Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

Impaired tumor rejection by memory CD8 T cells in mice with NKG2D dysfunction.

Cytotoxic T cells are important effectors for robust antitumor immune responses. However, tumor-infiltrating CD8 T cells are often functionally impaired. Insufficient antitumor activity of CD8 T cells can be due to a lack of costimulatory signals. NKG2D is such a costimulatory receptor on CD8 T cells that facilitates immunorecognition of stressed and malignant cells, promotes tumor rejection by NK and CD8 T cells and contributes to immunosurveillance of spontaneous malignancies. Previous reports suggested an involvement of NKG2D in establishing CD8 T cell-mediated antitumor memory. However, the significance of NKG2D for the generation and effector phase of memory CD8 T cell responses is largely unknown. To address these issues, we made use of a transgenic mouse model (H2-K(b)-MICA mice) where the human NKG2D ligand MICA is ubiquitously and constitutively expressed resulting in a severe dysfunction of NKG2D. Both, ovalbumin (OVA)-specific (H2-K(b)/OVA(257-264)) memory CD8 T cells arisen from the endogenous T cell pool and adoptively transferred OVA-specific OT-I memory cells were unable to control growth of an OVA-expressing lymphoma in H2-K(b)-MICA mice. While expansion of memory T cells in these mice on antigen challenge was not different from controls, CD8 memory T cells of H2-K(b)-MICA mice did not effectively eliminate tumor cells in vivo. Altogether, our data suggest that NKG2D has no major role in the generation and expansion of memory CD8 T cells, but rather substantially enhances the cytolytic effector responses of reactivated memory T cells and thereby contributes to an efficacious tumor rejection.

Interaction of C-type lectin-like receptors NKp65 and KACL facilitates dedicated immune recognition of human keratinocytes.

Many well-known immune-related C-type lectin-like receptors (CTLRs) such as NKG2D, CD69, and the Ly49 receptors are encoded in the natural killer gene complex (NKC). Recently, we characterized the orphan NKC gene CLEC2A encoding for KACL, a further member of the human CLEC2 family of CTLRs. In contrast to the other CLEC2 family members AICL, CD69, and LLT1, KACL expression is mostly restricted to skin. Here we show that KACL is a non-disulfide-linked homodimeric surface receptor and stimulates cytotoxicity by human NK92MI cells. We identified the corresponding activating receptor on NK92MI cells that is encoded adjacently to the CLEC2A locus and binds KACL with high affinity. This CTLR, termed NKp65, stimulates NK cytotoxicity and release of proinflammatory cytokines upon engagement of cell-bound KACL. NKp65, a distant relative of the human activating NK receptor NKp80, possesses an amino-terminal hemITAM that is required for NKp65-mediated cytotoxicity. Finally, we show that KACL expression is mainly restricted to keratinocytes. Freshly isolated keratinocytes express KACL and are capable of stimulating NKp65-expressing cells in a KACL-dependent manner. Thus, we report a unique NKC-encoded receptor-ligand system that may fulfill a dedicated function in the immunobiology of human skin.

NKp80 defines and stimulates a reactive subset of CD8 T cells.

NKp80, an activating homodimeric C-type lectin-like receptor (CTLR), is expressed on essentially all human natural killer (NK) cells and stimulates their cytotoxicity and cytokine release. Recently, we demonstrated that the ligand for NKp80 is the myeloid-specific CTLR activation-induced C-type lectin (AICL), which is encoded in the natural killer gene complex (NKC) adjacent to NKp80. Here, we show that NKp80 also is expressed on a minor fraction of human CD8 T cells that exhibit a high responsiveness and an effector memory phenotype. Gene expression profiling and flow cytometric analyses revealed that this NKp80(+) T-cell subset is characterized by the coexpression of other NK receptors and increased levels of cytotoxic effector molecules and adhesion molecules mediating access to sites of inflammation. NKp80 ligation augmented CD3-stimulated degranulation and interferon (IFN)gamma secretion by effector memory T cells. Furthermore, engagement of NKp80 by AICL-expressing transfectants or macrophages markedly enhanced CD8 T-cell responses in alloreactive settings. Collectively, our data demonstrate that NKp80 is expressed on a highly responsive subset of effector memory CD8 T cells with an inflammatory NK-like phenotype and promotes T-cell responses toward AICL-expressing cells. Hence, NKp80 may enable effector memory CD8 T cells to interact functionally with cells of myeloid origin at sites of inflammation.

Mutual activation of natural killer cells and monocytes mediated by NKp80-AICL interaction.

Receptors encoded by the natural killer (NK) cell gene complex (such as NKG2D) govern the reactivity of NK cells. However, the function and ligand(s) of the NK cell gene complex-encoded human NK cell receptor NKp80 remain elusive. Here we demonstrate that NKp80 binds to the genetically linked 'orphan' receptor AICL, which, like NKp80, is absent from rodents. We defined AICL as a myeloid-specific activating receptor that is upregulated by Toll-like receptor stimulation. AICL-NKp80 interactions promoted NK cell-mediated cytolysis of malignant myeloid cells. In addition, during crosstalk between NK cells and monocytes, NKp80 stimulated the release of proinflammatory cytokines from both cell types. Thus, by specifically bridging NK cells and myeloid cells, NKp80-AICL interactions may contribute to the initiation and maintenance of immune responses at sites of inflammation.