Analysis of radiotherapy-induced alteration of CD8+ T cells and PD-L1 expression in patients with uterine cervical squamous cell carcinoma

Radiotherapy induces an immune response in the cancer microenvironment that may influence clinical outcome. The present study aimed to analyse the alteration of CD8⁺ T-cell infiltration and programmed death-ligand 1 (PD-L1) expression following radiotherapy in clinical samples from patients with uterine cervical squamous cell carcinoma. Additionally, the current study sought to analyse the association between these immune responses and clinical outcomes. A total of 75 patients who received either definitive chemoradiotherapy or radiotherapy were retrospectively analyzed. CD8⁺ T-cell infiltration and PD-L1 expression were determined by immunohistochemistry using biopsy specimens before radiotherapy (pre-RT) and after 10 Gy radiotherapy (post-10 Gy). The PD-L1⁺ rate was significantly increased from 5% (4/75) pre-RT to 52% (39/75) post-10 Gy (P<0.01). Despite this increase in the PD-L1⁺ rate post-10 Gy, there was no significant association between both pre-RT and post-10 Gy and overall survival (OS), locoregional control (LC) and progression-free survival (PFS). On the other hand, the CD8⁺ T-cell infiltration density was significantly decreased for all patients (median, 23.1% pre-RT vs. 16.9% post-10 Gy; P=0.038); however, this tended to increase in patients treated with radiotherapy alone (median, 17.7% pre-RT vs. 24.0% post-10 Gy; P=0.400). Notably, patients with high CD8⁺ T-cell infiltration either pre-RT or post-10 Gy exhibited positive associations with OS, LC and PFS. Thus, the present analysis suggested that CD8⁺ T-cell infiltration may be a prognostic biomarker for patients with cervical cancer receiving radiotherapy. Furthermore, immune checkpoint inhibitors may be effective in patients who have received radiotherapy, since radiotherapy upregulated PD-L1 expression in cervical cancer specimens.     

Programmed cell death ligand-1 (PD-L1) expression combined with CD8 tumor infiltrating lymphocytes density in non-small cell lung cancer patients

Cancer immunotherapy targeting programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway has shown promising results in treatment of non-small cell lung cancer (NSCLC) patients. T cells play a major role in tumor-associated immune response. This study aimed to investigate PD-L1 expression alone and combined with CD8 tumor infiltrating lymphocytes (TILs) density in relation to clinicopathologic parameters and survival in NSCLC patients. Immunohistochemical analysis was used to evaluate PD-L1 expression and CD8 TILs density in 55 NSCLC patients. PD-L1 immunopositivity was detected in 36 (65.5%) of NSCLC cases. PD-L1 expression was significantly related to high tumor grade (p value?=?0.038) and low CD8 TILs density (p value?=?0.004), whereas no significant relations were detected between PD-L1 expression and tumor stage (p value?=?0.121), overall survival (OS) (p value?=?0.428) and progression-free survival (PFS) (p value?=?0.439). Among PD-L1/CD8 TILs density groups, PD-L1⁺/CD8^Low group was significantly associated with high tumor grade compared to PD-L1^?/CD8^high group (pairwise p?=?0.016). PD-L1⁺/CD8^Low group was significantly related to advanced tumor stage compared to PD-L1⁺/CD8^high and PD-L1^?/CD8^Low groups (pairwise p?=?0.001 and 0.013 respectively). PD-L1^?/CD8^high group exhibited the best OS and PFS whereas PD-L1⁺/CD8^low group had the poorest OS and PFS (p value?=?0.032 and 0.001 respectively). Assessment of PD-L1 combined with CD8 TILs density, instead of PD-L1 alone, suggested important prognostic relevance in NSCLC patients.     

