Worldwide, lung cancer has the highest mortality rate with small cell lung cancer (SCLC) making up 10%–15%.1 2 SCLC is known as an aggressive and recalcitrant disease in that it has fast doubling time, high growth fraction, early extensive dissemination, as well as no remarkable advances in therapeutic methods in the past three decades.2 3 SCLC can be classified into limited-stage SCLC (LS-SCLC) and extensive-stage SCLC (ES-SCLC), and the latter accounts for 60%–70%.4 ES-SCLC is principally treated with platinum-based chemotherapy with a median overall survival (OS) of approximately 9–12 months.45 Despite an up to 75% initial sensitivity to platinum-based chemotherapy, patients with ES-SCLC will inevitably develop resistance.4

Recently, immunotherapy has revolutionized cancer therapy due to promising antitumor activity and significantly improved OS.6 Notably, immunotherapy has shown therapy promise and has broken the situation of no superior treatment than chemotherapy in SCLC.7 8 IMpower133, a phase 3 clinical trial, demonstrated a better prognosis among patients who took administration of atezolizumab and chemotherapy versus those who received chemotherapy alone.7 Subsequent CASPIAN study obtained similar results.9 However, immunotherapy improves the survival of patients with SCLC by only 2 months. For patients with SCLC, little progress has been made in efficacious predictive biomarkers including programmed death-ligand 1 (PD-L1).10 Therefore, identification of patients who may benefit from immunotherapy is urgently needed.

Human leukocyte antigen (HLA), also called major histocompatibility complex, exerts critical roles in the interaction between tumor cells and the human immune system. HLA class I is one of the canonical HLA molecules, and the role of HLA class I-specific CD8-positive T cell responses in antitumor immune response has been well established.11–13 Canonically coded by HLA-DP, DQ, and DR, HLA class II is highly restricted to professional antigen-presenting cells (APCs) and participates in exogenous antigen presentation to CD4+ T cells.1114 Nonetheless, HLA class II expression on tumor cells and immune cells can be induced by interferon (IFN)-γ.15 16 Evidence showed HLA class II on tumor cells affected the tumor immunogenicity, tumor migration and invasion, cancer progression, immune response, and prognosis in numerous malignancies in vitro and in vivo.17–19 HLA class II on tumor-infiltrating lymphocytes (TILs) was related to antigen presentation, interactions with immune cells, intracellular signaling, and prognosis of patients with cancer.20 21 Besides, HLA class II on TILs had a controversial correlation with tumor metastasis to lymph nodes in gastric cancer.22 Compared with non-small cell lung cancer (NSCLC), reduced expression of HLA class II on TILs in SCLC was considered as a form of tumor immune escape.21 However, the implications of HLA class II on TILs in tumor immune microenvironment and prognosis in SCLC are not completely understood.

Herein, this study aims to analyze expression patterns of HLA class II on tumor cells and TILs by immunohistochemical (IHC) staining, and its association with clinical and immune indicators and recurrence-free survival (RFS) in SCLC. Additionally, we established an HLA class II-based immune risk model by least absolute shrinkage and selection operator (LASSO) regression and investigated the enrichment pathways and immune infiltration landscape associated with HLA class II in SCLC by bioinformatics analysis.

Immunotherapy has improved the survival of patients with SCLC, but little is known about efficacious predictive factors, which highlights the urgent need for better immune biomarkers to optimize therapeutic options. Herein, we analyzed the expression pattern of HLA class II and its connection with tumor immune microenvironment and prognosis. We detected positive HLA class II on few SCLC cells and nearly half of TILs. HLA class II on TILs was negatively correlated with lymph node metastasis and positively related to multiple immune markers and a longer RFS. Additionally, we established an HLA class II-based immune risk model that could more effectively predict the recurrence than tumor stage in SCLC. Using pathway analysis, we found signatures of transmembrane transportation, channel activity, and neuroactive ligand–receptor interaction were enriched in the high HLA class II expression group. And high-risk and low-risk patients with SCLC markedly differed in T follicular helper cells, activated memory CD4+ T cells, and resting dendritic cells. These findings indicate HLA class II plays a crucial role in tumor immune microenvironment and prognostic evaluation in patients with SCLC.

