Julia Steitz

Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO

CpG‐rich oligonucleotides (CpG‐ODN) bind to Toll‐like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG‐ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG‐ODN resulted in antigen‐specific T cell activation in local lymph nodes. In contrast, systemic application of CpG‐ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3‐dioxygenase (IDO) as indicated by the observation that CpG‐ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1‐methyl‐tryptophan (1‐MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG‐ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG‐ODN‐induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG‐ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down‐modulation of immune responses by CpG‐ODN may be possible in certain pathological conditions.

Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope

Enhanced green fluorescent protein (EGFP) is a novel marker gene product, which is readily detectable using techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. In the present studies, we have examined the immunogenicity of EGFP in murine models. A stable transfectant of the transplantable CMS4 sarcoma of BALB/c origin expressing EGFP, CMS4-EGFP-Zeo, was generated. Splenocytes harvested from mice immunized with a recombinant adenovirus expressing EGFP (Ad-EGFP) were restimulated in vitro with CMS4-EGFP-Zeo. Effector lymphocytes displayed strong cytotoxicity against CMS4-EGFP-Zeo, but not against mock-transfected CMS4-Zeo tumor cells. A number of candidate H2-Kd-binding peptides derived from the EGFP protein were chosen according to an epitope prediction program and synthesized. These peptides were tested for their ability to bind to H2-Kd molecules and stimulate IFNγ-production by splenocytes harvested from Ad-EGFP-immunized mice. Using this methodology, the peptide, HYLSTQSAL (corresponding to EGFP200–208) which strongly binds to H2-Kd molecules, was identified as a naturally occurring epitope of EGFP. These results should facilitate the use of EGFP as a model tumor antigen in BALB/c mice.

Immune Cell–Poor Melanomas Benefit from PD-1 Blockade after Targeted Type I IFN Activation

UNLABELLED Infiltration of human melanomas with cytotoxic immune cells correlates with spontaneous type I IFN activation and a favorable prognosis. Therapeutic blockade of immune-inhibitory receptors in patients with preexisting lymphocytic infiltrates prolongs survival, but new complementary strategies are needed to activate cellular antitumor immunity in immune cell-poor melanomas. Here, we show that primary melanomas in Hgf-Cdk4(R24C) mice, which imitate human immune cell-poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peritumoral injections of immunostimulatory RNA initiated a cytotoxic inflammatory response in the tumor microenvironment and significantly impaired tumor growth. This critically required the coordinated induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibody-mediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 receptors further prolonged the survival. These results highlight important interconnections between type I IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combination immunotherapies. SIGNIFICANCE Using a genetically engineered mouse melanoma model, we demonstrate that targeted activation of the type I IFN system with immunostimulatory RNA in combination with blockade of immune-inhibitory receptors is a rational strategy to expose immune cell-poor tumors to cellular immune surveillance.

Genetic immunization of mice with human tyrosinase‐related protein 2: Implications for the immunotherapy of melanoma

The melanosomal protein TRP2 expressed by melanocytes and most melanoma cells is an attractive, clinically relevant model antigen for the experimental development of melanoma immunotherapy in mice. A peptide shared by murine and human TRP2 can be recognized by melanoma‐reactive CTL in C57BL/6 mice, as well as in human melanoma patients. Previous experiments demonstrated that gene gun immunization of mice with plasmid DNA encoding autologous murine TRP2 was unable to induce protective immunity against B16 melanoma cells naturally expressing TRP2. In the present study, we investigated whether the use of cDNA encoding xenogeneic human TRP2, which is highly homologous to murine TRP2, would be more effective. Genetic immunization of mice with human TRP2 resulted in coat depigmentation as a sign of autoimmune‐mediated destruction of melanocytes and provided significant protection against metastatic growth of B16 melanoma. Induction of protective immunity was associated with TRP2‐reactive antibodies and CD8+ T cells. Furthermore, immunization with recombinant adenovirus was more effective than immunization with plasmid DNA using the gene gun. Our results provide new insights for the development of antigen‐specific immunotherapy of melanoma. Int. J. Cancer 86:89–94, 2000.

Dendritic cell-based genetic immunization in mice with a recombinant adenovirus encoding murine TRP2 induces effective anti-melanoma immunity.

 The induction of cellular immune responses to melanocyte‐specific enzymes such as the tyrosinase family of proteins is the goal of various clinical studies for the immunotherapy of melanoma. Tyrosinase‐related protein‐2 (TRP2) is an attractive model antigen for preclinical studies in C57BL/6 mice because it is naturally expressed by the murine B16 melanoma and can be recognized by self‐reactive cytolytic T lymphocytes (CTL). Here we describe efforts to develop genetic immunization with dendritic cells (DC) for the immunotherapy of melanoma in this clinically relevant system.

Adenovirus-transduced dendritic cells stimulate cellular immunity to melanoma via a CD4 + T cell-dependent mechanism

We previously showed that genetic immunization of C57BL/6 mice with recombinant adenovirus encoding human TRP2 (Ad-hTRP2) was able to circumvent tolerance and induce cellular and humoral immune responses to murine TRP2 associated with protection against metastatic growth of B16 melanoma. In the present study we compared delivery of Ad-hTRP2 with cultured dendritic cells (DC) and direct injections of Ad-hTRP2. We show that application of Ad-hTRP2 with cultured DC enhanced protective immunity to B16 melanoma cells. Most importantly, delivery of recombinant adenovirus with DC alters the character of the immune response resulting in preferential stimulation of strong cellular immunity in the absence of significant humoral immunity to the encoded antigen. Adoptive transfer of lymphocytes from mice immunized with Ad-hTRP2-transduced DC confirmed that cellular components of the immune response were responsible for rejection of B16 melanoma. The protective efficacy of Ad-hTRP2-transduced DC clearly depended on the presence of CD4+ T helper cells. Furthermore, AD-hTRP2-transduced DC, but not direct injection of Ad-hTRP2, were effective in the presence of neutralizing anti-adenoviral antibodies. These preclinical studies demonstrate the superiority of melanoma vaccines consisting of cultured DC transduced with recombinant adenoviruses encoding melanoma antigens.

