Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunoth

Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

Main

Results

Volumetric growth curves, total itIC counts, percent itIC subpopulations (CD3+ T cells, DCs, NK cells, macrophages (MΦ) and MDSC-like (MDSC) cells) and intratumoral CD3+CD4+ (CD4+) and CD3+CD8+ (CD8+) T cell counts of SW1 tumors (a–d, respectively) or of empty vector (EV)- or mFuk-expressing SW1 tumors (e–h, respectively) in C3H/HeN mice. Red triangle, initiated L-fuc (LF) supplementation. The growth curves show mean ± standard error of the mean (s.e.m.) from groups of mice as follows: n = 11 control and n = 10 L-fuc-fed mice (a), n = 4 mice per group (b,d), n = 7 mice per group (e), n = 3 mice per group (f,h)(b,d,f,h show mean ± s.e.m.). i, Association of melanoma-specific fucosylation and CD3+ T cell density (log2 scale) in a 40-patient melanoma tissue microarray (med = median fucosylation signal per melanoma cell). j, Box plots showing lower melanoma-specific fucosylation in male (n = 22) versus female (n = 18) patients. The minima and maxima represent the minimum and maximum tumor fucosylation values while the centra represent median values. P values shown are two-sided P values derived from the Spearman correlation test. k, Scatterplots show higher correlation (cor) between melanoma-specific fucosylation and CD3+ T cell density (log2 scale) in male (Spearman’s ρ = 0.43; P = 0.036) versus female (Spearman’s ρ = 0.25; P = 0.3367) patients. The gray bands highlight 95% confidence bands for the prediction line (based on linear regression). Volumetric growth curves for SW1 tumors in PBS (control)-injected (both (control and L-fuc) groups, n = 7 mice) (l), CD8+ T cell- (both groups, n = 6 mice) (m) or CD4+ T cell- (n) immunodepleted C3H/HeN mice (both groups, n = 7 mice). o, Comparison of intratumoral NK, DC, CD8+ T and CD4+ T cell subpopulations (absolute cell numbers) from tumors (n = 4 (control) and n = 3 (L-fuc)) (l), (for both groups, n = 4) (n). All error bars represent s.e.m. With the exception of e, for which one-way ANOVA was performed, two-sided t-tests were performed for all other analyses.

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We confirmed an essential role for tumor-specific fucosylation by overexpressing murine fucokinase (mFuk) in SW1 melanoma cells to exclusively increase tumor fucosylation. mFuk expression alone suppressed tumor growth and increased total itICs comparably to oral L-fuc administration alone. Again, CD4+ and CD8+ T cells were the most increased itICs (Extended Data Fig. 1l,m and Fig. 1e–h). These data indicate that melanoma-specific fucosylation is an essential determinant of L-fuc-triggered itIC induction and tumor suppression, regardless of any other physiological host effects that L-fuc may elicit (for example, microbiome or metabolic effects).

Correlations between tumor fucosylation and CD3+ T cells in humans were assessed by immunofluorescently analyzing a 40-patient melanoma microarray. Patients with higher-than-median tumor fucosylation levels exhibited significantly increased intratumoral CD3+ T cell densities (Fig. 1i). Intriguingly, average melanoma fucosylation levels were lower in male patients (Fig. 1j) but exhibited a stronger association with intratumoral CD3+ T cells (Fig. 1k).

These data indicate that melanoma fucosylation substantially shapes the itIC landscape, correlates with increased intratumoral CD3+ T cells in mice and humans and can be boosted by oral L-fuc to increase itICs and suppress BRAF- and NRAS-mutant melanomas.

