Patients who survive sepsis experience long-term immunoparalysis characterized by numerical and/or functional lesions in innate and adaptive immunity that increase the host’s susceptibility to secondary complications. The extent to which tumor development/growth is affected in sepsis survivors remains unknown. In this study, we show cecal ligation and puncture (CLP) surgery renders mice permissive to increased B16 melanoma growth weeks/months after sepsis induction. CD8 T cells provide partial protection in this model, and tumors from sepsis survivors had a reduced frequency of CD8 tumor-infiltrating lymphocytes (TILs) concomitant with an increased tumor burden. Interestingly, the postseptic environment reduced the number of CD8 TILs with high expression of activating/inhibitory receptors PD-1 and LAG-3 (denoted PD-1hi) that define a tumor-specific CD8 T cell subset that retain some functional capacity. Direct ex vivo analysis of CD8 TILs from CLP hosts showed decreased proliferation, IFN-γ production, and survival compared with sham counterparts. To increase the frequency and/or functional capacity of PD-1hi CD8 TILs in tumor-bearing sepsis survivors, checkpoint blockade therapy using anti–PD-L1/anti–LAG-3 mAb was administered before or after the development of sepsis-induced lesions in CD8 TILs. Checkpoint blockade did not reduce tumor growth in CLP hosts when therapy was administered after PD-1hi CD8 TILs had become reduced in frequency and/or function. However, early therapeutic intervention before lesions were observed significantly reduced tumor growth to levels seen in nonseptic hosts receiving therapy. Thus, sepsis-induced immunoparalysis is defined by diminished CD8 T cell–mediated antitumor immunity that can respond to timely checkpoint blockade, further emphasizing the importance of early cancer detection in hosts that survive sepsis.

This article is featured in In This Issue, p.583

Infectious pathogens are normally found in host barrier tissues, where immune responses are recruited and pathogen control is localized. Should the integrity of these barrier tissues become compromised, the pathogen can enter the circulation, resulting in harmful and/or lethal levels of pro- and anti-inflammatory cytokines/chemokines as the host fights to eradicate the systemic infection (13). Nearly 2 million patients in the U.S. are struck by sepsis annually, and this number is expected to increase in the future (4, 5). Improvements in identification and efficacious treatment options have dramatically increased the survival of patients during the acute phase of sepsis (5). However, this early management of sepsis has subsequently revealed that patients who survive the septic event have increased long-term mortality rates compared with nonseptic patients (6, 7). Currently, the greatest risk of mortality in sepsis patients after hospital discharge is the increased susceptibility to secondary complications (such as infection by opportunistic pathogens) because of reasons that are still largely unknown (810). This “chronic critical illness” diminishes sepsis survivors’ quality of life and results in high economic strain on the healthcare system, making this a pressing health issue (11, 12).

Global immune system paralysis (immunoparalysis) is a sequela of sepsis that largely defines the chronic critical illness observed clinically, and in experimental animal models it predisposes survivors to increased chronic disease burden and mortality (1316). Most research has sought to understand the implications of sepsis-induced immunoparalysis in response to secondary infections (1719), but little is known about the capacity of sepsis survivors to efficiently control other chronic diseases with capacity to diminish the long-term survival of this population (6, 20). Cancer is a significant chronic disease of sepsis survivors that accounts for mortality in 25% of patients a year after systemic infection (21), with similar mortality rates seen in patients after community-acquired pneumonia (22, 23). Importantly, the high rate of cancer-associated mortality seen in sepsis survivors occurs largely in patients with no previous history of malignancy, suggesting an increased rate of malignancy development/progression upon sepsis recovery. Given this, it is surprising how limited our knowledge is on how the chronic immunoparalysis phase of sepsis alters the host’s capacity to control/eradicate cancer and if defects in antitumor immunity can be therapeutically resolved using strategies that have benefited nonseptic cancer patients. The limited amount of data shows increased tumor progression weeks/months after sepsis induction, suggesting potential lesions in antitumor immune responses (24, 25). Alterations in the number and/or function of CD8 T cells (critical effector cells in tumor control/eradication) have not been directly examined in sepsis survivors, and there is a paucity of information regarding the extent to which defects in this T cell population underlie the exacerbated rates of cancer-associated mortality after sepsis.

