Immunogenicity of HLA Class i and II double restricted influenza a-derived peptides

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and-II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A02:01 and HLA-DRB101:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A02:01/HLADRB101:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.Fil: Pedersen, Sara Ram. Universidad de Copenhagen; DinamarcaFil: Christensen, Jan Pravsgaard. Universidad de Copenhagen; DinamarcaFil: Buus, Søren. Universidad de Copenhagen; DinamarcaFil: Rasmussen, Michael. Universidad de Copenhagen; DinamarcaFil: Korsholm, Karen Smith. Statens Serum Institute; DinamarcaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Claesson, Mogens Helweg. Universidad de Copenhagen; Dinamarc

The availability of a functional tumor targeting T-cell repertoire determines the anti-tumor efficiency of combination therapy with anti-CTLA-4 and anti-4-1BB antibodies

It has previously been found that combination therapy with anti-CTLA-4 and anti-4-1BB antibodies may enhance tumor immunity. However, this treatment is not efficient against all tumors, and it has been suggested that variations in tumor control may reflect differences in the immunogenicity of different tumors. In the present report, we have formally tested this hypothesis. Comparing the efficiency of combination antibody therapy against two antigenically distinct variants of the B16.F10 melanoma cell line, we observed that antibody therapy delayed the growth of a variant expressing an exogenous antigen (P<0.0001), while this treatment failed to protect against the non-transfected parental line (P = 0.1850) consistent with published observations. As both cell lines are poorly immunogenic in wild type mice, these observations suggested that the magnitude of the tumor targeting T-cell repertoire plays a major role in deciding the efficiency of this antibody treatment. To directly test this assumption, we made use of mice expressing the exogenous antigen as a self-antigen and therefore carrying a severely purged T-cell repertoire directed against the major tumor antigen. Notably, combination therapy completely failed to inhibit tumor growth in the latter mice (P = 0.8584). These results underscore the importance of a functionally intact T-cell population as a precondition for the efficiency of treatment with immunomodulatory antibodies. Clinically, the implication is that this type of antibody therapy should be attempted as an early form of tumor-specific immunotherapy before extensive exhaustion of the tumor-specific T-cell repertoire has occurred

Influenza infected HLA transgenic mice show no reactivity towards any of the immunogenic peptides.

<p>HLA transgenic mice were challenged intranasally with 4 LD₅₀ (B) or 0.4 LD₅₀ (A and C) of influenza A. IFNγ production from splenocytes was detected 10 days later using IFNγ ELISpot (A and B) or flow cytometry (C). In A and B, each bar represents mean ± SD of IFNγ spot forming units (SFU) of groups of 4–5 mice from which splenocytes were pooled before being stimulated <i>ex vivo</i> with peptide in four replicate cultures. In C, each bar represents mean ± SD of the number of IFNγ producing splenic CD8 T cells from 2 individual mice after <i>ex vivo</i> stimulation with peptide. Experiments were conducted 1–4 times depending of the peptide. Experiments were conducted 1–4 times, depending on the peptide.</p

No protection against influenza infection in mice immunized with the double restricted epitopes.

<p>HLA transgenic mice were immunized three times every second week with CAF09 and different combinations of double restricted immunogenic peptides or the known HLA-A*02:01 epitope M(58–66) (20nM). One week post last immunization, mice were intranasally challenged with 4 LD₅₀ (A), 0.4 LD₅₀ (B), or 0.04 LD₅₀ (C) of influenza A. As control groups, mice were either injected three times at two week intervals with CAF09 and Tris buffer (A and B) or were non-immunized (C) before influenza challenge. Influenza M gene expression in the lungs was measured by Q-PCR. Dots show numbers of M gene copies in the lungs per mice and horizontal lines depict means of 3–6 mice. Experiments were conducted once. Groups were tested using Mann-Whitney and ns = not significant compared to CAF09 + Tris (B) or non-immunized (C). Groups in A could not be tested by Mann-Whitney due to n = 3 in each group. Experiments were conducted once. Groups were tested using Mann-Whitney and ns = non significant compared to CAF09 + Tris (B) or non-immunized (C). Groups in A could not be tested by Mann-Whitney due to n = 3 in each group.</p

Immunization with the four double restricted epitopes induces both CTL and Th2 reactivity.

<p>Five groups of HLA transgenic mice were immunized intraperitoneally three times at two week intervals with CAF09 plus one of the four double restricted peptides or M(58–66) (20nM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145629#pone.0145629.ref021" target="_blank">21</a>]. One week after last immunization, IFNγ (A) or IL-4 (B) ELISpot was used to measure spot forming units (SFU) from splenocytes. Cells were pooled from each group and depleted for CD4 or CD8 positive cells before being stimulated <i>ex vivo</i> with peptide in four replicate cultures. Each bar represents mean ± SD of IFNγ (A) or IL-4 (B) spot forming units (SFU) of groups of 4 immunized mice. Experiments were conducted twice.</p

Induction of double restricted responses after peptide immunization of HLA transgenic mice.

<p>Eighteen groups of 3–5 mice were injected intraperitoneally three times at two week intervals with CAF09 plus one of the 9-11mer peptides (20nM). One week after the last immunization, IFNγ ELISpot was used to measure HLA-A*02:01 and/or HLA-DRB1*01:01 restricted CD8 and CD4 T cell responses. As a control, a group of mice (n = 4) was injected intraperitoneally three times at two week intervals with CAF09 plus the known HLA-A*02:01 binding peptide M(58–66) (21). Each bar represents mean ± SD of IFNγ spot forming units (SFU) of groups of 3–5 immunized mice from which splenocytes were pooled and depleted for CD4 or CD8 positive cells before being stimulated <i>ex vivo</i> with peptide in four replicate cultures. Experiments were conducted 1–5 times depending on the individual peptides.</p