CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours

Abstract

Most clinically applied cancer immunotherapies rely on the ability of CD8⁺ cytolytic T cells to directly recognize and kill tumour cells^1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment^4-6. The ability of CD4⁺ effector cells to contribute to antitumour immunity independently of CD8⁺ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified^7-10. Here, we describe a mechanism whereby a small number of CD4⁺ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8⁺ T cell targeting. The CD4⁺ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II⁺CD11c⁺ antigen-presenting cells. We show that T helper type 1 cell-directed CD4⁺ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4⁺ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4⁺ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8⁺ T cells and natural killer cells and advance cancer immunotherapies.

Fig. 1. A small population of CD4⁺ effector T cells can eradicate MHC-deficient and IFN-unresponsive melanomas that resist destruction by CD8⁺ cytotoxic T cells.

a, Density of CD8⁺ T cells infiltrating the tumour centre and the invasive margin of 20 human melanoma skin metastases and corresponding MHC-I and MHC-II expression, categorized into high, intermediate (int) and low expression. b, UMAP of single-cell transcriptomes from an extra set of 20 melanoma metastases in skin (n = 5), subcutis (n = 4) and lymph nodes (n = 11) annotated for melanoma, immune and stromal cell phenotypes. c,d, MHC-I (c) and MHC-II (d) gene set expression in single melanoma cells. e, ICB therapy responders in patients with high, intermediate and low MHC-I expression on melanoma cells. f, Structure of recombinant adenovirus Ad-PT. g, Experimental protocol for ACT immunotherapy of established tumours in mice (Cy, C, cyclophosphamide; V, Ad-PT; T, TCRtg Pmel-1 CD8⁺ or TRP-1 CD4⁺ T cells; I, innate stimuli, polyI:C and CpG) and time point for flow cytometric analyses. h,i, Kaplan–Meier survival curves of mice bearing established B16 melanomas and treated either with CD4 ACT or CD8 ACT (h) or with the indicated components of the CD4 ACT protocol (i). NT, non-treated; CR, complete responders. i, **P = 0.0084. j,k, Immune cell composition (n = 2 biologically independent samples) (j) and phenotype of endogenous and transferred (VT, CVTI, right columns) CD4⁺ T cells (k) in tumours treated as indicated (mean ± s.e.m. from n = 4 biologically independent samples). l–o, Graphical representation of the immune cell interaction phenotypes (left) and Kaplan–Meier survival curves (right) of mice bearing established Ciita-KO (l), Trp1-KO (m), CRISPR-ctrl (n) or Jak1-KO (o) melanomas and treated as indicated. o, ****P < 0.0001. p, Immune cell composition of tumours treated as indicated (mean ± s.e.m. from four biologically independent samples). q, Structure of recombinant adenovirus Ad-OVA. r, Graphical representation of the immune cell interaction phenotype of ovalbumin-expressing HCmel12 Jak1-KO cells (left) and Kaplan–Meier survival curves (right) of mice bearing established melanomas treated as indicated. ****P < 0.0001. Survival was statistically compared using a log-rank Mantel–Cox test. NS, not significant. Source Data

Fig. 2. CD4⁺ effector T cells interact with MHC-II-expressing CD11c⁺ antigen-presenting cells in clusters within the invasive tumour margin.