In this study, we observed that HLA class II was positively expressed on tumor cells in only 8.8% and on TILs in 44.1% of SCLC patients. The expression frequency of HLA class II on tumor cells varied among different types of cancers. Severe deficiency of HLA class II expression on tumor cells was also documented in other SCLC studies,21 32 hepatocellular carcinomas,33 ductal breast cancers,34 and acute myeloid leukemia.35 In our previous study of 72 Caucasian SCLC samples, no HLA class II was detected on tumor cells.21 The reasons for loss of HLA class II expression on tumor cells mainly included structural alterations (eg, mutations or rearrangements in HLA class II alleles) and deregulation of HLA class II antigen presentation machinery components induced by the promoter polymorphism or hypermethylation of HLA class II regulator CIITA.36 IFN-γ played a crucial role in the regulation of HLA class II expression to ensure an immune response against tumor cells.36 37 In vitro studies in SCLC and hepatocellular carcinomas showed the absence of HLA class II expression could not be rescued by IFN-γ induction,32 33 which to some extent could be considered as a form of antitumor immune escape and might partly explain their refractory features. Nonetheless, HLA class II was detectable in multiple other cancer types including NSCLC (30%),21 primary melanomas (50%–60%),38 thyroid papillary carcinomas (≈50%),39 pancreatic cancer (>70%),40 and colorectal cancers (21%–56%).41–43 As for HLA class II on TILs, our previous results revealed that HLA class II was positively expressed in only 15.3% Caucasian patients with SCLC.21 In the previous study including patients with NSCLC and SCLC, 11 (15.3%) of 72 patients with SCLC were positive for HLA class II with a cut-off value of 80%, markedly higher than 25% in this study. Given that the cut-off value was decided by the survival analysis of another NSCLC cohort with prognosis data rather than SCLC patients, the results may be incomparable. Overall, the differences of HLA class II expression level on tumor cells or TILs among a wide variety of tumors may be attributable to different research design, patient population, mechanisms of oncogenesis, molecular phenotypes, tumor origin, and various cut-off values for positivity.

We further analyzed the association of HLA class II with clinical factors. We found HLA class II expression on TILs was negatively related to lymph node metastasis, which indicated its protective function. Multiple studies have demonstrated that HLA class I was associated with lymph node metastasis in lung cancer,44 gastric cancer,22 45 and colorectal cancer.46 However, data about the involvement of HLA class II in lymph node metastasis is limited. Ogoshi et al22 found HLA-DR2, HLA-DR4, and HLA-DR6 on TILs had an opposite relation to lymph node metastasis in different histological subtypes and stages of patients with gastric cancers. Paradoxical to our result, lymph node metastasis showed a positive correlation with HLA-DR4 on TILs from the blood in gastric cancer47 and HLA-DR on tumor cells in lung cancer.48 In terms of the reasons for these discrepancies, we consider different histological types or subtypes, cancer stage, and expression location of HLA class II may influence the relationship with lymph node metastasis. Our study showed SCLC patients with positive HLA class II expression on TILs had less lymph node metastasis. We speculate that better recognition of HLA class II by CD4+ T cells may lead to the elimination of tumor cells and the prevention of migration and invasion. Future study is warranted to verify it and explore potential mechanisms.

In our current research, HLA class II on TILs was coexpressed with PD-1/PD-L1 on TILs and CD3, CD4, CD8, and FOXP3 on immune cells. It indicates that HLA class II on TILs may have a close connection with multiple immune markers. On the one hand, Kitamura et al49 demonstrated that downregulation of HLA class II expression induced by interleukin-6/STAT3 signaling activation hampered dendritic cells activating effector T cells in colorectal cancer. The impairment of HLA class II on TILs could account for the immunosuppression in NSCLC.50 On the other hand, considerable evidence supported T cells expressing HLA class II could function as APCs, also called T-APCs, and antigen presentation mediated by T-APCs could induce apoptosis or clonal anergy in activated T cells or cytotoxicity in resting T cells.20 Correspondingly, HLA class II-specific monoclonal antibodies were developed to treat hematological malignancy.51 Additionally, Costantini et al18 found HLA-DR mediated signaling increased the expression of adhesion receptors and PD-L1 and the activation of the JAK/STAT3 pathway, and promoted progression, migration, and invasion of melanoma cells. Taken together, HLA class II played an important role in tumor immune microenvironment, oncogenesis, and tumor progression. But it remains to be elucidated whether this association reflects the attraction of TILs by HLA class II or the upregulation of HLA class II-induced by IFN-γ secreted by TILs.