L-fuc triggers CD4± T cell-induced itICs and alters CD4± T cell biology

The contribution of CD4+ and CD8+ T cells to L-fuc-triggered tumor suppression was assessed by immunodepletion in the SW1 model. L-fuc reduced tumor growth by >50% in control and CD8+ T cell-depleted mice, whereas this effect was completely abrogated by CD4+ T cell depletion (Fig. 1l–n, immunodepletion confirmed by splenic profiling, and Extended Data Fig. 1n,o). Consistent with known roles for CD4+ T cells in recruiting and activating tumor-suppressive itICs13, CD4+ T cell depletion also blocked L-fuc-induced increases in total itICs, including intratumoral NK cells, DCs and CD8+ T cells, observed in control mice (Extended Data Fig. 1p and Fig. 1o). Similarly, in the SM1 model, CD4+ but not CD8+ T cell depletion abrogated L-fuc-triggered tumor suppression and increases in total itICs and itIC subpopulations (immunodepletion confirmed by splenic profiling, Extended Data Fig. 1q–w)).

Phosphoproteomic and fucosylated proteomic analyses revealed that L-fuc mechanistically regulates CD4+ T cell biology by significantly altering protein kinase A (PKA) and (to a lesser extent) actin signaling, potentially via integrin B5, an upstream regulator of both of these pathways14 that we discovered to be one of five proteins most highly bound to Aleuria aurantia lectin (AAL) (and likely fucosylated) in human peripheral blood monocyte (PBMC)-derived, CD3–CD28-activated CD4+ T cells, as well as Jurkat cells treated with L-fuc (Extended Data Fig. 2a–f). The fact that integrin, PKA and actin signaling have been reported to mediate T cell activation, motility and immune synapse formation15,16 suggests that L-fuc promotes T cell trafficking to the tumor, a notion confirmed using an SW1 melanoma C3H mouse model treated with or without FTY720 (an inhibitor of lymph node egress). Inhibition of lymph node egress completely abrogated L-fuc-triggered tumor suppression (Fig. 2a,b). Strikingly, L-fuc-triggered tumor suppression was associated with increases in intratumoral CD4+ T central and effector memory subpopulations that were abrogated by FTY720 (Fig. 2a,c (blue dashed boxes) and Supplementary Table 1), consistent with the role that PKA plays in regulating memory phenotype in T cells17. Intriguingly, oral L-fuc induced significant, albeit transient, increases in intratumoral monocyte-derived DCs (moDCs) and lymph node conventional DC2 (cDC2) cells, which can promote memory CD4+ T cell phenotypes and cross-talk with CD4+ T cells to mediate tumor suppression, respectively18,19,20 (Fig. 2a,c (orange dashed boxes) and Supplementary Table 1). Finally, L-fuc also transiently but significantly increased cytotoxic CD4+ T cells at the midpoint (day 28) of the experiment (Fig. 2d,e).

a, Immune subpopulation markers use to profile by flow cytometry. b, Volumetric growth curves for SW1 tumors in C3H/HeN mice fed without (control, n = 3 mice) or with (L-fuc, n = 10 mice) L-fuc and treated with FTY720 (control mice were administered FTY720 (FTY) (n = 10 mice); L-fuc-supplemented mice were administered FTY720 (L + F) (n = 10 mice)). FTY720 was administered at 20 µg per mouse every 2 d starting on day 12, just before the initiation of L-fuc administration. c, Pie charts showing ratios of intratumoral or lymph node (LN)-resident CD4+ or CD8+ T cell subpopulations as well as DC subtypes from mice at days 14, 28 and 42 (each pie chart represents 4–5 mice). Assessment of cytotoxic CD4+ T cell populations (CRTAM+) and cytotoxic CD8+ T cell populations (GrzB+) from tumors at day 28 (d) (n = 5 mice for all groups except L-fuc, where n = 4 mice) and day 42 (e) (n = 5 mice each for control and L-fuc groups, n = 4 mice each for FTY and L + F groups). NS, not significant. Corresponding raw flow cytometric data for these charts are shown in Supplementary Table 1. The tumor growth curves and column charts show mean ± s.e.m. per group of mice.