a,d, Experimental protocols to assess the distribution of adoptively transferred T cells (left) and graphics depicting the invasive tumour margin (right) of CRISPR-ctrl (a) or Jak1-KO (d) tumours. b,e, Arrest coefficient and mean speed of adoptively transferred Venus⁺ Pmel-1 CD8⁺ (red) and eGFP⁺ TRP-1 CD4⁺ T cells (green) in the stromal (S) and tumoural (T) compartment at the invasive margin (bars indicate the median) of CRISPR-ctrl (b) or Jak1-KO (e) tumours (75–842 cells examined from three independent experiments; ****P < 0.0001, ***P = 0.0007, **P = 0.0068, CD4⁺ versus CD8⁺ in stroma *P = 0.0204, CD4⁺ in stroma versus CD8⁺ in tumour *P = 0.0107 using a Kruskal–Wallis test with Dunn’s multiple comparison test). c,f, Representative intravital microscopic images (scale bars, 100 µm) and insets exemplifying 450 s motion tracks of Venus⁺ Pmel-1 CD8⁺ and eGFP⁺ TRP-1 CD4⁺ T cells at the stromal (S) and tumoural (T) area of the invasive tumour margin of CRISPR-ctrl (c) or Jak1-KO (f) melanomas. g, Experimental protocol to assess antigen-dependent interactions between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c⁺ immune cells. h, Arrest coefficient, mean speed and relative contact duration between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c-Venus cells (the bars indicate the median; 43–132 cells examined from three independent experiments; ****P < 0.0001, **P = 0.0022 with a Mann–Whitney U-test). i, Representative motion tracks of eGFP⁺ TRP-1 CD4⁺ T cells interacting with CD11c-Venus cells in CRISPR-ctrl and Trp1-KO melanomas. Scale bars, 20 µm. j, Experimental protocol to assess the impact of MHC-II blockade on antigen-dependent interactions between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c⁺ immune cells. k, Arrest coefficient, mean speed and relative contact duration between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c-Venus cells (the bars indicate the median; 203–273 cells examined from three independent experiments; ****P < 0.0001, ***P = 0.0003, with a Mann–Whitney U-test). l, Representative motion tracks of eGFP⁺ TRP-1 CD4⁺ T cells interacting with CD11c-Venus cells in CRISPR-ctrl Tyr-KO tumours with and without MHC-II blockade. Scale bars, 20 µm. Data were pooled from at least three independent mice and groups statistically compared using a one-way ANOVA with Tukey post hoc. Source Data

Fig. 3. CD4⁺ effector T cells and innate immune stimulation promote the recruitment and IFN-dependent activation of monocytes to eradicate established tumours.

a, Experimental protocol for scRNA-seq analyses of tumour-infiltrating CD11b⁺ Ly6G⁻ cells. b, Visualization and dimensionality reduction of single-cell transcriptomes from CD4 ACT-treated and non-treated (NT) mice using UMAP. c,d, UMAP plots with cell types assigned using SingleR (c) and z-score for the hallmark IFN gamma response gene set (MSigDB) of each cell (d). e, RNA velocity projected on UMAP plots for monocytes and macrophages (Mono–macs) of CD4 ACT-treated tumours. Arrows point towards the predicted course of cell maturation dynamics. Arrow sizes indicate the strength of predicted directionality. f, Immune cell composition of HCmel12 tumours treated as indicated (mean ± s.e.m. from n = 6 biologically independent samples). g,j, Experimental treatment protocol to investigate the impact of innate stimuli (g) or IFNγ-blockade (j) on myeloid cell activation and tumour control. h,k, Percentage of intratumoural iNOS⁺ mono–macs and neutrophils (mean ± s.e.m. from n = 5–7 biologically independent samples). h, ****P < 0.0001, *P = 0.0111, ***P = 0.0005; k, ****P < 0.0001, ***P = 0.0002). i,l, Kaplan–Meier survival curves of mice bearing established HCmel12 CRISPR-ctrl tumours, treated as indicated (i, ****P < 0.0001; l, **P = 0.0053). Means between groups were statistically compared using a one-way ANOVA with Tukey post hoc. Survival was statistically compared using log-rank Mantel–Cox test. NT, non-treated; C, cyclophosphamide; V, Ad-PT; T, TCRtg TRP-1 CD4⁺ T cells; I, innate stimuli, polyI:C and CpG; CR, complete responders. Source Data

Fig. 4. CD4⁺ effector T cells cooperate with activated iNOS-expressing tumouricidal monocytes and macrophages to orchestrate remote inflammatory cell death of MHC-deficient and IFN-unresponsive tumours.

a, Graphical representation of interaction phenotypes of indicated HCmel12 variants. b, Experimental treatment protocol to study the impact of chemical iNOS inhibition using L-NIL on CD4 ACT-mediated tumour control. c, Kaplan–Meier survival graphs of mice bearing established HCmel12 Ciita-KO melanomas (left) or HCmel12 Jak1-KO melanomas (right) and treated as indicated (NT, non-treated; CR, complete responders; **P = 0.0033). Survival was statistically compared using a log-rank Mantel–Cox test. Means between groups were statistically compared using a one-way ANOVA with Tukey post hoc. d, Experimental protocol to assess the ability of the inflammatory mediators TNF, IFNγ and the nitric oxide donor SNAP to induce melanoma cell death. e,f, Percentage of cell death in mouse (e) and human (f) melanoma cells treated as indicated (mean ± s.e.m. from 2–3 technical replicates). g, Graphical summary of inflammatory cell death induction of MHC-deficient and IFN-unresponsive tumours by CD4⁺ T cells in cooperation with iNOS-expressing myeloid cells. Source Data