Expansion of CD3+CD8+PD1+ T lymphocytes and TCR repertoire diversity predict clinical responses to adoptive cell therapy in advanced gastric cancer

The adoptive cell therapy (ACT) and delivery of ex vivo activated cellular products, such as dendritic cells (DCs), NK cells, and T cells, have shown promise for the treatment of gastric cancer (GC). However, it is unknown which cells can improve patient survival. This study was focused on the antitumour activity of a subset of these cellular products and their relationships with clinical outcomes. Nineteen patients were enrolled at the Capital Medical University Cancer Center, Beijing Shijitan Hospital, from June 1, 2013, to May 30, 2016. CD8⁺PD1⁺ T-cell sorting was carried out using flow cytometry, and the T-cell receptor (TCR) repertoire during ex vivo expansion for 15 days was analyzed by next-generation sequencing. After 15 days of culture, the number of CD8⁺ T cells had increased significantly, and the number of CD4⁺ T cells had increased correspondingly. After ex vivo expansion, CD8⁺ T cells exhibited significantly enhanced expression of PD-1, LAG-3, and TIM-3 but not 4-1BB. Survival analysis showed that patients with a pro/pre value of CD8⁺PD-1⁺ T cells >2.4 had significantly favorable overall survival (OS) (median OS time, 248 days versus 96 days, P=0.02) and progression-free survival (PFS) (median PFS time, 183 days vs. 77 days, P=0.002). The sorted CD8⁺PD-1⁺ T cells displayed enhanced antitumor activity and increased IFN-γ secretion after coculture with autologous tumor cell lines. TCR repertoire diversity was decreased after ex vivo expansion, which decreased the Shannon index and increased the clonality value. The prognosis of patients was significantly improved and was associated with the extent of CD8⁺PD-1⁺ T-cell expansion. In summary, this study showed that after ex vivo expansion for 15 days, CD8⁺PD-1⁺ T cells could be identified as tumor-reactive cells in patients treated for GC. Changing TCR species can predict the extent of CD3⁺CD8⁺PD1⁺ T-cell growth and the effect of ACT treatment.     

Tumor β-catenin expression is associated with immune evasion in non-small cell lung cancer with high tumor mutation burden

β-catenin expression by tumor cells suppressed dendritic cell recruitment to the tumor microenvironment in a melanoma model, resulting in fewer tumor-infiltrating lymphocytes. Immunohistochemistry was used in the present study to examine the association between the expression of β-catenin and tumor infiltrating lymphocytes and CD11c⁺ cells in 122 patients with non-small cell lung cancer (NSCLC), who underwent radical surgery. β-catenin was positive in 24% of NSCLC tumors compared with 59% of squamous cell carcinomas and 11% of adenocarcinomas. There was no significant association between the expression of β-catenin and the frequency of CD8⁺ cell infiltration into tumor tissues, including the stroma. Conversely, the infiltration of CD8⁺ cells into tumor nests was significantly lower in β-catenin-positive cases compared with that in negative β-catenin cases. Similarly, CD11c⁺ cell infiltration was significantly lower in the β-catenin-positive group. The β-catenin-positive group had shorter overall survival and recurrence-free survival times compared with that in the negative group. Furthermore, β-catenin-positive NSCLC had a high tumor mutation burden, but tended to have a low expression of programmed death-ligand 1. In conclusion, the expression of β-catenin in NSCLC was negatively associated with CD11c⁺ cells and cytotoxic T cell infiltration at the tumor site and had a tendency towards a poor prognosis.     

The predictive value of centre tumour CD8+ T cells in patients with hepatocellular carcinoma: comparison with Immunoscore

The increasing evidences suggest that Immunoscore(IS), a combinatorial density analysis of CD8⁺ and CD3⁺ cells in the centre and invasive margin of tumour (CT and IM), has an advantage over the currently used tumour staging methods in a variety of tumours; however, IS in hepatocellular carcinoma remains unreported. In this study, IS was performed on serial sections from two HCC cohorts (total 449) and compared with current tumour staging systems. Kaplan–Meier curves illustrate a positive association between a higher IS (IS≥2) and longer survival of HCC patients. Although the IS was highly related to the outcome of patients, however, IS seems not to be the optimal prognostic factor when compared with the CD8_CT. As noted, among CD8_CT, CD8_IM, CD3_CT, CD3_IM and IS, CD8_CT, as an independent indicator, demonstrated the highest prognostic impact on both DFS and OS in our Cox multivariate regression analysis (P< 0.0001). In our study, the minimum cut-off value was 93 CD8_CT cells per mm², to be used to divide the patients into CD8_CT^Hi group and CD8_CT^Lo group in clinical settings. Our results suggest that CD8_CT densities analysis notably improved the accuracy of survival prediction with convenience of clinical manipulation in HCC.     

CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcome

The Cancer Immunoediting concept has provided critical insights suggesting dual functions of immune system during the cancer initiation and development. However, the dynamics and roles of CD4⁺ and CD8⁺ T cells in the pathogenesis of breast cancer remain unclear. Here we utilized two murine breast cancer models (4T1 and E0771) and demonstrated that both CD4⁺ and CD8⁺ T cells were increased and involved in immune responses, but with distinct dynamic trends in breast cancer development. In addition to cell number increases, CD4⁺ T cells changed their dominant subsets from Th1 in the early stages to Treg and Th17 cells in the late stages of the cancer progression. We also analyzed CD4⁺ and CD8⁺ T cell infiltration in primary breast cancer tissues from cancer patients. We observed that CD8⁺ T cells are the key effector cell population mediating effective anti-tumor immunity resulting in better clinical outcomes. In contrast, intra-tumoral CD4⁺ T cells have negative prognostic effects on breast cancer patient outcomes. These studies indicate that CD4⁺ and CD8⁺ T cells have opposing roles in breast cancer progression and outcomes, which provides new insights relevant for the development of effective cancer immunotherapeutic approaches.     

Tumor immune microenvironment predicts the pathologic response of neoadjuvant chemoimmunotherapy in non–small-cell lung cancer

The clinical outcome of resectable non–small-cell lung cancer (NSCLC) patients receiving neoadjuvant chemoimmunotherapy is good but varies greatly. In addition, the pathological response after neoadjuvant chemoimmunotherapy is significantly associated with survival outcomes. The aim of this retrospective study was to identify which population of patients with locally advanced and oligometastatic NSCLC has a favorable pathological response after neoadjuvant chemoimmunotherapy. NSCLC patients treated with neoadjuvant chemoimmunotherapy were enrolled between February 2018 and April 2022. Data on clinicopathological features were collected and evaluated. Multiplex immunofluorescence was performed on pre-treatment puncture specimens and surgically resected specimens. In total, 29 patients with stages III and IV locally advanced or oligometastatic NSCLC who received neoadjuvant chemoimmunotherapy and R0 resection were enrolled. The results showed that 55% (16/29) of patients had a major pathological response (MPR) and 41% (12/29) of patients had a complete pathological response (pCR). In the stroma area of the pre-treatment specimen, the higher infiltration of CD3⁺PD-L1⁺ tumor-infiltrating lymphocytes (TILs) and the lower infiltration of CD4⁺ and CD4⁺FOXP3⁺ TILs were more likely to appear in patients with pCR. However, in the tumor area, the higher infiltration of CD8⁺ TILs was more likely to appear in patients with non-MPR. In the post-treatment specimen, we found increased infiltration of CD3⁺CD8⁺, CD8⁺GZMB⁺, and CD8⁺CD69⁺ TILs and decreased infiltration of PD-1⁺ TILs both in the stroma and tumor areas. Neoadjuvant chemoimmunotherapy achieved an MPR rate of 55% and induced greater immune infiltration. In addition, we observed that the baseline TILs and their spatial distribution correlate to the pathological response.     

CD8+ T cells inhibit metastasis and CXCL4 regulates its function

Background The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival.Methods We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well.Results We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8⁺ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8⁺ T-cell function. TCGA pan-cancer data confirmed that CD8^lowPlatelet^high patients have a significantly lower survival probability compared to CD8^highPlatelet^low.Conclusions CD8⁺ T cells inhibit metastasis. When the balance between CD8⁺ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8⁺ T-cell function.Subject terms: Tumour immunology, Metastasis     

Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells

CD8⁺ T cell-mediated immune response plays an important role in inhibiting progression of hepatocellular carcinoma (HCC). For strategic immunotherapy, it is critical to understand why some of the tumor cells escape from this immune attack. In this study, we investigated how HCC cells alter endogenous anti-tumor immunity and their related signaling pathways. We found that HCC cells, both in vitro and in vivo, substantially secret and express amphiregulin (AR). AR in turn activates immunosuppressive function of intratumoral CD4⁺Foxp3⁺ regulatory T cells (Tregs), a major inhibitor of CD8⁺ T cells. Using either lentiviral siRNA, or AR neutralizing antibody, we blocked the expression and function of AR to test the specificity of AR mediated activation of Tregs, Biochemical and cell biology studies were followed and confirmed that blocking of AR inhibited Tregs activation. In addition, we found that AR can trigger the activation of rapamycin complex 1(mTORC1) signaling in Tregs. The mTORC1 inhibitor rapamycin treatment led to compromise Treg function and resulted in enhancing anti-tumor function of CD8⁺ T cells. Blocking AR/EGFR signaling in Tregs with Gefitinib also enhanced anti-tumor immunity and decreased tumor size in a mouse xenograft tumor model. Taken together, our study suggested a novel mechanism of functional interaction between HCC and Tregs for regulating anti-tumor function of CD8⁺ T cells.     