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These data confirmed that CD4+ T cells play a key role in induction of itICs and suppression of melanomas by L-fuc, suggesting that L-fuc triggers key changes in CD4+ T cell signaling and biology at the tumor and lymph node levels that are important for tumor suppression. Importantly, the fact that mFuk expression alone in melanoma cells resulted in smaller tumors with increased itICs (Fig. 1e–h) suggests that melanoma-specific fucosylated protein(s) can also promote anti-tumor immunity, although the mechanism was unclear.

Fucosylated HLA-DRB1 induces itICs and melanoma suppression

To identify melanoma proteins that contributed to fucosylation-triggered, CD4+ T cell-mediated melanoma suppression, we subjected fucosylated proteins from human melanoma cells to liquid chromatography–mass spectrometric (LC–MS/MS) analysis followed by Ingenuity Pathway Analysis21 (Extended Data Fig. 3a, left). These analyses identified ‘Antigen presentation pathway’ as the only immune-related pathway, in which the MHC-I and MHC-II proteins HLA-A and HLA-DRB1, respectively, were identified as the only antigen-presentation and plasma membrane proteins with T cell-modulating functions22 (Extended Data Fig. 3a, right). We confirmed their expression in human melanocytes and melanoma cells by immunoblot (IB) analysis (Fig. 3a). Furthermore, lectin pulldown (LPD) using AAL and Ulex europaeus agglutinin I (UEA1) lectins, which bind to common core and terminal fucosylated glycans, respectively23,24,25,26,27,28, revealed association of both proteins with AAL (and to a lesser extent, UEA1), suggesting N′-linked core glycosylation–fucosylation (Fig. 3b). Finally, immunoprecipitation and IB analysis of V5-tagged HLA-A or HLA-DRB1 revealed direct recognition of HLA-DRB1 by AAL, indicating that a fraction of total HLA-DRB1 but not HLA-A is directly fucosylated in melanoma (Fig. 3c).

a, IB analysis of HLA-A and HLA-DRB1 levels in primary human melanocytes (HEMN) or the indicated human melanoma cell lines. b, LPD and IB analysis of patient-matched primary and metastatic cell line pairs WM793 and 1205Lu (left) and WM115 and WM266-4 (right) for HLA-A and HLA-DRB1. c, V5-immunoprecipitation and IB analyses of WM793 cells expressing (left) V5-tagged HLA-A or (right) V5-tagged HLA-DRB1. Volumetric growth curves for non-targeting control short hairpin RNA (shRNA) (shNT)- (d), H2K1-targeting shRNA (shH2K1)- (e) or H2EB1-targeting shRNA (shEB1)- (f) expressing SW1 tumors in C3H/HeN mice (n = 8 mice for each shNT group, n = 6 for each shH2K1 group and n = 6 and 7 for shEB1 and shEB1 with L-fuc groups, respectively). Flow cytometric comparison of total itIC counts (g) or the indicated subpopulations (h) from shNT- or shEB1-expressing tumors in d,f. n = 3 mice per group. For d–f, the red triangle indicates initiated L-fuc supplementation; growth curves and column charts show mean ± s.e.m. from each mouse group. In a–c, representative images are shown for n = 3 independent biological replicate experiments.

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To determine contributions of HLA-A or HLA-DRB1 to fucosylation-triggered anti-tumor immunity, we knocked down their C3H/HeN mouse orthologs H2K1 or EB1 (ref. 29), respectively, in SW1 tumors (Extended Data Fig. 3b) and assessed growth and itICs in vivo. Whereas L-fuc impaired control tumor growth, H2K1 knockdown suppressed tumor growth regardless of L-fuc (Fig. 3d,e), potentially reflecting tumor-protective, immunosuppressive roles of MHC-I proteins30,31. Notably, EB1 knockdown completely abolished L-fuc-triggered tumor suppression and induction of total itICs, including DC, CD8+ and CD4+ T cell subpopulations (Fig. 3f–h), similar to the effects elicited by CD4+ T cell depletion (Fig. 1l–o).