We also found HLA class II expressed on tumor cells did not correlate with clinical and other immune factors except for PD-1 on TILs. Similar results of insignificant correlation between HLA class II on tumor cells with CD4/CD8+ T cells was observed in intrahepatic cholangiocarcinoma.52 However, a significant association was observed between HLA class II expression and multiple immune molecules and cells, including PD-1/PD-L1 and CD8+ TILs in melanoma cells,18 53 CD4+ TILs in lymphomas and colorectal cancers,54 55 CD3+ TILs in mismatch repair-deficient cancers,56 and CD68+ and CD163+ tumor-associated macrophages in oropharyngeal cancer.57 As for SCLC, HLA class II on tumor cells had a limited association with immune markers, which might be ascribed to the extremely low level of HLA class II expression on tumor cells in SCLC in comparison with other types of cancers. Future larger sample-size clinical researches or fundamental investigation may further ascertain their association and potential mechanisms.

We observed RFS had no significant difference in SCLC patients with different expression status of HLA class II on tumor cells, and its rare expression limited further analysis. Nonetheless, RFS was significantly longer in SCLC patients positive for HLA class II on TILs as opposed to those negative, in accordance with our previous study.21 This favorable prognostic connection is not unique to SCLC, as it has recently been reported in a variety of solid and hematological tumors.39 57–60 In lymphoma and melanoma, HLA class II positivity on tumor cells was not only a favorable prognostic predictor but also an efficacious predictor of PD-1 blockade, thus producing the hypothesis of HLA class II-limited, CD4+ TILs-mediated action mechanism of PD-1 blockade.58 60 61 Additionally, the association of HLA class II with immune factors may partly account for its prognostic implication. Despite a lot of evidence supporting the favorable prognostic implications of positive HLA class II, other studies showed a discrepant correlation. HLA class II antigen was related to a poor prognosis in hepatocellular carcinoma and osteosarcomas.62 63 The inverse results may be induced by different oncogenesis mechanisms and heterogeneity of HLA class II subtypes. Overall, HLA class II has a definite link to RFS in SCLC, and its prognostic significance may be partly attributed to a close immune interaction.

Considering the close connection between HLA class II and immune markers and prognosis, we performed LASSO regression analysis among multiple immune variables to further analyze the influential factors of RFS in SCLC. And then we determined an HLA class II-based optimal risk model that categorized patients into high-risk and low-risk groups. High-risk patients with SCLC had a poorer prognosis, which was further confirmed in the public dataset. To our knowledge, it is the first time that we build an HLA class II-based immune risk model for prognostic evaluation in patients with SCLC, and it even has better predictive power than tumor stage.