Intraepithelial T cells and tumor proliferation

BACKGROUND:

The aim of the study was to determine whether tumor‐infiltrating lymphocytes and/or tumor mitotic activity could identify subgroups of patients with advanced serous epithelial ovarian cancer who would maximally benefit from aggressive surgical cytoreduction.

METHODS:

Snap‐frozen specimens from 134 consecutive patients with stage III or IV serous or poorly differentiated ovarian adenocarcinoma undergoing primary debulking surgery from a single US institution were characterized based on CD3⁺, CD8⁺, FoxP3⁺ tumor‐infiltrating lymphocytes, and Ki67 expression. Kaplan‐Meier survival curves were estimated and compared using a log‐rank statistic. A multivariate Cox model was used to estimate adjusted hazard ratios. Interactions were modeled using recursive partitioning based on maximal prognostic differentiation.

RESULTS:

Brisk intraepithelial CD8⁺ cells (P = .035) and low Ki67 expression (P = .042) portended prolonged survival. The T‐cell infiltration was more likely to occur in tumors with high proliferation index. Patients whose tumors exhibited low Ki67 expression and high intraepithelial CD8⁺ frequency had a 5‐year survival rate of 73.3%. Patients with aggressive tumor behavior, that is, whose tumors exhibited low frequency of intraepithelial CD8⁺ T cells or high Ki67 expression were more likely to draw benefit from aggressive surgical cytoreduction. Survival was similar for patients with brisk CD8⁺ T cells who had optimal or suboptimal debulking. Likewise, survival was similar for patients with low Ki67 expression who had optimal or suboptimal debulking.

CONCLUSIONS:

For the first time, these novel interactions of T cells, tumor proliferation index, and surgical treatment reveal that biological prognosticators may be useful for surgical decision making in ovarian cancer. Cancer 2009. © 2009 American Cancer Society.     

Prognostic value of CD57<Superscript>+</Superscript> T lymphocytes in the peripheral blood of patients with advanced gastric cancer

Background  Natural killer (NK)-like T cells comprising CD56⁺ T cells and CD57⁺ T cells belong to a subset of CD1d-independent NKT cells playing an important role in regulating immune responses. Although NK-like T cells are reported to increase in patients with advanced gastric carcinomas, it remains unknown how NK-like T cells are involved in disease progression in gastric cancer patients. Methods  The proportions of Th1 cells (interferon [IFN]-γ-producing CD4⁺ T cells), Th2 cells (IL-4-producing CD4⁺ T cells), and NK-like T cells (CD56⁺ T cells and CD57⁺ T cells) in the peripheral blood of 48 gastric cancer patients and 20 healthy controls were measured by two-color flow cytometry analysis and by intracellular cytokine analysis to investigate an association of these immune cells with the survival rate of gastric cancer patients. Results  Univariate analysis showed that Th1 cells and CD57⁺ T cells, as well as some clinicopathological factors, significantly influenced the survival rate. CD57-high (≧18%) patients survived for a significantly shorter period after surgery compared to CD57-low patients (P = 0.046; Kaplan-Meier, log-rank test) in the stage III–IV patients, but not in the stage I–II patients. Further, multivariate analysis showed that lymphatic invasion was a statistically significant independent risk factor in all the gastric cancer patients, but the proportion of CD57⁺ T cells as well as depth of tumor were statistically significant independent risk factors in patients with advanced carcinomas (stage III–IV). Conclusion  An increased proportion (≧18%) of CD57⁺ T cells in the peripheral blood of patients with advanced gastric carcinomas could indicate a poor prognosis.     

