UCPVax, a CD4 helper peptide vaccine, induces polyfunctional Th1 cells, antibody response, and epitope spreading to improve antitumor immunity

Abstract

The induction of an antitumor CD4⁺ T helper response is essential for the efficacy of therapeutic cancer vaccines. However, few vaccines are specifically designed to target CD4⁺ T cells in human cancers. Here, we characterize the immune mechanisms of UCPVax, a helper peptide vaccine derived from telomerase. Ex vivo immune profiling of peripheral blood from 60 patients with advanced lung cancer reveals that UCPVax selectively activates CD4⁺ T cells in vivo across a broad HLA-DR restriction. The vaccine elicits a synergistic immune triad, including cytokine polyfunctional CD4⁺ Th1 cells, epitope spreading, and antibody response, contributing to effective tumor control. Single-cell analysis further demonstrates that UCPVax drives CD4⁺ T cells toward effector memory and cytolytic differentiation. Thus, vaccine-induced CD4⁺ T cells trigger broad and durable antitumor immunity. These findings highlight UCPVax as an off-the-shelf helper platform to enhance therapeutic cancer vaccine efficacy. This study was registered at ClinicalTrials.gov: NCT02818426.

Keywords: CD4(+) T cells; antibody response; cancer vaccine; epitope spreading; helper peptide; lung cancer; polyfunctional T cell; telomerase.

Graphical abstract

Figure 1

UCPVax induces hTERT-reactive CD4⁺ T cells in a broad range of HLA class II contexts (A) Treatment plan of UCPVax. UCPVax includes UCP2 and UCP4 pan-HLA-DR binding helper peptides derived from telomerase (hTERT) as previously described (Adotévi et al.⁴¹). Each helper peptide was emulsified in the adjuvant Montanide ISA-51 VG and injected subcutaneously. Patients received 6 weekly injections (priming) following by booster vaccinations for 8 weeks (boost) for a maximum of 12 months. Blood samples were collected for immune readouts as indicated (created with BioRender.com). (B) Heatmap showing distribution of vaccine-induced UCP2- and UCP4-specific CD4⁺ T cells over time (n = 60). The frequency of immune responders is shown at the top of the heatmap, intensity of response is shown at the bottom, and low and high responders were defined according to the median of IFNγ spots for each peptide (median spots: UCP2 = 84 and UCP4 = 63). The ∗ denotes patients evaluated with a mix of UCP2+UCP4. (C) Distribution of HLA-DR-B1 allele frequencies in patient responders to UCP2 (n = 37) and UCP4 (n = 24). (D) Heatmap representing HLA-DR-B1 allele expression in responders to UCP2 and UCP4 and in non-responders. (E) PHBR scores for UCP2 and UCP4. Boxplot represents median ± 1^st and 3^rd quartiles, Mann-Whitney test. (F) PHBR score according to the intensity of anti-UCP2 or UCP4 CD4⁺ T cell responses. (G) HLA-DR restriction. Histograms representing example of UCP2- (left) and UCP4- (right) specific responses with or without indicated MHC class I and II blocking antibodies measured by ex vivo ELISpot. Results represent mean of triplicates + SD. See also Figure S1 and Tables S1 and S2.

