Persistence of peripheral CD8 + CD28− T cells indicates a favourable outcome and tumour immunity in first-line HER2-positive metastatic breast cancer

Background The contradictory role of CD8 + CD28− T cells in tumour immunity has been reported, while their biological and clinical significance in HER2-positive metastatic breast cancer (MBC) is still unknown. Methods HER2-positive MBC patients with no prior therapy in the metastatic setting were retrospectively recruited at two medical centres. Peripheral CD8 + CD28− T cells (pTCD8+CD28-) were detected at baseline and following therapeutic intervals. Progression-free survival (PFS) was compared according to pTCD8+CD28− levels. The molecular features of pTCD8+CD28− and its correlation with tumour immunity were also investigated. Results A total of 252 patients were enrolled, and the median follow-up time was 29.6 months. pTCD8+CD28− high at baseline has prolonged PFS compared to pTCD8+CD28− low (P = 0.001). Patients who maintained pTCD8+CD28− high had a longer PFS than those who kept pTCD8+CD28− low (P < 0.001). The enhanced pTCD8+CD28− level also indicates a longer PFS compared to pTCD8+CD28− low (P = 0.025). Here, pTCD8+CD28- was demonstrated as an antigen-experienced effector T cell. Higher IL-2 level (P = 0.034) and lower TGF-β level (P = 0.016) in the serum and highly infiltrated CD8 + CD28− T cells (P = 0.037) were also connected to pTCD8+CD28− high. Conclusions High pTCD8+CD28− level is associated with a favourable tumour immunity and a better PFS of HER2-targeting therapy in MBC patients.


INTRODUCTION
The human immune system plays an important role in the progression of cancer [1].The intimate interaction between lymphocytes and breast cancer development has been reported [2,3].Among various subpopulations of lymphocytes, activated CD8+ cytotoxic T lymphocytes (CTLs) serve as a potent mechanism through which the immune system eliminates tumours [4].The full activation of CTLs requires two fundamental signals: T-cell receptor (TCR) signalling and co-stimulatory signalling [5].During this process, CD28 is an indispensable molecule needed for initiating co-stimulatory signalling [6].CD28-mediated co-stimulatory signalling contributes to T-cell survival, proliferation, cytokine production, and metabolism [5].Alongside the maturation of CTLs, CD28 expression is progressively and irreversibly down-regulated [6].The CD8 + CD28− T cell (T CD8+CD28-) was first identified as a phenotype associated with memory/effector cells [7].T CD8+CD28-also expresses some natural killer cell-related receptors, which mediate TCRindependent cytotoxicity, suggesting its innate immune property [8].The increased level of T CD8+CD28-has been shown to be related to normal aging, chronic viral infections, and malignancies [8,9].Notably, studies have reported conflicting roles of T CD8+CD28-in various cancers.The high cytotoxic potential of T CD8+CD28-has been found in melanoma, head and neck cancer, cervical cancer and hepatocellular carcinoma [10][11][12][13].On the contrary, the regulatory or suppressive role of T CD8+CD28− has been identified in lung cancer, pleural mesothelioma, and glioblastoma [14][15][16].There is now a growing consensus that T CD8+CD28− displays an immunosuppressive function in many other cancers [17].Currently, only a few studies have reported on the role of T CD8+CD28− in breast cancer.Peripheral T CD8+CD28-(pT CD8+CD28-) has been identified as a prognostic factor for progression-free survival (PFS) in metastatic breast cancer (MBC) patients who received chemotherapy or adoptive T-cell therapy [18,19].These two studies both suggested a negative prognostic role of pT CD8+CD28− .However, the aforementioned studies primarily concentrated on the initial level of pTCD8 + CD28− and regrettably overlooked the dynamic alterations that occur in response to clinical intervention.Besides, the regimens, clinical subtypes, and therapeutic lines of these study cohorts were not strictly confined.These limitations may undermine our understanding of the multifaceted nature of pT CD8+CD28− , as our research revealed a varied prognostic significance of peripheral CTL according to molecular subtypes of MBC [20].Given the aforementioned unresolved issues, we conducted dynamic monitoring of pT CD8+CD28− in HER2-positive (HER2 + ) MBC patients who underwent first-line HER2-targeting therapy.Our objective was to shed light on the biological significance of pT CD8+CD28− .Surprisingly, we found a favourable prognostic role for pT CD8+CD28-in HER2-positive MBC, which differs from previous reports.