Pathogen-specific naive (Ag-inexperienced) and memory (Ag-experienced) CD8 T cells undergo an apoptosis-induced numerical reduction at early stages after sepsis induction (2629). The remaining naive and memory CD8 T cells exhibit impaired Ag-dependent and -independent functions characterized by diminished proliferation and effector cytokine production that contributes to the increased susceptibility of sepsis survivors to new and previously encountered infections (3034). It is unknown if the same sepsis-induced lesions observed in pathogen-specific CD8 T cell responses during the chronic immunoparalysis phase of sepsis occur in tumor-specific CD8 T cells because of the distinct biological context of these secondary complications. Thus, tumor models provide an additional research tool to further probe the fitness of CD8 T cell–mediated immunity following a septic event and provide further insight to the capacity of sepsis survivors to control chronic diseases with potential to diminish the long-term survival of these patients.

This report elucidates malignancy-specific lesions in the maintenance and function of tumor-infiltrating CD8 T cells that underlie the increased tumor growth and progression observed after sepsis. Our data demonstrate the long-lasting immunoparalysis state that develops in the wake of systemic bacterial infection is also defined by the inability of the host to generate optimal antitumor immunity, a notion with direct relevance to the healthcare of an increasing number of sepsis survivors.

C57BL/6 mice were purchased from National Cancer Institute (Frederick, MD) and maintained at University of Iowa animal facilities at the appropriate biosafety level according to the University of Iowa Animal Care and Use Committee and National Institutes of Health guidelines. Male and female mice >6 wk old were used in experiments; results were similar in both. B16 and B16-OVA cells were obtained from Dr. L. Norian (University of Alabama at Birmingham, Birmingham, AL). B16 cells were grown in DMEM with 4.5 g/l d-glucose, l-glutamine, 10% FCS (HyClone Laboratories) and supplementum complementum (made in-house). Cell lines were passaged every 2–3 d and/or when cell confluency was >80% in 75 cm2-tissue culture flask (DOT Scientific). Cells were not sequentially passaged longer than 3 wk. In vitro and in vivo tumor growth did not vary considerably throughout the study. For implantation, 2 × 104 B16 cells were injected s.c. in the hind flank at 100-μl volume with equal parts B16 medium and Matrigel Matrix (356234; Corning). Tumor progression was determined by measuring tumor length multiplied by width using an electronic digital caliper. Mice were sacrificed upon reaching any animal protocol threshold including tumor length of >15 mm or tumor ulceration. Metastatic modeling was performed by injecting 5 × 104 B16 cells i.v. in the tail vein in a total volume of 200 μl. Weight loss and survival of mice were monitored to determine disease progression.

Mice were anesthetized with ketamine/xylazine (University of Iowa, Office of Animal Resources), the abdomen was shaved and disinfected with povidone-iodine (Betadine) (Purdue Products), and laparotomy was performed. The distal third of the cecum was ligated with PERMAHAND Silk (Ethicon), punctured once using a 25-gauge needle, and a small amount of fecal matter was extruded to confirm cecal perforation. The cecum was returned to abdomen, the peritoneum was closed with 641G PERMAHAND Silk (Ethicon), and skin was sealed using surgical Vetbond (3M). Following surgery, 1 ml sterile PBS was administered s.c. as postsurgery fluid recovery. Bupivacaine (Hospira) was administered at the incision site, and flunixin meglumine (Phoenix Scientific) was administered for postoperative analgesia. This procedure created a septic state characterized by loss of appetite and body weight, ruffled hair, shivering, diarrhea, and periorbital exudate that normally results in a 0–10% mortality rate (35). Sham mice underwent surgery procedure omitting cecal ligation and puncture (CLP).

Mice were evaluated for pulmonary function using an unrestrained whole-body plethysmograph (Buxco Electronics, Wilmington, NC). Whole-body plethysmography parameters were assessed from days 19 to 26 after surgery as changes in respiration from baseline values and did not use methacholine administration. Pressure and volume changes in the chamber caused by respiration were averaged over a 5-min period and used to calculate enhanced pause (Penh), tidal volume, and respiratory rate (breaths per minute [BPM]).

