We selected 23 patients with CIN2 who received ALA-PDT at the Renji Hospital of Shanghai Jiaotong University based on the following inclusion criteria: (i) women between the ages of 18 and 50 years; (ii) histologically proven CIN2 before treatment; (iii) has completed ALA-PDT as previously administered (6); (iv) follow-up information at 6 months after treatment; and (v) having been infected with hrHPV. The exclusion criteria were as follows: (i) receiving other treatments such as laser therapy during ALA-PDT and (ii) having immune system and autoimmune diseases.

This study was approved by the Ethics Committee of Renji Hospital (CTR20210524) and was performed in accordance with the Declaration of Helsinki and national and international guidelines. Written informed consent was obtained from all the patients, and all patients were followed up for 6 months.

We obtained 23 pairs of archival formalin-fixed paraffin-embedded (FFPE) blocks at pre-treatment and at the 6-month follow-up period. We divided the outcomes of ALA-PDT for patients with CIN2 into four groups at the 6-month follow-up period: disappearance, regression, persistence, and progression of CIN2, and two groups for HPV: HPV clearance and HPV-positive.

The disappearance of CIN2 was defined as the disappearance of initial findings and a normal histology. The regression of CIN2 was defined as the reduction in the severity of the histological results (LSIL or condyloma-like) compared to the initial state histology. The persistence of CIN2 was defined as no change by colposcopy-directed biopsy. The progression of CIN2 was defined as the diagnosis of CIN3+ resulting from a biopsy guided by colposcopy. Moreover, HPV clearance was defined as patients with HPV-negative results and HPV-positive was defined as patients with hrHPV infection at 6 months follow-up.

FFPE tissue blocks were cut into 4 um slides and blank slides were deparaffinized in xylene and rehydrated with an ethanol series. Next, they were incubated with primary antibodies against CD45 (1:50, Santa Cruz sc-28369), CD3 (1:500, Abcam ab237707), CD8 (1:100, Cell Signaling Technology 70306), Granzyme B (GrB) (1:50, Cell Signaling Technology 46890), Chemokine receptor 3 (CXCR3) (1:500, Abcam ab288437), TCF1 (1:50, Cell Signaling Technology 2203), CD45RO (1:400, Cell Signaling Technology 55618), CD103 (1:50, Cell Signaling Technology 95835), and programmed cell death protein 1 (PD-1) (1:100, Cell Signaling Technology 86163) at 4°C overnight. The samples were then incubated with a fluorescence-coupled secondary antibody. Images were captured using a confocal laser scanning microscope (Leica TCS SP8 STED; Leica Microsystems GmbH, Wetzlar, Germany). The fluorescence integral density was measured using ImageJ software (v1.48; National Institutes of Health, USA).

Images were captured using a Pannoramic 250 digital slide scanner (3DHISTECH, Ltd., Budapest, Hungary). Ten areas representing the density of immune infiltrates in each sample were selected. Images of these ten areas were captured on each stained slide using a panoramic viewer at 40×. Cells were manually counted by two individual researchers using the cell counter plug-in in ImageJ software.

The mean cell count per image was then calculated. Cell counts were converted and presented as the number of cells per mm². Each case was assigned a random subject number to ensure that both researchers were unaware of the clinical characteristics of each case when analyzing the immune infiltrate.

The infiltration of CD8⁺ T cells in CIN2 was compared before and after ALA-PDT in patients with different outcomes at the 6-month follow-up. We used the median cell counts of CD45, CD3, CD8, GrB, CXCR3, CD103, CD45RO, TCF1, and PD-1 in patients with CIN2 (n = 23).

All results are expressed as mean ± standard deviation (SD). An unpaired Student’s t-test was performed for comparisons between two groups. All analyses were performed using the GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). Statistically significant differences between groups were defined as p < 0.05.

We selected 23 patients with CIN2 who underwent ALA-PDT. The mean age was 27.65 ± 4.35 years old (20–48 years old). At the 6-month follow-up, 16 of the 23 patients treated with ALA-PDT achieved complete hrHPV clearance. In addition, the CIN2 disappeared in 15 patients, regressed in 8, and persisted or progressed in 0. See Table 1 for an overview of patient characteristics.

