Associations of immune cell homing gene signatures and infiltrates of lymphocyte subsets in human melanomas: discordance with CD163+ myeloid cell infiltrates.

BACKGROUND: Immune cells in the tumor microenvironment have prognostic value. In preclinical models, recruitment and infiltration of these cells depends on immune cell homing (ICH) genes such as chemokines, cell adhesion molecules, and integrins. We hypothesized ICH ligands CXCL9-11 and CCL2-5 would be associated with intratumoral T-cells, while CXCL13 would be more associated with B-cell infiltrates. METHODS: Samples of human melanoma were submitted for gene expression analysis and immune cells identified by immunohistochemistry. Associations between the two were evaluated with unsupervised hierarchical clustering using correlation matrices from Spearman rank tests. Univariate analysis performed Mann-Whitney tests. RESULTS: For 119 melanoma specimens, analysis of 78 ICH genes revealed association among genes with nonspecific increase of multiple immune cell subsets: CD45+, CD8+ and CD4+ T-cells, CD20+ B-cells, CD138+ plasma cells, and CD56+ NK-cells. ICH genes most associated with these infiltrates included ITGB2, ITGAL, CCL19, CXCL13, plus receptor/ligand pairs CXCL9 and CXCL10 with CXCR3; CCL4 and CCL5 with CCR5. This top ICH gene expression signature was also associated with genes representing immune-activation and effector function. In contrast, CD163+ M2-macrophages was weakly associated with a different ICH gene signature. CONCLUSION: These data do not support our hypothesis that each immune cell subset is uniquely associated with specific ICH genes. Instead, a larger set of ICH genes identifies melanomas with concordant infiltration of B-cell and T-cell lineages, while CD163+ M2-macrophage infiltration suggesting alternate mechanisms for their recruitment. Future studies should explore the extent ICH gene signature contributes to tertiary lymphoid structures or cross-talk between homing pathways.

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of 'transformed self'. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.

Immune Cell Infiltration and Tertiary Lymphoid Structures as Determinants of Antitumor Immunity.

Limited representation of intratumoral immune cells is a major barrier to tumor control. However, simply enhancing immune responses in tumor-draining lymph nodes or through adoptive transfer may not overcome the limited ability of tumor vasculature to support effector infiltration. An alternative is to promote a sustained immune response intratumorally. This idea has gained traction with the observation that many tumors are associated with tertiary lymphoid structures (TLS), which organizationally resemble lymph nodes. These peri- and intratumoral structures are usually, but not always, associated with positive prognoses in patients. Preclinical and clinical data support a role for TLS in modulating immunity in the tumor microenvironment. However, there appear to be varied functions of TLS, potentially based on their structure or location in relation to the tumor or the origin or location of the tumor itself. Understanding more about TLS development, composition, and function may offer new therapeutic opportunities to modulate antitumor immunity.

The Barrier Molecules Junction Plakoglobin, Filaggrin, and Dystonin Play Roles in Melanoma Growth and Angiogenesis.

OBJECTIVE: To understand role of barrier molecules in melanomas. BACKGROUND: We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. METHODS: FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. RESULTS: Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. CONCLUSIONS: FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.

Patterns of immune-cell infiltration in murine models of melanoma: roles of antigen and tissue site in creating inflamed tumors.

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/ß-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/ß-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.

Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma.

The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.

Peripheral tissue homing receptors enable T cell entry into lymph nodes and affect the anatomical distribution of memory cells.

Peripheral tissue homing receptors enable T cells to access inflamed nonlymphoid tissues. In this study, we show that two such molecules, E-selectin ligand and α4ß1 integrin, enable activated and memory T cells to enter lymph nodes (LN) as well. This affects the quantitative and qualitative distribution of these cells among regional LN beds. CD8 memory T cells in LN that express these molecules were mostly CD62L(lo) and would normally be classified as effector memory cells. However, similar to central memory cells, they expanded upon Ag re-encounter. This led to differences in the magnitude of the recall response that depended on the route of immunization. These novel cells share properties of both central and effector memory cells and reside in LN based on previously undescribed mechanisms of entry.

Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells.

Lymphatic endothelial cells (LECs) induce peripheral tolerance by direct presentation to CD8 T cells (T(CD8)). We demonstrate that LECs mediate deletion only via programmed cell death-1 (PD-1) ligand 1, despite expressing ligands for the CD160, B- and T-lymphocyte attenuator, and lymphocyte activation gene-3 inhibitory pathways. LECs induce activation and proliferation of T(CD8), but lack of costimulation through 4-1BB leads to rapid high-level expression of PD-1, which in turn inhibits up-regulation of the high-affinity IL-2 receptor that is necessary for T(CD8) survival. Rescue of tyrosinase-specific T(CD8) by interference with PD-1 or provision of costimulation results in autoimmune vitiligo, demonstrating that LECs are significant, albeit suboptimal, antigen-presenting cells. Because LECs express numerous peripheral tissue antigens, lack of costimulation coupled to rapid high-level up-regulation of inhibitory receptors may be generally important in systemic peripheral tolerance.

Tertiary lymphoid structures in desmoplastic melanoma have increased lymphocyte density, lymphocyte proliferation, and immune cross talk with tumor when compared to non-desmoplastic melanomas.

Tertiary lymphoid structures (TLS) are ectopic lymphoid structures that can arise in human cancers and are associated with improved overall survival (OS) and response to immune checkpoint blockade (ICB) in several cancers, including non-desmoplastic metastatic melanoma (NDMM). Desmoplastic melanoma (DM) has one of the highest response rates to ICB, and we previously identified that primary DM (PDM) contains TLS. Despite the association of TLS with survival and ICB response, it is unknown whether TLS or associated markers of immune activity can differ between PDM and NDMM. We hypothesized that PDM would contain higher frequencies of TLS than NDMM, that T and B-cell densities and proliferation would be greater in TLS of PDM than TLS of NDMM, and that proliferation rates of T and B-cells in PDM TLS would be concordant with those of intratumoral lymphocytes. We found that four features of TLS in PDM distinguish them from TLS in NDMM. TLS were peritumoral in NDMM but intratumoral in PDM. CD8+ T-cell and CD20+ B-cell densities and proliferative fractions were higher in PDM TLS than NDMM TLS. Additionally, the proliferative fractions of T- and B-cells were concordant between the TLS and tumor site in PDM and discordant in NDMM. Collectively, these data suggest that TLS and associated immune markers can differ across melanoma subsets and suggest that PDM TLS may be more immunologically active and have enhanced immune cell trafficking between tumor and TLS compared to NDMM.

Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1d-dependent NKT cell activation.

Ischemia reperfusion injury results from tissue damage during ischemia and ongoing inflammation and injury during reperfusion. Liver reperfusion injury is reduced by lymphocyte depletion or activation of adenosine A2A receptors (A2ARs) with the selective agonist 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]- prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e). We show that NKT cells are stimulated to produce interferon (IFN)-gamma by 2 h after the initiation of reperfusion, and the use of antibodies to deplete NK1.1-positive cells (NK and NKT) or to block CD1d-mediated glycolipid presentation to NKT cells replicates, but is not additive to, the protection afforded by ATL146e, as assessed by serum alanine aminotransferase elevation, histological necrosis, neutrophil accumulation, and serum IFN-gamma elevation. Reduced reperfusion injury observed in RAG-1 knockout (KO) mice is restored to the wild-type (WT) level by adoptive transfer of NKT cells purified from WT or A2AR KO mice but not IFN-gamma KO mice. Additionally, animals with transferred A2AR-/- NKT cells are not protected from hepatic reperfusion injury by ATL146e. In vitro, ATL146e potently inhibits both anti-CD3 and alpha-galactosylceramide-triggered production of IFN-gamma by NKT cells. These findings suggest that hepatic reperfusion injury is initiated by the CD1d-dependent activation of NKT cells, and the activation of these cells is inhibited by A2AR activation.

