Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.

Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

LAG-3 is the third immune checkpoint pathway successfully targeted for cancer therapy. Although ineffective as a monotherapy, combination of LAG-3 and PD-1 blockade improves survival from advanced melanoma. In this issue of Cell, two studies in mice and a human clinical trial provide insights on LAG-3 in immune regulation.

OBJECTIVE: Immune checkpoint inhibitors have revolutionized the treatment of renal cell carcinoma (RCC), but many patients do not respond to therapy and the majority develop resistant disease over time. Thus, there is increasing need for alternative immunomodulating agents. The co-inhibitory molecule T-cell immunoglobulin and ITIM domain (TIGIT) may play a role in resistance to approved immune checkpoint inhibitors and is being investigated as a potential therapeutic target. The purpose of this study was to quantify TIGIT positivity in tumor-infiltrating T cells in RCC.
METHODS: We employed tissue microarrays containing specimens from primary RCC tumors, adjacent normal renal tissue, and RCC metastases to quantify TIGIT within tumor-infiltrating CD3+ T cells using quantitative immunofluorescent analysis. We also compared these results to TIGIT+ CD3+ levels in four other tumor types (melanoma, non-small cell lung, cervical, and head and neck cancers).
RESULTS: We did not observe significant differences in TIGIT positivity between primary RCC tumors and patient-matched metastatic samples. We found that the degree of TIGIT positivity in RCC is comparable to that in lung cancer but lower than that in melanoma, cervical, and head and neck cancers. Correlation analysis comparing TIGIT positivity to previously published, patient-matched spatial proteomic data by our group revealed a negative association between TIGIT and the checkpoint proteins PD-1 and LAG3.
CONCLUSIONS: Our findings support careful evaluation of TIGIT expression on T cells in primary or metastatic RCC specimens for patients who may be treated with TIGIT-targeting antibodies, as increased TIGIT positivity might be associated with a greater likelihood of response to therapy.

OBJECTIVE: Immune cells are reported to upregulate CD47 during infection, however, the role of CD47 in innate and adaptive immune cells remains unclear.
METHODS: To bridge this knowledge gap, we analysed our single cell (sc)-RNA dataset along with other publicly available sc-RNA datasets from healthy controls, people with HIV-1 (PWH) and COVID-19 patients. We characterized each immune cell based on low, intermediate, and high expression of CD47 .
RESULTS: Our analyses revealed that CD47 high pDCs and monocytes exhibited relatively higher expression of IFN-α regulatory genes, antiviral interferon-stimulated genes (ISGs) and MHC-I associated genes compared to CD47 inter. and CD47 low cells. Furthermore, CD47 high NK and CD8+ T cells showed higher expression of antiviral ISGs, as well as genes encoding for cytotoxic markers like granzyme B, perforin, granulysin, interferon gamma and NKG7. Additionally, CD47 high CD8+ T cells expressed higher levels of PD-1 and LAG-3 genes. Lastly, we found that CD47 high B cells had enriched expression of genes involved in cell activation and humoral responses.
CONCLUSIONS: Overall, our analyses revealed that innate and adaptive immune cells expressing elevated activation and functional gene signatures also express higher CD47 levels.

BACKGROUND: Lymphocyte activation gene 3 (LAG-3) has been considered as the next generation of immune checkpoint and a promising prognostic biomarker of immunotherapy. As with programmed cell death protein-1/programmed death-ligand 1 and cytotoxic T-lymphocyte antigen-4 inhibitors, positron emission tomography (PET) imaging strategies could benefit the development of clinical decision-making of LAG-3-related therapy. In this study, we developed and validated 68Ga-labeled cyclic peptides tracers for PET imaging of LAG-3 expression in bench-to-bedside studies.
METHODS: A series of LAG-3-targeted cyclic peptides were modified and radiolabeled with 68GaCl3 and evaluated their affinity and specificity, biodistribution, pharmacokinetics, and radiation dosimetry in vitro and in vivo. Furthermore, hu-PBL-SCID (PBL) mice models were constructed to validate the capacity of [68Ga]Ga-CC09-1 for mapping of LAG-3+ lymphocytes infiltrates using longitudinal PET imaging. Lastly, [68Ga]Ga-CC09-1 was translated into the first-in-human studies to assess its safety, biodistribution and potential for imaging of LAG-3 expression.
RESULTS: A series of cyclic peptides targeting LAG-3 were employed as lead compounds to design and develop 68Ga-labeled PET tracers. In vitro binding assays showed higher affinity and specificity of [68Ga]Ga-CC09-1 in Chinese hamster ovary-human LAG-3 cells and peripheral blood mononuclear cells. In vivo PET imaging demonstrated better imaging capacity of [68Ga]Ga-CC09-1 with a higher tumor uptake of 1.35±0.33 per cent injected dose per gram and tumor-to-muscle ratio of 17.18±3.20 at 60 min post-injection. Furthermore, [68Ga]Ga-CC09-1 could detect the LAG-3+ lymphocyte infiltrates in spleen, lung and salivary gland of PBL mice. In patients with melanoma and non-small cell lung cancer, primary lesions with modest tumor uptake were observed in [68Ga]Ga-CC09-1 PET, as compared with that of [18F]FDG PET. More importantly, [68Ga]Ga-CC09-1 delineated the heterogeneity of LAG-3 expression within large tumors.
CONCLUSIONS: These findings consolidated that [68Ga]Ga-CC09-1 is a promising PET tracer for quantifying the LAG-3 expression in tumor microenvironment, indicating its potential as a companion diagnostic for patients stratification and therapeutic response monitoring in anti-LAG-3 therapy.

