Background

Treatment with monocolonal anti-CD4 antibodies has long been used to deplete regulatory T cells (Tregs) and elicit antitumor immunity.1–3 In contrast to Treg-depleting antibodies, such as anti-CTLA-4, anti-CD25, or anti-CCR44 5 antibodies against CD4 completely deplete Foxp3+ Tregs in tumors and throughout tissues.6 Preclinical studies have shown that anti-CD4 elicits robust and persistent tumor Ag-specific CD8+ T cell responses in mouse tumor models as a monotherapy and in combination with immune checkpoint blockade (ICB).2 7 8 Such responses appear largely independent of CD4+ T cell help.9 10 Antibody-mediated CD4+ T cell depletion can also be achieved in humans, with a humanized defucosylated IgG1 anti-CD4 mAb affording disease stabilization and shrinkage in a small study of patients with advanced solid tumors.11 These results highlight the potency and feasibility of CD4 depletion therapy, both preclinically and clinically. However, the underlying mechanisms of anti-CD4 efficacy remain incompletely understood.

Though the success of anti-CD4 is widely attributed to the depletion of CD4+ Foxp3+ Tregs,2 3 12 the removal of other CD4-expressing cells may also play an important role. One potential mechanism is through the creation of homeostatic space. We and others have shown that host CD8+ T cells expand and increase expression of CD44 in mice treated with anti-CD4.8 9 Indeed, in lymphopenic RAG knockout mice, homeostatic proliferation is known to promote CD8+ T cell activation and reactivity against tumor antigens.13 14 Accordingly, T cell receptor (TCR) repertoire analysis in gastric cancer patients treated with anti-CD4 mAb revealed that pre-existing and new CD8+T cell clones expand in blood following anti-CD4 treatment. The degree of clonal expansion was correlated with the extent of CD4 depletion, which was speculated to depend on “space” created by CD4+ T cell removal.15 Homeostatic proliferation is dependent on IL-7 and IL-15,16 17 although it is unknown whether homeostatic space or associated cytokines support tumor-Ag specific CD8+ T cell priming during anti-CD4 treatment.

A second putative mechanism involves the removal of the Foxp3-negative CD4+ conventional (Tconv) cell b. Indeed, IL-10-producing CD4+T cells such as type 1 regulatory T cells (Tr1s)18 and CCR8+CD25+Foxp3− Tconv cells,19 both mediate tumor immune suppression. On the other hand, CD4+ Th1 cells which produce IFN-γ can clearly contribute to antitumor immunity.20 Anti-CD4 efficiently removes all Tconv cell subsets, although it is not known if this is beneficial or detrimental for the tumor-specific CD8+ T cell response. It is also unclear whether ICB treatments that preserve CD4+ cells elicit more robust or long-lived tumor-specific CD8+ T cell responses compared with anti-CD4 therapy.

Finally, anti-CD4 may function through targeting CD4-expressing antigen-presenting cells (APCs). Indeed, CD4 is expressed by subpopulations of macrophages and plasmacytoid dendritic cells (pDCs) and by most conventional type 2 DCs (cDC2s). cDC2s are known to prime CD4+ T cell responses, and targeted depletion of Tregs in Foxp3-DTR mice promotes cDC2 migration and generation of cytotoxic CD4+ T cell responses against melanoma.21 In tumor-bearing mice depleted of total CD4+ T cells, it is not known whether codepletion of CD4-expressing cDC2s may skew in favor of type 1 DCs (cDC1s) which promote CD8+ T cell responses.22

Here, we investigate the underlying mechanisms whereby anti-CD4 treatment induces CD8+ T cell responses against cancer. We compare anti-CD4 with dual ICB (anti-PD-1 + anti-CTLA-4) to define how these contexts afford different properties to de novo primed tumor Ag-specific CD8+ T cells. We also uncouple anti-CD4 efficacy from the creation of homeostatic space and the depletion of CD4-expressing APCs. Instead, we find that the removal of total CD4+ T cells—including both Tregs and Tconv cells—is critical for priming robust and persistent CD8+ T cell responses in tumor-bearing hosts. Extending relevance to human cancer, we show that CD4+ subsets with immunosuppressive characteristics are dominant in melanoma tumors from patients even following neoadjuvant ICB therapy. This work underscores the importance of depleting total CD4+ T cells in future cancer clinical trials.

