IL-15/NKG2D signaling-mediated activation of CD8⁺ T cell bypass pathways in sepsis-induced immune injury

Introduction

Sepsis is a systemic inflammatory response with a high mortality rate that increases significantly if the disease progresses to septic shock (1). Sepsis continues to top the list of fatal complications among intensive care unit (ICU) patients. Despite continuous updates to bundled therapy guidelines, the death rate of sepsis in the ICU remains as high as 30–50% (2,3). The application of bundled therapy strategies, such as early antibiotics and fluid resuscitation, has reduced the mortality rate of sepsis to some extent (4,5). Still, drug-resistant infections, immune disorders, and the lack of individualized treatment plans all limit the prognostic improvement of sepsis (6). The immune response is a key driver in both sepsis initiation and trajectory (7). In the early stages of systemic inflammatory response syndrome (SIRS), if pathogens are eliminated by innate immunity in time, immune balance can be quickly restored, and the body can recover to normal in a short period (8). In the early stages of sepsis, the innate immune system is activated, leading to an excessive inflammatory response (9). As the disease progresses, the immune system may turn to a suppressed state (9), and this state makes sepsis patients more prone to adverse prognoses such as secondary infections and death.

CD8⁺ T lymphocytes exhibit a variety of functions, including cytotoxic effects that enable them to kill target cells and cells expressing specific antigens (10). They also contribute to the immune response by promoting antibody synthesis and T lymphocyte proliferation (11). These cells are crucial regulators of both humoral and cellular immunity and are vital for maintaining homeostasis (11). Furthermore, CD8⁺ memory T cell subsets are particularly important in combating secondary infections in sepsis patients (12). Despite the extensive body of basic and clinical research on the role of CD8⁺ T cells in sepsis, the specific contributions of their various subsets remain relatively understudied.

Interleukin 15 (IL-15) is a 14–15 kDa glycoprotein predominantly secreted by dendritic cells, macrophages, monocytes, endothelial cells, interstitial cells, and renal epithelial cells (13). As a pleiotropic cytokine, IL-15 receptors are widely expressed on the surface of various immune cells, where they play a complex, dual regulatory role in maintaining innate immune responses: not only does IL-15 inhibit neutrophil apoptosis and modulate their phagocytic function (14,15), but it also enhances the phagocytic efficiency and cytokine secretion of macrophages, while simultaneously promoting the maturation and survival of dendritic cells (16). However, precisely because of these extensive and potent immune-activating capabilities, the specific role of IL-15 in the pathogenesis of sepsis remains elusive to this day. Due to its benefits to various immune cells, IL-15 has become an attractive option for immunotherapy of sepsis (17). In infectious diseases, IL-15 can enhance the body’s immune defense and help clear pathogens (18). However, excessive IL-15 production may also lead to immunopathological damage (19). NKG2D (Natural Killer Group 2 Member D, gene name: KLRK1) is widely expressed on diverse immune cells, such as natural killer (NK) cells, CD8⁺ T cells, γδ T cells, and some NK T cells (20). NKG2D on the surface of different cell types plays different but synergistic roles in the immune response (21). Furthermore, previous studies have shown that IL-15 can promote the expression of NKG2D (22,23).

Against this background, this study aims to investigate whether the IL-15/NKG2D signaling mediates bypass initiation of memory CD8⁺ T cells, thereby exacerbating sepsis-induced immune damage. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0206/rc).

Methods

Animal experiments

SPF grade 6–8-week-old male C57BL/6 mice, weighing between 18–22 g, were purchased from the Experimental Animal Center of DASHUO [SCXK (Chuang) 2025-0030]. The animal experiments were conducted at Tianjin Medical University General Hospital. All animal experiments were performed under a project license (No. IRB2021-YX-220-01) granted by the Medical Ethics Committee of Tianjin Medical University General Hospital, in compliance with the Chinese Guidelines for Animal Welfare. These mice were divided into four groups: a control group, a model group (cecal slurry infection model), a cecum ligation and puncture (CLP) group, and a CLP + IL-15 group, with n=6 in each group. Apart from the control group, the remaining mice received injections of cecal contents to activate the immune system and establish the model. Animal experiments reference prior expert consensus initiatives (24).

The model group of mice was used to establish an immune system-activated mouse model following previous methods (25). Twelve-week-old C57BL/6 mice (n=6) were sacrificed by cervical dislocation, and the cecum was excised in its entirety. The cecal contents were squeezed out and mixed with sterile water (concentration 250 µg/µL). The mixture was passed through an 860 µm pore size filter and subsequently combined with an equal volume of phosphate buffered saline (PBS) containing 30% glycerol (final concentration 125 µg/µL). The mixture was aliquoted and stored at −80 ℃. The frozen cecal serous fluid was swiftly thawed in a 37 ℃ water bath. It was injected intraperitoneally into 6-8 week-old wild-type male C57BL/6 mice at a dose of 0.9 mg/g (based on mouse body weight). Injection time points were: days 1, 4, 7, and 10.