CD8 T cells activated in distinct lymphoid organs differentially express adhesion proteins and coexpress multiple chemokine receptors.

Previous work from this laboratory showed that generation of memory CD8 T cells by different immunization routes correlates with control of tumors growing in distinct sites. We hypothesized that effector CD8 T cell expression of adhesion proteins and chemokine receptors would be influenced by activation in different secondary lymphoid organs. In this report, CD8 T cells were activated by immunization with bone marrow-derived dendritic cells via i.p., i.v., or s.c. routes. Three distinct populations of activated CD8 T cells arise in mesenteric, axillary/brachial, and mediastinal lymph nodes and spleen based on differential expression of alpha4beta7 integrin, E-selectin ligand, and alpha4beta1 integrin, respectively. In contrast, three subsets of CD8 T cells defined by differential expression of P-selectin ligand and chemokine receptors were induced irrespective of activation site. The majority of activated CD8 T cells expressed CXCR3, with one subset additionally expressing P-selectin ligand, and another subset additionally expressing CCR3, CCR4, CCR5, CCR6, and CCR9. In the mesenteric lymph node, a fourth subset expressed CCR9 and CXCR3 in the absence of CCR5. Similar homing receptor profiles were induced in the same sites after localized vaccinia immunization. Homing receptor expression on CD8 T cells activated in vitro was distinct, revealing influences of both dendritic cells and the lymphoid microenvironment. Collectively, these results identify previously undescribed populations of activated CD8 T cells based on adhesion protein expression and coexpression of chemokine receptors that arise after activation in distinct secondary lymphoid organs.

Mechanisms of spatial and temporal development of autoimmune vitiligo in tyrosinase-specific TCR transgenic mice.

Generalized vitiligo is thought to have an autoimmune etiology and has been correlated with the presence of CD8 T cells specific for melanocyte differentiation Ag. However, limited animal models for the disease have hampered its understanding. Thus, we generated TCR transgenic mice that recognize an epitope of the melanocyte protein, tyrosinase. These animals develop vitiligo with strikingly similar characteristics to the human disease. Vitiligo develops temporally and spatially, with juvenile lesions forming bilaterally in head and facial areas, and only arising later in the body of adult animals. Vitiligo is entirely dependent on CD8 T cells, whereas CD4 T cells exert a negative regulatory effect. Importantly, CD8 T cells can be pervasively present in the skin in the steady state without inducing vitiligo in most areas. This points to developmental differences in melanocyte susceptibility and/or immunological effector mechanisms over time, or in different body locations. Disease is strongly dependent on both IFN-gamma and CXCR3, whereas dependence on CCR5 is more limited, and both CCR4 and perforin are dispensable. Genetic ablation of CXCR3 or IFN-gamma also resulted in scarce CD8 T cell infiltration into the skin. Our results identify unexpected complexity in vitiligo development and point toward possible therapeutic interventions.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition.

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1(H) versus HA-1(R)) in the HMHA1 gene. The HA-1(H) peptide is restricted by HLA-A2 and is immunogenic in HA-1(R/R) into HA-1(H) transplants, while HA-1(R) has been suggested to be a "null allele" in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1(R) variant. To understand these findings, we determined the structure of an HLA-A2-HA-1(H) complex to 1.3A resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1(H)-specific T-cells bound HA-1(H) peptide with moderate affinity but failed to bind HA-1(R), indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy.

The activation and recruitment of CD4(+) T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4(+) T cells, we have investigated phosphopeptides, a new category of tumor-derived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4(+) T cell recognition. We first demonstrated the fine specificity of human CD4(+) T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome "fingerprints." We then generated HLA-DRbeta1*0101-restricted CD4(+) T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4(+) T cells provides potential new targets for cancer immunotherapy.

Strategies and challenges in eliciting immunity to melanoma.