ResultsAnti-CD4 is more potent than dual ICB at inducing tumor-specific CD8+ effector T cells that disseminate and persist as memory

We and others have shown that anti-CD4 treatment breaks CD8+ T cell tolerance to shared melanoma/melanocyte antigens in B16 tumor-bearing mice.2 23 While primary B16 tumors are resistant to anti-CD4 treatment, mice generate systemic CD8+ T cell responses which mediate concomitant immunity against melanoma rechallenge on the opposite flank.2 B16 is a poorly immunogenic tumor that is also resistant to anti-CTLA-4 and anti-PD-1 dual ICB therapy.24 Compared with anti-CD4 treatment, dual ICB afforded better primary B16 tumor growth control (figure 1A,B). Thus, we sought to determine if dual ICB also induced stronger CD8+ T cell priming against a tumor-expressed antigen. Tumor-specific CD8+ T cell responses were tracked by adoptively transferring 104 naïve, congenically marked (Thy1.1+) TCR transgenic ‘pmel’ cells specific for gp10025-33, as we have previously described.7

Figure 1

Priming, dissemination, and persistence of tumor Ag-specific CD8+ T cells is induced by treatment with anti-CD4 but not dual ICB (anti-CTLA-4 + anti-PD-1). (A) Naïve pmel CD8+T cells were transferred into mice 1 day prior to implanting intradermal B16 tumors. Mice were untreated or treated with either anti-CD4 or anti-PD1+ anti-CTLA-4 on days 4 and 10 after tumor inoculation. Flow cytometric analysis was performed on day 12 after tumor injections. (B) Tumor growth curves. (C) Proportion of CD8+ T cells in tumors on day 12, gated out of live lymphocytes. (D) Proportion of CD44hi Thy1.1+ pmel cells, gated out of live CD8+ T cells, in the indicated tissues on day 12, compared between treatment groups. (E) Normalized (relative to anti-CD4) proportion of CD8+ T cells in skin (gated on live lymphocytes) on day 12. (F) T cells from day 12 lymph nodes were restimulated for intracellular cytokine staining, and proportions of IFNγ,TNF-α, Gzmb, and IL-2-producing cells (gated on live CD8+Thy1.1+ pmel cells) were analyzed; gated on live CD8+Thy1.1+ pmel cells. (G) CD44hi Thy1.1+ pmel cells gated out of live CD8+ T cells, across tissues, at a memory timepoint (30 days after tumor excision surgery). (H) Proportion of CD8+ T cells in skin 30 days postsurgery; gated out of live CD45+lymphocytes. Each experiment was repeated at least two times with similar results and n≥4 mice per group; n.s. signifies a p>0.05. Data are pooled from two (C–F) or three (G, H) experiments. Each symbol represents an individual mouse, and flow plots depict representative mice; Bars signify the mean with error bars depicting SEM. For experiments with two groups, a paired t-test was used to determine statistical significance, and for those with more than two groups, a one-way ANOVA with multiple comparisons was used. ANOVA, analysis of variance; ICB, immune checkpoint blockade; TDLN, tumor draining lymph node; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

On day 12, we found that dual ICB and anti-CD4 gave rise to similar proportions of pmel cells and total CD8+ T cells in B16 tumors (figure 1C,D). However, in tumor draining lymph nodes (TDLNs), anti-CD4 induced threefold higher proportions of pmel cells compared with dual ICB (figure 1D). Similarly, in the spleen, where essentially no pmel cells accumulated without treatment, anti-CD4 induced a threefold larger proportion of pmel cells than dual ICB (figure 1D). Extending this analysis to mice-bearing MC38 tumors expressing OVA as a model tumor neoantigen, we also observed stronger priming of OT-1 CD8+ T cells in TDLNs and greater dissemination to spleen with anti-CD4 as compared with dual ICB (online supplemental figure 1). Further, in the B16 model, anti-CD4 induced robust pmel (and total CD8+ T cell) infiltration in the skin where cognate Ag is expressed, whereas dual ICB elicited a minimal skin response (figure 1D,E). Together, these results show that CD4 depletion drives stronger priming and dissemination of tumor Ag-specific CD8+ T cells than dual ICB.