CLP (26) surgery was used to induce sepsis models in mice in the CLP group and CLP + IL-15 group for 14 days. Solid food was withheld for 6–12 hours (26) before surgery (but water was allowed) to reduce intestinal contents and facilitate cecal manipulation. Under isoflurane anesthesia, the cecum was exteriorized via a standard laparotomy and ligated at the ileocecal junction (1/2). A 20-G sterile needle was used to puncture the head and tail of the ligated cecum, and the contents were squeezed out along the perforation site, yielding 0.3 mL. The cecum and squeezed contents were returned to the abdominal cavity, and the abdominal wall incision was sutured layer by layer. In the sham surgery group, only the cecum was opened and freed. The cecum was placed externally for 1–2 minutes without ligation or perforation, and the cecum was directly returned to the abdominal cavity, with the abdominal wall incision sutured layer by layer. Mice in the CLP + IL-15 group were intraperitoneally injected with a solution of 1.5 µg IL-15 SA (eBiosciences, 34-8151-85) diluted to 200 µL with sterile PBS after CLP surgery. Samples were collected from the mice 14 days later for subsequent studies.

Enzyme linked immunosorbent assay

Plasma was collected from each group of mice and centrifuged for supernatant. Serum interleukin (IL)-1β, IL-10, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) levels were assayed per the manufacturer’s protocol. Mouse IL-1β ELISA Kit (ZC-37974) and Mouse TNF-α ELISA Kit (ZC-39024) were purchased from Shanghai ZCIBIO Technology Co., Ltd. Mouse IL-10 ELISA Kit (YJ037873) and Mouse IFN-γ ELISA Kit (YJ002277) were purchased from Shanghai Yuanju Biotechnology Co., Ltd. Absorbance value at a wavelength of 450 nm (OD450) values were read on a microplate reader and used to determine sample concentrations.

Biochemical index testing

Whole blood was collected from mice and allowed to coagulate following a 2-hour room-temperature incubation. The coagulated sample was centrifuged at 3,000 rpm for 5 minutes to obtain serum. The levels of AST, ALT, LDH, BUN and CREA were quantitatively determined using kits for alanine aminotransferase (ALT) (140124014, Mindray), aspartate aminotransferase (AST) (140224007, Mindray), lactate dehydrogenase (LDH) (142723008, Mindray), blood urea nitrogen (BUN) (A013-2-1, Nanjing Jiancheng Bioengineering Institute) and creatinine (CREA) (141124014, Mindray) according to the manufacturer’s instructions. The levels of these indicators were measured using a biochemical analyzer (BS-460, Mindray).

Hematoxylin and eosin (H&E) staining

After completing the experiment, the spleen, lung, liver, and kidney of mice were fixed with 10% formaldehyde solution. Next, these tissues were dehydrated and cleared with ethanol, and then embedded in liquid paraffin. The tissues were embedded, and paraffin blocks were prepared using a paraffin embedding machine. These paraffin blocks were sectioned, cut into 4 µm thick sections, dewaxed, stained with hematoxylin (H9627, Sigma Aldrich, Missouri, USA) and eosin (YE2080, Bomeibio, Anhui, China), and mounted with neutral resin. The morphological characteristics of the spleen, lung, liver, and kidney tissues were then carefully observed using an optical microscope.

Immunohistochemical assay

Immunohistochemistry was performed to detect CD8 protein expression in mouse liver, lung, kidney, and spleen tissues. After dewaxing and rehydrating paraffin-embedded sections of each tissue, antigen retrieval was performed using sodium citrate buffer. Following a rinse, the sections were blocked with 5% BSA for 30 minutes at 37 ℃ in the dark, and then incubated overnight at 4 ℃ with a CD8 antibody (1:100, Ab217344, Abcam, Cambridge, UK). After rinsing, the sections were incubated with a secondary antibody (1:100, GB23303, Servicebio, Hubei, China) for 1 hour at 37 ℃, followed by DAB staining and hematoxylin counterstaining; differentiation was subsequently performed using 0.5% hydrochloric acid-ethanol. After repeated rinsing, the sections were dehydrated through a graded series of ethanol (1 minute each) and cleared in xylene (5 minutes, twice), then mounted using neutral gum. Images were captured using a microscope, and the expression of the target protein was analyzed using ImageJ software.

Isolation of primary spleen cells

After rinsing the spleen tissues of each group of mice in a 60-mm glass petri dish containing 2 mL of pre-cooled PBS, cut them into small pieces with ophthalmic scissors, gently grind the tissues with a grinder, collect the cell suspension, filter it through a 200-mesh cell sieve, centrifuge at 300 g for 5 min and remove the supernatant, wash twice with PBS, centrifuge at 300 g for 5 min, discard the supernatant, add 3 times the volume of erythrocyte lysis buffer, lyse at 25 ℃ for 5 min, wash twice with PBS, centrifuge and collect the cell pellet for flow cytometry analysis.