MATERIALS AND METHODS Patient enrollment and study design
BC patients were recruited at Peking University Cancer Hospital and Jin Xiang People's Hospital between January 2014 and September 2020.The criteria for BC patient selection are summarised in Supplementary Table S1.Oestrogen Receptor (ER) and Progesterone Receptor (PgR) positivity was evaluated by immunohistochemistry (IHC), using antibodies against ER (CONFIRM anti-Oestrogen Receptor (ER)(SP1), Ventana Medical Systems, Inc., Arizona, USA) and PgR (CONFIRM TM anti-Progesterone Receptor (PR)(1E2), Ventana Medical Systems, Inc., Arizona, USA).IHC staining against HER2 (VENTANA anti-HER2/neu (4B5), Roche Diagnostics, Mannheim, Germany) was scored according to intensity as 0, 1+ , 2+ or 3 + ; sample with scores of 0 ~1+ was considered HER2 − , while sample with a score of 3+ was considered HER2 + .HER2 amplification of 2+ samples were confirmed by FISH.Eligible HER2 + MBC patients were grouped according to the regimen they received.The peripheral lymphocyte (pL) was detected at baseline and subsequent follow-up visits until failure of first-line treatment.The flow diagram of the cohort selection is shown in Fig. 1.All procedures involving human participants met with the criteria of the Peking University Cancer Hospital ethical committee (Ethic No. 2016KT47) and Jin Xiang People's Hospital ethical committee (Ethic No. 2021092301).

Patients' follow-up
For each enrolled patient, there is one pre-treatment follow-up and then a routine post-treatment follow-up every 2 or 3 therapeutic cycles for imaging-based medical assessments to monitor disease progression.The primary endpoint of this study was PFS, which was defined as the time from the start of therapy to disease progression (PD) or the last date of follow-up.Patients alive without an event as of the PD date were censored at the last study follow-up date (September 30, 2020).Treatment efficacy was evaluated by diagnostic imaging per Response Evaluation Criteria in Solid Tumours (RECIST) 1.1 [21].

Tumour-infiltrating lymphocytes (TILs) evaluation
Two experienced pathologists independently performed the TILs evaluation on full whole-slide sections stained with hematoxylin & eosin.These sections were obtained from needle biopsies of metastatic lesions in patients with HER2 + MBC.TILs were assessed using the guidelines of the international TIL working group [22].Briefly, stromal TILs were measured as a percentage of immune cells in stromal tissue within the tumour that showed a mononuclear immunological infiltrate.The level of TILs was analysed as a continuous measurement ranging from 0 to 100%.

Confocal immunofluorescence assay and infiltrated CD8 + CD28− T cell
For immunofluorescent staining, sections were blocked and incubated overnight at 4 °C with primary antibodies.The dilution of the primary antibodies against CD8 (ab237709, Abcam, Cambridge, UK) and CD28 (ab193350, Abcam, Cambridge, UK) was both 1:50.After washing in PBS, sections were incubated with fluorescence-conjugated secondary antibodies at a dilution of 1:200 (ab6717, Abcam, Cambridge, UK; bs-0296P-PE-Cy3, Bioss, Beijing, China) for 1 hour at room temperature.Sections were then washed and counterstained with nuclear dye 4,6-diamino-2phenylindole (DAPI).The resulting images were visualised and captured on a confocal microscope (FV1200, Olympus, Japan).The infiltrated CD8 + CD28-T cells in tumour tissue were evaluated by two independent pathologists.Every ten fields per slide were selected to calculate the percentage of target cells in all nucleated cells within the tumour nests and tumour stroma.The percentage of infiltrated CD8 + CD28-T cells ranged from 0 to 100%.