It remained possible that CD8+ T cells induced by anti-CD4 were functionally inferior to those induced by dual ICB, especially due to the absence of CD4+ Tconv cells which promote CD8+ T cell memory.25 Interestingly, however, anti-CD4 and dual ICB induced similar proportions of pmel cells in TDLNs that were capable of coproducing IFN-γ and TNF-α (figure 1F). Moreover, while a significantly higher proportion of pmel cells from dual ICB-treated mice produced granzyme B (figure 1F), more pmel cells from anti-CD4 treated mice produced IL-2 (figure 1F), which is associated with memory potential.26 To assess whether CD8+ T cell responses primed by dual ICB treatment could generate memory, tumors were surgically excised, and pmel responses were assessed 30 days later. We have previously shown that anti-CD4 treated mice develop melanoma-associated vitiligo which promotes resident and circulating memory T cells that afford long-term melanoma protection in skin, lungs, liver, and lymph nodes.7 27 28 While dual ICB also induced vitiligo, the depigmentation was weaker and less widespread (online supplemental figure 2A). Moreover, in contrast to anti-CD4 treatment, pmel cells primed during dual ICB treatment largely failed to persist 30 days postsurgery (figure 1G,H), which similar to what we have previously shown in untreated tumor-excised mice.7 29 Among polyclonal CD44highCD62Llow CD8+ T cells in TDLNs, anti-CD4 induced a greater proportion of CD103+CD69+ TRM cells, of which a greater proportion were also CXCR6+CD127+ (online supplemental figure 2B). Together these data demonstrate that anti-CD4 and dual ICB induce distinct responses, with the absence of CD4+ T cells paradoxically promoting greater programming, dissemination, and persistence of tumor Ag-specific memory CD8+ T cells.

To better understand the intrinsic differences in CD8+ T cells primed by anti-CD4 versus dual ICB treatment, we also conducted single-cell RNA sequencing (scRNAseq) on endogenous Ag-experienced (CD44high CD62Llow/intermediate) CD8+ T cells sorted from day 12 TDLNs (online supplemental figure 3), with paired TCR-sequencing to define expanded clonotypes. UMAP projection of 5204 cells sorted from combined untreated, anti-CD4, and dual-ICB treated mice resolved 6 distinct clusters (figure 2A). The largest cluster, termed “C0-MPEC” resembled memory precursor effector cells (MPECs), expressing high Il7r and effector transcripts including Nkg7, Klrk1, and Klf2 (figure 2B). On the other hand, “C1-Stem-Like” had the highest levels of Tcf7, Ccr7, Sell, and Jun (figure 2B). “C2-Tpex” similarly expressed stem-associated transcripts, but also Slamf6, Pdcd1, Ctla4 and Lag3, as well as Ifng, Tnf, and Il2 (figure 2B), suggesting a progenitor exhausted state.30 “C3-Prolif-Eff” expressed proliferation-associated genes, such as Birc5, Stmn1 and Mcm3 as well as Gzma and Gzmb, whereas “C4-Exhausted” had the highest expression of Tox and high Mki67 and Il2rb (figure 2B). The smallest cluster, “C5-Recent-Act” had high Tnfrsf9 (4-1BB), Cd69, Il2ra, and Nr4a1 (figure 2B), indicative of activated T cells.31 32 Altogether, these TDLN clusters resembled known stages of CD8+ T cell activation previously described in the setting of cancer.32