Spleen and lymph nodes were mashed through a 70-μm filter with the plunger flange of a 1-ml syringe. Splenocytes were treated with ACK lysis buffer for 3 min; lymph nodes did not undergo an RBC lysis step. Tumors were cut into small pieces and placed in DMEM plus 10% FCS plus supplementum complementum plus 0.8 mg/ml collagenase type I (Worthington Biochemical) plus 60 U/ml of DNase and placed in a 37°C shaker at 300 RPM for 45–60 min. Tumor samples were mashed through a 70-μm cell strainer using the plunger flange of a 1-ml syringe, and cell suspensions were treated with ACK. After washing, samples were filtered through a 70-μm cell strainer one additional time prior to Ab labeling in 96-well flat bottom plates. Cells were labeled with the following fluorescently labeled mAb: CD8α (53-6.7), CD69 (H1.2F3), PD-1 (J43), LAG-3 (eBioC9B7W), IFN-γ (XMG1.2), Thy1.1 (HIS51), Thy1.2 (53-2.1), Ki67 (B56), MART-1 (MLANA/1409R). Samples requiring intracellular labeling were subsequently treated with Cytofix/Cytoperm (BD Biosciences) for 10 min at 4°C and washed in Perm/Wash (BD Biosciences) before intracellular labeling. For Ki67 analysis, the Foxp3 Staining Fixation and Permeabilization Set (eBioscience) was used. Samples were run on a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software. Mitochondrial Membrane Potential Dye Mito Flow Reagent (Cell Technology) was used following the manufacturer’s protocol; cell fixation was not used prior to flow cytometry. Activated caspase 3/7 and propidium iodide labeling were performed using Vybrant FAM Caspase-3 and -7 Assay Kit for flow cytometry (Invitrogen) and used following the manufacturer’s protocol.

For checkpoint blockade experiments, mice received 200 μg of anti–PD-L1 (10F.9G2) and 200 μg anti–LAG-3 (prepared from hybridoma clone C9B7W) mAb, or control groups received equal amounts of rat IgG every 3 d, as described previously (36).

Flow cytometric data of tumor samples were manually gated on CD8+ cells using FlowJo software and exported to specifically analyze the CD8 tumor-infiltrating lymphocyte (TIL) population. These individual files were uploaded to Cytobank (http://www.cytobank.org), a cloud-based computational platform, and input into Spanning-Tree Progression Analysis of Density-Normalized Events (SPADE) algorithm, as described previously (37). Clustering channels included CD8α, PD-1, LAG-3, and IFN-γ. Forward light scatter, side light scatter, and time were not used for clustering. SPADE was run with a 200-node target and 10% downsampling. After SPADE trees were generated, neighboring nodes were manually gated into three distinct populations based on PD-1 expression. The parameter shown is LAG-3 (PE) in the blue–yellow color scheme using median metric and a symmetric scale that was global among samples. Maximum node size was manually increased to more easily appreciate differences in cell distribution between nodes. The number of cells in individual nodes is represented by the size of the node, and the range (rounded to an integer) is shown.

Tumor sections were collected from mice, fixed, stained with DAPI (Sigma-Aldrich), CD8-biotin (53-6.7), and streptavidin–FITC (catalog no. 554060) and then imaged on a Leica SP8 microscope (Leica) using a 25×, 0.95 numerical aperture water-immersion objective with coverslip correction. High-resolution confocal stacks sampled with 1-μm z-spacing were acquired at an acquisition rate of 40 frames per second to provide image volumes of 170/388 × 170/388 × 30–54 μm3 and transformed into volume-rendered three-dimensional composite images. Sequences of image stacks were further processed with Imaris software (Bitplane).

Statistical analyses were performed using GraphPad Prism software version 7 (GraphPad Software Inc). Data are shown as ±SEM. Bar graphs, tumor growth graphs, and survival curves were analyzed using an unpaired t test, two-way ANOVA, and log rank (Mantel–Cox) tests, respectively.