The ability of CD8+ T cells to recognize melanoma tumors has led to the development of immunotherapeutic approaches that use the antigens CD8+ T cells recognize. However, clinical response rates have been disappointing. Here we summarize our work to understand the mechanisms of self-tolerance that limit responses to currently utilized antigens and our approach to identify new antigens directly tied to malignancy. We also explore several aspects of the anti-tumor immune response induced by peptide-pulsed dendritic cells (DCs). DCs differentially augment the avidity of recall T cells specific for self-antigens and overcome a process of aberrant CD8+ T-cell differentiation that occurs in tumor-draining lymph nodes. DC migration is constrained by injection route, resulting in immune responses in localized lymphoid tissue, and differential control of tumors depending on their location in the body. We demonstrate that CD8+ T-cell differentiation in different lymphoid compartments alters the expression of homing receptor molecules and leads to the presence of systemic central memory cells. Our studies highlight several issues that must be addressed to improve the efficacy of tumor immunotherapy.

Tumor necrosis factor receptor regulation of peripheral node addressin biosynthetic components in tumor endothelial cells.

Tumor-associated tertiary lymphoid structures are ectopic lymphoid aggregates that have considerable morphological, cellular, and molecular similarity to secondary lymphoid organs, particularly lymph nodes. Tumor vessels expressing peripheral node addressin (PNAd) are hallmark features of these structures. Previous work from our laboratory demonstrated that PNAd is displayed on intratumoral vasculature of murine tumors, and its expression is controlled by the engagement of lymphotoxin-α3, secreted by effector CD8 T cells, with tumor necrosis factor receptors (TNFR) on tumor endothelial cells (TEC). The goals of the present work were: 1) to identify differences in expression of genes encoding the scaffolding proteins and glycosyl transferases associated with PNAd biosynthesis in TEC and lymph node blood endothelial cells (LN BEC); and 2) to determine which of these PNAd associated components are regulated by TNFR signaling. We found that the same genes encoding scaffolding proteins and glycosyl transferases were upregulated in PNAd+ LN BEC and PNAd+ TEC relative to their PNAdneg counterparts. The lower level of PNAd expression on TEC vs LN BEC was associated with relatively lower expression of these genes, particularly the carbohydrate sulfotransferase Chst4. Loss of PNAd on TEC in the absence of TNFR signaling was associated with lack of upregulation of these same genes. A small subset of PNAd+ TEC remaining in the absence of TNFR signaling showed normal upregulation of a subset of these genes, but reduced upregulation of genes encoding the scaffolding proteins podocalyxin and nepmucin, and carbohydrate sulfotransferase Chst2. Lastly, we found that checkpoint immunotherapy augmented both the fraction of TEC expressing PNAd and their surface level of this ligand. This work points to strong similarities in the regulation of PNAd expression on TEC by TNFR signaling and on LN BEC by lymphotoxin-ß receptor signaling, and provides a platform for the development of novel strategies that manipulate PNAd expression on tumor vasculature as an element of cancer immunotherapy.

NK cells reduce anergic T cell development in early-stage tumors by promoting myeloid cell maturation.

Introduction: Studies of NK cells in tumors have primarily focused on their direct actions towards tumor cells. We evaluated the impact of NK cells on expression of homing receptor ligands on tumor vasculature, intratumoral T cell number and function, and T cell activation in tumor draining lymph node. Methods: Using an implantable mouse model of melanoma, T cell responses and homing receptor ligand expression on the vasculature were evaluated with and without NK cells present during the early stages of the tumor response by flow cytometry. Results: NK cells in early-stage tumors are one source of IFNγ that augments homing receptor ligand expression. More significantly, NK cell depletion resulted in increased numbers of intratumoral T cells with an anergic phenotype. Anergic T cell development in tumor draining lymph node was associated with increased T-cell receptor signaling but decreased proliferation and effector cell activity, and an incomplete maturation phenotype of antigen presenting cells. These effects of NK depletion were similar to those of blocking CD40L stimulation. Discussion: We conclude that an important function of NK cells is to drive proper APC maturation via CD40L during responses to early-stage tumors, reducing development of anergic T cells. The reduced development of anergic T cells resulting in improved tumor control and T cell responses when NK cells were present.