Figure 2

Compared with dual ICB, anti-CD4 induces a more diverse proliferating clonal repertoire, a reduction in progenitor-exhausted cells, and an enhancement in TCF7-expressing memory precursors. Endogenous CD44hi CD62Llo CD8+ T cells and CD44hi Thy1.1+ pmel cells were hashtag-labeled, FACS sorted and pooled (see online supplemental figure 1) from tdLNs of five untreated, five anti-CD4-treated, and five dual ICB-treated mice on day 12. Hashtag sequences were used to identify cells from each treatment group. Gene expression was determined by single-cell RNA sequencing (scRNAseq) of 460, 325, and 460 pmel cells and 1436, 1250, and 1355 endogenous effector cells from the no treat, anti-CD4 and Dual ICB groups, respectively. Using the 10X Genomics platform. The 10X Cellranger VdJ pipeline was used to determine TCR α and β-chain CDR3 sequences. (A) UMAP plots displaying 5204 cells from the treatment groups combined, with both RNA and TCR sequencing. Each dot represents a single cell. (B) DotPlot depicting cluster-defining genes. (C) UMAP plots depicting clustering by treatment group. (D) Plots depicting clonally expanded endogenous CD8+ T cells, by respective treatment group, superimposed on the overall UMAP (in gray). Colors depict overall level of clonal expansion, as specified in the legend; frequency of expanded clonotypes in each of the treatment groups is shown at right. (E) UMAP plot of pmel cells (in green), by treatment group, superimposed on the overall umap; frequency of pmel cells in each of the clusters from (A) is shown, at right. (F) Pseudobulk analysis of gene expression comparing pmel cells from each of the three different treatment groups, depicting relative expression of effector and memory-associated transcripts. (G) Blended FeaturePlot illustrating the overlap between Tcf7 and Gzma expression across clusters. Heatmap depicts colors that represent the extent of overlapping expression of each transcript. The colors in the upper right-hand corner depict the cells with the highest expression of each transcript that are simultaneously overlapping. Colors closer to each axis depict inverse expression of each transcript. (H) Flow cytometry analysis of Tcf1 and Tbet expression on pmel cells taken from tdLNs of anti-CD4 versus dual-ICB treated mice on day 12. Data are pooled from two independent experiments with similar results; one-way ANOVA with multiple comparisons was used to determine statistical significance, with n.s. indicating a p>0.05. For A-G, the analysis was done once. ANOVA, analysis of variance; ICB, immune checkpoint blockade; TDLN, tumor draining lymph node. *p<0.05, **p<0.01, ****p<0.0001.

We next compared CD8+ T cell clustering and clonal expansion between the treatment groups (figure 2C). Whereas cells from untreated mice were limited to the left side of the UMAP in the C1-Stem-like, C2-Tpex, and C5-Recent-Act clusters, cells from anti-CD4 treated mice tended to populate the right of the UMAP in the C3-Prolif-Eff and C0-MPEC clusters. In contrast, cells from dual ICB mice were more intermediate in their distribution across all clusters in the UMAP, and cells from all three groups contributed to the C4-Exhausted cluster (figure 2C). Assessing the level of T cell clonal expansion between the groups, we found that untreated mice had the lowest frequency of clonotypes that had expanded and most of these were only minimally expanded (figure 2D). In contrast, anti-CD4 treatment resulted in the largest frequency of expanded clonotypes, which mainly fell within the C3-Prolif-Eff and C0-MPEC clusters (figure 2D). Dual ICB also induced expansion of clonotypes in these two clusters, with some very highly expanded clonotypes (figure 2D). Taken together, these results suggest that both anti-CD4 and dual ICB induce the proliferation and activation of effector cells and memory precursors in TDLNs, but that the response is more clonally diverse with anti-CD4 treatment.

In addition to endogenous polyclonal CD8+ T cells, we also sorted pmel cells for scRNAseq (figure 2E). Comparison across the treatment groups revealed that pmel cell UMAP clustering mirrored that of polyclonal antigen-experienced CD8+ T cells (figure 2E). Moreover, pseudobulk analysis of transcripts from pmel cells from untreated TDLNs revealed high stem-associated transcripts (eg, Tcf7 and Slamf6), and lower expression of effector transcripts (eg, Gzma, Klf2, Eomes, and Tbx21), whereas those from anti-CD4 treated mice had higher levels of activation and memory-associated markers including Cxcr3, Icos, Il2, as well as high Fabp5 and intermediate Tbx21 (figure 2F), which is associated with Trm formation.33 Interestingly, compared with dual ICB treated mice, pmel cells from anti-CD4 treated mice expressed higher Tcf7 and Slamf6, but lower Gzma (figure 2F), with Tcf7 and Gzma demonstrating inverse correlation (figure 2G). Flow cytometry confirmed a twofold higher expression of TCF-1, and twofold lower expression of Tbet in pmel cells from mice treated with anti-CD4 compared with dual ICB (figure 2H), consistent with the identification of clonally expanded MPEC populations in mice treated with anti-CD4 (figure 2D). These data are consistent with the interpretation that anti-CD4 promotes stem-like memory precursors, whereas dual ICB induces more differentiated effector T cells.

