{
  "abstract": "Background Tertiary lymphoid structures (TLS) are associated with improved responses to immune checkpoint inhibitors (ICI) in several cancers including clear cell renal cell carcinoma (ccRCC) and soft tissue sarcoma (STS). 1 2 Yet, many TLS-positive tumors remain resistant, suggesting the presence of active immunosuppressive mechanisms.3 Methods To investigate ICI resistance in TLS-positive tumors, we performed multi-modal profiling of ccRCC samples using bulk, single-cell, and spatial transcriptomics, bulk and spatial metabolomics, and multiplex immunofluorescence. Findings were validated in four independent ICI-treated cohorts (total n=1,107), with additional support from in vitro B cell experiments and two murine models of STS.Results Differential gene expression analysis between ccRCC TLS-positive tumours from responding (R) and non-responding (NR) patients highlighted the enrichment of four GABA receptors-associated genes in NR: GABBR1, GNAI1, ADCY2 and APBA1, which we collectively named ‘GABA ccRCC signature’. Indeed, this signature was highly upregulated in non-responders ( figure 1a). Stratifying TLS-positive tumors using this signature—by tertiles or median—identified a ‘GABA-high’ group with higher NR rates and shorter progression-free survival (figure 1b-c). This pattern was not observed in TLS-negative tumors or VEGFR-targeted TKI-treated patients. We validated these results in three additional metastatic ccRCC ICI and/or TKI treated cohorts (analysis of a total of 1,107 ccRCC patients). Multi-modal integration identified proximal tubule-like tumor cells as the primary source of GABA, especially in proximity to immature TLS in NR ccRCC patients. Transcriptional analysis of TLS in NR revealed impaired immune activation, reduced IgG production, diminished antigen presentation, overexpression of GABA receptors, and elevated TCA cycle activity (figure 1d). Spatial metabolomics confirmed accumulation of TCA cycle intermediates in TLS located near GABA-producing tumor regions. Moreover, in-vitro experiments on human B cells, activated to generate PBs and PCs, showed that GABA exposure inhibits their proliferation and secretion of immunoglobulins. In vivo, combining anti-PD1 with 3-mercaptopropionic acid (3-MPA), an inhibitor of GABA synthesis, enhanced antitumor activity versus anti-PD1 alone. In the MCA-OVA subcutaneous model, this combination led to significantly delayed tumor growth (p=0.021; figure 1e-f), which was further validated in a TLS-rich intraperitoneal model.Conclusions We identify GABA as a novel immunosuppressive metabolite that mediates ICI resistance in TLS-positive tumors. These findings suggest that GABA production is a key tumor-intrinsic resistance mechanism, and targeting the GABAergic axis may restore TLS function and enhance ICI efficacy. Our results offer new biomarkers for patient stratification and rational combinations for immunotherapy development in TLS-positive cancers.References Fridman WH, Petitprez F, Meylan M, Chen TW-W, Sun C-M, Roumenina LT, Sautès-Fridman C. B cells and cancer: to B or not to B? Journal of Experimental Medicine 2020;218:e20200851.Vanhersecke L, Brunet M, Guégan J-P, Rey C, Bougouin A, Cousin S, Le Moulec S, Besse B, Loriot Y, Larroquette M, et al. Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression. Nat Cancer. 2021;2:794–802.Italiano A, Bessede A, Pulido M, Bompas E, Piperno-Neumann S, Chevreau C, Penel N, Bertucci F, Toulmonde M, Bellera C, et al. Pembrolizumab in soft-tissue sarcomas with tertiary lymphoid structures: a phase 2 PEMBROSARC trial cohort. Nat Med. 2020;28:1199–1206.Abstract 457 Figure 1a, GSVA scores of GABA ccRCC signature in TLS+ tumours (R vs NR). b, Response rates; c, PFS by GABA score. d, Differentially expressed genes in mature vs immature TLS (R vs NR). e, In-vivo overview. f, Tumour growth under indicated treatments.",
  "authors": [
    {
      "affiliations": [
        "Universite Paris Cite, Sorbonne Universite, Paris, France"
      ],
      "name": "Isaias Hernandez-Verdin"
    },
    {
      "affiliations": [
        "Cordeliers Research Centre, Paris, France"
      ],
      "name": "Anne Calvez"
    },
    {
      "affiliations": [
        "Universite Paris Cite, Sorbonne Universite, Paris, France"
      ],
      "name": "Cheng-Ming Sun"
    },
    {
      "affiliations": [
        "Universite Paris Cite, Sorbonne Universite, Paris, France"
      ],
      "name": "Antoine Bougouin"
    },
    {
      "affiliations": [
        "Limoges University, Limoges, Haute-Vienne, France"
      ],
      "name": "Pierre Hirt"
    },
    {
      "affiliations": [
        "Limoges University, Limoges, Haute-Vienne, France"
      ],
      "name": "Eric Pinaud"
    },
    {
      "affiliations": [
        "Karolinska Institutet, Stockholm, Stockholm, Sweden"
      ],
      "name": "Matthew Smith"
    },
    {
      "affiliations": [
        "Karolinska Institutet, Stockholm, Stockholm, Sweden"
      ],
      "name": "Craig Wheelock"
    },
    {
      "affiliations": [
        "Hôpital Européen Georges-Pompidou, Paris, France"
      ],
      "name": "Virginie Verkarre"
    },
    {
      "affiliations": [
        "Hôpital Européen Georges-Pompidou, Paris, France",
        "Centre de Recherche des Cordeliers, Paris, France"
      ],
      "name": "Yann-Alexandre Vano"
    },
    {
      "affiliations": [
        "Universite Paris Cite, Sorbonne Universite, Paris, France"
      ],
      "name": "Asmat Ullah"
    },
    {
      "affiliations": [
        "Institut Bergonié, Bordeaux, Gironde, France"
      ],
      "name": "Lucile Vanhersecke"
    },
    {
      "affiliations": [
        "Explicyte, Bordeaux, France"
      ],
      "name": "Alban Bessede"
    },
    {
      "affiliations": [
        "Explicyte, Bordeaux, France"
      ],
      "name": "Jean-Philippe Guégan"
    },
    {
      "affiliations": [
        "Gustave Roussy, Villejuif, Ile-de-France, France"
      ],
      "name": "Laurence Albiges"
    },
    {
      "affiliations": [
        "Gustave Roussy, Villejuif, Ile-de-France, France"
      ],
      "name": "Sylvere Durand"
    },
    {
      "affiliations": [
        "Gustave Roussy, Villejuif, Ile-de-France, France"
      ],
      "name": "Guido Kroemer"
    },
    {
      "affiliations": [
        "Institut du Cerveau-Paris Brain Institute-ICM, Paris, Paris, France",
        "Sorbonne Université, Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière-Charles Foix, Paris, Paris, France"
      ],
      "name": "Agusti Alentorn"
    },
    {
      "affiliations": [
        "Institut Bergonié, Bordeaux, Gironde, France"
      ],
      "name": "Antoine Italiano"
    },
    {
      "affiliations": [
        "Hôpital Européen Georges-Pompidou, Paris, France"
      ],
      "name": "Stephane Oudard"
    },
    {
      "affiliations": [
        "Universite Paris-Cite, Paris, France"
      ],
      "name": "Catherine Sautes-Fridman"
    },
    {
      "affiliations": [
        "Cordeliers Research Centre, Paris, France"
      ],
      "name": "Wolf H Fridman"
    }
  ],
  "title": "457 GABA promotes resistance to immunotherapy of patients with TLS-positive tumours",
  "uid": "5ba40711-2399-5f3c-bc20-398d191856e3"
}