Sepsis-induced immunoparalysis begins upon resolution of the cytokine/chemokine storm (38, 39), and the extent to which sepsis increases the susceptibility to tumor growth at various times after sepsis induction is not well defined. To experimentally test this, C57BL/6 (B6) mice underwent laparotomy followed by ligation and puncture of the distal cecum to induce intra-abdominal, polymicrobial sepsis (CLP group) or laparotomy alone (Sham group) (Fig. 1A). Two days after CLP surgery, at the time when the cytokine storm has resolved (Fig. 1B) and lymphopenia has been established (∼90% decline in PBL cellularity (Fig. 1C), mice were challenged s.c. with 2 × 104 B16 melanoma cells in the hind flank. B16 is a stringent low-immunogenicity tumor that results in 100% mortality in unmanipulated wild-type hosts (40), and all mice eventually succumbed to tumor. However, mortality (defined as >15 mm in tumor dimension or tumor ulceration) was greatly increased in mice that experienced CLP-induced sepsis compared with sham controls (Fig. 1D, 1E).

FIGURE 1.

Tumor progression is increased early after sepsis induction. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 2 d later. (B) Levels of IL-6 in the serum at indicated time after surgery. (C) Total cellularity in 1 ml of peripheral blood before and 2 d after surgery. (D) Tumor size and (E) percentage survival at indicated time point after B16 injection. (F) Experimental design. Mice received sham or CLP surgery followed by i.v. injection of 5 × 104 B16 cells in the tail vein 2 d later. (G) Weight loss and (H) percentage survival at indicated day after surgery. In a similar, independent experiment (I) weight loss and (J) percentage survival at indicated day after surgery. (K) Penh (airway obstruction) (L) respiratory rate (BPM), and (M) tidal volume (milliliters) were analyzed in whole-body plethysmography machines at indicated day after surgery. (N) Representative images of lungs harvested from mice sacrificed 26 d after surgery. Number of mice per group are shown. Tumor size ± SEM are shown. Data are representative of at least two independent experiments. Lung function, tumor growth, and survival curves analyzed using unpaired t test, two-way ANOVA, and log rank (Mantel–Cox) tests, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 1.

Tumor progression is increased early after sepsis induction. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 2 d later. (B) Levels of IL-6 in the serum at indicated time after surgery. (C) Total cellularity in 1 ml of peripheral blood before and 2 d after surgery. (D) Tumor size and (E) percentage survival at indicated time point after B16 injection. (F) Experimental design. Mice received sham or CLP surgery followed by i.v. injection of 5 × 104 B16 cells in the tail vein 2 d later. (G) Weight loss and (H) percentage survival at indicated day after surgery. In a similar, independent experiment (I) weight loss and (J) percentage survival at indicated day after surgery. (K) Penh (airway obstruction) (L) respiratory rate (BPM), and (M) tidal volume (milliliters) were analyzed in whole-body plethysmography machines at indicated day after surgery. (N) Representative images of lungs harvested from mice sacrificed 26 d after surgery. Number of mice per group are shown. Tumor size ± SEM are shown. Data are representative of at least two independent experiments. Lung function, tumor growth, and survival curves analyzed using unpaired t test, two-way ANOVA, and log rank (Mantel–Cox) tests, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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To extend this observation, B6 mice received an i.v. challenge of 5 × 104 B16 cells 2 d after sham or CLP surgery to determine to what extent sepsis influenced the capacity to control the establishment of lung tumors (Fig. 1F). Initially, morbidity (weight loss) and survival were followed to show that sepsis increases the rate of cancer-associated mortality compared with sham controls (Fig. 1G, 1H). In a second experiment, we evaluated lung function and lung tumor formation as readouts of disease progression. Lungs were harvested when cancer-associated weight loss and mortality first occurred in sepsis survivors, 26 d after surgery (Fig. 1I, 1J). Readouts of disease progression obtained prior to host sacrifice showed that lung function was negatively impacted, as demonstrated by increased airway obstruction (Penh), decreased respiratory rate (BPM), and decreased tidal volume in tumor-bearing CLP hosts compared with sham controls (Fig. 1K–M). Gross lung histology from mice that had not yet succumbed to cancer revealed an average of 8.6 metastatic nodules in sham controls, whereas sepsis survivors showed a nearly confluent layer of metastatic tumors that were too numerous to accurately count (Fig. 1N). These data collectively demonstrate that the early immunoparalysis phase of sepsis is characterized by greatly diminished control of tumor growth and increased host mortality.

