Neoantigen-specific T cell help outperforms non-specific help in multi-antigen DNA vaccination against cancer.

CD4+ T helper antigens are essential components of cancer vaccines, but the relevance of the source of these MHC class II-restricted antigens remains underexplored. To compare the effectiveness of tumor-specific versus tumor-unrelated helper antigens, we designed three DNA vaccines for the murine MC-38 colon carcinoma, encoding CD8+ T cell neoantigens alone (noHELP) or in combination with either "universal" helper antigens (uniHELP) or helper neoantigens (neoHELP). Both types of helped vaccines increased the frequency of vaccine-induced CD8+ T cells, and particularly uniHELP increased the fraction of KLRG1+ and PD-1low effector cells. However, when mice were subsequently injected with MC-38 cells, only neoHELP vaccination resulted in significantly better tumor control than noHELP. In contrast to uniHELP, neoHELP-induced tumor control was dependent on the presence of CD4+ T cells, while both vaccines relied on CD8+ T cells. In line with this, neoHELP variants containing wild-type counterparts of the CD4+ or CD8+ T cell neoantigens displayed reduced tumor control. These data indicate that optimal personalized cancer vaccines should include MHC class II-restricted neoantigens to elicit tumor-specific CD4+ T cell help.

Multiantigen pan-sarbecovirus DNA vaccines generate protective T cell immune responses.

SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell-based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8+ and CD4+ T cell responses to shared epitopes. Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2-transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness.

Genome-wide CRISPR screens define determinants of epithelial-mesenchymal transition mediated immune evasion by pancreatic cancer cells.

The genetic circuits that allow cancer cells to evade immune killing via epithelial mesenchymal plasticity remain poorly understood. Here, we showed that mesenchymal-like (Mes) KPC3 pancreatic cancer cells were more resistant to cytotoxic T lymphocyte (CTL)-mediated killing than the parental epithelial-like (Epi) cells and used parallel genome-wide CRISPR screens to assess the molecular underpinnings of this difference. Core CTL-evasion genes (such as IFN-γ pathway components) were clearly evident in both types. Moreover, we identified and validated multiple Mes-specific regulators of cytotoxicity, such as Egfr and Mfge8. Both genes were significantly higher expressed in Mes cancer cells, and their depletion sensitized Mes cancer cells to CTL-mediated killing. Notably, Mes cancer cells secreted more Mfge8 to inhibit proliferation of CD8+ T cells and production of IFN-γ and TNFα. Clinically, increased Egfr and Mfge8 expression was correlated with a worse prognosis. Thus, Mes cancer cells use Egfr-mediated intrinsic and Mfge8-mediated extrinsic mechanisms to facilitate immune escape from CD8+ T cells.

OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum.

Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy.

Effective combination of liposome-targeted chemotherapy and PD-L1 blockade of murine colon cancer.

Therapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient drug penetration and rapid drug clearance and toxicity can be improved by using a liposomal platform. Drug resistance for instance against pemetrexed, can be reduced by combination with docetaxel. Here, we developed a specific liposomal formulation to simultaneously deliver docetaxel and pemetrexed to enhance efficacy and safety. Hydrophobic docetaxel and hydrophilic pemetrexed were co-encapsulated into pH-sensitive liposomes using a thin-film hydration method with high efficiency. The physicochemical properties, toxicity, and immunological effects of liposomes were examined in vitro. Biodistribution, anti-tumor efficacy, and systemic immune response were evaluated in vivo in combination with PD-L1 immune checkpoint therapy using two murine colon cancer models. In cellular experiments, the liposomes exhibited strong cytotoxicity and induced immunogenic cell death. In vivo, the treatment with the liposome-based drug combination inhibited tumor development and stimulated immune responses. Liposomal encapsulation significantly reduced systemic toxicity compared to the delivery of the free drug. Tumor control was strongly enhanced when combined with anti-PDL1 immunotherapy in immunocompetent mice carrying syngeneic MC38 or CT26 colon tumors. We showed that treatment with liposome-mediated chemotherapy of docetaxel and pemetrexed combined with anti-PD-L1 immunotherapy is a promising strategy for the treatment of colon cancers.

Cancer-specific T helper shared and neo-epitopes uncovered by expression of the MHC class II master regulator CIITA.

We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral- and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses, which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important targets for cancer immunotherapy.

Upconversion nanoparticle platform for efficient dendritic cell antigen delivery and simultaneous tracking.