Consistent with roles of HLA-DRB1 in CD4+ T cell activation32,33,34, our findings demonstrate that HLA-DRB1 is expressed and fucosylated in melanoma and required for L-fuc-triggered CD4+ T cell-mediated itIC induction and melanoma suppression.

Fucosylation regulates HLA-DRB1 localization and immunological effects

We reasoned that determining how HLA-DRB1 is regulated by fucosylation would provide important insight into its crucial role in L-fuc-triggered anti-tumor immunity. Using NetNGlyc version 1 and NetOGlyc version 4 (https://services.healthtech.dtu.dk)35, we predicted N- and O-linked glycosylation sites at Asn48 (N48) and Thr129 (T129), respectively, which are conserved sites within constant regions of human and mouse HLA-DRB1 (Fig. 4a, top)29,36. Importantly, EB1 exhibits ~80% sequence homology with HLA-DRB1 and contains the conserved glycosylation–fucosylation site at N46 (ref. 29). Modeling of HLA-DRB1 interactions with prominent binding partners HLA-DM or CD4–TCR suggests that fucosylation of neither site affects interaction interfaces or peptide loading or presentation (Fig. 4a, bottom).

Nano-LC–MS/MS analysis of HLA-DRB1 immunoprecipitated from WM793 cells identified the fragment FLEYSTSECHFFNGTER as glycosylated–fucosylated at N48 with the predicted glycan HexNAc(4)Hex(3)Fuc(1) (Fig. 4b and Extended Data Fig. 4a). We mutated N48 or T129 to Gly or Ala, respectively, to abolish and verify fucosylation37,38,39. Unlike wild-type (WT) or the T129A ‘glycofucomutant’ HLA-DRB1, the HLA-DRB1N48G glycofucomutant did not bind to AAL in LPD assays (Fig. 4c), confirming fucosylation at N48 on an N-linked glycan.

To determine how fucosylation might regulate HLA-DRB1, we assessed its subcellular localization in WM793 cells that were pharmacologically modulated for fucosylation by treatment with 2F-peracetyl-fucose (a FUT inhibitor (FUTi)40) versus vehicle (dimethylsulfoxide, DMSO; control). Cells treated with FUTi exhibited dimmer, more central, endoplasmic reticulum (ER)-colocalization of HLA-DRB1 than vehicle-treated cells, suggesting less accumulation at the cell surface (Fig. 4d). Furthermore, flow cytometry revealed that cell surface fucosylation and HLA-DRB1 both decreased or increased after FUTi or L-fuc treatments, respectively, whereas mRNA and protein levels remained unchanged; thus fucosylation promotes cell surface localization of HLA-DRB1 (Fig. 4e and Extended Data Fig. 4b). Finally, global proteomic profiling to identify interactors that might mediate fucosylation-regulated cell surface localization of HLA-DRB1 revealed that N48 glycosylation–fucosylation promotes binding to calnexin, which has been reported to mediate maturation and trafficking of MHC-II complexes to the surface41 (Extended Data Fig. 5a–d).

To assess how HLA-DRB1 glycosylation–fucosylation contributes to tumor suppression and itICs, we compared control- or EB1-knocked-down SW1 tumors reconstituted with WT or glycofucomutant (N46G) EB1 (confirmation of knockdown reconstitution and fucosylation by IB and LPD, respectively, in Extended Data Fig. 5e). Abrogation of L-fuc-induced itIC and tumor growth suppression by EB1 knockdown was rescued by reconstitution with only WT but not glycofucomutant EB1, demonstrating that glycosylation–fucosylation of EB1 or HLA-DRB1 is essential for L-fuc-triggered itIC induction and melanoma suppression (Fig. 4f and Extended Data Fig. 5f,g). This is consistent with our finding that loss of glycosylation–fucosylation of HLA-DRB1 or EB1 abrogates its cell surface localization and impairs its ability to induce anti-tumor immunity. Thus, despite the other fucosylated proteins identified in melanoma cells (Extended Data Fig. 3), these data confirm that the N48 glycosylation–fucosylation of HLA-DRB1 is a key regulator of anti-melanoma immunity and tumor suppression. Despite other potential host physiological effects of dietary L-fuc (for example, microbiome, metabolome, etc.), these data confirm that L-fuc-induced itIC increases and melanoma suppression are critically mediated by melanoma-intrinsic expression and fucosylation of HLA-DRB1, which promotes its cell surface accumulation to trigger CD4+ T cell-mediated anti-tumor immune responses.