Extended Data Fig. 1. Landscapes of MHC expression, distribution of tumour infiltrating CD8+ T cells, and single cell transcriptomes of human melanoma metastases.

a, Representative immunohistochemical stains of human melanoma skin metastases with high, intermediate and low expression of MHC-I. b, Left: Overviews of representative immunohistochemistry stainings for CD8 and magnifications of 0.1 mm² squares in the tumour center and at the tumour invasive margins that were evaluated for the number of infiltrating CD8+ T cells; Right: graphical illustration of the three distinct patterns of immune infiltration observed. c, Representative immunohistochemical stains of human melanoma skin metastases with high, intermediate and low expression of MHC-II. d, MHC-I and (e) MHC-II gene set expression in single melanoma cells of an additional set of human melanoma metastases in skin (n = 5), subcutis (n = 4), and lymph nodes (n = 11), categorised into ICB therapy responders and non-responders. Median MHC-I gene set expression was used to categorize tumours according to high (>0.75), intermediate (0.25 - 0.75) and low (<0.25) expression levels. f, Uniform manifold approximation and projection (UMAP) clustering of the immune cell compartment in human melanomas annotated as indicated. g, MHC-I (top) and MHC-II (bottom) gene set expression in the indicated immune cell subpopulations.

Extended Data Fig. 2. Establishment of an experimentally tractable adoptive cell transfer model to compare CD4+ and CD8+ T-cell effector functions against tumours and eradication of established MHC-II-deficient melanomas through indirect antigen recognition on MHC-II+ tumour-infiltrating immune cells.

a, Experimental protocol to assess the in vivo expansion of adoptively transferred CD8+ and CD4+ T cells. b, Representative flow cytometric contour plots with 0.5 x 10⁶ transferred cells (top) and quantitation of Pmel-1 CD8+ and TRP-1 CD4+ TCRtg T-cell expansion in peripheral blood, lymph nodes and spleen 7 days after ACT (bottom, mean ± SEM from n = 2-4 biologically independent samples; ****p < 0.0001, **p = 0.0010, *p = 0.0146 by one-way ANOVA with Tukey’s post-hoc test). c,e, Experimental protocols for adoptive cell transfer (ACT) immunotherapy of established tumours in mice and flow cytometric analyses of tumour-infiltrating immune cells. d,f, Individual tumour growth curves of established B16 melanomas treated as indicated. g, t-SNE heatmaps of multiparametric flow cytometry for B16 melanoma single cell suspensions showing the indicated markers (top) and corresponding annotation (bottom) of TRP-1 CD4+ T-cells (GFP⁺), immature monocytes (CD11b⁺ Ly6C^hi), mature monocytes (CD11b⁺ Ly6C^lo), mature macrophages (CD11b⁺ F4/80⁺), dendritic cells (CD11b⁺ MHC-II⁺ CD11c⁺), endogenous lymphocytes (CD11b^- CD11c^-), and neutrophils (CD11b⁺ Ly6G⁺). h, Annotated t-SNE plots quantifying the immune cell composition of B16 melanomas treated as indicated. i, Representative flow cytometric contour plots for the phenotyping of endogenous and transferred (VT, CVTI, green) CD4+ T-cells from mice treated as indicated. j, Representative flow cytometric histograms for MHC-II expression on indicated melanoma cells cultivated in the presence or absence of IFNγ. k, Representative western blot analysis for TRP-1 expression for the indicated melanoma cells (n = 2 biologically independent samples). Beta-actin was used as a loading control. For uncropped images see source data table. l, Graphical representation of direct and (m) indirect recognition of melanoma cells by CD4+ T-cells (left) and representative flow cytometry histograms showing IFNγ⁺ TRP-1 CD4+ T-cells following stimulation by the indicated melanoma cells (right). n, Experimental protocol and (o,p) individual tumour growth curves of established B16 melanomas treated as indicated. q, Injection of a tumour cell mixture consisting of ~75% HCmel12 CRISPR-ctrl cells and ~25% HCmel12 Trp1-KO cells (top) and treatment protocol (bottom). r, Individual tumour growth curves of mice bearing established melanomas and treated as indicated (left) and proportion of HCmel12 CRISPR-ctrl and HCmel12 Trp1-KO cells from escaping tumours (right). s, Experimental treatment protocol for depletion of CD8+ T-cells during CD4 ACT. t, Individual tumour growth curves and Kaplan-Meier survival graph of mice bearing established HCmel12 CRISPR-ctrl melanomas treated as indicated. Means between groups were statistically compared using one-way ANOVA with Tukey’s post-hoc test. Survival was statistically compared using a log-rank Mantel-Cox test. NT, non-treated; C, cyclophosphamide; V, Ad-PT; T, TCRtg TRP-1 CD4+ T-cells; I, innate stimuli, polyI:C and CpG; CR, complete responders. Source Data