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.

BACKGROUND: Anti-programmed death-1 (PD-1)/anti-PD-ligand-1 (PD-L1) pathway inhibition is a standard regimen for advanced urothelial carcinoma (UC); however, its limited efficacy has been reflected in reported medium response rates. This study explored the role of next-generation coinhibitory receptors (IRs; lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain 3 (TIM-3), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT)) and their ligands (LGs) in the response to PD-(L)1 blockade therapy and the oncological outcomes in patients with UC.
METHODS: We investigated metastatic UC cases who underwent PD-(L)1 therapy (cohort 1: n=348, cohort 2: n=89, and cohort 4: n=29) or advanced UC cases involving surgery (cohort 3: n=293 and cohort 5: n=90). We assessed the mRNA expression profiles and corresponding clinical information regarding IRs and LGs using cohorts 1, 2, and 3. Additionally, we elucidated the spatial features of these targeted markers using multiplex immunohistochemistry (mIHC) on formalin-fixed paraffin-embedded samples from cohorts 4 and 5. Survival, differential expressed gene, and Gene Set Enrichment analyses were performed. For mIHC, quantitative analyses were also performed to correlate immune and tumor cell densities with patient survival.
RESULTS: LAG-3 expression was strongly associated with the responsiveness of PD-(L)1 blockade compared with the expression of TIM-3 and TIGIT. In tumors with high LAG-3 levels, the increased expression of fibrinogen-like protein 1 (FGL1) had a significantly negative effect on the response to PD-(L)1 blockade and overall survival. Moreover, high FGL1 levels were associated with elevated CD4+ regulatory T-cell gene signatures and the upregulation of CD39 and neuropilin-1, with both indicating CD8+ T-cell exhaustion. mIHC analyses revealed that patients with stromal CD8+LAG-3+cellshigh-tumor FGL1+cellshigh exhibited a significant negative correlation with survival rates compared with those with stromal CD8+LAG-3+cellshigh-tumor FGL1+cellslow.
CONCLUSIONS: LAG-3 expression and high FGL1 coexpression are important predictive factors of adverse oncological outcomes related to the presence of immunosuppressive contextures. These findings are hypothesis-generating, warranting further mechanistic and clinical studies aimed to evaluate LAG-3/FGL1 blockade in UC.

BACKGROUND: Lymphocyte activation gene 3 (LAG-3) is expressed on activated immune cells and has emerged as a promising target for immune checkpoints blockade. However, conflicting findings have been reported regarding the association between LAG-3 expression in tumors and patient prognosis, indicating the need for further investigation into the significance of LAG-3 expression levels in tumor therapies. In this study, 68Ga-NOTA-XH05, a novel peptide-based positron emission tomography (PET) tracer targeting LAG-3, was constructed to non-invasively detect LAG-3 expression in melanoma after CpG oligonucleotide (CpG) treatment and explore the relationship between LAG-3 expression and therapeutic effect.
METHODS: The tracer 68Ga-NOTA-XH05 was identified by high-performance liquid chromatography after being prepared and purified. Cell uptake and blocking essays were performed to verify the specificity of the tracer in vitro. The expression of LAG-3 in B16-F10 subcutaneous tumors was monitored by flow cytometry, and its correlation with the tracer uptake was analyzed to evaluate the tracer specificity. PET imaging and biodistribution studies were conducted after CpG treatment of unilateral or bilateral B16-F10 subcutaneous tumor models to assess the ability of 68Ga-NOTA-XH05 in monitoring immunotherapy efficacy and the abscopal effect of CpG.
RESULTS: Following purification, 68Ga-NOTA-XH05 exhibited high radiochemical purity and specificity. Flow cytometry analysis revealed a positive correlation between LAG-3 expression in tumors and the uptake of 68Ga-NOTA-XH05. In B16-F10 bearing mice treated with CpG, PET imaging using 68Ga-NOTA-XH05 demonstrated a higher tumor to blood ratio (TBR) compared with the control group. Furthermore, TBR values obtained from CpG-treated mice allowed for differentiation between responders and non-responders. In a bilateral subcutaneous tumor model where only right-sided tumors were treated with intratumoral injection of CpG, TBR values of left-sided tumors were significantly higher than those in the control group, indicating that 68Ga-NOTA-XH05 could effectively monitor the systemic effect of local CpG injection.
CONCLUSIONS: Our findings highlight the detection capability of 68Ga-NOTA-XH05 in assessing LAG-3 expression levels within tumors and evaluating response to immunotherapy, thereby suggesting promising clinical translational prospects.