Oral L-fuc augments anti-PD1-mediated melanoma suppression

Expression of MHC-II reportedly correlates with increased anti-PD1 efficacy42,43. Indeed, patients who failed anti-PD1 therapy exhibited relative >45% reduced cell surface MHC-II but not MHC-I (Extended Data Fig. 5h). As anti-PD1 efficacy can be limited by itIC abundance44, particularly of CD4+ T and memory CD4+ T cells10,45,46,47,48,49, we tested whether the ability to increase CD4+ T cell-mediated itIC induction and tumor suppression using oral L-fuc could be leveraged to augment anti-PD1 efficacy. In the SW1 model, oral L-fuc suppressed tumors as much as anti-PD1 but did not enhance efficacy of anti-PD1 therapy (~50–60%; Fig. 5a, left). By contrast, in the SM1 model, L-fuc was less tumor suppressive than anti-PD1 therapy alone but rather augmented durable suppression in combination with anti-PD1 therapy (Fig. 5a, right). Importantly, we also found that L-fuc does not alter cell surface levels of programmed cell death ligand 1 (PD-L1) in mouse or human melanoma cells (Extended Data Fig. 6a), suggesting that the L-fuc-associated tumor suppression in these models is attributed to determinants beyond the PD1–PD-L1 axis.

a, Volumetric growth curves for SW1 tumors in C3H/HeN mice (left) and SM1 tumors in C57BL/6 mice (right) fed with or without L-fuc and treated with PBS (control) or anti-PD1 therapy (concurrent initiation of L-fuc with or without anti-PD1 therapy (red triangle)). The tumor growth curves show mean ± s.e.m. per mouse group. For each group, n = 7 mice except PBS with L-fuc and anti-PD1 therapy with L-fuc groups, which each have eight mice. b, Volumetric growth curves for SM1 tumors in C57BL/6 mice fed with or without L-fuc and treated with PBS (control) or anti-PD1 therapy (PD1) (concurrent initiation of L-fuc with or without anti-PD1 therapy (red triangle)). The tumor growth curves show mean ± s.e.m. from ≥7 mice per group. At day 7 (before administration of L-fuc or anti-PD1 therapy, n = 3 mice), day 21 (endpoint for tumors of control-treated mice, n = 5 mice per group), day 31 (endpoint for tumors of L-fuc-treated mice, n = 5 mice per group) and day 63 (endpoint for tumors of anti-PD1-treated mice, n = 5 mice per group), the primary tumors (tumor) and draining lymph nodes of 4–5 mice per treatment group were analyzed by flow cytometry for intratumor levels of CD4+ and CD8+ T subpopulations (naive or terminal; stem central, central or effector memory) and DC subpopulations (cDC1, cDC2 and moDC) as in Fig. 2. Proportions of CD4+, CD8+ and DC subpopulations in each organ at each time point are represented by the color-coded pie charts (each pie chart represents 4–5 mice). Absolute numbers of the subpopulations per 106 cells of tumor or tissue homogenate at each time point are represented in the color-coded column charts. Corresponding raw flow cytometric data for these charts are shown in Supplementary Table 2. Column charts show mean ± s.e.m. from each mouse group.