Upconversion nanoparticles (UCNPs) represent a group of NPs that can convert near-infrared (NIR) light into ultraviolet and visible light, thus possess deep tissue penetration power with less background fluorescence noise interference, and do not induce damage to biological tissues. Due to their unique optical properties and possibility for surface modification, UCNPs can be exploited for concomitant antigen delivery into dendritic cells (DCs) and monitoring by molecular imaging. In this study, we focus on the development of a nano-delivery platform targeting DCs for immunotherapy and simultaneous imaging. OVA 254-267 (OVA24) peptide antigen, harboring a CD8 T cell epitope, and Pam3CysSerLys4 (Pam3CSK4) adjuvant were chemically linked to the surface of UCNPs by amide condensation to stimulate DC maturation and antigen presentation. The OVA24-Pam3CSK4-UCNPs were thoroughly characterized and showed a homogeneous morphology and surface electronegativity, which promoted a good dispersion of the NPs. In vitro experiments demonstrated that OVA24-Pam3CSK4-UCNPs induced a strong immune response, including DC maturation, T cell activation, and proliferation, as well as interferon gamma (IFN-γ) production. In vivo, highly sensitive upconversion luminescence (UCL) imaging of OVA24-Pam3CSK4-UCNPs allowed tracking of UCNPs from the periphery to lymph nodes. In summary, OVA24-Pam3CSK4-UCNPs represent an effective tool for DC-based immunotherapy.

Combinatorial therapeutic approaches of photodynamic therapy and immune checkpoint blockade for colon cancer treatment.

Photodynamic therapy (PDT) has shown impressive therapeutic effects on various types of cancers by reactive oxygen species (ROS) generation and induction of immune responses. However, under certain conditions, the immune responses induced by PDT are not always sufficient to eradicate the remaining tumor cells. On the other hand, the photosensitizer indocyanine green (ICG) can mediate PDT under near-infrared (NIR) illumination, thereby enhancing the penetration depth of the excitation light into the tumor. We found that ICG is rapidly taken up in vitro by colorectal MC38 and CT26 tumor cells and it promotes PDT-mediated cell-killing effects. Our results furthermore revealed that ICG induces immunogenic cell death (ICD), as dendritic cells (DCs) were found to engulf ICG-PDT-treated tumor cells and undergo phenotypic maturation. ICG accumulated in tumors 2 h after administration, as measured by fluorescence and photoacoustic imaging. Considering the advantages of ICG as a photosensitizer, we sought to design a therapy that combines PDT and immune checkpoint blockade to maximize tumor control. To this end, a 25% thermosensitive polymer 407 hydrogel was included as a co-delivery platform for this treatment scheme. NIR-PDT under 808 nm irradiation in combination with cytotoxic T-lymphocyte-associated protein 4 (CTLA4)/programmed death-ligand 1 (PD-L1) checkpoint blockade prolonged survival rate of colorectal tumor-bearing mice by inducing a series of immune responses, like the phagocytosis of tumor debris by macrophages and DCs, and induction of acute inflammation, leukocyte infiltration, maturation and activation of DCs. Altogether, our work presents a NIR-triggered PDT strategy in combination with immune checkpoint blockade. Compared to a single treatment, the combination treatment increased efficiency to inhibit solid tumor growth and improved the survival rate of tumor-bearing mice.

A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection.

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8+ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8+ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8+ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

Lipid A analog CRX-527 conjugated to synthetic peptides enhances vaccination efficacy and tumor control.

Adjuvants play a determinant role in cancer vaccination by optimally activating APCs and shaping the T cell response. Bacterial-derived lipid A is one of the most potent immune-stimulators known, and is recognized via Toll-like receptor 4 (TLR4). In this study, we explore the use of the synthetic, non-toxic, lipid A analog CRX-527 as an adjuvant for peptide cancer vaccines. This well-defined adjuvant was covalently conjugated to antigenic peptides as a strategy to improve vaccine efficacy. We show that coupling of this TLR4 agonist to peptide antigens improves vaccine uptake by dendritic cells (DCs), maturation of DCs and T cell activation in vitro, and stimulates DC migration and functional T cell priming in vivo. This translates into enhanced tumor protection upon prophylactic and therapeutic vaccination via intradermal injection against B16-OVA melanoma and HPV-related TC1 tumors. These results highlight the potential of CRX-527 as an adjuvant for molecularly defined cancer vaccines, and support the design of adjuvant-peptide conjugates as a strategy to optimize vaccine formulation.