Extended Data Fig. 3. Comparative evaluation of CD4+ and CD8+ T-cell effector functions against IFN-unresponsive tumours lacking MHC-I and MHC-II.

a, Representative flow cytometric histograms for MHC-I and MHC-II expression of indicated melanoma cells in the presence or absence of IFNγ. b, Graphical representation of the interaction phenotype of the indicated melanoma cells (left) and experimental treatment protocol (right). c, Individual tumour growth curves of mice bearing established melanomas and treated as indicated. d, t-SNE heatmaps of multiparametric flow cytometry for HCmel12 melanoma single cell suspensions showing the indicated markers (top) and corresponding annotation (bottom) of TRP-1 CD4+ T-cells (GFP⁺), Pmel-1 CD8+ T-cells (Venus⁺), immature monocytes (CD11b⁺ Ly6C^hi), mature monocytes (CD11b⁺ Ly6c^lo), mature macrophages (CD11b⁺ F4/80⁺), dendritic cells (CD11b⁺ MHC-II⁺ CD11c⁺ F4/80^-), endogenous lymphocytes (CD11b^- CD11c^-), and neutrophils (CD11b⁺ Ly6G⁺). e, Quantification of the immune cell composition of HCmel12 CRISPR-ctrl and Jak1-KO tumours treated as indicated. f, Recombinant adenovirus Ad-OVA (top) and experimental protocol (bottom) to assess the in vivo expansion of adoptively transferred ovalbumin-specific CD8+ and CD4+ T-cells. g, Representative flow cytometric contour plots with 0.5 x 10⁶ transferred T-cells and quantitation of OT-I CD8+ and OT-II CD4+ TCRtg T-cell expansion in peripheral blood 7 days after ACT (mean ± SEM from n = 9 biologically independent samples, ****p < 0.0001 using a two-tailed paired t-test). h, Experimental protocol and (i) individual tumour growth curves of mice bearing established HCmel12-OVA Jak1-KO tumours, treated as indicated. Means between groups were statistically compared using a two-tailed paired t-test. Source Data

Extended Data Fig. 4. CD4+ effector T-cells show a different spatial distribution and migratory behaviour in tumour tissues when compared to CD8+ effector T-cells.

a, Macroscopic phenotype and graphical representation for the generation of amelanotic HCmel12 CRISPR-ctrl or Jak1-KO Tyr-KO tagBFP cell lines (top) and experimental treatment protocols (bottom). b,c, Representative fluorescence image for the distributions of Venus+ Pmel-1 CD8+ T-cells and eGFP+ TRP-1 CD4+ T-cells in indicated HCmel12 variants. d,e, Diagrammatic representation of the Venus+ Pmel-1 CD8+ T-cell and eGFP+ TRP-1 CD4+ T-cell distribution in a whole tumour cryosection of HCmel12 Tyr-KO CRISPR-ctrl (d) or Jak1-KO (e) melanomas (left) and corresponding quantitation (right) at the invasive margin (IM) and in the tumour centre (TC) (mean ± SEM for n = 6-7 biologically independent samples; CD4+ IM vs CD4+ TC **p = 0.0051, CD4+ IM vs CD8+ IM **p = 0.0035, CD4+ TC vs CD8+ TC **p = 0.068 using a one-way ANOVA with Tukey post-hoc). f, Diagrammatic representation and experimental protocol for treatment of HCmel12 Jak1-KO Tyr-KO OVA-tagBFP cells. g, Cell density (mean ± SEM from 7 biologically independent samples; CD4+ IM vs CD8 IM * p = 0.0122, CD4+ TC vs CD8+ IM * p = 0.0173, CD8 TC vs CD8 IM *p = 0.121 using a one-way ANOVA with Tukey post-hoc) at the IM and in the TC and arrest coefficient (***p = 0.0005, **p = 0.0046 using a Kruskal-Wallis test with Dunn’s multiple comparison test) and mean speed (**p = 0.0012, *p = 0.0499 using a Kruskal-Wallis test with Dunn’s multiple comparison test) of adoptively transferred of adoptively transferred Venus+ OT-I CD8+ and dsRed+ OT-II CD4+ T-cells in the stromal (S) and tumoural (T) compartment at the invasive margin (11-794 cells examined from 3 independent experiments; bar indicates the median). Source Data