Anti-CD4 efficacy does not depend on creating homeostatic space but requires the depletion of CD4+ Tconv cells

Considering the unique ability of anti-CD4 to induce tumor-specific CD8+ T cell memory, we next sought to understand its underlying mechanisms of efficacy. We have previously shown that anti-CD4 treatment induces acute homeostatic proliferation, in association with CD44 expression by host endogenous CD8+ T cells, with a return to normal CD8+ T cell proportions within 2 weeks.9 To determine if homeostatic proliferation is required for tumor-specific CD8+ T cell priming, we instead treated with anti-CD4 beginning 14 days prior to inoculating B16 tumors and pmel cells, to afford a setting in which the window of homeostatic space had closed prior to the initiation of priming (figure 3A). Interestingly, tumor growth (figure 3B), and pmel cell proportions across TDLNs, spleen, and skin were similar in mice treated on day −14 as compared with day −1 (figure 3C), suggesting a lack of dependence on homeostatic space. As both IL-7 and IL-15 are known to drive CD8+ T cell homeostatic proliferation,16 17 we separately used monoclonal antibodies to the IL-7 receptor (CD127) or IL-15, to neutralize the action of these cytokines during anti-CD4 treatment. However, acute blockade of neither IL-7 nor IL-15 diminished pmel cell priming (figure 3D) or subsequent memory formation (online supplemental figure 4). Taken together, these results reveal that the creation of homeostatic space is not required for anti-CD4 treatment efficacy. In contrast, antibody blockade of IL-2 receptor subunits CD122 and CD25 abrogated pmel cell priming, revealing dependence on IL-2 (figure 3E). This was notable given the role of CD4+ helper T cells as dominant producers of IL-2,34 but consistent with our observation that pmel cells produce their own IL-2 in response to anti-CD4 treatment (figure 1F).

Figure 3

IL-2 is required, but homeostatic space, IL-7, and IL-15 are all dispensible, for tumor Ag specific CD8+ T cell priming during anti-CD4 therapy. (A) As depicted, for B, C, mice were either left treated, or treated with anti-CD4 beginning either 14 days or 1 day(s) prior to transfer of 104 naïve pmel cells and B16 tumor cell inoculation on day 0. Proportion of CD44hi Thy1.1+ pmel cells out of live CD8+ T cells was analyzed across tissues, twelve days post tumor inoculation. (B) Tumor sizes on day 12. (C) Proportions of CD44hi Thy1.1+ pmel cells (gated on live CD8+ T cells) were analyzed across tissues on day 12. For (D, E) Pmel cells and B16 tumors were transferred and implanted into mice and mice were treated with anti-CD4 in addition to either PBS or neutralizing antibodies against CD127 or IL-15 (D) or CD25 or CD122 (E) on days 4 and 10 after tumor inoculation. Representative flow cytometry plots of proportions of pmel Thy1.1+ cells (gated out of live CD8+ T cells) across tissues from each treatment group are depicted with bar graphs adjacent to them. Bar graphs show mean and SEM of data. All experiments were repeated at least twice with n≥3 mice per group; data from each panel are pooled from two independent experiments. Flow plots depict representative mice; bars represent means and error bars represent SEM. One-way ANOVA with multiple comparisons was used to determine statistical significance for experiments with more than two groups. Paired t-test was used to determine statistical significance in experiments with two groups; n.s. indicates p>0.05. ANOVA, analysis of variance; TDLN, tumor draining lymph node; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

