

Vaccine sites are depleted of naïve and enriched for effector memory CD8⁺ T cells compared to peripheral blood. Phenotypes of CD8⁺ T cells (T_CD8) were determined by multi-parameter flow cytometry staining with CCR7 and CD45RA. Cells were sorted by their ability to bind to tetramers relevant to the HLA type of the patient. Gating was performed on live, singlet, CD8⁺/CD4^neg/CD14^neg/CD19^neg, then tetramer^pos or tetramer^neg lymphocyte populations. The figure depicts percentages of CD8⁺ T cells staining with naïve (Naïve, CCR7⁺/CD45RA⁺) and effector memory (EM, CCR7^neg/CD45RA^neg) phenotypes. a T_naive of tetramer^neg cells. Values from vaccine site and/or peripheral blood at each time point are shown for each individual patient with a unique Subject ID. b T_naive of tetramer^pos cells. c T_EM of tetramer^neg cells. d T_EM of tetramer^pos cells

Fig. 3

Marked activation, antigen specificity and cellular dysfunction in vaccine sites. CD69 expression and staining with MHC-peptide tetramers in live, singlet, CD8⁺/CD4^neg/CD14^neg/CD19^neg lymphocyte populations were evaluated by multi-parameter flow cytometry. Functionality was assessed by IFN-γ response to vaccine peptides in direct ELISpot assay. CD69 Expression. a Upper (group 1C) and lower (group 2C) panels display representative flow cytometry from the VSME and time-matched PBMC for individual patients. b Percentages of CD8⁺ T cell populations staining positive for CD69 are shown for individual patient samples. Tetramer staining. Cells were sorted by their ability to bind MHC-peptide tetramers relevant to the HLA type of the patient. c Upper (group 1C) and lower (group 2C) panels display representative flow cytometry from the VSME and time-matched PBMC for representative patients. Total numbers of cells evaluated per assay from the VSME were lower than in PBMC. d Tetramer^pos proportion of CD8⁺ T cell populations for individual patients. e Ratio of ELISpot reactivity to tetramer. The number of cells producing IFN-γ in response to vaccine peptides was compared to the number of tetramer positive cells per 10⁵ CD8⁺ T cells at each time point. Ratios of IFN- γ⁺ to tetramer^pos cells are depicted for individual patients

Fig. 4

In vaccine sites, chemokine receptor CXCR3 expression is elevated. Live, singlet, CD14^neg/CD19^neg cells were identified and gated on CD4⁺, CD8⁺/Tetramer^pos or CD8⁺/Tetramer^neg populations. T_CD8 = CD8⁺ T cells. T_CD8 = CD8⁺ T cells. T_CD4 = CD4⁺ T cells.CXCR3 expression among a tetramer^neg and b tetramer^pos T_CD8 and c T_CD4 are shown for individual patients. d CXCR3 populations in a representative group 2C patient. Each grey histogram is a negative control (FMO-CXCR3)

Fig. 5

Retention integrin expression is elevated in vaccine sites. Gating was performed on live, singlet, CD8⁺/CD4^neg/CD14^neg/CD19^neg, then tetramer^pos or tetramer^neg lymphocyte populations. T_CD8 = CD8⁺ T cells. a Epithelial retention integrin αEβ7 among tetramer^neg cells. Percentages of tetramer^neg T_CD8 populations staining positive for αEβ7 are shown for individual patients. b αEβ7 among tetramer^neg cells. c Collagen retention integrin α1β1 among tetramer^neg cells. Percentages of tetramer^neg T_CD8 populations staining positive for α1β1 are shown for individual patients. d Collagen retention integrin α1β1 among tetramer^pos cells) e αEβ7 and α1β1 staining among tetramer^neg cells and f tetramer^pos cells. Panels display representative flow cytometry from T_CD8 from vaccine site biopsies and time-matched PBMC for a representative group 2C patient