Genetic immunization with a melanocytic self-antigen linked to foreign helper sequences breaks tolerance and induces autoimmunity and tumor immunity

Mechanisms maintaining peripheral tolerance to self-antigens present a major obstacle for the development of antigen-specific melanoma vaccines, presumably because self-antigens are not able to stimulate a CD4 T-helper response. Using the melanosomal enzyme tyrosinase-related protein 2 (TRP2) expressed by melanocytes and most melanoma cells as a model self-antigen, we investigated whether linkage with a foreign immunogenic protein providing strong CD4 helper sequences would be able to circumvent tolerance and enhance the induction of antigen-specific tumor immunity. We found that genetic immunization of mice with cDNA encoding a fusion protein between enhanced green fluorescent protein (EGFP) from jellyfish and autologous murine TRP2 (EGFP.mTRP2) resulted in the stimulation of TRP2-reactive T cells in vivo. Importantly, immunization with EGFP.mTRP2 effectively protected mice against metastatic growth of B16 melanoma in the lungs and was associated with fur depigmentation as a sign of autoimmune-mediated destruction of melanocytes. Our results show that tumor vaccines consisting of self-antigens linked to immunogenic helper sequences can be successfully applied to the immunotherapy of melanoma and provide a scientific basis for the translation of this strategy in future clinical investigations.

Absence of CD26 expression on skin-homing CLA+ CD4+ T lymphocytes in peripheral blood is a highly sensitive marker for early diagnosis and therapeutic monitoring of patients with Sézary syndrome.

Patients with Sézary syndrome (SS) show clonal expansion in the peripheral blood of skin‐homing CD4+ T‐helper cells expressing cutaneous lymphocyte antigen (CLA). However, an increase of CLA+ CD4+ T cells can also be observed in various inflammatory dermatoses. To facilitate early diagnosis and therapeutic monitoring of SS using flow cytometry, we evaluated the expression of CD7 and CD26 on the CLA+ CD4+ lymphocyte subset. Peripheral lymphocytes from 7 patients with SS, 16 patients with mycosis fungoides (MF) and 11 healthy controls were analysed by flow cytometry for the expression of CD4, CD7, CD26, CLA and CCR4. In addition, a longitudinal study was performed over 16 months in two patients with SS. Absence of CD7 and CD26 on CLA+ CD4+ T cells was highly specific for SS. Importantly, the absence of CD26 on CLA+ CD4+ T cells was very sensitive for SS, at 100% in our patient cohort. The number of CD26− CLA+ CD4+ T cells closely correlated with therapeutic interventions in the longitudinal analysis of two patients over more than 1 year. We conclude that the absence of CD26 expression on skin‐homing CLA+ CD4+ T‐helper cells is a very sensitive and highly specific parameter for early diagnosis and therapeutic monitoring of patients with SS.

Initiation and regulation of CD8+T cells recognizing melanocytic antigens in the epidermis: implications for the pathophysiology of vitiligo.

Antigen-specific CD8+T lymphocytes play an important role in defense against cutaneous microbial infection and skin cancer as well as in the pathophysiology of autoimmune skin disease such as lupus erythematodes and vitiligo. We have explored the role of CD8+ cytotoxic T lymphocytes (CTL) in an experimental mouse model of vitiligo, a pigmentation disorder characterized by focal loss of melanocytes in the skin. Using genetic immunization techniques we found that pigment cells in the epidermis can be destroyed by CD8+ T cells specifically recognizing a single H2-Kb-binding peptide derived from the model melanocytic self antigen tyrosinase-related protein 2 (TRP2), a melanosomal enzyme involved in pigment synthesis. Experimental evidence suggests that peripheral tolerance of pigment cell-specific cytotoxic CD8+T cells is regulated in two steps. In the induction phase, stimulation and expansion of these T cells in vivo strictly depends on CD4+ T cell help. In the effector phase, autoimmune destruction of melanocytes in the skin depends on local inflammation facilitating the migration of T cells into the epidermis and supporting effector functions. Our results suggest that accidental stimulation of CD8+ CTL recognizing MHC class I-binding peptides derived from melanocytic proteins in the context of an inflammatory skin disease may play an important role in the pathophysiology of vitiligo. Further investigations will address the role of chemokines, chemokine receptors and adhesion molecules in this experimental system and will reveal the role of keratinocytes and Langerhans cells in regulating cutaneous CD8+ T cell responses.

Effective induction of anti-melanoma immunity following genetic vaccination with synthetic mRNA coding for the fusion protein EGFP.TRP2

RNA-based genetic immunization represents an alternative novel strategy for antigen-specific cancer vaccines. In the present paper we investigate the use of synthetic messenger RNA in an experimental melanoma model. We show that gene gun-based immunization using synthetic RNA mediates gene expression in the epidermis and effectively induces antigen-specific cellular and humoral immunity in mice in vivo. Importantly, bombardment of the skin with RNA coding for the melanocytic self-antigen TRP2 linked to the immunogenic protein EGFP was associated with protection against experimentally induced B16 melanoma lung metastases and vitiligo-like fur depigmentation. Our results provide a scientific basis for clinical trials using synthetic mRNA encoding melanocytic antigens linked to immunogenic helper proteins for vaccination of patients with melanoma.