EILSA detection for serum level of IFN-γ, IL-2,TNF-α and TGF-β
The serum samples were collected from patients with empty stomach in the morning.Blood was collected in EDTA tube at 1600 × g for 10 min at 4 °C, 1 h after collection.It was then transferred into tubes and kept at −80 °C for further use.Human serum IFN-γ, IL-2, TNF-α and TGF-β antigens were measured using commercially available kits: Human IFN-γ ELISA (EHC102g, QuantiCyto ® , China), Human IL-2 Instant ELISA TM (BMS221INST, Thermo Scientific Inc., US), Human TNF alpha Instant ELISA TM (BMS2231INST, Thermo Scientific Inc., USA) and Human TGF-β1 ELISA (KGEHC107b, Jiangsu KeyGEN BioTECH Ltd, China) respectively.Assays were performed according to the manufacturer's protocol.All samples were assayed using an automated immunoassay analyzer AIA-system (TOSOH Corp., Tokyo, Japan).Plasma cfDNA extraction and NGS testing cfDNA was extracted from plasma samples using QIAamp circulating nucleic acid kit.In all, 5-20 ng of the extracted cfDNA were prepared for library construction, including end-repair, dA-tailing, adaptor ligation, and PCR amplification.The library was hybridised overnight with the panel probes.Unbound fragments were then washed away.The quantity and quality of the purified cfDNA were checked using Qubit fluorimeter and Bioanalyzer 2100.Paired-end sequencing with 2 × 150 bp reads was performed using the Illumina sequencing platform.cfDNA testing from plasma was conducted in China (Huidu Shanghai Medical Sciences Ltd.) using the PredicineCARE assay targeting 152 genes.Detailed information can be found in Supplementary Fig. S2.

Single-cell preparation and sequencing
The collected pT CD8+CD28-and pT CD8+CD28+ were separately stored in the sCelLive TM Tissue Preservation Solution (Singleron Biotechnologies, Nanjing, China) and then transported to the Singleron lab on ice as soon as possible.
The sample was stained with trypan blue (Sigma, Shanghai, China) and microscopically evaluated for cell viability.Single-cell suspensions at 1 × 10 5 cells/mL in PBS (HyClone, Shanghai, China) were prepared and loaded onto microfluidic devices and scRNA-seq libraries were constructed according to the GEXSCOPE ® protocol using the GEXSCOPE ® Single-Cell RNA Library Kit (Singleron Biotechnologies) and Singleron Matrix ® Automated single-cell processing system (Singleron Biotechnologies).Individual libraries were diluted to 4 ng/µL and pooled for sequencing.Pools were sequenced on Illumina novaseq6000 with 150-bp paired-end reads.

Statistical analysis
Clinical data were obtained from the patient electronic medical recording system.The relationships between pT CD8+CD28-levels, mutation prevalence and other clinical characteristics were assessed using the Chi-square test or Fisher exact test, Student t test or Mann-Whitney U test, and Pearson correlation tests accordingly.The training and validation sets were allocated to each case using computer-generated randomised numbers.All analyses were conducted by SPSS 19.0 version software (IBM Inc., NY, USA).Missing data was excluded from the analysis.The cut-off value of pT CD8+CD28-level regarding PFS was calculated using the software of X-tile 3.6.1 version reported by Camp RL et al. [23] Kaplan-Meier survival analysis and the log-rank test were used to compare PFS between different patient cohorts.The Cox proportional hazard regression model was utilised to estimate the hazard ratio (HR) and 95% confidence interval (CI) of the proportion of pT CD8+CD28-in peripheral blood, while adjusting for confounding factors.All statistical tests were two-sided with a significance level set at 0.05.

Basic characteristics of the study cohort
A total of 252 HER2 + MBC patients were enrolled in this study.
The clinical characteristics of the cohort are presented in Table 1.In summary, the median follow-up time of the HER2 + MBC cohort was 29.6 months (range: 3.7-95.0months), and the median age at diagnosis was 52 years (range: 25-82).At the time of treatment, primary stage IV breast cancer was observed in 67 patients (26.6%), while recurrent breast cancer was present in 185 patients (71.9%).All individuals of the cohort received first-line therapy.Out of the 252 patients, 151 (60.0%) received chemotherapy plus trastuzumab, 49 (19.4%) received chemotherapy in combination with trastuzumab and pertuzumab, 23 (9.1%) received chemotherapy plus tyrosine kinases inhibitors (TKIs) and 29 (11.5%) received chemotherapy alone.In those who received TKIs, all had previously undergone adjuvant and/or neoadjuvant anti-HER2 therapy involving trastuzumab and/or pertuzumab.