Flow cytometry for T cell population

The primary spleen cells were resuspended in 100 µL PBS. CD3 (Biolegend, 100248, California, USA), CD8 (Biolegend, 100712), CD44 (Biolegend, 103029), CD122 (Biolegend, 123207), NKG2D (Biolegend, 115605), CD38 (Biolegend, 102707), and HLA-DR (eBioscience, 11-9952-42) were added to each tube. The cells were kept at 4 ℃ in the dark for 30 minutes, then centrifuged at 300 g for 5 minutes, and the supernatant was aspirated. Following a PBS wash, the cells were spun at 300 g for 5 minutes, and the supernatant was removed. The cells were resuspended in 300 µL PBS and analyzed. First, CD3⁺/CD8⁺ positive T cells were selected. Then, CD44^high/CD122⁺ cells were selected from this background cell population. Next, CD44^low/high/NKG2D⁺ was filtered, and in this context, CD44^high/NKG2D^(−/+)/CD38⁺/HLA-DR was further segmented.

Additionally, primary spleen cells were resuspended in 100 µL PBS, and CD3, CD8, CD44, and NKG2D were added to each tube. The cells were incubated at 4 ℃ in the dark for 30 minutes, then centrifuged at 300 g for 5 minutes, and the supernatant was discarded. After washing with PBS, the cells were centrifuged at 300 g for 5 min, and the supernatant was discarded. Add 200 μL of fixation buffer to each test tube, incubate the cells at room temperature for 20 min, centrifuge at 350 g for 5 min, and discard the supernatant. Each tube was washed twice with 250 µL of Intracellular Straining Perm Wash Buffer, centrifuged at 350 g for 5 min, and resuspended in 100 µL of Intracellular Straining Perm Wash Buffer. Granzyme B (Biolegend, 515407), INF-γ (Biolegend, 505806), and TNF-α (Biolegend, 506321) were added to each tube, and the cells were incubated at 4 ℃ for 30 min. After centrifugation, the supernatant was discarded, and each tube was washed with 250 µL of PBS. After centrifugation, the supernatant was eliminated, and the cells were re-dissolved in 300 µL of PBS for analysis. CD44^high/NKG2D^(+/−)/Granzyme B, CD44^high/NKG2D^(+/−)/INF-γ, and CD44^high/NKG2D^(+/−)/TNF-α were screened for observation of CD8⁺ T cell secretion of inflammatory factors.

Sorting of CD3⁺CD8⁺CD44^high T cells

After rinsing the spleen tissue in a 60-mm glass petri dish containing 2 mL of pre-chilled PBS, the tissue was cut into small pieces with ophthalmic scissors and gently ground with a grinder. The cell suspension was collected, filtered through a 200-mesh filter, centrifuged at 300 g for 5 min, and the supernatant was discarded. The spleen was washed twice with PBS to prepare a single-cell suspension. After erythrocyte lysis, the suspension was labeled with anti-CD3 (Biolegend, 100248), CD8 (Biolegend, 100712), and CD44 (Biolegend, 103029) antibodies at 4 ℃ in the dark for 30 min. After washing with PBS, the suspension was processed, and fragmented cells were excluded by FSC-ASSC-A gating. Single cells were selected (FSC-HESC-A) for further screening of CD3⁺CD8⁺ double-positive populations. Finally, the CD44^high subset was sorted for subsequent cell experiments.

Flow cytometry detection of the response of CD3⁺CD8⁺CD44^high T cells to IL-15

Sorted CD3⁺CD8⁺CD44^high cells from model mice and model mice + CLP were centrifuged at 250 g for 5 min, the supernatant was poured off, and the pellet was gently resuspended in an appropriate volume of 1640 medium (SenBeiJia, BC-M-017). The suspension was diluted to 4×10⁵ cells per well, and 2 mL aliquots were transferred into 6-well plates for culture at 37 ℃ with 5% CO₂. The experimental groups were: T cells from model mice (CD3⁺CD8⁺CD44^high), T cells from model mice + CLP (CD3⁺CD8⁺CD44^high), T cells from model mice + CLP (CD3⁺CD8⁺CD44^high) + IL-15, and T cells from model mice + CLP (CD3⁺CD8⁺CD44^high) + IL-15 + anti-NKG2D. T cells from model mice with CLP (CD3⁺CD8⁺CD44^high) were stimulated for 12 h with 20 ng/mL IL-15 and 20 ng/mL IL-15 + 10 µg/mL anti-NKG2D, respectively. According to the grouping, the supernatant was aspirated into corresponding numbered centrifuge tubes for later use. The cells were centrifuged a washed with PBS. Cells were pelleted in a 1.5 mL tube (250 g, 5 min) and the supernatant removed. The cells were resuspended in 500 µL of PBS, labeled with CD38 (Biolegend, 102707), HLA-DR (eBioscience, 17-9952-42), and NKG2D (Biolegend, 115711) antibodies, and reacted at room temperature in the dark for 30 min. Following centrifugation at 250 g for 5 min, the cells were washed twice with PBS and re-suspended in 500 µL PBS for subsequent analysis.