Concurrent infiltration by CD8+ T cells and CD4+ T cells is a favourable prognostic factor in non-small-cell lung carcinoma

The purpose of this study was to clarify the relationship between the number of tumour-infiltrating T lymphocytes and the clinicopathological features and clinical outcome in patients with non-small-cell lung cancer (NSCLC). Tissue specimens from 109 patients who underwent surgical resection for NSCLC were immunohistochemically analysed for CD4 and CD8 expression. Patients were classified into two groups according to whether their tumours exhibited a 'high' or 'low' level of CD8(+) or CD4(+) lymphocyte infiltration. Although the level of infiltration by CD8(+) T cells alone had no prognostic significance, the survival rate for patients with both 'high' CD8(+) and 'high' CD4(+) T-cell infiltration was significantly higher than that for the other groups (log-rank test, P=0.006). Multivariate analysis indicated that concomitant high CD8(+) and high CD4(+) T-cell infiltration was an independent favourable prognostic factor (P=0.0092). In conclusion, the presence of high levels of both CD8(+) T cells and CD4(+) T cells is a significant indicator of a better prognosis for patients with NSCLC, and cooperation between these cell populations may allow a significantly more potent antitumour response than either population alone.     

The prognostic value of peripheral CD4+CD25+ T lymphocytes among early stage and triple negative breast cancer patients receiving dendritic cells-cytokine induced killer cells infusion

Objective

This study aimed to assess the prognostic value of CD4⁺CD25⁺ T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy.

Methods

217 patients participated in the follow-up study. CD4⁺CD25⁺ proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4⁺CD25⁺ proportion more than 5% and less than or equal to 5% in peripheral T cells.

Results

Peripheral CD4⁺CD25⁺ T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4⁺CD25⁺ proportion of T cells was related with the median overall survival by a shorten of 51 months (p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4⁺CD25⁺proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients.

Conclusion

Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4⁺CD25⁺T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.     

Circulating and tumor-infiltrating Tim-3 in patients with colorectal cancer

T-cell exhaustion represents a progressive loss of T-cell function. The inhibitory receptor PD-1 is known to negatively regulate CD8⁺ T cell responses directed against tumor antigen, but the blockades of PD-1 pathway didn''t show the objective responses in patients with colorectal cancer (CRC). Thus, further exploring the molecular mechanism responsible for inducing T-cell dysfunction in CRC patients may reveal effective strategies for immune therapy. This study aims to characterize co-inhibitory receptors on T cells in CRC patients to identify novel targets for immunotherapy. In this study, peripheral blood samples from 20 healthy controls and 54 consented CRC patients, and tumor and matched paraneoplastic tissues from 7 patients with advanced CRC, subjected to multicolor flow cytometric analysis of the expression of PD-1 and Tim-3 receptors on CD8⁺ T cells. It was found that CRC patients presented with significantly higher levels of circulating Tim-3⁺PD-1⁺CD8⁺ T cells compared to the healthy controls (medians of 3.12% and 1.99%, respectively, p = 0.0403). A similar increase of Tim-3⁺PD-1⁺CD8⁺ T cells was also observed in the tumor tissues compared to paraneoplastic tussues. Tim-3⁺PD-1⁺CD8⁺ T cells in tumor tissues produced even less cytokine than that in paraneoplastic tissues. Functional ex vivo experiments showed that Tim-3⁺PD-1⁺CD8⁺ T cells produced significantly less IFN-γ than Tim-3⁻PD-1⁻CD8⁺ T cells, followed by Tim-3⁺PD-1⁻CD8⁺ T cells, and Tim-3⁻PD-1⁺CD8⁺ T cells, indicating a stronger inhibition of IFN-γ production of Tim-3⁺CD8⁺ T cells. It is also found in this study that Tim-3⁺PD-1⁺CD8⁺ T cell increase in circulation was correlated with clinical cancer stage but not histologic grade and serum concentrations of cancer biomarker CEA. Our results indicate that upregulation of the inhibitory receptor Tim-3 may restrict T cell responses in CRC patients, and therefore blockage of Tim-3 and thus restoring T cell responses may be a potential therapeutic approach for CRC patients.     