Figure 2

Expansion of cytokine polyfunctional triple CD4⁺ Th1 cells after UCPVax vaccination (A) Vaccine-induced UCP-specific CD4⁺ Th1 cells by ex vivo ICS assay in 48 patients. Left, schematic representation of UCPVax-expanded Th1 subset: mono-functional Th1, polyF double+ Th1, and polyF triple+ Th1 cells. Right, cytometry dot plots showing representative patients of each subset. (B) Nested pie chart showing the proportion (median) of each subset of UCP-specific Th1 cells (n = 48). (C) Pie chart showing the repartition of patients according to UCPVax-expanded Th1 cell subsets (n = 34). (D) Representative dot plots of patients with UCP-specific polyF triple+ Th1 cells. (E) Histograms showing rate of polyF triple+ Th1 cells in vaccinated patients (n = 18). Mean ± SD. (F) Count of polyF triple+ Th1 cells in the blood per mm³. (G) Representative examples of cytometry dot plots showing UCP-specific CD4⁺ T cells producing IFNγ, IL-2, and TNF-α at individual secretion and simultaneously along vaccination. (H) Representative examples of stacked overlays of CXCR3, CXCR5, and CCR6 marker expression gated in UCP-specific polyF triple+ Th1 cells. (I) Overall survival (OS) in patients according to polyF Th1 subsets; p value, two-tailed log rank test. (J) Table showing median OS with 95% confidence interval and survival rate at 12 months in overall population and according to polyF Th1 subsets. See also Figures S2 and S3 and Table S3.

Figure 3

Vaccine-induced CD4⁺ Th1 cells display activated and memory differentiation (A) Experimental design of scRNA-seq of UCP-specific CD4 and bulk CD4 cells performed in four patients, P23, P05, and pooled samples from P20 and P37. (B) UCell scoring of T helper polarization gene signatures in single-cell dataset from bulk CD4 T cells at baseline (BSL; P23) and from vaccine-specific CD4 cells from P23, P05, and P20–P37. (C) Average gene expression of CD4 helper-related transcription factors. (D) Uniform manifold approximation and projection (UMAP) analysis of UCP-specific CD4 T cell differentiation clusters (colors correspond to the cell cluster identified), pooled post-vaccine specific samples from all four patients. (E) Proportion of each cell cluster from post-vaccine samples. (F) Average gene expression of activation and cycling markers. (G) Average gene expression of cytotoxic markers. (H) UMAP visualization by the cell trajectory analysis by monocle3 pseudotime. The scale from 0 to 20 means from the least differentiated state to the most differentiated state. (I) Left, flow cytometry dot plots of pHLA-DR1-UCP2 and pHLA-DR1-UCP4 multimer staining in a representative patient. Right, percentage of pHLA-DR1-UCP2/UCP4 multimer+ CD4⁺ T cells (n = 6) at BSL and after vaccine. (J) Representative flow cytometry dot plots showing expression of T cell differentiation markers CCR7 and CD45RA in pHLA-DR1-UCP2 multimer+ cells. Right, histogram (mean ± SD) showing the percentage of naive T (CCR7+CD45RA−), T effector memory (CCR7−CD45RA−), T central memory (CCR7+CD45RA+), and terminally differentiated effector memory T cells TEMRA (CCR7−CD45RA+) cells in pHLA-DR1-UCP2 multimer+ cells or ICS (n = 10 patients). (K) Top, representative dot plots of activation markers CD39, ICOS, 4-1BB, and OX40 expression gated on pHLA-DR1 multimer+ cells and bottom on IFNγ+ UCP-specific CD4⁺ T cells by ex vivo ICS assay. (L) Percentage of CD39, ICOS, 4-1BB, and OX40 + UCP-specific CD4⁺ T cells by ICS or pHLA-DR1 multimer (n = 10). Mean ± SD See also Figure S5.

Figure 4

UCP-specific CD4⁺ T cells are cytolytic and oligoclonal (A) Flow cytometry dot plots showing expression of cytotoxic makers granzyme B, perforin, SLAMF-7, and TRAIL in IFNγ+ UCP-specific CD4⁺ T cells in representative patients. (B) Percentage of each cytotoxic marker+ in vaccine-expanded specific CD4⁺ T cells, mean ± SD (n = 9 patients). (C) CD107a degranulation assay. Post-vaccine PBMCs were co-cultured with HLA-DR-matched L-DR-cell line loaded or not with UCP2/4 during 15 h before CD107a staining. (D) Example flow cytometry dot plot of CD107a expression in two representative patients. (E) Percentage of CD107a+ CD4⁺ T cells after co-culture (n = 9 patients). Mann-Whitney test. (F) Experimental design of TCR-β sequencing of UCP-specific CD4 and bulk CD4 cells performed in four patients: P23, P05, P20, and P37. (G) Number of total clonotypes in UCP-specific CD4 and bulk CD4 fractions. (H) Relative abundance of small, medium, large, and hyperexpanded clonotypes in UCP-specific CD4 and bulk CD4 fractions at baseline. (I) Number of clonotypes occupying 50% of the repertoire. (J) Repertoire overlaps between the patients’s UCP-specific T cells. (K) Tracking of most frequent (top 10) clonotypes in UCP-specific CD4 and bulk CD4 fractions. See also Figure S5.