As the primary rationale for treating with anti-CD4 is to deplete Tregs, we next examined whether targeted elimination of Foxp3+ Tregs was sufficient to induce a systemic CD8+ T cell response of the type afforded by anti-CD4. To selectively deplete Tregs, we administered diphtheria toxin (DT) to Foxp3-DTR mice and assessed the priming of pmel cells relative to wild-type (WT) untreated and CD4-depleted mice (figure 4A; online supplemental figure 5). While targeted Foxp3+ Treg depletion resulted in better control of primary tumors (online supplemental figure 6A), similar proportions of pmel cells accumulated in tumors of Treg-depleted and CD4-depleted mice (figure 4B). Furthermore, in TDLNs, Treg and CD4 depletion led to similar proportions of pmel cells (figure 4B), indicating that, in contrast to dual ICB treatment, Treg depletion is required for robust CD8+ T cell priming. However, targeted Foxp3+ Treg depletion did not promote pmel cell accumulation in spleen or skin (figure 4B), indicating that depletion of total CD4-expressing cells is required for systemic dissemination of tumor-specific CD8+ T cell responses. Treg depletion promoted polyclonal CD8+ T cell access to skin, but the absence of pmel cells in skin underscored the Ag-irrelevant nature of this response (figure 4C). Finally, we investigated whether pmel cells primed in Treg depleted mice could persist as memory. As targeted Foxp3+ Treg depletion causes fatal autoimmunity in mice,35 only two mice survived 1 month following tumor excision. However, surviving mice neither developed vitiligo nor sustained detectable pmel cell populations (online supplemental figure 6B). Thus, the promotion of systemic and durable CD8+ T cell responses in tumor-bearing mice required the depletion of total CD4-expressing cells.

Figure 4

Removal of Ag-specific CD4+ T cells is required to induce systemic CD8+ T cell responses against tumor antigens. (A) Congenically marked Thy1.1+ pmel cells were transferred into Foxp3-DTR and WT B6 mice 1 day prior to tumor inoculation. WT mice were either untreated or treated with anti-CD4 on days 4 and 10 after tumor inoculation. Foxp3-DTR mice were treated for five consecutive days with DT beginning on day four post tumor injection. Tissues were harvested for flow cytometry analysis on day 12 after tumor inoculation. (B) Proportion of Thy1.1+ pmel cells; gated on live CD8+ T cells across tissues on day 12. (C) Proportion of total CD8+ T cells, gated on live CD45+ lymphocytes in skin and tumor on day 12. (D) RAG knockout mice were reconstituted with polyclonal CD8+ T cells and congenically marked pmel cells along with either polyclonal CD4+ T cells or OTII CD4+ T cells 1 day prior to tumor implantation. Tumors were left to grow for 12 days prior to analyzing pmel cell priming and dissemination by flow cytometry. (E) Proportion of CD44hi Thy1.1+ (TDLN, Spleen, Skin) or CD44hi (tumor) pmel cells, gated on live CD8+ T cells on day 12. (F) Proportion of total CD8+T cells, gated on live lymphocytes in skin and tumor on day 12. Experiments were repeated twice with n≥4 mice per group. Data are pooled from two independent experiments with similar results. Flow plots depict representative mice; bars represent means and error bars represent SEM One-way ANOVAs with multiple comparisons for experiments with three groups, and paired t-tests for experiments with two groups were used to determine statistical significance. ANOVA, analysis of variance; DT, diphtheria toxin; TDLN, tumor draining lymph node; WT, wild-type; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