There are no data that rigorously define the duration of immunoparalysis following a septic event, but recent data suggest it lasts for at least 60 d (in mice), as CLP surgery resulted in mice being more susceptible to tumor growth and development (25). To independently determine the duration of chronic immunoparalysis that increases tumor-associated mortality in sepsis survivors, B6 mice were challenged with B16 tumors at multiple times distal to surgery, and subsequent cancer progression was monitored (Fig. 2A). At the time of tumor inoculation (days 12, 46, or 106 postsurgery) sepsis-induced lymphopenia was resolved, and the number of PBL was indistinguishable from sham controls (Fig. 2B, 2D, 2F). Importantly, tumor growth was accelerated in the survivors of CLP-induced sepsis, and cancer-associated mortality was increased compared with sham controls at all time points examined (Fig. 2C, 2E, 2G). These data further extend our knowledge on the breadth of sepsis-induced immunoparalysis that has a profound impact proximal to the septic event and lingering impairments in chronic disease control evident >100 d later. These data show sepsis leads to a long-lasting impairment in the ability to control tumor growth and suggest a diminished capacity to mount protective antitumor immune responses even at times when sepsis-induced lymphopenia had been resolved.

FIGURE 2.

The chronic immunoparalysis phase of sepsis is characterized by increased tumor progression. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank at 12, 46, or 106 d later. (B) Total cellularity in 1 ml of peripheral blood before, 2, and 12 d after surgery. (C) Tumor size at indicated time point after B16 injection was given 12 d after surgery. (D) Total cellularity in 1 ml of peripheral blood before, 2, and 46 d after surgery. (E) Tumor size at indicated time point after B16 injection was given 46 d after surgery. (F) Total cellularity in 1 ml of peripheral blood before, 2, and 106 d after surgery. (G) Tumor size at indicated time point after B16 injection was given 106 d after surgery. Survival at final tumor measurement is shown in parentheses for each group. Tumor size ± SEM is shown. Data shown are of three independent experiments. Tumor growth graphs analyzed using unpaired two-way ANOVA tests. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 2.

The chronic immunoparalysis phase of sepsis is characterized by increased tumor progression. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank at 12, 46, or 106 d later. (B) Total cellularity in 1 ml of peripheral blood before, 2, and 12 d after surgery. (C) Tumor size at indicated time point after B16 injection was given 12 d after surgery. (D) Total cellularity in 1 ml of peripheral blood before, 2, and 46 d after surgery. (E) Tumor size at indicated time point after B16 injection was given 46 d after surgery. (F) Total cellularity in 1 ml of peripheral blood before, 2, and 106 d after surgery. (G) Tumor size at indicated time point after B16 injection was given 106 d after surgery. Survival at final tumor measurement is shown in parentheses for each group. Tumor size ± SEM is shown. Data shown are of three independent experiments. Tumor growth graphs analyzed using unpaired two-way ANOVA tests. *p < 0.05, ***p < 0.001, ****p < 0.0001.

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CD8 T cells provide partial protection against B16 tumors (Supplemental Fig. 1) (40) and sepsis has a capacity to severely diminish CD8 T cell–mediated immunity (19). Thus, we reasoned suboptimal CD8 T cell–mediated antitumor immunity may be one explanation for the increased susceptibility to tumor growth in sepsis survivors. Before examining antitumor CD8 T cell–mediated responses in sepsis survivors, CD8 TILs were first examined in B16 tumors implanted in nonseptic mice to determine how the phenotype of CD8 T cells changes with tumor development. Naive B6 mice received 2 × 104 (1×) and 2 × 105 (10×) B16 cells into the left and right flank, respectively (Fig. 3A). This model allows for simultaneous monitoring of CD8 T cells in a shared circulation and in tumors of different sizes (Fig. 3B). Importantly, within the tumor, the frequency of CD8 TILs negatively correlated with tumor size (Fig. 3C), suggesting tumor development is indeed associated with the extinction of antitumor CD8 T cell immunity.

FIGURE 3.

