Page 196 - 2021_03-Haematologica-web

R. Li et al.
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C
D
Figure 2. Cross-presentation of Dickkopf-1 (DKK1) -long peptide (LP) efficiently primes DKK1-specific CD8+ T cells in vivo. Shown are CD8+ T-cell responses induced
-LP or HLA-A*0201-restricted P20 short peptide. (A) DKK1-P20-specific tetramer staining showing the frequency of DKK1 P20-specific CD8+ T cells in the spleen of a HLA-A*0201-transgenic mouse. Representative results from one of three mice are shown. (B) DKK1-P66v-specific tetramer staining showing the frequency of DKK1 P66v-specific CD8+ T cells in the spleen of an HLA-A*0201-transgenic mouse. These results
in HLA-A*0201-transgenic mice after immunization with DKK1
3-76
3-76
+
-LP was cross-presented and efficiently primed DKK1-P66v-specific CD8 T cells in HLA-A*0201-transgenic mice. Representative results from
indicate that DKK1
one of three mice are shown. (C) Cytolytic activity of CD8+ T cells isolated from mice immunized with DKK1
LP against unpulsed dendritic cells (DC) or DC pulsed +
with DKK1
3-76
LP or P20 short peptide. Representative results of three experiments are shown. (D) Cytolytic activity of CD8 T cells isolated from mice immunized with
DKK1
3-76
LP against U266 and ARP-1 human myeloma cell lines, and K562. Representative results of three experiments are shown. *P<0.05; **P<0.01.
3-76 3-76
the main IFN-g-secreting cells, because anti-HLA-DR mAb significantly reduced the percentage of IFN-g-secreting CD4+ cells (Figure 5C). Moreover, HLA-A2 antibody sig- nificantly reduced the percentages of IFN-g-secreting CD8+ T cells in cultures after repeated stimulations with DKK13-76-LP. The results showed that DKK13-76-LP-specific CD8+ T-cell responses were HLA*0201-restricted (Figure 5D). Taken together, the results demonstrate that human DKK1-specific CTL and Th1 cells can be induced by DKK13-76-LP ex vivo.
Finally, we determined whether the DKK13-76-LP could induce DKK1-specific HLA-A*0201-restricted and HLA- DR*4-restricted T-cell responses from MM patients. After 3-4 weeks of stimulating patient-derived PBMC with DKK13-76-LP in vitro, the frequency of DKK1-specifc CD8+ CTL and CD4+ Th1 cells was detected by intracellular IFN-g staining. Figure 6A is a representative flow cytome- try analysis showing the percentages of IFN-g-secreting CD8+ and CD4+ T cells after re-stimulation with DKK1 -
patients by in vitro repeated stimulations with DKK1 -LP 3-76
and these DKK1-specific T cells killed autologous patient (MM1) myeloma cells, primary myeloma cells from another HLA-A*0201+ patient (MM2), and HLA-A*0201+ myeloma cell line U266. No killing was observed on HLA- A*0201- primary myeloma cells (MM3) or myeloma cell line ARP-1, normal B cells, or K562 cells (to exclude NK- cell activity) (Figure 6C).
Discussion
In this study, we identified a 74aa DKK13-76-LP thast
contains multiple epitopes for CD4+ and CD8+ T cells and
explored the potential of using this LP for immunotherapy
of human MM. We showed that DKK1-specific CTL,
detected by DKK1 short peptide (P20 and P66v)-HLA-
3-76 LP from a MM patient (MM1). Low percentages of IFN-g-
detected by IFN-g secretion and CSFE-dilution assay, can
secreting CD8+ and CD4+ T cells were observed in T-cell cultures re-stimulated with unpulsed DC or DC pulsed with DKK1-P20 short peptide. Figure 6B shows the per- centages of IFN-g-secreting CD4+ and CD8+ T cells from a total of ten patients with MM (with different MHC back- grounds) after a 4-week in vitro stimulation of blood T cells with DKK13-76-LP-pulsed autologous DC. Furthermore, we generated CD8+ T-cell lines from HLA-A*0201+
be induced by (cross)-presentation of DKK1 -LP in vitro 3-76
and in vivo. We also verified the presence of DKK1-specific Th1 responses in MM patients.
Recent studies evaluating the CTL repertoire of HPV-16 e6 and e7 oncogenic protein showed complete and lasting regression of end-stage cervical cancer patients after melanoma antigen A3 (MAGE-A3) vaccination with pep- tide21,22,29-31 After immunization, a new wave of antigen- specific CTL clones arose in the peripheral blood, provid-
A*0201 tetramer staining, and DKK1-specific Th cells,
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haematologica | 2021; 106(3)