Good syndrome presenting with CD8+ T-Cell large granular lymphocyte leukemia

Good Syndrome is an adult-onset combined immunodeficiency defined by hypogammaglobulinemia, low or absent number of B cells, T cell deficiency and thymic tumor. We have characterized CD8+ T cells from a patient with Good syndrome that presented with CD8+T-cell large granular lymphocytic leukemia (LGL). Characterization of peripheral blood CD8+ T cells revealed that majority of CD8+ T cells were terminally differentiated effector memory phenotype (T_EMRA; CD8+CCR7-CD45RA+), and were PD-1^high (CD279), ICOS^low (CD278), and granzyme^high. Almost all CD8+ T cells were IFN-γ+. CD8 Treg (CD8+CD183+CCR7+CD45RA-) were decreased. T_EMRA phenotype along with CD279^high, demonstrates that these are exhausted CD8+ T cells. This phenotype along with CD278^low may also explain severe T cell functional deficiency in our patient. In the present patient, T-LGL appears to be a clonal expansion of CD279+granzyme+IFN-γ+CD8+T_EMRA cells. To best of our knowledge this is the first case of CD8+T-cell LGL leukemia associated with Good syndrome.     

Tertiary lymphoid structures show infiltration of effective tumor-resident T cells in gastric cancer

Several studies have reported that tissue-resident memory T cells (TRM cells) or tertiary lymphoid structures (TLSs) are associated with a good prognosis. The aim of this study was to clarify the association of TRM cells and TLSs in the tumor immune microenvironment in gastric cancer (GC). We performed immunohistochemical and immunofluorescence staining to detect the presence of CD103⁺ T cells and to assess the association between CD103⁺ T cells and TLSs. CD103⁺ T cells were observed in the tumor epithelium accompanied by CD8⁺ T cells and were associated with a better prognosis in GC. Furthermore, CD103⁺ T cells were located around TLSs, and patients with CD103^high had more rich TLSs. Patients who had both CD103^high cells and who were TLS-rich had a better prognosis than patients with CD103^low cells and who were TLS-poor. Moreover, for patients who received PD-1 blockade therapy, CD103^high and TLS-rich predicted a good response. Flow cytometry was performed to confirm the characteristics of CD103⁺CD8⁺ T cells and showed that CD103⁺CD8⁺ T cells in GC expressed higher levels of PD-1, granzyme B, and interferon-γ than CD103⁻CD8⁺ T cells. Our results suggested that CD103⁺CD8⁺ cells in GC are correlated with TLSs, resulting in enhanced antitumor immunity in GC.     

Clonal expansion of bone marrow CD8+ T cells in acute myeloid leukemia patients at new diagnosis and after chemotherapy

CD8⁺ T cells are crucial adaptive immune effectors and express receptors (T cell receptors, TCRs) that specifically recognize and eradicate tumor cells. The diversity of the TCR repertoire is generated by specialized genetic diversification mechanisms, which lead to an extremely variable TCR repertoire that is capable of recognizing a wide range of antigens. However, the variations in CD8⁺ TCR diversity and their clinical implications in acute myeloid leukemia (AML) patients remain unknown. CD8⁺ T cells were enriched from 10 healthy donors and 31 AML patients at diagnosis and after chemotherapy, and TCRβ deep sequencing was performed to analyze CD8⁺ T cell clonal expansion and TCR repertoire diversity. Diminished TCR repertoire diversity and increased T cell clone expansion were noted in the bone marrow of AML patients. In relapsed patients, T cells were found to be more clonally expanded after chemotherapy than at new diagnosis. Moreover, significantly more expanded TCRβ clonotypes were noted in CD8⁺ PD-1⁺ T cells than in CD8⁺ PD-1^- T cells regardless of the time of examination. Our systematic T cell repertoire analysis may help better characterize CD8⁺ T cells before and after chemotherapy in AML, which may provide insights into therapeutic strategies for hematological malignancies.     

Myeloid-Derived Suppressor Cells in Nonmetastatic Urothelial Carcinoma of Bladder Is Associated With Pathologic Complete Response and Overall Survival