The prognostic value of baseline pT CD8+CD28-level
The 252 patients with HER2 + MBC were randomly allocated into two groups: the training set (n = 126) and the validation set (n = 126).Using X-tile software, we evaluated the prognostic potential of each pL subtype in terms of first-line PFS within the training set.Consequently, only the pT CD8+CD28− level demonstrated a significant association with first-line PFS in HER2 + MBC patients (Supplementary Fig. S3).The optimal cut-off value for pT CD8+CD28- was determined to be 18.0%(ranging from 16.5 to 19.0%) with a significant P value of 0.028, as shown in Fig. 2a.This identified cut-off value was subsequently validated in the independent validation set (P = 0.033) (Fig. 2b).Apart from pT CD8+CD28-level, other baseline characteristics that may contribute to first-line PFS were also assessed through univariate analysis.These characteristics included the primary lymph node stage (P = 0.017), the presence of uncommon metastatic sites (P = 0.001) and the utilisation of HER2targeted therapy (P = 0.034) (Supplementary Table S2).The final Cox regression model in multivariate analysis was adjusted to include all variables that showed statistical significance in the aforementioned univariate analysis.Consistent with expectations, the Cox regression model confirmed that the baseline pT CD8+CD28− level served as an independent prognostic factor for first-line PFS among HER2 + MBC patients, and high pT CD8+CD28-level (≥18.0%) was associated with a prolonged median PFS (mPFS) compared to low level (10.5 vs. 17.4 months, P = 0.002) (Fig. 2c).
The prognostic value of dynamic pT CD8+CD28− levels Considering the aforementioned results, we sought to determine the relationship between the dynamic alteration of pT CD8+CD28- and first-line PFS.Based on variations in pT CD8+CD28-, patients who underwent anti-HER2-based therapy were categorised into four groups: high-level, low-level, reducing and enhancing groups respectively.The high-level group consistently maintained a high level of pT CD8+CD28-throughout the entire therapeutic course, while the low-level group consistently maintained a low level.The reducing group initially exhibited a high level but subsequently showed a low level at each follow-up visit, whereas the enhancing group demonstrated the opposite pattern (Fig. 3a).Consequently, we included a total of 139 patients who met these criteria in Kaplan-Meier analysis regarding first-line median PFS.Among them, the high-level group (n = 71) had the longest mPFS of 15.5 months, while the low-level group (n = 25) had the shortest mPFS of 7.7 months.Meanwhile, both the reducing group (n = 6, mPFS = 11.4 months) and the enhancing group (n = 37, mPFS = 11.1 months) displayed similar mPFS durations.In summary, we demonstrated that the dynamic changes in pT CD8+CD28-levels were also associated with first-line in patients with HER2 + who received anti-HER2-based therapy (P = 0.000427) (Fig. 3b).However, it is important to note that this prognostic value was observed within a limited sample size (n = 139); therefore, further validation in an independent cohort is warranted for future studies.

Association of pT CD8+CD28-level with other clinical characteristics
In addition to assessing the prognostic value of pT CD8+CD28-, we also investigated its correlation with other clinical characteristics in the cohort.The baseline pT CD8+CD28-level exhibited a significant negative correlation with bone metastasis (coefficient = −0.189,P = 0.003), lymph node metastasis (coefficient = −0.152,P = 0.016), and the number of metastatic sites (coefficient = −0.200,P = 0.001) (Table 2).Furthermore, there was a negative correlation between changes in pT CD8+CD28-and bone metastasis (coefficient = −0.218,P = 0.006) (Supplementary Table S3).Notably, both bone and lymph node are all critical immune-related organs.Compared to health controls, HER2 + MBC patients exhibited a unique positive correlation between pT CD8+CD28-and total T cells (coefficient = 0.134, P = 0.033) as well as natural killer T cells (coefficient = 0.143, P = 0.024) (Table 3).These findings, however, require further validation and should be cautiously interpreted due to the low coefficients.

