Robust PD‐1 blockade compromises clonal affinity of stem‐like CD8 + T cells

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CD8+ T cells encountering persistent antigen, such as those responding to cancer or chronic viral infection, acquire a dysfunctional state known as “exhaustion.” Exhausted T cells are characterized by decreased proliferation and dampened effector function, as well as sustained expression of multiple inhibitory receptors (also known as immune “checkpoints”), including programmed cell death protein 1 (PD-1). T-cell exhaustion remains a major contributor to defective anti-tumor immunity, which can lead to tumor outgrowth and disease progression. Therapeutic immune checkpoint blockade, which aims to reinvigorate exhausted CD8+ T-cell responses, has shown promising results in promoting tumor clearance and is now a front-line therapy for multiple malignancies, including metastatic melanoma and non-small cell lung cancer.1, 2 While the exact mechanism of exhausted T-cell reinvigoration remains a topic of intense study, recent evidence has implicated specific populations of antigen-specific stem-like CD8+ T (TSL, or precursor/progenitor of exhausted T cells3, 4) cells that reside in the tumor-draining lymph nodes (tdLN) to be critical in mediating clinical responses to immune checkpoint blockade.3, 4 Importantly, the mechanisms by which TSL cells are maintained within the tdLN microenvironment in the face of persistent antigen, and how immune checkpoint blockade affects these dynamics remains largely unknown. In a recent study in Nature, Hor et al.5 now demonstrate a key role for antigen-presenting niches in tdLN that are indispensable for the proliferation, maintenance and antigen-affinity evolution of TSL cells. These niches, rich in type 1 conventional dendritic cells (cDC1), play a crucial role in balancing stimulatory and inhibitory signals. Using a preclinical model of Kras/P53 lung adenocarcinoma expressing the ovalbumin antigen (KP-OVA), and in combination with fluorescent XCR1 reporter mice and elegant 3D imaging techniques, the authors identified the persistence of a small population of antigen-specific T cells forming clusters in association with cDC1 in the T-cell zone of the tdLN post-priming. These clustered T cells expressed high levels of stem-like (SLAMF6 and TCF1) and activation-associated (PD-1 and BATF) markers, confirming their identities as TSL cells. In contrast, effector T cells (TCF1−, TEFF) during the same time point were mainly found in the periphery of the tdLN. Given that persistent T-cell receptor (TCR) signaling is thought to drive terminal effector differentiation of T cells,6 the authors next tested whether differential antigen signaling occurs within these tdLN niches. Using nuclear translocation of the nuclear factor for activated T cells 1 (NFAT1) as a proxy of antigen signaling, the authors found increased nuclear NFAT1 in TSL within these cDC1-associated clusters, suggesting that these tdLN niches represented sites of ongoing antigen presentation and signaling. Moreover, TSL cells within these cDC1-enriched niches were associated with active proliferation. Indeed, depletion of cDC1 post-priming resulted in marked and specific reduction in TSL cells within tdLN, highlighting the crucial role of antigen presentation by cDC1 and active TCR signaling in the maintenance of antigen-specific TSL cells. The authors then asked whether and how TCR affinity affects TSL cell fate. This is an important question as it reveals, at least in part, whether TSL cell fate decision is a preceding factor or a consequence of localization in cDC1-enriched niches. To address this question, the authors investigated the polyclonal CD8+ T-cell response using the KP-OVA model. Devising a TCR affinity index by normalizing H-2Kb-SIINFEKL tetramer staining intensity to CD3 expression, the authors observed a positive correlation between TCR affinity index and expression of TSL markers such as PD-1 and SLAMF6 among antigen-specific T cells localized in cDC1-enriched niches, indicating an enrichment of TSL cells among high-affinity T cells. Moreover, TCR affinity steadily increased over time, indicating a gradual increase in average clonal affinity among TSL cells localized in cDC1-enriched niches and suggesting a role for tdLN niches in driving clonal selection. Challenging the notion that prolonged antigen stimulation drives terminal effector differentiation, these findings suggest that TSL cell fate is initially promoted by high TCR affinity during priming and maintained by further antigen signaling within cDC1-enriched niches post-priming. Using a combination of techniques, the authors demonstrated that inhibitory PD-1 signaling not only dictated clonal affinity evolution but was also critical in maintaining high-affinity TSL cells in cDC1-enriched tdLN niches. Indeed, while TSL cells expressed high levels of the inhibitory receptor PD-1, cDC1 expressed high levels of PD-L1 and PD-L2, ligands of PD-1. Imaging analysis revealed close interactions between TSL and cDC1, as well as PD-1/PD-L1-enriched microclusters at the cell–cell interface. Given that PD-1/PD-L1 interactions at the immunological synapse potently inhibit TCR signaling via the recruitment of SHP1/2 phosphatases,7 the combination of ongoing antigen presentation and strong inhibitory signals provides a highly selective environment whereby only TSL cells with high TCR affinity can overcome the activation threshold and are thus maintained. Accordingly, as TSL cells continually differentiate to generate TEFF cells, this selection provides a mechanism to ensure the generation of high-affinity TEFF cells in combating ongoing antigen insult. This concept parallels that of B-cell selection within germinal centers8 and is in line with the observed increase in TSL clonal affinity over time. Consistent with this idea, blockade of the PD-1 signaling axis, via either combined inhibition of PD-L1/2 (but not PD-L1 alone) or PD-1, resulted in the selective loss of high-affinity TSL clones. Differentiation into TEFF cells and activation-induced cell death were the main reasons for the loss of high-affinity TSL cells following robust PD-1 inhibition. These data demonstrate the importance of inhibitory PD-1 signals within cDC1-enriched tdLN niches in counterbalancing stimulatory TCR signals to maintain long-term high-affinity antitumor immunity. This study raises important clinical implications. While immune checkpoint blockade has revolutionized tumor immunotherapy, many patients respond poorly or develop resistance to treatment over time.2 A shift in the clonal repertoire and pruning of high-affinity TSL cells following robust PD-1 blockade, as revealed in this study, could be a potential mechanism underpinning reduced responsiveness to successive treatments. This idea aligns with previous observations on melanoma patients given PD-1 blockade therapy that each consecutive responding wave of T cells consisted of different clones9 and is in line with the notion of “clonal replacement.”10 Notably, this shift in clonal repertoire and loss of high-affinity TSL clones were not observed when either PD-L1 or PD-L2 alone was blocked, suggesting the presence of an optimal therapeutic window where T-cell reinvigoration can be achieved without compromising TSL cell fitness and their long-term maintenance. While combined anti-PD-L1/2 therapies are not typically used clinically, this study provides evidence to caution against excessive blockade of immune checkpoints, for example, simultaneously targeting both PD-1 and CTLA-4, which is commonly used in clinics.1, 2 Overall, this study elucidates a key mechanism underpinning TSL cell survival and clonal selection within the tdLN microenvironment and demonstrates a key role for inhibitory PD-1 signals in fine-tuning the TCR repertoire in chronic T-cell responses. This work has strong implications for the optimization of checkpoint-based immunotherapy. We thank Thomas Gebhardt for insightful discussions and providing critical feedback to this manuscript. Carlson Tsui: Wri

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