YAC-1 cell apoptosis detection

The YAC-1 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences. It was cultured in YAC-1 cell-specific medium (CM-0246, Procell system) containing 10% fetal bovine serum (FCS), with the addition of 0.1 IU·L⁻¹ penicillin and 100 mg·L⁻¹ streptomycin. The culture environment was 37 ℃ in a 5% CO₂ incubator, with the medium changed every 2 days. Logarithmically growing cells were selected for the study and assigned to the following groups: a: YAC-1 cells; b: YAC-1 cells + T cells from model mice (CD3⁺CD8⁺CD44^high); c: YAC-1 cells + T cells from model mice + CLP (CD3⁺CD8⁺CD44^high); d: YAC-1 cells + T cells from model mice + CLP (CD3⁺CD8⁺CD44^high) + IL-15; e: YAC-1 cells + T cells from model mice + CLP (CD3⁺CD8⁺CD44^high)+ IL-15 + anti-NKG2D. The T cell treatment method was the same as described above. Effector T cells after drug intervention were co-cultured with CFSE (gcpbio, GC14056)-labeled YAC-1 cells at a ratio of 10:1 for 6 h. According to the grouping, the supernatant was aspirated into corresponding numbered centrifuge tubes for later use. The tubes were centrifuged at 250 g for 5 min, the supernatant was discarded, and the cells were washed with an appropriate amount of PBS. The suspension was then transferred to a 1.5 mL conical EP tube, centrifuged at 250 g for 5 min, and the supernatant was discarded to obtain the cell pellet. The cells were resuspended in 500 µL of PBS, incubated with Annexin V/PI (eBioscience, 11-9952-42) at room temperature in the dark for 15 min, centrifuged at 250 g for 5 min, washed twice with PBS, and then resuspended in 500 µL of PBS for analysis.

Statistical analysis

Data are presented as mean (x) ± standard deviation (SD) and analyzed with SPSS 21.0. Statistical analysis was performed using SPSS 21.0. The statistical significance of differences between two groups was determined using a t-test. Differences greater than or equal to the difference between two groups were assessed by one-way analysis of variance (ANOVA); P<0.05 was deemed significant.

Results

Exogenous IL-15 exacerbates multi-organ damage and inflammatory response in sepsis model mice

As illustrated in Figure 1A,1B, relative to the control group, AST and ALT levels were increased in mice with cecal ligation and puncture (CLP). Exogenous IL-15 stimulation based on CLP showed a further tendency to increase AST and ALT levels. Regarding LDH, levels in the model mice were higher than in the control group. Compared with the model group, LDH increased after CLP surgery and continued to grow after exogenous IL-15 stimulation (Figure 1C). Next, CREA-S and BUN were measured. Compared with the control group, CREA-S and BUN in the model mice showed an increasing trend. CREA-S and BUN continued to increase in the model group mice after CLP and CLP + IL-15 intervention (Figure 1D,1E). Regarding the levels of TNF-α, IL-1β, IL-10, and IFN-γ, compared with the control group, the levels of these four pro-inflammatory factors were increased in the model group, the CLP group, and the CLP + IL-15 group. Versus the model group, the levels of these four pro-inflammatory factors were increased in the CLP group and the CLP + IL-15 group. Compared with the CLP group, the levels of these four pro-inflammatory factors were increased in the CLP + IL-15 group mice (Figure 1F). Histopathological observations revealed that, compared to the control group, the spleens of CLP and CLP + IL-15 mice showed mild to moderate multifocal lymphocytic necrosis, primarily in the lymphoid nodule region. Lymphocyte nuclei were pyknotic and fragmented, with increased eosinophilicity in the cytoplasm. In the CLP + IL-15 group, macrophage vacuolation was observed in the red pulp of the spleen, manifesting as large, well-defined vacuoles within the cytoplasm. Additionally, mild to moderate neutrophilia was observed in the septa of the lung tissues of both CLP and CLP + IL-15 mice. The liver tissue of CLP + IL-15 mice showed mild focal granulomatous inflammation. The center of the granuloma contained necrotic hepatocytes and cell debris, while the middle layer consisted of numerous infiltrated or necrotic neutrophils. The periphery of the granuloma was surrounded by proliferating fibrous tissue with neutrophil infiltration. Necrotic hepatocytes and a small number of mononuclear cells were also observed infiltrating the periphery of the granuloma. Furthermore, mild multifocal tubular degeneration/necrosis was observed in the renal cortex of mice in the CLP + IL-15 group, characterized by increased basophilicity of the renal tubules and visible necrotic epithelial cells (Figure 1G).

