Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap.
We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+T cell help in the antitumor effects of the anti-PD-1+LAG-3 combination.
The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatory CD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+T cell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFNγ release but, conversely, increased numbers of activated CD8+T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors.
Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+T cell polarization, which in turn, impacted cytotoxic CD8+T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.
© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

CTLA-4 exists as membrane (mCTLA-4) and soluble (sCTLA-4) forms. Here, we show that effector-type regulatory T cells (Tregs) are main sCTLA-4 producers in basal and inflammatory states with distinct kinetics upon TCR stimulation. Mice specifically deficient in sCTLA-4 production exhibited spontaneous activation of Th1, Th17, Tfh, and Tc1 cells, autoantibody and IgE production, M1-like macrophage polarization, and impaired wound healing. In contrast, sCTLA-4-intact mCTLA-4-deficient mice, when compared with double-deficient mice, developed milder systemic inflammation and showed predominant activation/differentiation of Th2, M2-like macrophages, and eosinophils. Consistently, recombinant sCTLA-4 inhibited in vitro differentiation of naïve T cells towards Th1 through CD80/CD86 blockade on antigen-presenting cells, but did not affect Th2 differentiation. Moreover, sCTLA-4-intact mCTLA-4-deficient Tregs effectively suppressed Th1-mediated experimental colitis whereas double-deficient Tregs did not. Thus, sCTLA-4 production by Tregs during chronic inflammation is instrumental in controlling type 1 immunity while allowing type 2 immunity to dominate and facilitate inflammation resolution.

Background Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma it remains unclear whether their mechanisms of action and resistance overlap. Methods We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion and ELISPOT assays were used to demonstrate the unique role of CD4+ T cell help in the anti-tumor effects of the anti-PD-1+LAG-3 combination. Tetramer assays confirmed the loss of CD8+ tumor-reactive T cells in brain tumors resistant to the anti-PD-1+LAG-3 combination. Results The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+ regulatory CD4+ cells (Tregs), fewer activated CD4+ T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+ T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+ T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+ T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+ T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+ T cell depletion was associated with fewer activated (CD69+) CD8+ T cells, impaired IFNγ release and increased numbers of myeloid-derived suppressor cells (MDSCs) but, conversely, increased numbers of activated CD8+ T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. Analysis of relapsing melanoma brain metastases from anti-PD-1+LAG-3 treated mice showed an increased accumulation of MDSCs and a loss of gp100+ tumor reactive CD8+ T cells. An analysis of the inferred cell-cell interactions from the scRNA-seq data suggested the MDSCs interacted with multiple subsets of T cells in a bi-directional manner. Conclusions Together these studies suggest that these two clinically relevant ICI combinations have differential effects upon CD4+ T cell polarization, which in turn, impacted cytotoxic CD8+ T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

Although PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells and a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Tumor-infiltrating NK cells that express PD-1 were highly associated with the expression of CXCR6. Furthermore, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wild-type mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells. These data demonstrate that there may be a role for the PD-1/PD-L1 axis in tumor-infiltrating NK cells in vivo.
© 2022 The Author(s).

Compelling evidence suggests a crucial role for Foxp3+ regulatory T cells (Tregs) in the control of atherosclerosis. Although suppression of pro-inflammatory CD4+ T cell immune responses is supposed to be important for athero-protective action of Foxp3+ Tregs, few studies have provided direct evidence for this protective mechanism. We investigated the impact of Foxp3+ Treg depletion on CD4+ T cell immune responses and the development of atherosclerosis under hypercholesterolemia. We employed DEREG (depletion of regulatory T cells) mice on an atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr -/-) background, which carry a diphtheria toxin (DT) receptor under the control of the foxp3 gene locus. In these mice, DT injection led to efficient depletion of Foxp3+ Tregs in spleen, lymph nodes and aorta. Depletion of Foxp3+ Tregs augmented CD4+ effector T cell immune responses and aggravated atherosclerosis without affecting plasma lipid profile. Notably, the proportion of pro-inflammatory IFN-γ-producing T cells were increased in spleen and aorta following Foxp3+ Treg depletion, implying that Foxp3+ Tregs efficiently regulate systemic and aortic T cell-mediated inflammatory responses under hypercholesterolemia. Unexpectedly, Foxp3+ Treg depletion resulted in an increase in anti-inflammatory IL-10-producing T cells, which was not sufficient to suppress the augmented proinflammatory T cell immune responses caused by reduced numbers of Foxp3+ Tregs. Our data indicate that Foxp3+ Tregs suppress pro-inflammatory CD4+ T cell immune responses to control atherosclerosis under hypercholesterolemia.
© 2022 The Author(s).