Pyroptosis-inducing active caspase-1 as a genetic adjuvant in anti-cancer DNA vaccination.

Pyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase-1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory cytokines such as Interleukin-1 beta (IL-1ß). Here, we evaluated whether pyroptosis could be exploited in DNA vaccination by incorporating a constitutively active variant of caspase-1 to the antigen-expressing DNA. In vitro, transfection with constitutively active caspase-1 DNA induced pro-IL-1ß maturation and IL-1ß release as well as gasdermin D-dependent cell death. To test active caspase-1 as a genetic adjuvant for the induction of antigen-specific T cell responses, mice were vaccinated intradermally with a DNA vaccine consisting of the active caspase-1 plasmid together with a plasmid encoding an ovalbumin-derived CD8 T cell epitope. Active caspase-1 accelerated and amplified antigen-specific CD8 T cell responses when administered simultaneously with the DNA vaccine at an equimolar dose. Moreover, upon challenge with melanoma cells expressing ovalbumin, mice vaccinated with the antigen vaccine adjuvanted with active caspase-1 showed significantly better survival compared to the non-adjuvanted group. In conclusion, we have developed a novel genetic adjuvant that for the first time employs the pyroptosis pathway to improve DNA vaccination against cancer.

Cost-minimisation analysis of rectal cancer neoadjuvant chemoradiotherapy based on fluoropyrimidines (capecitabine versus 5-fluorouracil).

OBJECTIVES: The current standard treatment for patients with rectal cancer stage II-III is neoadjuvant chemoradiotherapy followed by surgery. Neoadjuvant chemoradiotherapy can be performed with 5-fluorouracil (5-FU) or capecitabine (CPC) considered to be equivalent therapies. Medication cost is higher for CPC than for 5-FU, however, the administration of continuous 5-FU intravenous infusion is related to other costs such as those associated with outpatient facilities or central venous catheter insertion. METHODS: This retrospective study analysed the direct sanitary costs associated with the treatments and their complications from a hospital perspective. Costs in patients treated with 5-FU or CPC were measured between January 2010 and July 2018 at Mataró Hospital. The aim of this study was to perform a cost-minimisation analysis between the two treatments. We aimed to assess the cost associated with the complications related to each drug and the economic impact of applying the most efficient option. RESULTS: Ninety-eight patients were analysed: 32 were treated with CPC and 66 with 5-FU. Treatment cost was significantly higher for 5-FU than for CPC (2560.86±99.17 and 563.10±9.52 respectively, P=0.0001). No significant differences were found in the costs associated with treatment complications between groups (148.21±934.91 and 41.41±102.50 euros respectively, P=0.322). CONCLUSIONS: Considering the clinical equivalence shown in the available trials and previous reviews, the most efficient treatment is neoadjuvant chemoradiotherapy with CPC. Complications associated with the treatments did not significantly modify these results. Other studies gave similar results both in the neoadjuvant and adjuvant context, reaffirmed in this study.

Bioorthogonal protein labelling enables the study of antigen processing of citrullinated and carbamylated auto-antigens.

Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation.

Distinct antigen uptake receptors route to the same storage compartments for cross-presentation in dendritic cells.

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.

Autophagy regulates long-term cross-presentation by murine dendritic cells.

Autophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endolysosomal compartments and thereby sustain MHCI antigen cross-presentation to CD8+ T cells. In the current study, we investigated the role of autophagy in long-term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5-/- DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross-presentation. This augmented antigen cross-presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long-term MHCI cross-presentation.

Immunogenicity of rat-neu+ mouse mammary tumours determines the T cell-dependent therapeutic efficacy of anti-neu monoclonal antibody treatment.

The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2+ breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu+ tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours.

Effective chemoimmunotherapy by co-delivery of doxorubicin and immune adjuvants in biodegradable nanoparticles.