Figure 5

Targeted CD4⁺ T cells with UCPVax promotes specific antibody response and epitope spreading (A) Schematic design of vaccine-induced antibody (Ab) response by ELISA in plasma. (B) UCP-specific IgG1 Ab titer (ng/mL) before (pre) and after priming vaccinations (post-vacc) (n = 50). (C) Boxplot showing anti-UCP IgG1 titer Ab (ng/mL) pre- and post-vaccinations (n = 50). Data are presented as median ± 1^st and 3^rd quartiles. Mann-Whitney test. (D) Graphs showing evolution of anti-UCP IgG response during vaccination in 10 representative patients. (E) Pie charts representing the distribution of patients with positive Ab response (POS; n = 31) on the left and without Ab response (NEG; n = 19) on the right according to the UCP CD4⁺ Th1 response by ELISpot; positive in red and negative in blue. (F) Overall survival (OS) according to anti-UCP Ab response (n = 50). p value, two-tailed log rank test. (G) Schematic design of epitope-spreading assessment pre- and post-vaccination by IFNγ ELISpot after 6 days. IVS of PBMCs with mixture of MHC class I and II peptides derived from indicated tumor-associated antigens (TAAs) (n = 41). (H) Representative IFNγ spot wells from two patients positive for epitope spreading. (I) Pie chart representing number of patients with (POS) or without (NEG) epitope-spreading induction. (J) Left, expansion of T cells against indicated TAA in the IFNγ ELISPOT assay. Right, distribution of specific T cells against class II epitope from TAA and class I peptides from hTERT. (K) Frequency of patients with epitope spreading according to anti-UCP CD4⁺ T cell response after vaccination. (L) Heatmap showing diversity of epitope-spreading response according to intensity of anti-UCP CD4⁺ T cells (n = 25). (M) OS according to epitope spread response. p values, two-tailed log rank test.

Figure 6

UCPVax-mediated immune triad is associated with optimal tumor control (A) Unsupervised principal-component analysis (PCA), including UCP-specific CD4⁺ T cells (IFNγ spots), polyF triple+ Th1 cells, anti-UCP Ab response, and epitope spreading. (B) 3D clustering graph (k-means) showing individual patient according to vaccine-induced immune response subtype. (C) Graph showing HLA-DR alleles and OS of the 7 patients with vaccine-induced immune triad. (D) Change in target lesion from baseline (BSL) evaluated by RECIST1.1 in one patient with complete response (P23). (E) Kinetic of vaccine-induced UCP-specific CD4 and antibody responses since inclusion in P23. (F) Tracking of the most frequent clonotype (top 10) of UCP-specific clones from BSL, priming, and 4.5 years post-vaccine in P23. Red: preserved clonotypes along the time. (G) Predominant signaling pathway on T cells pre- versus post-vaccination by CellChat. Top signaling pathways enriched at BSL (orange) and more enriched after vaccination (blue). In bold: pathways that are statistically significant. (H) Chord diagram showing the cell-cell communication mediated by TGF-β and IL-10 signaling. Arrows and edge color indicate direction (source: target). Edge thickness indicates the sum of weight key signals between populations. See also Figure S6 and Tables S4 and S5.