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To clarify how the combination of L-fuc and anti-PD1 therapy enhanced suppression, we characterized immune cell profiles in the tumors and lymph nodes of SM1 tumor-bearing mice over a time course of treatment with L-fuc, with or without anti-PD1 therapy. Administration of L-fuc (1) alone increased intratumoral CD4+ T central and effector memory cells, an effect that was increased when combined with anti-PD1 therapy (Fig. 5b (blue dashed boxes) and Supplementary Table 2) and (2) initially expanded intratumoral cDC2 cells, followed by later expansion of cDC2 cells and moDCs in the lymph nodes when combined with anti-PD1 therapy (Fig. 5b (orange dashed boxes) and Supplementary Table 2). In addition to expanding the absolute numbers of intratumoral CD4+ and CD8+ T cells at the endpoint (day 63), the combination of L-fuc and anti-PD1 therapy increased the relative percentage of intratumoral CD8+ T central memory cells (Fig. 5b (green dashed box) and Supplementary Table 2). Thus, L-fuc can suppress some melanomas as effectively as anti-PD1 therapy, whereas, in others, it can enhance efficacy, which is associated with increased intratumoral CD4+ T central and effector memory subpopulations and lymph node cDC2 and moDC populations, consistent with the effects of L-fuc observed in Fig. 2.

Clinical implications of tumor fucosylation and fucosylated HLA-DRB1

Given the potent enhancement of anti-PD1 efficacy by oral L-fuc administration in mice, we investigated whether tumor fucosylation or total/fucosylated HLA-DRB1 might correlate at all with responsiveness to anti-PD1 therapy in human patient biopsies, as the identification of preliminary correlations might support their subsequent development into predictive biomarkers for anti-PD1 responsiveness. To this end, we devised a new technique: we modified the conventional proximity ligation assay (PLA)50 to facilitate immunofluorescent visualization of fucosylated HLA-DRB1 by applying anti-HLA-DRB1 antibody together with biotinylated AAL, which has previously been successfully used to stain tissues specifically for core-fucosylated glycans51 (Fig. 6a). This technique, lectin-mediated PLA (L-PLA), revealed cytoplasmic and/or membranous localization of endogenous fucosylated HLA-DRB1 in melanoma cells (Fig. 6b) that is lost upon FUTi treatment (Fig. 6c), confirming L-fuc-stimulated cell surface localization of HLA-DRB1 (Fig. 4d,e and Extended Data Fig. 4b). The cytoplasmic and/or ’vesicular-appearing’ staining is consistent with HLA-DRB1 that was fucosylated in the endoplasmic reticulum (ER)–Golgi and is en route to the surface via the secretory pathway. In applying this technique further to formalin-fixed, paraffin-embedded (FFPE) melanoma tissue specimens, we observed similar staining patterns for fucosylated HLA-DRB1 (Fig. 6d,e), which were completely abolished by washing the tissue with L-fuc, confirming specificity for fucosylated HLA-DRB1 (Fig. 6‍f).‍

To assess correlations of (1) tumor-specific fucosylation and total/fucosylated HLA-DRB1 of individual tumor cells and (2) intratumoral numbers CD4+ T cells with responder status to single-agent anti-PD1 therapy, we implemented L-PLA on primary melanoma biopsies from two distinct responder and two non-responder patients followed by single-cell segmented signal quantitation (Fig. 7a,b). Tumors of responders clearly contained tumor cell populations with high levels of fucosylation and total HLA-DRB1 as compared to non-responders (Fig. 7b(i,ii)). Although the tumor of only one of two responders contained melanoma cells with increased levels of fucosylated HLA-DRB1 compared with those of the non-responders (Fig. 7b(iii)), this trend mirrored that of intratumoral CD4+ T cell counts (Fig. 7b(iv)), consistent with the role for fucosylated HLA-DRB1 in CD4+ T cell-mediated tumor suppression.

