Abstract:

Objective To investigate the relationship between the levels of T-cell immunoglobulin and mucin domain containing protein-3 （TIM-3） and programmed death-1 （PD-1） on the surface of peripheral blood and tumor tissue of CD4⁺ T cells and the efficacy of immunotherapy in cervical cancer，and to analyze the mediating role of macrophage polarization indicators CD68，CD163，arginase-1 （Arg-1），and inducible nitric oxide synthase （iNOS）. Methods A total of 111 patients with cervical cancer treated at 982nd Hospital of Joint Support Force of the People's Liberation Army of China from April 2024 to January 2025 were selected as study subjects. All patients received radical concurrent chemoradiotherapy combined with pembrolizumab immunotherapy，the levels of TIM-3，PD-1，and macrophage polarization indicators were compared between patients with different treatment responses. The levels of TIM-3 and PD-1 were also compared among patients with different clinicopathological characteristics. The influence of macrophage polarization indicators on the levels of TIM-3 and PD-1 was investigated using multiple linear regression analysis. A multivariate generalized linear mixed model （GLMM） was constructed to analyze the effects of TIM-3 and PD-1 on immunotherapy efficacy. Receiver operator characteristic （ROC） curves were used to evaluate the predictive value of TIM-3 and PD-1 for immunotherapy efficacy. Restricted cubic spline analysis was performed to examine the dose-response relationship between TIM-3/PD-1 levels and immunotherapy efficacy. The Bootstrap method was used to analyze the mediating effect of macrophage polarization indicators on the relationship between TIM-3/PD-1 levels and immunotherapy efficacy. Results The overall efficiency rate among 111 patients was 73.87% （82/111）. There were statistically significant differences in the levels of TIM-3，PD-1，CD68，CD163，Arg-1，iNOS of peripheral blood and tumor tissues between effective （n=82） and ineffective patients （n=29） （all P<0.001）. There were statistically significant differences in the levels of TIM-3 and PD-1 of peripheral blood between patients with and without lymph node metastasis （Z=-1.97，P=0.049； Z=2.02，P=0.043）； there were statistically significant differences in the levels of PD-1 of peripheral blood between patients with different differentiation degrees （Z=2.59，P=0.010）； there were statistically significant differences in the levels of TIM-3 of peripheral blood between patients at different International Federation of Gynecology and Obstetrics （FIGO） stages （H=6.65，P=0.036）. There were statistically significant differences in the levels of TIM-3 and PD-1 of tumor tissue between patients with and without lymph node metastasis，as well as among patients with different maximum tumor diameters （Z=-2.52，P=0.012； Z=2.53，P=0.011； Z=-2.83，P=0.005； Z=2.10，P=0.036）； there were statistically significant differences in the levels of TIM-3 of tumor tissue between patients with varying depths of infiltration （Z=-2.43，P=0.015）. After adjusting for other covariates，CD68 （β=1.16，P=0.017； β=1.12，P=0.016），CD163 （β=1.84，P<0.001； β=2.05，P<0.001），Arg-1 （β=1.92，P<0.001； β=1.70，P<0.001），and iNOS （β=-1.59，P<0.001； β=-1.69，P=0.012） were linearly associated with TIM-3 and PD-1 levels of peripheral blood，the trends in CD68，CD163，Arg-1，iNOS and the levels of TIM-3 and PD-1 of tumor tissue were consistent with those in peripheral blood. Medium and high levels of TIM-3 and PD-1 of peripheral blood and tumor tissue were independent risk factors for inefficacy of immunotherapy in cervical cancer （all P<0.05）. The area under the curves （AUCs） for predicting the ineffectiveness of immunotherapy in cervical cancer by the levels of TIM-3 and PD-1 of peripheral blood were 0.80 and 0.84，respectively； the AUCs for the levels of TIM-3 and PD-1 of tumor tissues were 0.86 and 0.91，respectively. The AUCs for the combined peripheral blood indicators （TIM-3+PD-1），the combined tumor tissue indicators （TIM-3+PD-1），and the total combined （all indicators from peripheral blood+tumor tissue） were 0.95，0.96，and 0.98，respectively. The total combined predicted AUC was higher than that of other indicators （Z=6.26，P<0.001； Z=6.04，P=0.001； Z=5.49，P=0.006； Z=5.11，P=0.010； Z=4.21，P=0.019； Z=3.43，P=0.025）. There was a nonlinear dose-response relationship between the levels of TIM-3 and PD-1 in peripheral blood and tumor tissue and immunotherapy inefficacy in cervical cancer. The lowest risk points were 4.78%，15.61%，and 2.87，3.29 points，respectively. The results of the mediation analysis indicated that CD68，CD163，Arg-1，and iNOS played a mediating role in the relationship between the levels of TIM-3 and PD-1 in peripheral blood and tumor tissue and the efficacy of immunotherapy in cervical cancer. Conclusions The levels of TIM-3 and PD-1 in peripheral blood and tumor tissue are higher in patients with ineffective immunotherapy for cervical cancer than those in responders，and their high levels are independent risk factors for immunotherapy inefficacy. The macrophage polarization indicators CD68，CD163，Arg-1，iNOS have a linear correlation with the levels of TIM-3 and PD-1，and they play a mediating regulatory role in the relationship between TIM-3，PD-1 and the efficacy of immunotherapy.

Key words: Uterine cervical neoplasms, Treatment outcome, Programmed death-1, Macrophage polarization