CD4-positive T-helper cell responses to the PASD1 protein in patients with diffuse large B-cell lymphoma

Abstract

Background: Vaccine development targeting the novel immunogenic Per ARNT Sim Domain containing 1 (PASD1) cancer testis antigen represents an attractive therapeutic approach for the significant number of patients with diffuse large B-cell lymphoma who are refractory to conventional treatment. Since CD4-positive T helper cells have crucial roles in promoting and maintaining immune responses to tumor antigens, the presence of a CD4-positive T-helper immune response to the PASD1 antigen in patients with diffuse large B-cell lymphoma was investigated in the current study.

Design and methods: Thirty-one patients with diffuse large B-cell lymphoma (25 with de novo, five with transformed and one with T-cell-rich B-cell lymphoma) were studied. Five immunogenic PASD1 peptides predicted to bind to several major histocompatibiliy complex, class II DR beta 1 alleles were identified using web-based algorithms. Peripheral blood mononuclear cells from patients were used to investigate the immunogenicity of these DR beta 1-restricted peptides in vitro using both gamma-interferon release enzyme-linked immunospot and cytolytic assays.

Results: Two of the five PASD1 peptides, PASD1(6) and PASD1(7), were shown to be immunogenic in 14 out of 32 patients studied in a gamma-interferon release assay. CD4-positive T-helper cell lines from two patients raised against PASD1 peptides were able to lyse cell lines derived from hematologic malignancies expressing endogenous PASD1 protein.

Conclusions: This is the first report of a CD4-positive T-helper response to the PASD1 protein in patients with lymphoma. The immunogenic peptides described here represent valuable additional candidates for inclusion in a vaccine to treat patients with PASD1-positive diffuse large B-cell lymphoma whose disease is refractory to conventional therapies.

Figure 1.

Diagram to show the positions of the CD4 PASD1 peptides investigated here across the PASD1a and PASD1b protein isoforms.

Figure 2.

Interferon-γ ELISPOT studies showing CD4 Th responses to PASD1 peptides of patients 4 and 47. (A) A study monitoring the CD4 Th responses of these two patients at the time of diagnosis and 1 year after-diagnosis showed the presence of significant immune interferon-γ responses to PASD1(6) and PASD1(7) peptides even after 1 year in remission (P<0.05). In contrast, no significant responses to the control HIV peptide or medium only were observed. (B) CD4 Th cell lines were cultured from patient 4 and patient 47 and CD4⁺ cell populations were obtained using a magnetic purification step. After overnight incubation with either PASD1(6) or PASD1(7) peptides, the purified CD4⁺ cells from both patient 4 and 47 responded to these PASD1 peptides. No significant response was detected to the irrelevant control peptide or to medium only. The removal of the CD4⁺ cells or the addition of anti–HLA-DR antibody abrogated the interferon-γ response to both peptides. The results are the mean ± SD and were from triplicate cultures.

Figure 3.

Cytolytic activity of PASD1-specific CD4 Th cell lines derived from two DLBCL patients 4 and 47. The cytolytic activity of CD4 Th cell lines raised from patient 4 (DRB1*0102,*0301) and patient 47 (DRB1*0301,*0801) against PASD1(6) (A) and PASD1(7) (B) peptides was tested in an 18 h ⁵¹Cr release assay on the PASD1-positive Thiel (DRB1*0401,*1101) and OCI-ly3 (DRB1-0301) cell lines. Significant lysis was observed only in those cultures containing the PASD1-positive Thiel or OCI-Ly3 cells. This was in contrast to the lack of lysis observed of the PASD1-negative SUDHL-6 (DRB1*0101,*0401) cell line (P<0.001). Similarly, significant lysis was also observed for CD4 Th cell lines raised from patient 47 against (C) the PASD1(6) and (D) PASD1(7) peptides. The results are the mean ± SD and were from triplicate cultures.

Figure 4.

Double immunofluorescent labeling of the cultured CD4⁺ T-cell lines. In a) the majority of cultured cells express both CD3 and CD4 antigens (arrow). Heterogeneity was observed in CD4 expression. An example of a CD3⁺ cell CD4⁻ T cell is shown (arrowhead). b) Shows the presence of a small population of CD8⁺ cells (green) c) Co-expression of CD45R0 on the majority of CD4⁺ cells can be observed. An example of a CD4⁺ CD45RO⁻ cell is arrowed. d) Only the occasional cell was CD56⁺ (arrowhead) confirming the presence of only a small number (<1%) of natural killer cells in the cultured cells.