Association of pT CD8+CD28-with the tumour immunity
To ascertain the relationship between pT CD8+CD28-levels and patients' tumour immunity, we initially compared the TILs infiltration in individuals with high and low levels of pT CD8+CD28-.
To further substantiate the association between pT CD8+CD28- and tumour immunity, we investigated the variations of pT CD8+CD28-mediated by immunotherapy.Regrettably, immunotherapy is not currently employed as a standard regimen for HER2 + MBC patients.Alternatively, we evaluated our hypothesis in the context of metastatic triple-negative breast cancer (mTNBC) patients who are eligible for immunotherapy.We enrolled five mTNBCs patients received combination therapy of anti-PD1 and multiple antigen-specific cell therapy (MASCT) at different therapeutic lines.Specifically, patients with PD-L1-positive tumours underwent a dose-escalation study using intravenous camrelizumab (3 mg/kg, every 2 weeks), along with intravenous infusion of T cells and dendritic cells (DCs) every 27-36 days.Meanwhile, the baseline and subsequent follow-up assessments until disease progression revealed a decrease in peripheral levels of CD8 + PD1 + T cells (Fig. 5d, left) and an increase in pT CD8+CD28- levels (Fig. 5d, middle) across all five patients.In contrast, the fluctuation in peripheral CD3 + CD8 + T-cell levels did not demonstrate a consistent pattern, thereby ruling out the possibility of false-positive amplification of pT CD8+CD28-due to T-cell infusion (Fig. 5d, right).

DISCUSSION
Despite the dramatic clinical improvements during the past two decades, resistance to HER2-targeted therapy remains virtually inevitable [24].Therefore, there is an urgent need for a robust predictor of therapeutic efficacy.Numerous studies have reported biomarkers associated with the effectiveness of anti-HER2 therapy [25,26].Recently, immune-related factors have garnered much attention among a range of biomarkers.In 2023, Hills RK and his colleagues reported the prognostic value (but not the predictive value) of TILs in early breast cancer patients receiving trastuzumab [27].In metastatic setting, TILs also exhibit a prognostic value for OS, as evidenced by a retrospective analysis of the CLEOPATRA trial [28].These findings suggest a connection between tumour immunity and the overall outcome of the patients.The detection of peripheral lymphocytes offers advantages such as real-time monitoring, minimal invasiveness, and homogeneity compared to needle biopsy-based detection.Here, we demonstrated the prognostic value of pT CD8+CD28-in MBC patients receiving anti-HER2-based therapy.At baseline, patients with pT CD8+CD28-high had significantly longer mPFS than those with pT CD8+CD28-low (Fig. 2a-c).In subgroup analysis, the same result was reproduced in patients who received trastuzumab plus chemotherapy (Fig. 2d).Although similar trends were noted in other subgroups, none of these reached statistical significance (Fig. 2e-g).This may be partly due to the limited sample size and the insufficient follow-up time period.Moreover, the dynamic monitoring of pT CD8+CD28- also showed a prognostic value comparable to the baseline pT CD8+CD28-level (Fig. 3).It is noteworthy that even among patients with pT CD8+CD28-low at baseline, those who exhibited an increased pT CD8+CD28-level upon HER2-targeting therapy also gained more benefit than those maintain pT CD8+CD28-low (7.7 vs. 11.1 months, P = 0.025) (Fig. 3).Evidence have demonstrated the anti-tumour effect of trastuzumab induced antibody-dependent cellular cytotoxicity (ADCC) [29].Therefore, we hypothesize that pT CD8+CD28-level may reflect tumour immunity of the patients, thus aiding in the identification of individuals who are more likely to benefit from HER2-targeted therapy.Numerous studies have highlighted the suppressive role of CD8 + CD28-T cells in  regulating tumour immunity, suggesting that the presence of cell surface markers CD8+ and CD28-signifies a senescent or exhausted phenotype of T cell [30].According to our data, however, high level of pT CD8+CD28-appears to indicate a positive tumour immunity in HER2 + MBC.First, we found an intensive expression of perforin and granzyme B in pT CD8+CD28-, suggesting its cytotoxic function (Fig. 4a).In addition, TCR clonality and transcriptome analysis further revealed that pT CD8+CD28-served as an antigen-experienced effector T cell in HER2 + MBC (Fig. 4b, c).
In peripheral blood, pT CD8+CD28-level showed a positive correlation with total T cells and nature killer T-cell level (Table 3).In metastatic lesions, we noticed a trend indicating that pT CD8+CD28- level was positively associated with TILs infiltration, particularly CD8 + CD28− T-cell infiltration (Fig. 5a, b).The maintenance of terminally differentiated effector T cells is well-known to be dependent on IL-2 and can be disrupted by transforming growth factor-β (TGF-β) [31,32].Our findings support this notion, as we found a significant association between a higher serum level of IL-2 (P = 0.034) and a lower level of TGF-β (P = 0.016) with pT CD8+CD28-high (Fig. 5b).These evidence were consistent with what we found at cellular level.Furthermore, we also observed the anti-PD1 therapy induced upregulation of pT CD8+CD28-level, although this result was achieved in TNBC patients (Fig. 5).This clinical phenomenon further strengthens our hypothesis regarding the close relationship between pT CD8+CD28-and tumour immunity.Still, our hypothesis needs more detailed investigation and concrete evidence.Collectively, we demonstrated that a high level of pT CD8+CD28-is associated with enhanced tumour immunity in HER2 + MBC.This observation provides partial insight into why patients with an intensive or increasing level of pT CD8+CD28-upon anti-HER2-based therapy could gain more benefit.There are several limitations of present study.Firstly, this retrospective study has a limited number of patients in most therapeutic subgroups.Perspective studies designed to verify the predictive value of pT CD8+CD28-for each therapeutic subgroup are necessary.Secondly, the observed increase in pT CD8+CD28-level following anti-PD1 therapy in mTNBC patients should also be validated in HER2 + MBC patients.Finally, the comprehensive mechanisms underlying the prognostic role of pT CD8+CD28-and its effect on tumour progression remain unclear, necessitating further exploration.