In the final immune risk model, PD-L1, CD4, and FOXP3 were protective factors of RFS in patients with SCLC. PD-L1 on tumor cells or TILs was higher among early-stage SCLC compared with metastatic patients,64 and multiple authors demonstrated an association with better outcome in SCLC.64–68 However, limited studies reported contrary results.69 70 Different antibodies, cut-off values, and study design may cause the discrepancy. In our study, CD4+ TILs were closely associated with HLA class II and better prognosis in SCLC. CD4+ TILs recognition of HLA class II-restricted antigen could activate antitumor immunity.11 CD4+ T cells activation promoted differentiation into effector T cells and regulatory T (Treg) cells. The number of effector T cells in LS-SCLC was significantly more than that in ES-SCLC, and the high ratio of effector T cells to Treg cells predicted a long-term survival.71 FOXP3 is a specific biomarker of Treg cells, and its role in cancers is conflicting.72 Especially for SCLC, FOXP3+TILs presented inconsistent associations with prognosis. In a retrospective SCLC study, FOXP3+TILs were an independent positive prognostic factor in patients with non-metastatic SCLC at stage I–III, but not in metastatic patients.64 Our study also exhibited a positive association of FOXP3 with prognosis among patients with surgically resected SCLC at stage I–III. However, Kasahara et al73 found an insignificant correlation between FOXP3 and OS in patients with both LS-SCLC and ES-SCLC. The authors determined the cut-off value of FOXP3 based on survival analysis, but no specific value was provided. As for patients with metastatic SCLC, a similar insignificant association with survival was observed in another study that performed FOXP3+TILs IHC staining on brain metastasis specimens of patients with SCLC.65 Wang et al74 included 65 SCLC patients without stage information and demonstrated patients with a higher ratio of FOXP3/CD45 cells (measured as Tregs) had a worse median OS (120 vs 410 days). Considering its short OS, it may mainly consist of metastatic patients. The heterogeneous association of FOXP3 with prognosis in SCLC may be partly attributable to non-uniform cut-off values, tumor stage, and detection locations (primary vs metastatic). Additionally, FOXP3-positive T cells were constituted by three subpopulations: CD45RA(+)FOXP3(lo), CD45RA(−)FOXP3(hi), and CD45RA(−)FOXP3(lo).75 The former two were suppressive, and the third was non-suppressive T cells characterized by FOXP3 instability and inflammatory cytokines secretion. An abundance of inflammatory CD45RA(−)FOXP3(lo) T cells was correlated with a better prognosis in colorectal cancer.76 Therefore, the distinct predominant subpopulations of FOXP3+TILs during the dynamic process of tumor formation and progression may account for the different influences on prognosis in SCLC.

Furthermore, with bioinformatics methods, we investigated the implications of HLA class II and the immune risk model composed of HLA class II, PD-L1, CD4, and FOXP3 in immune infiltration and prognosis. After observing those SCLC patients with high HLA class II expression had a better prognosis versus low-expressed patients, we first performed GO and KEGG enrichment analyses to explore the potential biological pathways associated with HLA class II. Enriched pathways in the high-expression group involved transmembrane transport, channel activity, and ligand–receptor interaction. It indicates that HLA class II expression is associated with signaling transduction, as demonstrated in previous studies where ligation of HLA class II could transduce multiple intracellular signals such as protein kinase C membrane translocation and promote calcium influx.20 77 Then we did CIBERSORT analysis to delineate the immune infiltration landscape among high-risk and low-risk patients with SCLC. We found high-risk patients seemed to possess a closer interaction with immune cells and had significantly lower proportions of activated memory CD4+ T cells and resting dendritic cells versus those with low-risk. These differentially infiltrated immune cells correlated closely with antigen presentation and immune response. Collectively, we demonstrated a close relationship between HLA class II molecules, tumor immune microenvironment, and prognosis. Multiple studies mentioned that, except for the well-established role of tumor neoantigen-specific CD8+ T cells in antitumor immunity, HLA class II presenting neoantigens to CD4+ T cells also exerted a crucial function in tumor eradication and response to immunotherapy.58 78 79 Despite that, some cancer patients responded to immunotherapy even without detectable HLA class II,58 indicating that the mechanisms of HLA class II in immune regulation and antitumor immunity remain to be elaborated.

Finally, we acknowledge several limitations in our research. First of all, lack of representativeness may present as this study is single-center and retrospective. Second, given that there are no clear and standard cut-off values for HLA class II and multiple immune indicators, we determined the optimal cut-off values for immune markers by survival analysis. The repeatability and clinical utility of the optimal cut-off values need more studies to verify. Third, we established an immune risk model after cross-validation in the LASSO regression. Further validation and refinement are warranted in the future studies. Finally, not enough long follow-up duration restrains from comparing OS, but RFS already showed a sufficiently significant difference.

In this study, we detected positive HLA class II on few SCLC cells and nearly half of TILs. And we found positivity of HLA class II on TILs was negatively associated with lymph node metastasis and positively related to multiple immune markers and a better RFS. Furthermore, we established an HLA class II-based immune risk model, which can not only more effectively predict the recurrence than tumor stage but also identify immune infiltration heterogeneity in high-risk and low-risk patients with SCLC. Taken together, these findings indicate that HLA class II plays a crucial role in tumor immune microenvironment and prognostic evaluation in SCLC patients. Future studies are warranted to reveal the mechanism behind it.