Figure 1 Effects of exogenous IL-15 on organ damage in septic mice. (A) AST. (B) ALT. (C) LDH. (D) CREA-S. (E) BUN. (F) Levels of TNF-α, IL-1β, IL-10, and IFN-γ. (G) H&E staining of spleen, lung, liver, and kidney tissues (scale bar =50 µm). The arrows indicate apoptotic lymphocytes. Compared with the control group, *P˂0.05, **P˂0.01, and ***P<0.001. Compared with the model group, ^#P˂0.05, ^##P˂0.01, and ^###P˂0.001. Compared with the CLP group, ^&&P˂0.01, and ^&&&P˂0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CLP, cecal ligation and puncture; CREA, creatinine; H&E, hematoxylin and eosin; IFN, interferon; IL, interleukin; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor-alpha.

IL-15 promotes CD8⁺ T cell infiltration in the major organs of mice in a CLP-induced sepsis model

Immunohistochemical staining was used to assess CD8⁺ T cell infiltration in the liver, spleen, kidney, and lung tissues of mice in each group, followed by quantitative analysis (Figure 2A,2B). In the Control group, only minimal levels of CD8⁺-positive signals were observed within the various organs. Compared to the Control group, the percentage of CD8⁺-positive areas in the liver, spleen, kidney, and lung tissues of mice in the Model group and the CLP group showed varying degrees of increase, suggesting that peripheral infiltration and local activation of immune cells occur during a septic state. In the CLP + IL-15 group, the degree of CD8⁺ T cell infiltration in each organ was elevated compared to that in the Model and CLP groups. These results indicate that the exogenous administration of IL-15 can significantly enhance the recruitment and infiltration of CD8⁺ T cells in the major visceral organs of CLP model mice.

Figure 2 Immunohistochemical analysis of CD8⁺ T-cell infiltration in the liver, spleen, lung, and kidney of mice in each group. (A) Representative images of immunohistochemical staining in the mouse liver, spleen, kidney, and lung. (B) Quantitative statistical bar graph showing the percentage of CD8-positive areas in the mouse liver, spleen, kidney, and lung. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001. Compared with the CLP group, ^&&&P<0.001. CLP, cecal ligation and puncture; IL, interleukin.

IL-15 promotes NKG2D expression in memory CD8⁺ T cells

Single-cell suspensions were extracted and isolated from the spleen for T-cell detection. CD3-labeled T cells were first used, followed by screening for CD8⁺ T cells (Figure 3A). CD44^high was used to screen for memory T cells, and compared with the control group, CD44^high T cells in the spleen of model mice increased. The proportion of CD44^high T cells was further increased in CLP-operated and CLP + IL-15 mice (Figure 3B). Further verification of NKG2D expression in memory T cells showed that CLP and CLP + IL-15 stimulated mouse spleens showed increased expression in both high-density and low-density CD44 cell populations (Figure 3C). Next, the proportion of CD38⁺/HLA-DR⁺ T cells was examined in NKG2D-positive (NKG2D⁺) and NKG2D-negative (NKG2D⁻) cells. As shown in Figure 3D,3E, compared with the control group, the expression of CD38⁺/HLA-DR⁺ was increased in NKG2D-positive (NKG2D⁺) T cells in the model group, CLP, and CLP + IL-15-stimulated mice. In NKG2D⁻ cells, the proportion of exogenous IL-15-stimulated CD38⁺/HLA-DR⁺ T cells was also increased.

Figure 3 Effects of exogenous IL-15 on immune cell populations in septic mice. (A) Screening for CD3⁺/CD8⁺ T cells. (B) CD44^high/CD122⁺ T cells. (C) CD44^low/high/NKG2D⁺ T cells. (D) CD44^high/NKG2D⁻/CD38⁺/HLA-DR T cells. (E) CD44^high/NKG2D⁺/CD38⁺/HLA-DR T cells. Compared with the control group, *P˂0.05, **P˂0.01, and ***P<0.001. Compared with the model group, ^##P˂0.01 and ^###P˂0.001. Compared with the CLP group, ^&P˂0.05 and ^&&&P˂0.001. CLP, cecal ligation and puncture; IL, interleukin.