Chemoimmunotherapy is an emerging combinatorial modality for the treatment of cancers resistant to common first-line therapies, such as chemotherapy and checkpoint blockade immunotherapy. We used biodegradable nanoparticles as delivery vehicles for local, slow and sustained release of doxorubicin, two immune adjuvants and one chemokine for the treatment of resistant solid tumors. Methods: Bio-compatible poly(lactic-co-glycolic acid)-PEG nanoparticles were synthesized in an oil/water emulsion, using a solvent evaporation-extraction method. The nanoparticles were loaded with a NIR-dye for theranostic purposes, doxorubicin cytostatic agent, poly (I:C) and R848 immune adjuvants and CCL20 chemokine. After physicochemical and in vitro characterization the nanoparticles therapeutic efficacy were carried-out on established, highly aggressive and treatment resistant TC-1 lung carcinoma and MC-38 colon adenocarcinoma models in vivo. Results: The yielded nanoparticles average size was 180 nm and -14 mV surface charge. The combined treatment with all compounds was significantly superior than separate compounds and the compounds nanoparticle encapsulation was required for effective tumor control in vivo. The mechanistic studies confirmed strong induction of circulating cancer specific T cells upon combined treatment in blood. Analysis of the tumor microenvironment revealed a significant increase of infiltrating leukocytes upon treatment. Conclusion: The multi-drug loaded nanoparticles mediated delivery of chemoimmunotherapy exhibited excellent therapeutic efficacy gain on two treatment resistant cancer models and is a potent candidate strategy to improve cancer therapy of solid tumors resistant to first-line therapies.

A poly-neoantigen DNA vaccine synergizes with PD-1 blockade to induce T cell-mediated tumor control.

The combination of immune-stimulating strategies has the potency to improve immunotherapy of cancer. Vaccination against neoepitopes derived from patient tumor material can generate tumor-specific T cell immunity, which could reinforce the efficacy of checkpoint inhibitor therapies such as anti-PD-1 treatment. DNA vaccination is a versatile platform that allows the inclusion of multiple neoantigen-coding sequences in a single formulation and therefore represents an ideal platform for neoantigen vaccination. We developed an anti-tumor vaccine based on a synthetic DNA vector designed to contain multiple cancer-specific epitopes in tandem. The DNA vector encoded a fusion gene consisting of three neoepitopes derived from the mouse colorectal tumor MC38 and their natural flanking sequences as 40 amino acid stretches. In addition, we incorporated as reporter epitopes the helper and CTL epitope sequences of ovalbumin. The poly-neoantigen DNA vaccine elicited T cell responses to all three neoantigens and induced functional CD8 and CD4 T cell responses to the reporter antigen ovalbumin after intradermal injection in mice. The DNA vaccine was effective in preventing outgrowth of B16 melanoma expressing ovalbumin in a prophylactic setting. Moreover, the combination of therapeutic DNA vaccination and anti-PD-1 treatment was synergistic in controlling MC38 tumor growth whereas individual treatments did not succeed. These data demonstrate the potential of DNA vaccination to target multiple neoepitopes in a single formulation and highlight the cooperation between vaccine-based and checkpoint blockade immunotherapies for the successful eradication of established tumors.

PD-L1 blockade engages tumor-infiltrating lymphocytes to co-express targetable activating and inhibitory receptors.

BACKGROUND: The clinical benefit of immunotherapeutic approaches against cancer has been well established although complete responses are only observed in a minority of patients. Combination immunotherapy offers an attractive avenue to develop more effective cancer therapies by improving the efficacy and duration of the tumor-specific T-cell response. Here, we aimed at deciphering the mechanisms governing the response to PD-1/PD-L1 checkpoint blockade to support the rational design of combination immunotherapy. METHODS: Mice bearing subcutaneous MC-38 tumors were treated with blocking PD-L1 antibodies. To establish high-dimensional immune signatures of immunotherapy-specific responses, the tumor microenvironment was analyzed by CyTOF mass cytometry using 38 cellular markers. Findings were further examined and validated by flow cytometry and by functional in vivo experiments. Immune profiling was extended to the tumor microenvironment of colorectal cancer patients. RESULTS: PD-L1 blockade induced selectively the expansion of tumor-infiltrating CD4+ and CD8+ T-cell subsets, co-expressing both activating (ICOS) and inhibitory (LAG-3, PD-1) molecules. By therapeutically co-targeting these molecules on the TAI cell subsets in vivo by agonistic and antagonist antibodies, we were able to enhance PD-L1 blockade therapy as evidenced by an increased number of TAI cells within the tumor micro-environment and improved tumor protection. Moreover, TAI cells were also found in the tumor-microenvironment of colorectal cancer patients. CONCLUSIONS: This study shows the presence of T cell subsets in the tumor micro-environment expressing both activating and inhibitory receptors. These TAI cells can be targeted by combined immunotherapy leading to improved survival.