a, Representative images of one anti-PD1-treated Moffitt patient tumor subjected to immunofluorescent staining for the two indicated panels of markers. b, Dot plots showing single-cell distribution of total fucosylation (AAL) (i), total (ii) and fucosylated (iii) HLA-DRB1 staining intensities per melanoma cell and percent CD4+ T cells (of total cells) within tumors of two responder (R; patients (P)1 and 2) and two non-responder (NR; patients 3 and 4) Moffitt patients (iv). AU, arbitrary units. c, Box plots showing mean tumor cellular (MTC; means derived from single tumor cell intensities) fucosylation (left), total (center) and fucosylated (right; fuco-HLA-DRB1) HLA-DRB1 staining intensities of anti-PD1 responder (red dots) and non-responder (blue dots) patients from MGH (n = 32) (top) or the MDACC (n = 11) (bottom). In all box plots, the middle line indicates the median, the lower and upper hinges represent the first and third quartiles and whiskers (from the hinges) extend to 1.5× the interquartile range (IQR). d, Percent intratumoral CD4+ T cells (of total cells) plotted against corresponding average MTC fucosylated HLA-DRB1 values for each patient in the MGH (top) and MDACC (bottom) cohorts. P values shown are two-sided P values derived from the Spearman correlation test.

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We assessed potential associations between tumor fucosylation, total/fucosylated HLA-DRB1, CD4+ T cells and responder status in expanded cohorts of patients with melanoma treated with anti-PD1 therapy. Levels of tumor fucosylation and total and fucosylated HLA-DRB1 in tumor cells were generally higher in anti-PD1 responders than in non-responders from Massachusetts General Hospital (MGH; n = 31; Fig. 7c, top) and the MD Anderson Cancer Center (MDACC; n = 11; Fig. 7c, bottom). Total tumor fucosylated HLA-DRB1 exhibited weak or no association with tumoral CD4+ T cells (Fig. 7d, top and bottom), although the association was modestly increased when restricted to CD4+ T cells localized at the periphery of the tumors (Extended Data Fig. 6b,c; absolute CD4+ T numbers in Supplementary Table 3). Importantly, we found that some specimens containing divergent tumor–stroma content did exhibit divergent correlation strengths (for example, core biopsies containing only tumor versus non-core biopsies containing substantial stroma). For example, a ‘highly correlated’ anti-PD1 responder (non-core biopsy) containing substantial tumor–stromal interface exhibited correlated high levels of fucosylated HLA-DRB1 and CD4+ T cells, whereas a ‘non-correlated’ responder (a core biopsy) did not (Extended Data Fig. 6d), suggesting that variable stromal content within biopsies may have at least partially undermined the strength of the correlations that we assessed.

The lack of significant correlation may also be attributed to the dynamic relationship between fucosylated HLA-DRB1 and CD4+ T cell infiltration that is further weakened by suboptimal inclusion criteria and/or patient stratification. Comparison of these markers in five patient-matched tumors before and after anti-PD1 therapy revealed no significant correlation in total HLA-DRB1 levels. However, before treatment, tumor cell fucosylation was significantly higher in the complete responder versus partial responders and non-responders; this dropped to the equivalently lower levels of the other patients after treatment. With the exception of one non-responder, the complete responder also exhibited significantly increased fucosylated HLA-DRB1 in tumor cells before treatment (Extended Data Fig. 6e). The consistent trends in tumor fucosylation and fucosylated tumor HLA-DRB1 observed across the three independent cancer center cohorts appear to support potential utility but importantly point to the need for further study in expanded patient pre-treatment biopsy cohorts that are controlled for a number of specific clinical variables, which will be discussed below.

Discussion

The consistent trends that we observed in higher tumor fucosylation and fucosylated HLA-DRB1 across anti-PD1 responders versus non-responders between the three independent cancer center cohorts support their potential utility as biomarkers of anti-PD1 responsiveness. However, further analyses in expanded patient biopsy cohorts are clearly needed. Considering the variable tumor-suppressive effects of L-fuc observed in our anti-PD1-treated SM1 and SM1 mouse models, there are likely similar biological and clinical variables in patients that must be further explored and that may have precluded statistical significance in our small analyses.