Extended Data Fig. 5. CD4+ effector T-cells cluster with MHC-II-expressing CD11c+ immune cells at the invasive margin of mouse melanomas.

a, Graphical representation of the interaction phenotype of the indicated HCmel12 variants and (b) experimental protocol to study antigen-specific interactions between eGFP+ TRP-1 CD4+ T-cells and CD11c+ cells in CD11c-Venus mice. c,d, Representative immunofluorescence images of MHC-II-stained cryosections from a (c) CRISPR-ctrl and a (d) Trp1-KO melanoma (mean ± SEM from n = 3-5 biologically independent samples). The dashed lines indicate the tumour border. e, Diagrammatic representation of MHC-II expression and interactions between eGFP+ TRP-1 CD4+ T-cells and CD11c-Venus antigen-presenting cells in CRISPR-ctrl and Trp1-KO melanomas. f, Density of eGFP+ TRP-1 CD4+ T-cells at the invasive margin (IM) and in the tumour centre (TC) of indicated tumours (mean ± SEM from n = 3-5 biologically independent samples, **p = 0.0037, CRISPR-ctrl IM vs Trp1-KO IM *p = 0.0267, CRISPR-ctrl IM vs TC *p = 0.0112). Means between groups were statistically compared using a one-way ANOVA with Tukey post-hoc. g,h Intravital 2P-microscopy images of three eGFP+ TRP-1 CD4+ T-cells and their distance to CD11c-Venus cells over time. Source Data

Extended Data Fig. 6. CD4+ effector T-cells cluster with antigen-presenting dendritic cells and macrophages at the invasive margin of a human melanoma.

Multiple iterative labeling by antibody neodeposition (MILAN) of a human melanoma obtained from (Pozniak, J., et al., 2022). An overview (a) over the whole tumour and a selected area of the tumour margin (b) are shown with multiple label combinations selected from the published panel. Insets (1-3) show exemplary sites of CD4+ T-cell juxtaposition with different myeloid subtypes expressing MHC-II, CD11c, and/or CD68. c, combinatorial overlays of different T-cell markers (white) with myeloid cell markers (red) in the insets (1-3).

Extended Data Fig. 7. CD4 ACT therapy predominantly recruits immature monocytes into the tumour microenvironment and drives the acquisition of IFN-activated effector phenotypes.

a, Differentially expressed genes comparing samples from CD4 ACT-treated versus non-treated (NT) mice. Genes with –log Q-values >200 are shown in orange. b, Gene set enrichment analysis for the “GOBP_RESPONSE_TO_TYPE_I_INTERFERON” (top) and “GOBP_INTERFERON-GAMMA_MEDIATED_SIGNALING_PATHWAY” (bottom) gene sets. c, UMAP plots with Leiden clusters for monocytes and macrophages of CD4 ACT treated tumours. d, Corresponding expression levels and expression cell fractions of selected signature genes for the individual Leiden clusters and (e) pseudotime inference using slingshot. f, Heatmap of differentially expressed genes along the pseudotime trajectory of the indicated Leiden clusters representing the monocyte-macrophage (mono-mac) effector differentiation path. g, Experimental protocol (left), and t-SNE heatmaps of multiparametric flow cytometry for HCml12 melanoma single cell suspensions showing the indicated markers (right). h, Left: Corresponding annotation of immune of immature monocytes (CD11b+ Ly6Chi), mature monocytes (CD11b+ Ly6clo), mature macrophages (CD11b+ F4/80+ iNOS-), iNOS+ mono-macs (CD11b+ Ly6Chi iNOS+), dendritic cells (CD11b+ MHC-II+ CD11c+ F4/80-), endogenous lymphocytes (CD11b- CD11c-), and neutrophils (CD11b+ Ly6G+). Right: Annotated t-SNE plots quantifying the immune cell composition of HCmel12 melanomas at the indicated time points.