BackgroundMyeloid-derived suppressor cells (MDSC) have immunosuppressive activity and enhance tumor progression. We hypothesized that lower blood MDSC would correlate with pathologic complete response and better outcomes in nonmetastatic urothelial carcinoma (UC).Patients and MethodsBefore cystectomy, blood MDSC were measured in whole blood (WB) and peripheral blood mononuclear cells using flow cytometry. MDSC were defined as CD33⁺/HLA-DR⁻. MDSC subtypes were polymorphonuclear MDSC (CD15⁺/CD14⁻), monocytic (M)-MDSC (CD15⁻/CD14⁺), and uncommitted (UnC) MDSC (CD15⁻/CD14⁻). The Wilcoxon rank sum test was used to compare MDSC between pathologic complete response groups. The optimal cutoff points for MDSC were identified using recursive partitioning analysis with cross-validation. The Cox proportional hazard model was used to associate MDSC and other clinical factors with recurrence-free survival and overall survival (OS).ResultsOverall, 109 patients were included: 86% men with median (range) age of 67 (30-88) years, 76% with pure UC, 29% intravesical therapy, and 41% neoadjuvant chemotherapy. Twenty-one patients (19%) had pT0N0 and 23 (24%) < pT2N0. Median (range) follow-up time was 17.4 (0.4-42.4) months. Total MDSC and polymorphonuclear MDSC percentage in peripheral blood mononuclear cells was significantly lower in patients with pT0N0 disease (P = .03). One- and 2-year OS rates were 94% (95% confidence interval [CI], 90-99) and 83% (95% CI, 75-93), respectively. In the multivariate Cox model after adjusting for age and gender, patients with higher WB M-MDSC and UnC-MDSC had shorter OS (optimal cutoff points by recursive partitioning analysis, hazard ratio = 7.5 [95% CI, 2.5-22.8], P = .0004; hazard ratio = 3.4 [95% CI, 1.0-11.0], P = .046, respectively).ConclusionIn patients with nonmetastatic UC of bladder, higher WB M-MDSC and UnC-MDSC before cystectomy had negative prognostic value. Prospective validation is warranted.     

Human effector T cells derived from central memory cells rather than CD8T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy

Adoptive cell therapy provides an attractive treatment of cancer, and our expanding capacity to target tumor antigens is driven by genetically engineered human T lymphocytes that express genes encoding tumor-specific T cell receptors (TCRs). The intrinsic properties of cultured T cells used for therapy were reported to have tremendous influences on their persistence and antitumor efficacy in vivo. In this study, we isolated CD8⁺ central memory T cells from peripheral blood lymphocytes of healthy donors, and then transferred with the gene encoding TCR specific for tumor antigen using recombinant adenovirus vector Ad5F35-TRAV-TRBV. We found effector T cells derived from central memory T cells improved cell viability, maintained certain level of CD62L expression, and reacquired the CD62L⁺CD44^high phenotype of central memory T cells after effector T cells differentiation. We then compared the antitumor reactivity of central memory T cells and CD8⁺T cells after TCR gene transferred. The results indicated that tumor-specific TCR gene being transferred to central memory T cells effectively increased the specific killing of antigen positive tumor cells and the expression of cytolytic granule protein. Furthermore, TCR gene transferred central memory T cells were more effective than TCR gene transferred CD8⁺T cells in CTL activity and effector cytokine secretion. These results implicated that isolating central memory T cells rather than CD8⁺T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer.     

TCR repertoire intratumor heterogeneity of CD4+ and CD8+ T cells in centers and margins of localized lung adenocarcinomas

Intratumor heterogeneity (ITH) of T cell receptor (TCR) repertoire in different T-cell subsets and locations in lung adenocarcinomas was unclear. Here, we investigated percentages and TCR repertoire of freshly isolated CD4⁺ and CD8⁺ tumor infiltrating lymphocytes (TILs) in tumor centers and margins by flow cytometry on 80 tumor samples from 20 patients and high-throughput TCR sequencing on 27 and 25 samples of CD4⁺ and CD8⁺ TILs from seven patients. Our results demonstrated that amount and TCR repertoire diversity of CD4⁺ TILs were significantly higher than those of CD8⁺ TILs and moreover substantial ITH regarding amount and TCR repertoire of CD4⁺ and CD8⁺ TILs were observed. Additionally, ITH of CD4/CD8 T-cell ratio and CD8⁺ TIL repertoire across center regions was lower than that across margin regions. The amount and TCR repertoire ITH of CD4⁺ and CD8⁺ TILs and mean clonality of CD8⁺ TILs in tumor centers were associated with relapse. Our study provides insights into amount and TCR repertoire ITH of CD4⁺ and CD8⁺ TILs in tumor centers and margins as well as corresponding association with prognosis in lung adenocarcinoma patients, suggesting potential clinical significance of TCR repertoire.