Frequency and phenotype of CD8 TILs are dependent on tumor size. (A) Experimental design. Mice received s.c. injection of 2 × 104 (1×) and 2 × 105 (10×) B16 cells in left or right hind flank, respectively. (B) Tumor size 14 d after B16 injection. (C) Frequency of CD8+ cells among TILs in tumor samples. (D) Representative histogram of PD-1 expression on CD8 T cells in tumor (left) or draining inguinal lymph nodes (iLN) (right) from 1× (black) or 10× (blue) tumors. (E) Frequency of CD8 TILs based on PD-1 expression. (F) Correlation of the frequency of PD-1hi CD8 TILs to tumor size. (G) Frequency of CD8+ cells among draining iLN. (H) Frequency of CD8 T cells, based on PD-1 expression. (I) Correlation of the frequency of PD-1hi CD8 T cells in the iLN to tumor size. (J) Experimental design. Mice received adoptive transfer of Thy1.1/1.2 naive P14 cells followed by lymphocytic choriomeningitis virus–Armstrong (LCMV-Arm) infection. Eight days later mice received adoptive transfer of Thy1.1/1.1 memory OT-I CD8 T cells by retro-orbital injection and a day later received s.c. injection of 2 × 104 B16-OVA cells in the hind flank. (K) Representative gating of tumor-specific OT-I and virus-specific P14 CD8 T cells in spleen (top) and tumor (bottom). (L) Representative expression of LAG-3 and PD-1 on OT-I (left column) and P14 (right column) CD8 T cells in spleen (top) and tumor (bottom) samples. Bar graphs analyzed using unpaired t test. **p < 0.01, ***p < 0.001. NS, not significant.

FIGURE 3.

Frequency and phenotype of CD8 TILs are dependent on tumor size. (A) Experimental design. Mice received s.c. injection of 2 × 104 (1×) and 2 × 105 (10×) B16 cells in left or right hind flank, respectively. (B) Tumor size 14 d after B16 injection. (C) Frequency of CD8+ cells among TILs in tumor samples. (D) Representative histogram of PD-1 expression on CD8 T cells in tumor (left) or draining inguinal lymph nodes (iLN) (right) from 1× (black) or 10× (blue) tumors. (E) Frequency of CD8 TILs based on PD-1 expression. (F) Correlation of the frequency of PD-1hi CD8 TILs to tumor size. (G) Frequency of CD8+ cells among draining iLN. (H) Frequency of CD8 T cells, based on PD-1 expression. (I) Correlation of the frequency of PD-1hi CD8 T cells in the iLN to tumor size. (J) Experimental design. Mice received adoptive transfer of Thy1.1/1.2 naive P14 cells followed by lymphocytic choriomeningitis virus–Armstrong (LCMV-Arm) infection. Eight days later mice received adoptive transfer of Thy1.1/1.1 memory OT-I CD8 T cells by retro-orbital injection and a day later received s.c. injection of 2 × 104 B16-OVA cells in the hind flank. (K) Representative gating of tumor-specific OT-I and virus-specific P14 CD8 T cells in spleen (top) and tumor (bottom). (L) Representative expression of LAG-3 and PD-1 on OT-I (left column) and P14 (right column) CD8 T cells in spleen (top) and tumor (bottom) samples. Bar graphs analyzed using unpaired t test. **p < 0.01, ***p < 0.001. NS, not significant.

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To further explore this possibility, we examined the expression of activating/inhibitory receptors on CD8 TILs. PD-1 expression on Ag-specific CD8 T cells can define exhausted cells (41). However, it is important to note that CD8 T cells expressing PD-1 have also been in recent contact with cognate Ag (42), and T cells with an exhausted phenotype still retain some protective effector functions (4348). Thus, the intensity of PD-1 expression (i.e., negative, intermediate, and high) was determined to further subset the CD8 TIL compartment to further appreciate the dynamic changes in activation and maintenance of these cells. Highly activated PD-1hi CD8 TILs comprised the majority of CD8 TILs in the less progressed 1× tumor but were nearly undetectable in the more progressed 10× tumors, suggesting reduced CD8 TIL activation occurs with increasing tumor burden (Fig. 3D–F). Alterations in composition and phenotype of CD8 TILs were specific to the tumor microenvironment, as CD8 T cells in tumor-draining lymph nodes did not show similar phenotypic alterations (Fig. 3G–I). Thus, the phenotype of CD8 TILs changes predictably with tumor progression (Fig. 3), potentially because of the importance of this subset in the early tumor control.