IL-15 promotes bypass activation in memory CD8⁺ T cells by inducing NKG2D expression

As for CD44^highNKG2D^(−/+) Granzyme B⁺(%), compared with the control group, Granzyme B expression was increased in the Model, CLP, and CLP + IL-15 groups. Compared with the model mouse group, Granzyme B expression continued to increase in the CLP and CLP + IL-15 groups. Compared with the CLP group, exogenous IL-15 stimulation induced Granzyme B expression (Figure 4A). As for CD44^highNKG2D⁽⁺⁾IFN-γ⁺/TNF-α⁺(%), compared with the control group, the expression of IFN-γ⁺/TNF-α⁺ was increased in the Model, CLP, and CLP + IL-15 groups. Compared with the model mouse group, the expression of IFN-γ⁺/TNF-α⁺ continued to increase in the CLP and CLP + IL-15 groups. Compared with the CLP group, exogenous IL-15 stimulation induced the expression of IFN-γ⁺/TNF-α⁺ (Figure 4B,4C). Furthermore, IFN-γ and TNF-α in CD44^highNKG2D-T cells were not affected by CLP surgery or exogenous IL-15 stimulation (Figure 3B,3C).

Figure 4 Effects of exogenous IL-15 on secreted factors of CD8⁺ T cells. (A) CD44^high/NKG2D^(+/−)/Granzyme B T cells. (B) CD44^high/NKG2D^(+/−)/IFN-γ T cells. (C) CD44^high/NKG2D^(+/−)/TNF-α T cells. Compared with the control group, **P˂0.01 and ***P<0.001. Compared with the model group, ^##P˂0.01 and ^###P˂0.001. Compared with the CLP group, ^&&&P˂0.001. CLP, cecal ligation and puncture; IFN, interferon; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

In vitro experiments confirmed that IL-15 mediates bypass activation of memory CD8⁺ T cells in sepsis by inducing NKG2D expression

Compared with the model group selected subgroup (CD3⁺CD8⁺CD44^high), both the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) and the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 group showed increased CD38⁺/HLA-DR⁺ expression. Compared with the CLP group selected subgroup (CD3⁺CD8⁺CD44^high), the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 group showed increased CD38⁺/HLA-DR⁺ expression. However, compared with the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 group, the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 + anti-NKG2D group showed decreased CD38⁺/HLA-DR⁺ expression (Figure 5A). Furthermore, compared to the model group selected subgroup (CD3⁺CD8⁺CD44^high), both the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) and the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 group showed increased NKG2D+ expression. Compared to the CLP group selected subgroup (CD3⁺CD8⁺CD44^high), the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 group showed increased NKG2D⁺ expression. However, compared to the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 group, the CLP group selected subgroup (CD3⁺CD8⁺CD44^high) + IL-15 + anti-NKG2D group showed decreased NKG2D⁺ expression (Figure 5B).

Figure 5 Effect of IL-15 on CD38⁺/HLA-DR⁺ T cells via NKG2D. (A) CD38⁺/HLA-DR⁺ T cells. (B) NKG2D⁺ T cells. Compared with a, ***P<0.001. Compared with b, ^##P<0.01, ^###P<0.001. Compared with d, ^&&&P<0.001.

IL-15 enhances the killing function against target YAC-1 cells via NKG2D

The YAC-1 cell line is a lymphoma cell line isolated from AKR mice. It was stained and screened using the CFSE probe (Figure 6A). Next, the cytotoxic effect of T cells on YAC-1 cells was examined. Compared with untreated YAC-1 cells, the proportion of YAC-1 cells treated with CLP-selected T cell subsets (CD3⁺CD8⁺CD44^high) + IL-15 increased. Compared with the YAC-1 cells + T cells from model mice + CLP (CD3⁺CD8⁺CD44^high) + IL-15 group, the addition of anti-NKG2D reduced YAC-1 cell death (Figure 6B). These experimental results indicate that IL-15-activated CD8⁺ T cells are activated and have a cytotoxic effect.

Figure 6 Killing effector T cells against YAC-1 cells after IL-15 stimulation. (A) CFSE-labeled YAC-1 cells. (B) YAC-1 cell apoptosis. Compared with a, ***P<0.001. Compared with d, ^###P<0.001.

Discussion

Sepsis is a life-threatening multi-organ dysfunction caused by a dysregulated host response to infection (1). The main pathophysiological features of sepsis are an excessive systemic inflammatory response and organ dysfunction, which can be induced by bacteria, fungi, or other pathogens (6,27). Immunotherapy helps alleviate the condition of sepsis patients, including reversing immunosuppression and immunodeficiency, and promoting the proliferation and activation of immune cells (28). This study focused on the immune cell population in septic mice. Intraperitoneal injection of cecal contents exposed the mice to bacteria and inflammatory mediators, activating their immune system (25). These mice were then stimulated using the classic sepsis modeling method (CLP) (29). After modeling, the mice exhibited lethargy, increased respiratory rate, curled up in corners, and increased eye and nasal secretions. These findings indicate that the sepsis mouse model was successfully established and can be used for basic medical research. By employing a combined design involving the initiation of immune priming via cecal content injection alongside a standardized CLP procedure, this study successfully overcame the limitations associated with the excessively rapid immune responses typically observed in SPF-grade mice. Consequently, it achieved a high-fidelity recapitulation of the “two-hit” pathogenesis characteristic of human sepsis in an animal model. This highly biomimetic model design provides a robust foundation, backed by a strong potential for clinical translation, for subsequent investigations into how IL-15 modulates immune function and enhances survival rates in sepsis.