To analyze changes in the variety and quantity of immune cells after ALA-PDT, we performed immunofluorescence staining. CD45 is a receptor-linked protein tyrosine phosphatase that is expressed in all leukocytes; thus, we first examined the expression of CD45 in tissues. We observed a small amount of CD45⁺ immune cell infiltration in untreated CIN2, and lesions contained significantly (p < 0.001) higher numbers of CD45⁺ leukocytes after ALA-PDT (Figures 1A, B). According to a previous study (12), a better understanding of HPV tumor–host immune system interactions is mainly related to T cell-based immunity directed toward the elimination of viral-infected cells and prevention of pathogenesis. Thus, to determine the effect of ALA-PDT on T cell-based immunity, these paired cases were studied by co-staining with CD3 and CD8. We found an increase in the number of CD3⁺ T cells and CD8⁺ T cell infiltration in CIN2 compared to that before ALA-PDT (Figures 1A, B). Moreover, at the 6-month follow-up, the number of CD8⁺ T cell subsets was significantly higher in the HPV clearance group than in the HPV-positive group (Figure 1C). The number of CD8⁺ T cells was also significantly higher in the CIN2 disappearance group than that in the regression group (Figure 1D).

CD8⁺ T cells attack HPV-infected cells by releasing GrB, a marker used to identify activated cytotoxic T cells (13). Therefore, we evaluated the expression of GrB in cytotoxic CD8⁺ T cells to understand their activation states and found an increase in the percentage of CD8⁺ T cells expressing GrB in post-treatment samples (Figures 1E, F). Furthermore, the proportion of CD8⁺ T cells expressing GrB was significantly higher in the HPV clearance group than that in the HPV-positive group (Figure 1G). In addition, the CIN2 disappearance group showed higher GrB expression in CD8⁺ T cells than the regression group (Figure 1H). These data indicate that ALA-PDT can activate CD8⁺ T-cell immunity to eliminate abnormal cervical cells.

Understanding why ALA-PDT promotes CD8⁺ T cell entry into lesions is an important advancement in efforts to harness the ability of the immune system to fight CIN2. CXCR3 is critical for the trafficking and activation of CD8⁺ T cells (14), thus we detected the change in CXCR3 expression on CD8+ T cells after ALA-PDT. Our data showed a remarkable increase in CXCR3 expression on CD8⁺ T cells after ALA-PDT compared to that before treatment in patients with CIN2 (Figures 2A, B). Furthermore, at the end of the 6-month follow-up period, the proportion of CD8⁺ T cells expressing CXCR3 was significantly higher in the HPV clearance group than in the HPV-positive group (Figure 2C). In addition, the CIN2 disappearance group exhibited increased CXCR3 expression in CD8⁺ T cells compared to the regression group (Figure 2D). These data suggest that ALA-PDT increases CD8⁺ T cell infiltration by promoting CXCR3 expression.

Memory CD8⁺ T cells are capable of persisting and functioning throughout cervical lesions and can rapidly differentiate into effector CD8⁺ T cells upon pathogen re-encounter to eliminate HPV infection. Our data showed a significant increase in the infiltration of CD8⁺ T cells after ALA-PDT; therefore, we investigated whether ALA-PDT promotes the infiltration of CD8⁺ T cells by regulating the CD8⁺ memory T cell subset.

Tissue-resident memory T (T_RM) cells were identified as a subset of memory T cells (15, 16), and we first detected the infiltration of CD8⁺ T_RM cells into the paired samples by co-staining for CD8, CD45RO, and CD103. As shown in Figures 3A, B, cervical lesions showed a higher number of CD8⁺ T_RM cells after ALA-PDT. Furthermore, the density of CD8⁺ T_RM cells was higher in the HPV clearance group than that in the HPV-positive group (Figure 3C). In addition, an increase in CD8⁺ T_RM cell infiltration was observed in the CIN2 disappearance group compared to that in the regression group (Figure 3D).

Stem-like cell memory T (T_SCM) cells constitute another subset of memory T cells (17), and we further examined the distribution of CD8⁺ T_SCM cells by co-staining for CD8, CD45RO, and TCF1. Our data revealed that the number of CD8⁺ T_SCM cells did not show any obvious change after ALA-PDT (Figures 3E, F). Moreover, there was no significant difference in the number of CD8⁺ T_SCM cells between the HPV clearance and HPV-positive groups (Figure 3G). In addition, no significant difference was observed in the number of CD8⁺ T_SCM cells between the CIN2 disappearance and regression groups (Figure 3H).

PD-1 has been identified as a negative regulator of T cell activity (18). To better understand the mechanism by which ALA-PDT promotes CD8⁺ T immunity, we determined the proportion of CD8⁺ T cells expressing PD-1. After ALA-PDT, cervical lesions with a decreased percentage of CD8⁺ T cells expressing PD-1 were detected compared with those before treatment (Figures 4A, B). Moreover, the HPV clearance group showed a reduction in the proportion of CD8⁺ T cells expressing PD-1 compared to that in the HPV-positive group after ALA-PDT (Figure 4C). In addition, the CIN2 disappearance group showed a decreased percentage of CD8⁺ T cells expressing PD-1 compared with the regression group (Figure 4D).