These data from nonseptic, tumor-bearing hosts suggest the importance of examining CD8 T cells in the tumor to discover sepsis-induced lesions in antitumor immunity that may not be evident at distal sites. To formally confirm that the tumor microenvironment controls the phenotype of CD8 TILs, immune mice that contained TCR-transgenic (TCR-Tg), tumor-specific (OT-I), and virus-specific (P14) CD8 T cells were challenged with B16 cells expressing OVA protein (B16-OVA) (Fig. 3J). PD-1 and LAG-3 expression on the OT-I and P14 TCR-Tg CD8 T cells from the spleen or tumors was analyzed 30 d after tumor challenge. The data clearly revealed that tumor Ag recognition was required for the PD-1hi phenotype because upregulation of PD-1/LAG-3 occurred only on tumor-specific OT-I T cells residing inside the tumor and not in distal secondary lymphoid organs such as spleen (Fig. 3K, 3L). Thus, in nonseptic mice, the abundance of CD8 TILs and expression of activating/inhibitory receptors on these cells is dynamically regulated during B16 tumor growth. These parameters were subsequently examined in sepsis survivors to determine to what extent the chronic immunoparalysis phase of sepsis alters the efficacy of CD8 TIL responses.

It is unknown when patients that survive sepsis are most susceptible to de novo tumor burden during the chronic immunoparalysis phase. However, it seemed most stringent to characterize antitumor immune responses in sepsis survivors at the time when lymphopenia has resolved and cell numbers returned to basal, presepsis levels that occur∼7 d after CLP induction (33). To reveal potential sepsis-induced lesions in CD8 TILs from sepsis survivors, mice underwent sham or CLP surgery and 10 d later (when PBL cellularity returned to presepsis levels) challenged with B16. After 13 d, tumors were harvested for flow cytometric analysis (Fig. 4A). Reduced frequency of CD8 TILs concomitant with increased tumor burden was observed in sepsis survivors (Fig. 4B, 4C). Highly activated, tumor-specific PD-1hi CD8 TILs that were more prevalent in small tumors (seen in Fig. 3) were markedly absent in sepsis survivors, correlating with the increased tumor size in these hosts (Fig 4D, 4E). Direct ex vivo analysis of CD8 TILs (Fig. 4F) revealed the frequency of IFN-γ+ CD8 TILs directly correlated with the amount of PD-1 expressed, suggesting PD-1 marks tumor-infiltrating CD8 T cells that are still in close contact with tumor cells and possess detectable levels of antitumor immunity (Fig. 4G). In stark contrast, CD8 TILs from sepsis survivors did not express high levels of PD-1 and had diminished IFN-γ production that resulted in 10-fold fewer CD8 TILs with defined effector functions (Fig. 4G, 4H). Together, these data suggest the increased tumor progression seen in sepsis survivors during the chronic immunoparalysis phase of sepsis is characterized by diminished Ag-dependent CD8 TIL responses.

FIGURE 4.

Diminished frequency and effector cytokine production of CD8 TILs underlie the increased tumor progression in sepsis survivors. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 10 d later. After 13 d, tumor samples were harvested for histological and flow cytometric analysis. (B) Tumor size 13 d after inoculation. (C) Frequency of CD8+ cells among TILs. (D) Representative gating of PD-1 expression on CD8 TILs in sham (black) or CLP (red) hosts. (E) Correlation of PD-1hi expression on CD8 TILs to tumor size. (F) Representative effector cytokine production of PD-1hi CD8 TILs direct ex vivo in sham (black) or CLP (red) hosts. (G) Frequency of IFN-γ + CD8 TILs grouped by PD-1 expression. (H) Number of IFN-γ+ CD8 TILs (out of 100 TILs) in tumor samples. Data are representative of at least two independent experiments. Bar graphs, tumor growth, and survival curves analyzed using unpaired t test, two-way ANOVA, and log rank (Mantel–Cox) tests, respectively. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