Extended Data Fig. 8. Robust IFNγ-dependent eradication of established melanomas requires local adjuvant innate immune stimulation.

a, Experimental treatment protocol to address the impact of innate stimuli on myeloid cell activation and tumour control. b, Cell density (left) and representative contour plots quantifying the relative iNOS expression (right) in Ly6C^hi mono-macs (NT vs CVTI *p = 0.0109) and neutrophils (NT vs CVTI *p = 0.0114, CVTI vs CVT *p = 0.0474) in tumours 5 days post-ACT, treated as indicated (mean ± SEM from n = 6-7 biologcally independent samples). c, Individual tumour growth curves in mice bearing established melanomas and treated as indicated. d, Experimental treatment protocol to address the impact of local innate stimuli on tumour control and (e) individual growth curves of mice bearing contralateral HCmel12 tumours, treated as indicated. f, Experimental treatment protocol to address the impact of IFNγ-blockade. g, Cell density (left) and relative iNOS expression (right) in Ly6C^hi mono-macs and neutrophils in tumours 5 days post-ACT, treated as indicated (mean ± SEM from n = 6-7 biologically independent samples, *p = 0.0109). h, Individual tumour growth curves of mice bearing established melanomas and treated as indicated. Means between groups were statistically compared using a one-way ANOVA with Tukey post-hoc. Source Data

Extended Data Fig. 9. Chemical iNOS inhibition and antibody-mediated cell depletion of monocytes and neutrophils in vivo as well as treatment with the nitric oxide donor SNAP in vitro suggest a role for iNOS expressing myeloid cells in the control of established MHC-deficient and IFN-unresponsive melanomas.

a,b, Individual tumour growth curves of established HCmel12 Ciita-KO or HCmel12 Jak1-KO melanomas treated as indicated (L-NIL, iNOS-inhibitor). c, Experimental treatment protocol for antibody-mediated depletion of neutrophils and inflammatory monocytes. d, Individual tumour growth curves of established melanomas treated as indicated. e, Left: Representative gating strategy to evaluate the depletion of monocytes and neutrophils. Right: Flow cytometric percentages of monocytes and neutrophils in the blood 5 and 12 days post-CD4 ACT (mean ± SEM from 4-6 biologically independent samples). f, Experimental protocol to assess the ability of the inflammatory mediators TNF, IFNγ and the nitric oxide donor SNAP to induce melanoma cell death. g, h, Representative flow cytometric contour plots to assess cell death of mouse and human melanomas treated as indicated. Source Data

Extended Data Fig. 10. Spatial organisation and dynamics of T-cell effector functions in tumour tissues.

a, Graphical representation of direct antigen recognition and induction of cytolytic cell death. CD8+ and CD4+ effector T-cells can recognise their antigens as peptide epitopes presented by MHC-molecules on tumour cell surfaces and initiate direct killing through the release of cytolytic granules. b, Graphical representation of indirect antigen recognition and remote induction of inflammatory cell death. CD4+ effector T-cells also efficiently recognise tumour antigen on the surface of antigen-presenting cells (APC) including monocyte-derived dendritic cells (Mo-DC) and engage tumouricidal effector cells of the monocyte-macrophage lineage (Mono-Mac effectors) to initiate indirect cell death through the release of pro-inflammatory mediators. c, Spatial organisation and dynamics of direct induction of cytolytic cell death. CD8+ effector T-cells briskly infiltrate tumour tissues, where they directly interact with tumour cells (left), while CD4+ effector T-cells directly interact with tumour cells mainly near the invasive margin (right). d, Spatial organisation and dynamics of remote induction of inflammatory cell death. CD4+ effector T-cells cluster locally at the tumour invasive margin, where they indirectly recognise tumour antigen phagocytosed, processed and presented by dendritic cells. Activated CD4+ T-cells secrete IFNγ leading to the recruitment and activation of monocytes into the tumour tissue. Recruited monocytes phenotypically develop along differentiations path towards IFN-activated antigen-presenting (monocyte-derived dendritic cells, Mo-DCs) and tumouricidal effector phenotypes (monocyte-macrophage effector cells, Mono-Mac effectors). Mo-DCs additionally activate CD4+ T-cells and amplify monocyte recruitment, activation and differentiation. Innate immune stimulation promotes the Th1-directed differentiation of CD4+ T-cells and increases the tumouricidal functions of Mono-Mac effectors. CD4+ T-cell-derived IFNγ sensitises IFN-responsive melanoma cells for TNF-induced cell death. Myeloid cell-derived nitric oxide (NO) contributes to inflammatory cell death of IFN-unresponsive melanoma cells. Taken together, the induction of remote inflammatory cell death by CD4+ T-cells and tumouricidal myeloid cells eradicate IFN-responsive as well as IFN-unresponsive, MHC-deficient tumours that evade direct recognition and cytolytic killing.