Slingluff, C. L. | bioRxiv Science

bioRxiv2025

Antibody Responses to Melanoma Helper Peptide Vaccines May Enhance Antigen Opsonization Through Formation of Immune Complexes and are Modulated by Vaccine Adjuvants

BackgroundVaccines targeting melanoma antigens can elicit CD8+ T cell responses, but a growing body of work suggests CD4+ T cells also play a role in tumor control. Induction of CD4+ cells may also support B cells in producing tumor antigen-specific antibodies (Abs). We investigated Abs induced by vaccination with a cocktail of six class II MHC-restricted melanoma peptides (6MHP) and the effect of adjuvant type on Ab isotypes. We hypothesized that the vaccines would induce Abs that respond to different epitopes on individual peptides and that IgG subclass distribution varies with different vaccine adjuvants. MethodsSera from patients who received a 6MHP vaccine were evaluated with enzyme-linked immunosorbent assays to map epitopes for polyclonal Ab responses to synthetic melanoma peptides (n=8). IgG subclasses of Ab responses to 6MHP were assessed in patients who received one of 4 adjuvants (Incomplete Freunds Adjuvant (IFA) alone, IFA + polyICLC, IFA + systemic mCy, or IFA + polyICLC + systemic mCy) to characterize IgG subclass distribution (n=2-14). Comparisons were evaluated using a Mann-Whitney rank sum test. ResultsEpitope mapping revealed that at least 50% of patients exhibiting Ab responses to melanoma peptides had responses to two or more epitopes on the same peptide, suggesting polyclonal antibody responses. Serum evaluation for IgG isotypes showed predominant induction of IgG1 and IgG3. Mean total IgG was highest when IFA and polyICLC were used in combination. Patients who received TLR3 agonist polyICLC had significantly higher concentrations of total IgG, IgG1, and IgG3 compared to patients who did not receive polyICLC. ConclusionsThese findings suggest that vaccine-induced Abs may respond to multiple epitopes within the same peptide, which may support creation of large antigen-Ab complexes, with promise to facilitate antigen uptake and presentation. Abs were predominantly IgG1 and IgG3, which are optimal for binding complement and supporting Ab-dependent cellular cytotoxicity. The findings also show that adding polyICLC to IFA can significantly enhance Ab responses. Collectively, this work underscores the immunologic potential of peptide-induced Abs and the importance of adjuvant selection in cancer vaccine design.

cancer biology↗

bioRxiv2024

Spatial analysis reveals combinative role for natural killer and CD8 T cells in antitumor immunity despite profound MHC class I loss in non-small cell lung cancer

BackgroundMHC class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. MethodsWe used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFN{gamma}-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis ResultsSpatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (DFS) (HR=0.58, p=0.064) and over-all survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1x10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFN{gamma}+/- NK cells and T cells. We discovered that both IFN{gamma}+ NK and CD8 T cells were more frequently associated with other IFN{gamma}+ lymphocytes in comparison to IFN{gamma}- NK cells and CD8 T cells (p<1x10-30). Moreover, IFN{gamma}+ lymphocytes were most often found clustered near MHC-I+ tumor cells. ConclusionsTumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Co-association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFN{gamma}. Frequent co-localization of IFN{gamma}+ NK cells with other IFN{gamma}+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated. What is already known on this topicMHC-I loss occurs frequently in NSCLC and corresponds with waning immunity in the tumor microenvironment (TME). NK cells recognize "missing-self" targets and could be leveraged to target NSCLC tumors with MHC-I loss. While NK cell presence at tumor margins has been documented in NSCLC, they were shown to lose function in this environment. What this study addsWe developed spatial analysis pipelines leveraging the local heterogeneity of the TME at single cell resolution to test whether NK cells and T cells together contribute antitumoral immunity in NSCLC. We discovered that a high density of tumor-infiltrating NK cells corresponded with DFS, and this association was increased in patients with high coincident CD8 T cells, especially those in central tumor. Intriguingly, both cell types were found clustered together in MHC-I-bearing tumors, especially when both expressed IFN{gamma}, suggesting coordinated lymphocyte activities may enhance immune control of NSCLC. How this study might affect research, practice, or policyThis study provides a rationale for developing novel immunotherapies that simultaneously increase NK and T cell anti-tumoral immunity. Associations linking NK cells with patient survival and increased immune effector activity in NSCLC, even in MHC-I-deficient tumors, further highlights the need to devise and deploy NK cell activating strategies which may be highly efficacious in CD8 T cell refractory NSCLC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/581048v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1e383c0org.highwire.dtl.DTLVardef@1dee268org.highwire.dtl.DTLVardef@1e21bd4org.highwire.dtl.DTLVardef@1910a9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Phospho-β-catenin expression in primary and metastatic melanomas and in tumor-free visceral tissues

{beta}-catenin ({beta}cat) is an important downstream effector in the Wnt signaling pathway and plays an important role in the development and progression of many cancers including melanoma. {beta}cat expression is regulated by GSK-3{beta}-mediated phosphorylation at positions 33, 37 and 41. In normal cells, phosphorylation at these sites triggers proteasomal degradation, which in turn prevents accumulation of free cytoplasmic {beta}cat. In cancer cells, stabilized {beta}-catenin translocates into the nucleus, where it associates with TCF/Lef proteins to activate transcription of genes that promote tumorigenesis and metastasis. It has been suggested that nuclear phospho-{beta}cat (p{beta}cat) staining may be diagnostically useful in differentiating primary from metastatic melanoma. Also, a p{beta}cat peptide (residues 30-39, p33) is naturally presented by melanoma cells as a T-cell target. We evaluated the expression of pS33-{beta}cat in primary and metastatic melanoma tissues by immunohistochemistry. pS33-{beta}cat was detected in primary and metastatic melanomas and was most commonly cytoplasmic and almost never exclusively nuclear. Interestingly, staining with pS33-{beta}cat and pS33/37/T41-{beta}cat antibodies was most intense in mitotic melanoma cells, consistent with prior studies demonstrating changes in the level of {beta}cat during cell division. We observed no significant differences in p{beta}cat staining location or intensity between primary and metastatic melanomas, suggesting that p{beta}cat may have limited diagnostic or prognostic utility in melanoma. However, the high expression in dividing cells suggests promise as an immunotherapeutic target.

pathology↗