To confirm an immunosuppressive role for Ag-specific CD4+ T cells independent of anti-CD4 treatment, we separately implanted B16 tumors in RAG knockout mice that had been reconstituted with either polyclonal WT CD4+ T cells or TCR transgenic OTII T cells (figure 4D). In the latter group, both Tregs and Tconv cells were present, but neither could engage tumor Ag. Pmel cells were cotransferred to track the CD8+ T cell response (figure 4D). Pmel cell priming in TDLNs trended to be higher in mice with Ag-irrelevant OT-II CD4+ T cells, although the difference did not reach significance (figure 4E). Importantly, however, pmel cell responses in spleen and skin (figure 4E) and the proportion of total CD8+ T cells in skin and tumor (figure 4F) were significantly higher in mice reconstituted with OT-II as compared with WT CD4+ T cells. These data support the conclusion that antigen-specific CD4+ T cells naturally suppress the generation of tumor Ag-specific CD8+ T cell responses. Interestingly, the pmel cell proportion in tumors was not enhanced by the elimination of Ag-specific CD4+ T cells, consistent with our finding that anti-CD4 has a minimal effect on primary tumor growth and a greater effect on systemic immunity (figure 1B,C). Altogether, the above results demonstrate that the induction of tumor-specific CD8+ T cell priming on anti-CD4 treatment requires the elimination of antigen-specific Tconv responses, in addition to Tregs.

Depletion of CD4+ Tconv cells is required for cDC1-mediated priming of Ag-specific CD8+ T cells in tumor-bearing mice

Prior studies have shown that targeted Foxp3+ Treg depletion facilitates increased activation of migratory type 2 conventional DCs (cDC2s) in tumor-bearing mice, thus facilitating a potent CD4-mediated response against B16 melanoma.21 As cDC2s are known to express CD4,36 this presented the possibility that anti-CD4 may also function, in part, by depleting cDC2s. Indeed, varying proportions of APCs, including pDCs, macrophages, and cDC2s, express CD4 (figure 5A), and staining with a distinct anti-CD4 clone revealed their efficient depletion (online supplemental figure 7, figure 5A). Unexpectedly, however, anti-CD4 treatment did not reduce total cDC2 accumulation and instead led to an increase in the total number of Sirp1a+ cDC2s in TDLNs, comparable to Foxp3-DTR mice who received targeted Treg depletion (figure 5B,C). Despite this, anti-CD4 treatment promoted increased accumulation of cDC1s in TDLNs, which were more mature as evidenced by higher expression of CD86 (figure 5B,D). Importantly, elevated CD86hi cDC1 populations were not observed following Foxp3-targeted Treg depletion (figure 5D), or dual ICB treatment (figure 5E), indicating that anti-CD4 was unique in promoting accumulation of CD86hi cDC1s in TDLNs. Additionally, CD4 depletion induced negligible priming of pmel cells in tumor-bearing Batf3-knockout mice, indicating that cDC1s are required for the anti-CD4-induced CD8+ T cell response (figure 5F).

Figure 5

cDC1-dependent priming of tumor-specific CD8+ T cells does not require depletion of CD4-expressing antigen-presenting cells. WT B6 mice were either untreated or treated with anti-CD4 on days 4 and 10 post intradermal B16 tumor implantation. TDLNs were digested and assessed by flow cytometry to identify various myeloid cell populations (A–D). (A) Flow plots of CD4+ non-T cells in WT tdLNs on day 12. (B) Flow plots showing cDC1 and cDC2s in tdLNs on day 12. Populations are gated out of live singlets, lymphocytes, F480−/CD19− cells, CD11b±CD11c+/−, MHCII+Ly6C− cells. Xcr1+Sirp1a− cells are cDC1s and Xcr1−Sirp1a+ cells are cDC2s. (C) Proportion of and total cDC2s per tdLN on day 12. (D) Proportion and total of cDC1s per tdLN on day 12. CD86 expression on cDC1s on day 12. (E) Mice were treated as described in figure 1A. Normalized (relative to no treat) number of cDC1s per TDLN and CD86 expression on cDC1s in TDLNs from untreated, anti-CD4 treated and dual ICB-treated mice. (F) Congenically marked pmel cells were transferred into WT B6 and Batf3 knockout mice 1 day prior to B16 implantation. Mice were treated with anti-CD4 on days 4 and 10 post tumor injection and then tissues were harvested on day 12 to assess priming by flow cytometry. Proportions CD44hi Thy1.1+ pmel cells out of CD8+ T cells across tissues in WT and Batf3 KO mice on day 12. (G) Naïve, congenically marked pmel cells were transferred into RAG knockout mice lacking CD4+ T cells. For two groups, intradermal B16 tumors were implanted 1 day later. Anti-CD4 treatment was given to deplete CD4