An activation to memory differentiation trajectory of tumor-infiltrating lymphocytes informs metastatic melanoma outcomes.

There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, we applied advanced functional genomics to interrogate 9,000 human tumors and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.

N-glycosylation enhances presentation of a MHC class I-restricted epitope from tyrosinase.

We recently demonstrated that the mechanism of processing of an HLA-A*0201-restricted peptide epitope, Tyr(369)(D), derived from the membrane protein tyrosinase, involves retrotranslocation of glycosylated molecules from the endoplasmic reticulum to the cytosol, removal of an N-linked carbohydrate from Asn(371) by peptide N-glycanase, proteolysis by the proteasome and other proteases, and retransport of the resulting peptides into the endoplasmic reticulum for association with HLA-A*0201. Carbohydrate removal results in deamidation of Asn(371) to aspartic acid. The asparagine-containing homolog of this peptide, Tyr(369)(N), is not presented by tyrosinase-expressing cells, and this has been presumed to be due to quantitative glycosylation of Asn(371). Although examining cytosolic intermediates that accumulated in human melanoma cells treated with proteasome inhibitors, we were surprised to find both molecules that had been deglycosylated by peptide N-glycanase and a large number of molecules that had not been previously glycosylated. The failure of Tyr(369)(N) to be processed and presented from these latter molecules may be partially due to a process of deamidation independent of glycosylation. However, we also established that proteasomes degrade tyrosinase molecules that are still glycosylated, giving rise to a set of discrete intermediates that are not observed when unglycosylated molecules are degraded. We propose that Tyr(369)(N) fails to be presented because unglycosylated tyrosinase is degraded rapidly and relatively nonselectively. In contrast, glycosylation alters the selectivity of tyrosinase processing by the proteasome, enhancing the production or survival of Tyr(369)(D).

Targeting allergen to FcgammaRI reveals a novel T(H)2 regulatory pathway linked to thymic stromal lymphopoietin receptor.

BACKGROUND: The molecule H22-Fel d 1, which targets cat allergen to FcgammaRI on dendritic cells (DCs), has the potential to treat cat allergy because of its T-cell modulatory properties. OBJECTIVE: We sought to investigate whether the T-cell response induced by H22-Fel d 1 is altered in the presence of the T(H)2-promoting cytokine thymic stromal lymphopoietin (TSLP). METHODS: Studies were performed in subjects with cat allergy with and without atopic dermatitis. Monocyte-derived DCs were primed with H22-Fel d 1 in the presence or absence of TSLP, and the resulting T-cell cytokine repertoire was analyzed by flow cytometry. The capacity for H22-Fel d 1 to modulate TSLP receptor expression on DCs was examined by flow cytometry in the presence or absence of inhibitors of Fc receptor signaling molecules. RESULTS: Surprisingly, TSLP alone was a weak inducer of T(H)2 responses irrespective of atopic status; however, DCs coprimed with TSLP and H22-Fel d 1 selectively and synergistically amplified T(H)2 responses in highly atopic subjects. This effect was OX40 ligand independent, pointing to an unconventional TSLP-mediated pathway. Expression of TSLP receptor was upregulated on atopic DCs primed with H22-Fel d 1 through a pathway regulated by FcgammaRI-associated signaling components, including src-related tyrosine kinases and Syk, as well as the downstream molecule phosphoinositide 3-kinase. Inhibition of TSLP receptor upregulation triggered by H22-Fel d 1 blocked TSLP-mediated T(H)2 responses. CONCLUSION: Discovery of a novel T(H)2 regulatory pathway linking FcgammaRI signaling to TSLP receptor upregulation and consequent TSLP-mediated effects questions the validity of receptor-targeted allergen vaccines.