Sepsis triggers an uncontrolled systemic inflammatory response network, releasing large amounts of cytokines and inflammatory mediators that directly or indirectly damage tissues and cells (30). At the cellular level, when cells or tissues are damaged, ischemic, or die, the integrity of the cell membrane is disrupted, and large amounts of LDH are released from the cytoplasm into the bloodstream, leading to elevated serum LDH levels (31). At the tissue level, sepsis patients rarely experience single-organ dysfunction; multiple organs are usually affected (32). Clinically, the six principal organ systems-cardiovascular (microcirculation included), respiratory, renal, neurologic, hematologic, and hepatic-can be used to assess and monitor the progression of sepsis (32). First, liver damage is highly likely to occur in the early stages of sepsis. This is due to the liver’s multifunctional metabolic processes and unique anatomical location, making it susceptible to the effects of substances such as intestinal endotoxins (33). Patients with sepsis accompanied by liver damage have a poorer prognosis (33,34). The spleen, an immune organ, is significantly damaged in sepsis, primarily manifested as the destruction of lymphoid tissue (35). Additionally, mild to moderate neutrophilia (i.e., inflammatory cell infiltration) is observed in the septa of the lung tissue, a common pathological basis for acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) (36). Furthermore, renal tissue shows increased basophilicity in the renal tubules and visible necrotic epithelial cells (37). In this study, mice treated with CLP and exogenous IL-15 exhibited significant lesions in the spleen, liver, lungs, and kidneys, along with a large release of pro-inflammatory factors (TNF-α, IL-1β, IL-10, IFN-γ). Increased LDH levels indicated cellular damage. These histopathological findings, corroborated by elevated serum biochemical indicators (AST/ALT, CREA-S/BUN), collectively demonstrate the successful establishment of a multi-organ injury model of sepsis (CLP), with exogenous IL-15 stimulation exacerbating the inflammatory response and substantial damage in these organs, particularly revealing more severe pathological features in the liver and kidneys.

T lymphocytes play a crucial role in the acquired immune system by regulating the immune response in sepsis patients, promoting both cellular and humoral immunity (38). In sepsis, dysregulation of the acquired immune response includes abnormal responses of CD4⁺ T and CD8⁺ T cells, excessive release of inflammatory factors, and massive activation of CD4⁺ T cells, manifested as a decrease in the proportion of CD4⁺ T lymphocytes and a decrease in the CD4⁺/CD8⁺ T cell ratio (39,40). This decrease in the ratio may be an important manifestation of acquired immune abnormalities (39,40). After infection, the immune system recognizes antigens, and naive T cells rapidly differentiate into effector T cells. Most effector T cells migrate to the site of infection to fight pathogens (41). After the infection is cleared, most effector T cells die, and some differentiate into memory T cells (42). Furthermore, CD44 plays a crucial multiple role in T cell adhesion and migration localization, enhanced activation signal transduction, and the construction and rapid response of long-term immune memory (43). In this study, mice treated with CLP (cecal ligation and perforation, a common mouse sepsis model) and CLP + IL-15 (IL-15 injection after CLP surgery) showed a further increase in the proportion of CD44^high T cells. This indicates that both model manipulation and IL-15 stimulation can promote the generation or expansion of memory T cells. In addition, Motegi et al. (44) reported that IL-15-induced CD8⁺CD122⁺ T cells can enhance antibacterial and antitumor immune responses. We also observed the induction of CD122 by IL-15 in CD8⁺CD44⁺ T cells.

Interestingly, studies by Guo et al. (45) and Yajima et al. (46) demonstrated that the knockout or knockdown of IL-15 improved survival rates in septic mice; conversely, the administration of exogenous IL-15 or its presence in IL-15 transgenic mice exacerbated the condition and reduced survival rates, thereby indicating that IL-15 plays a pathogenic role in aggravating sepsis. In contrast, studies by Inoue et al. (15), Saito et al. (47), and Yang (48) found that IL-15 therapy improved survival rates and alleviated symptoms in septic mice, thereby exhibiting protective characteristics.

