Therapeutic vaccination with a trivalent T-cell receptor (TCR) peptide vaccine restores deficient FoxP3 expression and TCR recognition in subjects with multiple sclerosis

Abstract

Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund's adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naïve subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both 'native' CD4+ CD25+ and 'inducible' CD4+ CD25- peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.

Figure 1

Frequencies of T-cell receptor (TCR) peptide-reactive peripheral blood mononuclear cells (PBMC) from TCR tripeptide-vaccinated subjects with multiple sclerosis (MS). Reactivity to the tripeptide mixture of complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 was assessed in seven MS subjects in terms of proliferation responses using the limiting dilution assay and in terms of secretion of interleukin (IL)-10 or interferon (IFN)-γ using the enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). Blood samples were collected from the subjects prior to vaccination and at weeks 8/9, 12, 24 and 48 of the trial and cultured as described in the Materials and methods. Data are presented as mean frequency +mn; standard deviation for MS subjects at entry, at maximum postvaccination response, and at exit from the trial. ELISPOT frequencies represent the sum of responses to each of the three vaccinating TCR CDR2 peptides.

Figure 2

T-cell receptor (TCR) vaccination restored forkhead box (Fox) P3 message and protein expression. Peripheral blood mononuclear cells (PBMC) were collected from subjects with multiple sclerosis (MS) prior to vaccination and at the indicated time-points during treatment with TCR tripeptides, as well as from age- and gender-matched healthy control (HC) donors. The cells were sorted into CD4⁺ CD25⁺ and CD4⁺ CD25^– populations, and mRNA was extracted and evaluated for expression of FoxP3 and hypoxanthine guanine phosphoribosyl transferase (HPRT)1 genes. Sorted cells from the same subjects were also evaluated for FoxP3 and HPRT-1 protein by western blots. (a) FoxP3 expression in CD4⁺ CD25⁺ T cells showed a significant increase after vaccination. Note reduced FoxP3 expression in the CD4⁺ CD25⁺ cells from MS subjects versus HC donors prior to vaccination, and significantly enhanced expression to levels even higher than those in HC donors at week 12 and in all subsequent weeks tested during the vaccination procedure. (b) FoxP3 expression in CD4⁺ CD25^– T cells increased significantly after vaccination. FoxP3 expression was much lower in the CD4⁺ CD25^– population and was not different between MS subjects and HC donors prior to vaccination, but was significantly enhanced to levels higher than those in the HC donors in the MS subjects during vaccination with TCR tripeptides. (c) FoxP3 protein showed a significant increase after vaccination. FoxP3 protein levels were reduced in CD4⁺ CD25⁺ T cells from MS subjects relative to those in HC donors prior to vaccination, but were restored to levels higher than those in HC donors in MS subjects during vaccination. Error bars represent the standard error of the mean.

Figure 3

Forkhead box (Fox) P3 message expression was enhanced in a significant proportion (13/16; 81%P < 0·001) of vaccinated subjects with multiple sclerosis (MS). FoxP3 message levels (normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT)1) are shown for 13 MS subjects who had increased expression (a) and three MS subjects (19%) who had decreased expression (b) over the course of treatment with T-cell receptor (TCR) tripeptides.

Figure 4

Vaccination with T-cell receptor (TCR) tripeptides reduced reactivity to myelin oligodendrocyte glycoprotein (MOG) 145–160 peptide while maintaining or increasing reactivity to a recall antigen [herpes simplex virus (HSV)]. Peripheral blood mononuclear cells (PBMC) from two subjects with multiple sclerosis (MS) (subjects 5126 and 5127) were cultured in replicate wells of 200 000 cells with or without HSV antigen or MOG 145–160 peptide, and evaluated for proliferation responses in terms of uptake of ³H-Tdy. Wells were determined to be reactive to the antigen if the counts per minute (c.p.m.) exceeded 2 standard deviations of the mean c.p.m. of the unstimulated control wells. Significance was determined by comparing the rates of positive wells at entry and exit using Fisher's exact test (**P < 0·01; ***P < 0·001).

Figure 5

T-cell receptor (TCR) peptide vaccination increased the number of V gene sequences recognized by peripheral blood mononuclear cells (PBMC). (a) PBMC from three subjects with multiple sclerosis (MS) were evaluated prior to vaccination and at exit (week 52) for proliferation responses (determined by a t-test for triplicate wells stimulated with medium or the given peptide) to a panel of 113 AV and BV CDR2 peptides. The increase in the total number of peptides recognized by each of the three subjects after vaccination was significant (*P < 0·05; **P < 0·01; ***P < 0·001). (b) The number of unique peptides recognized by the MS subjects was reduced at entry compared with healthy control (HC) donors but increased significantly after vaccination.

Figure 6

Identity of the AV and BV CDR2 peptides recognized by the three healthy control (HC) donors and three subjects with multiple sclerosis (MS) before and after vaccination. The amino acid sequences of the indicated peptides are listed in Vandenbark et al. Multiple boxes indicate recognition by more than one HC donor or MS subject.