CONCLUSIONS
Our study has identified the novel prognostic value of pT CD8+CD28- for anti-HER2-based therapy in MBC patients.Specifically, patients with pT CD8+CD28-high at baseline exhibited prolonged PFS.Patients with consistent or enhanced pT CD8+CD28-levels upon the treatment also indicate a better PFS.In addition, we observed a positive correlation between pT CD8+CD28-levels and tumour immunity, which can be enhanced by immunotherapy.

DATA AVAILABILITY
Data generated during this study are available from the corresponding author upon reasonable request.

MATERIALS AVAILABILITY
Materials generated during this study are available from the corresponding author upon reasonable request.

Fig. 1
Fig. 1 Flow chart of patient selection and classification according to the therapeutic regimens.H trastuzumab, P pertuzumab, TKIs tyrosine kinase inhibitors, pts patients.

Fig. 2
Fig. 2 The prognostic role of baseline pTcd8+cd28-in HER2 + MBC patients regarding first-line PFS.Determine the prognostic role of pTcd8+cd28− in training set (a) and retest in the validation set (b). Cox regression analysis was performed for pTcd8+cd28−, including other factors contributing to the PFS.The HR values for each factor derived from the multivariate analysis are also presented (c).The prognostic role of pTcd8+cd28− in the chemotherapy plus trastuzumab subgroup (d), in the chemotherapy plus trastuzumab & pertuzumab subgroup (e), in the chemotherapy plus TKIs subgroup (f) and in chemotherapy-alone subgroup (g); The prognostic role of pTcd8+cd28− in patients who received anti-HER2 based therapy (h).pTcd8+cd28− peripheral CD8 + CD28-T cell, TKIs tyrosine kinase inhibitors.

Fig. 3
Fig. 3 The prognostic value of dynamic change of pTcd8+cd28-regarding first-line PFS.The diagram for grouping dynamic change of pTcd8+cd28− (a).The prognostic role of dynamic change of pTcd8+cd28− was assessed using K-M analysis (b).

Table 1 .
Clinical characteristics of the study cohort (n = 252).
b Multiple lesions occurred in the same organ only count once.c Tyrosine kinase inhibitors.X. Liu et al.