Building upon prior research demonstrating that IL-15 significantly promotes NKG2D expression and that a tightly coupled mechanism exists between IL-15 receptor signaling and the NKG2D activation pathway (22,23), we discovered that either the overexpression or exogenous supplementation of IL-15 upregulates NKG2D expression on the surface of downstream effector cells (constituting an overactivation of the IL-15/NKG2D). Consequently, this exacerbates sepsis-induced organ injury and intensifies pathological processes such as localized tissue cytotoxic damage and the triggering of a cytokine storm. After the pathogens or antigens of acute infection are cleared, the maintenance of memory CD8⁺ T cells relies on the co-maintenance of IL-7 and IL-15 (49,50). In addition, NKG2D is mainly expressed in NK cells, NKT cells, CD8⁺ T cell subsets, and γδ T cell subsets (51). Evidence suggests that in chronic viral infection and the tumor microenvironment, IL-15 can synergistically promote CD8⁺ T cell antigen-independent activation with NKG2D signaling (52). Furthermore, CD38 and HLA-DR are specific cell surface markers, and their expression can reflect the activation status of T cells (53). Our study showed that NKG2D expression in the mouse spleen was increased in both the CLP and CLP + IL-15 groups, in both high-density CD44 cell populations (i.e., memory T cells) and low-density CD44 cell populations (naive or effector T cells). IL-15 enhances T cell activation by increasing the CD38⁺/HLA-DR⁺ cell ratio; this effect is present in both NKG2D-positive and negative cells, but its activation effect on NKG2D⁺ cells may be stronger or more clinically significant. Overall, IL-15 may influence the immune response and disease progression in a sepsis model by promoting the number and activation of CD8⁺ memory T cells (via NKG2D and activation markers).

In 1996, Tough et al. (54) discovered that a large number of clonal T cells proliferated and activated after heterologous viral infection, speculating that T cells could proliferate in an antigen-nonspecific, T cell receptor (TCR)-independent manner. They also found that type I interferon (IFN-I) could induce TCR-independent T cell proliferation, which they termed bystander activation or bypass activation. Further studies revealed that memory T cells were the main activating cells in bypass activation of T cells. In addition to IFN-I, other cytokines, such as IL-15, have been shown to have the potential to induce CD8⁺ T cell bypass activation (55,56). In addition, the natural killer (NK) cell activation receptor, natural killer group 2 member D (NKG2D), is expressed on the surface of CD8⁺ T cells as a co-stimulatory signal receptor. It mediates the cytotoxic effects of CD8⁺ T cells by binding to ligands such as MHC class I chain-related protein A (MICA) and MICB, participating in the body’s defense or causing tissue damage in various inflammatory environments (57). Studies on the pathogenesis of various diseases, including autoimmune diseases, have suggested that IL-15 can promote increased NKG2D expression, and the two may interact through signaling pathway molecules, thereby mediating bypass activation of CD8⁺ T cells and generating an immune response (58,59). Currently, the IL-15/NKG2D has been shown to participate in the pathogenesis of various infectious diseases by bypassing the activation of CD8⁺ T cells (60). Our study also demonstrated that the IL-15/NKG2D signaling mediates bypass activation of memory CD8⁺ T cells.

Previous studies have shown that gene expression changes when naive T cells differentiate into memory cells (61). For example, naive CD8⁺ T cells do not encode IFN-γ, perforin, and granzyme B, but these are persistently expressed in effector and memory CD8⁺ T cells (50). Upon exposure to antigens, these factors are rapidly released. Due to the high levels of messenger RNA transcription in memory cells, memory CD8⁺ T cells produce these proteins much faster than naive CD8⁺ T cells (62). In this study, exogenous IL-15 significantly enhanced the expression of granzyme B, IFN-γ, and NF-α. The enhancing effect of IL-15 on cytokine production occurred only in the NKG2D⁺-expressing memory T cell subset, confirming that IL-15 promotes bypass activation by inducing NKG2D expression. In vitro experiments with primary mouse CD3⁺CD8⁺CD44^high T cells showed that anti-NKG2D antibodies blocked NKG2D function, and these antibodies also blocked the enhancing effect of IL-15 on T cell activation. This directly demonstrates that IL-15-enhanced memory CD8⁺ T cell activation is dependent on the NKG2D signaling pathway. IL-15 induces NKG2D expression and enhances the activation marker CD38⁺/HLA-DR⁺. We confirmed that IL-15-promoted NKG2D expression ultimately translates into functional enhancement—namely, more efficient killing of native target cells, YAC-1. In the context of sepsis, this IL-15-mediated NKG2D-dependent bypass activation and enhanced killing function may be one of the important mechanisms for the body to fight infection and stress responses, as it allows T cells to rapidly exert killing and pro-inflammatory functions even in the absence of new antigen presentation.

Conclusions

In a septic CLP mouse model and in vitro experiments, exogenous IL-15 was found to not only exacerbate systemic inflammatory responses but also cause multi-organ damage. IL-15 significantly promoted the initiation of memory CD8⁺ T cells, CD44^high, specifically through the induction of NKG2D expression to achieve bypass activation. In conclusion, IL-15-dependent NKG2D signaling pathway-dependent promotion of excessive initiation and cytotoxic function of memory CD8⁺ T cells is one of the important reasons for the aggravated multi-organ damage in septic mice.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0206/rc

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Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0206/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. IRB2021-YX-220-01) granted by the Medical Ethics Committee of Tianjin Medical University General Hospital, in compliance with the Chinese Guidelines for Animal Welfare.

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