In terms of biological variables, how T cell biology is regulated by fucosylation, for example, has heretofore been unclear. Reported divergent effects of fucosylation on T cell activation versus exhaustion (that is, via regulation of PD-L1 expression) point to FUT-specific expression and roles that remain to be elucidated54,55,56. The fact that L-fuc does not alter the cell surface levels of PD-L1 in human or mouse melanoma cells (Extended Data Fig. 6a), suggesting that the discrepant tumor suppression by single-agent versus combination L-fuc and anti-PD1 therapy in our SW1 and SM1 mouse models (Fig. 5a), is attributed to determinants beyond the PD1–PD-L1 axis. Indeed, our global fucosylated and phosphoproteomic analyses suggest that fucosylation in CD4+ T cells impacts integrin β5, PKA and actin signaling (Extended Data Fig. 2) and that this is associated with increased intratumoral T cell presence and memory phenotypes in our models (Figs. 2 and 5), consistent with previous reports that those functions are regulated by those pathways in T cell biology15,16,17,57. The fact that L-fuc can increase CD4+ T central memory cells also partially explains how it can augment anti-PD1 efficacy, which is associated with the presence of these cells10. How L-fuc may regulate these signaling pathways and enrich for CD4+ T memory subsets within the tumor microenvironment and, furthermore, how L-fuc alters DC biology and induces their intratumoral accumulation (Figs. 2 and 5) may contribute to anti-tumor immune responses and tumor suppression in this context are unclear and warrant further lines of study. In addition, sex might be a determinant, as melanoma fucosylation levels are lower but correlate more strongly with intratumoral CD3+ T cells in male versus female patients (Fig. 1j,k). Reduced melanoma fucosylation, which is expected to lower itICs, might explain increased lethality in male patients (American Cancer Society Facts and Figures, 2022).

The availability of pre-treatment anti-PD1 tumor tissue specimens for this study was extremely limited. Thus, the specimens that we acquired were subject to clinical variability that may have undermined statistical robustness in our analyses. Subsequent studies investigating tumor fucosylation, total/fucosylated HLA-DRB1 and CD4+ T cells as biomarkers will need to factor for clinical variables including therapies received before anti-PD1 therapy and pre-existing medical conditions as well as time from biopsy to anti-PD1 treatment. Because we were unable to control for these confounders in our specimens, it is unclear how they may have impacted tumor and HLA-DRB1 fucosylation and CD4+ T cell biology and thus the strength of correlations between these markers and responsiveness to treatment. Likewise, the importance and contribution of the immune environment of the peri-tumoral stroma in this setting remains to be elucidated, as some of our biopsies contained stroma, whereas other tumor core biopsies did not. Prior studies focusing on tumor–immune interactions and immunotherapies (including anti-PD1 therapy) have highlighted the importance of analyzing biomarker staining patterns at tumor–immune and tumor–stromal interfaces contained within biopsies, as these are areas of enriched immunological activity and signaling58,59,60. Indeed, our observation of ‘highly correlated’ and ‘non-correlated’ anti-PD1 responder biopsies containing disparate amounts of tumor stroma highlight how lack of sufficient stroma in tumor biopsies likely undermined the statistical robustness of the correlations between fucosylated HLA-DRB1 and CD4+ T cells (Extended Data Fig. 6d). The acquisition of such biopsy specimens that are controlled for the variables detailed above is an important consideration for subsequent studies. Our findings highlight the need for a prospective clinical trial with defined protocols for collection of monotherapy anti-PD1 pre-treatment biopsies at defined time points proximal to therapy and clear biopsy protocols to yield tumor specimens that contain substantial intact stromal interface.