Intralymphatic dendritic cell vaccination induces tumor antigen-specific, skin-homing T lymphocytes

Abstract

Purpose: The identification of tumor antigens recognized by cytotoxic and T helper lymphocytes has led to the development of specific cancer vaccines. Immunization with tumor antigen-pulsed dendritic cells has proved effective at eliciting elevated levels of tumor antigen-specific T cells in patient blood, but objective clinical responses remain rare, suggesting that vaccine-induced T cells are not trafficking optimally to site(s) of tumor burden. Accumulating evidence from animal models suggests that route of immunization can have a substantial influence on the subsequent migration of primed, activated T cells in vivo.

Experimental design: In a clinical trial designed to elicit more effective cytotoxic T-cell mediated antitumor responses, metastatic melanoma patients were immunized directly via a peripheral intralymphatic route with autologous dendritic cells pulsed with HLA-A*0201-restricted melanoma-associated peptide antigens derived from MART-1 and gp100.

Results: Within 10 days of intralymphatic dendritic cell vaccination, four of six patients developed dramatic and diffuse erythematous rashes in sun-exposed areas of skin that showed extensive T-cell infiltration. CTLs grown from rash biopsies were strongly enriched for tumor antigen-specific T cells that had elevated expression of cutaneous lymphocyte antigen and chemokine receptor-6, consistent with a skin-homing phenotype. Of note, the only patient in the study with cutaneously localized disease showed a significant regression of metastatic lesions following the development of a surrounding rash.

Conclusions: The evidence presented here is consistent with immunization studies in animal models and supports the concept that T cells are "imprinted" in peripheral lymph node sites to express specific ligands and chemokine receptors that allow them to migrate to skin. Furthermore, the preferential migration of the T cells to sun-exposed cutaneous sites suggests that inflammation plays a critical role in this migration. These observations suggest that further study of the effects of immunization route and inflammation on T-cell migration in humans is warranted, and could lead to vaccination approaches that would more reliably direct trafficking of activated T cells to diverse sites of metastatic disease.

Figures

Fig. 1

Development of rash in patients following intralymphatic dendritic cell infusion. HLA-A*0201-positive stage IV melanoma patients were infused intralymphatically with autologous, CD40 ligand – activated, monocyte-derived dendritic cells pulsed with the melanoma-associated peptides MART-126-35(27L) and gp100209-217(210M). A and B, the majority of treated patients developed a diffuse, erythematous rash in sun-exposed areas of skin 7 to 10 days following dendritic cell infusion. Representative photographs of patients 1 and 2.

Fig. 2

Skin rash biopsies contain extensive T-cell infiltrates. Histologic analysis of two representative patient biopsies of noninvolved (A–D) and rash-involved (E–H) skin. A, B, E, and F, H&E staining of skin tissue reveals dramatically increased lymphocytic infiltrates in rash sections as compared with normal skin. C, D, G, and H, immunohistochemical staining with anti-CD3 antibody shows that T cells constitute the majority of infiltrating lymphocytes in rash sections.

Fig. 3

Rash-derived CD8+ T cells recognize immunizing tumor antigens with high avidity. A, T cells cultured from skin rash biopsies of patients 1 and 2 contained 93% and 98% CD8+ cells, respectively, after 14 days of expansion in 6,000 IU/mL IL-2. B, rash-derived T cells show immune reactivity against HLA-A2-restricted melanoma tumor antigens MART-126-35(27L), gp100209-217(210M), and HLA-matched allogeneic melanoma lines, as measured by IFN-γ release. C, cultured T cells were incubated overnight with T2 cells pulsed with titrated doses of gp100209-217(210M) peptide and IFN-γ production was measured by ELISA on cultured supernatants. Peptide doses as low as 100 pmol/L were recognized by CD8+ T cells derived from both patients. These experiments were done twice with similar results.

Fig. 4

Tumor antigen – specific T cells show preferential migration to inflamed skin. HLA-A*0201 tetramer analysis of pretreatment PBMC, patient PBMC collected 10 to 12 days post-dendritic cell infusion, and skin rash – derived T cells cultured in 6,000 IU/mL IL-2. Pretreatment and posttreatment PBMC from patient 1 (A) and patient 2 (B) show very low frequencies of CD8+ T cells that stain specifically with HIV-1gag77-85 (negative control), gp100209-217(210M), or MART-126-35(27L)/HLA-A*0201 fluorescently conjugated tetramers. In contrast, cultured T cells derived from rash skin biopsies contained significantly higher percentages of T cells specific for the immunizing gp100 and/or MART-1 peptides. These experiments were done twice with representative data shown.

Fig. 5

Rash-derived T cells express the skin-homing markers chemokine receptor-6 and cutaneous lymphocyte antigen. A, flow cytometric analysis of rash-derived T cells cultured in 6,000 IU/mL IL-2 from patients 1 and 2 stained with monoclonal antibodies against skin-homing markers chemokine receptor-6 (CCR6) and cutaneous lymphocyte antigen (CLA). B, PBMC from patient 1 were stimulated in vitro with MART-1 peptide – pulsed dendritic cells for 21 days before staining for chemokine receptor-6 and cutaneous lymphocyte antigen and analyzing by flow cytometry. In contrast to antigen-specific rash-derived T cells generated in vivo by dendritic cell vaccination, in vitro dendritic cell – stimulated CD8+ T cells failed to show significant up-regulation of skin-homing markers. These experiments were done twice with representative results shown.

Fig. 6

E-selectinis up-regulated on rash endothelium and mediates adhesion to cutaneous lymphocyte antigen on IL-2-cultured, rash-derived T cells. A and B, immunohistochemical analysis of a representative patient biopsy showing E-selectin expression in noninvolved (A) and rash-involved (B) skin. Arrows in B indicate E-selectin expression. C to E, simulating the physiologic shear flow stresses present in the microvascular environment, a laminar flow of suspended, rash-derived T cells from patient 2 was passed through a flow chamber coated with bovine serum albumin control protein (C) or recombinant E-selectin/immunoglobulin chimeric protein (Glycotech; D). Cell tethering and adhesion were observed under an inverted phase-contrast microscope. E, numbers of T cells bound within 7 to 11 minutes of flow with either bovine serum albumin or E-selectin/immunoglobulin chimeric protein coated on the flow chamber. EDTA, which interferes with E-selectin/cutaneous lymphocyte antigen binding, was used as a negative control in this experiment. Representative data from three experiments.

Fig. 7

Regression of cutaneous lesion following development of rash. A to C, photographs document the regression of a cutaneous metastatic melanoma nodule in patient 2 following intralymphatic infusion of peptide-pulsed dendritic cells. By day 12 posttreatment, a rash developed surrounding the cutaneous tumor nodule, which showed a complete regression over the following 3 weeks.