Diminished frequency and effector cytokine production of CD8 TILs underlie the increased tumor progression in sepsis survivors. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 10 d later. After 13 d, tumor samples were harvested for histological and flow cytometric analysis. (B) Tumor size 13 d after inoculation. (C) Frequency of CD8+ cells among TILs. (D) Representative gating of PD-1 expression on CD8 TILs in sham (black) or CLP (red) hosts. (E) Correlation of PD-1hi expression on CD8 TILs to tumor size. (F) Representative effector cytokine production of PD-1hi CD8 TILs direct ex vivo in sham (black) or CLP (red) hosts. (G) Frequency of IFN-γ + CD8 TILs grouped by PD-1 expression. (H) Number of IFN-γ+ CD8 TILs (out of 100 TILs) in tumor samples. Data are representative of at least two independent experiments. Bar graphs, tumor growth, and survival curves analyzed using unpaired t test, two-way ANOVA, and log rank (Mantel–Cox) tests, respectively. *p < 0.05, ***p < 0.001, ****p < 0.0001.

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The reduced frequency of functional CD8 TILs in sepsis survivors suggested reduced accessibility to tumor Ag, which was confirmed using immunofluorescent confocal microscopy that indicated weak infiltration of CD8 TILs inside the tumor (Fig. 5A, 5B). Specifically, the composition of CD8 TILs in sepsis survivors appeared reduced and was relegated to diminutive immunological niches in the tumor periphery. In addition, it was possible reduced expression of MHC class I (MHC I) on B16 cells might also contribute to the inefficient recognition of tumor Ags by CD8 TILs. At baseline, B16 cells expressed low levels of H-2Kb in vitro that could be greatly increased by exogenous IFN-γ (Supplemental Fig. 2). Thus, the 10-fold reduction in IFN-γ produced by CD8 TILs (seen in Fig. 4) from sepsis survivors could result in minimal expression of MHC I on B16 cells, creating an unfavorable environment for CD8 T cell recognition/activation/function. Indeed, 14 d after tumor inoculation direct ex vivo expression of H-2Kb on B16 tumor cells was greatly diminished in sepsis survivors compared with nonseptic counterparts (Fig. 5C, 5D). Thus, the inability of CD8 TILs to penetrate the tumor and recognize tumor-derived Ags via MHC I likely contributes to the limited CD8 T cell–mediated antitumor immunity during the sepsis-induced immunoparalysis phase.

FIGURE 5.

The immunoparalysis phase of sepsis creates an unfavorable tumor microenvironment for Ag recognition. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 10 d later. After 14 d, tumor samples were harvested for histological and flow cytometric analysis. (B) Representative confocal microscopic images of CD8+ (green) and nucleated cells (blue) in tumors of mice that received sham (top) or CLP (bottom) surgery. Increased magnification (×6) of each image is seen in the right column. DAPI staining was used. (C) Representative H-2Kb expression on MART-1–expressing B16 cells direct ex vivo in mice that received sham (black) or CLP (red) surgery. (D) Summary of H-2Kb mean fluorescence intensity (MFI). Data are representative of at least two independent experiments. Bar graphs analyzed using unpaired t test. **p < 0.01.

FIGURE 5.

The immunoparalysis phase of sepsis creates an unfavorable tumor microenvironment for Ag recognition. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 10 d later. After 14 d, tumor samples were harvested for histological and flow cytometric analysis. (B) Representative confocal microscopic images of CD8+ (green) and nucleated cells (blue) in tumors of mice that received sham (top) or CLP (bottom) surgery. Increased magnification (×6) of each image is seen in the right column. DAPI staining was used. (C) Representative H-2Kb expression on MART-1–expressing B16 cells direct ex vivo in mice that received sham (black) or CLP (red) surgery. (D) Summary of H-2Kb mean fluorescence intensity (MFI). Data are representative of at least two independent experiments. Bar graphs analyzed using unpaired t test. **p < 0.01.

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To further document impairments in CD8 TILs after sepsis, readouts of cellular fitness were conducted that examined proliferative capacity, mitochondrial membrane potential, and cell survival. As shown previously, frequency of the PD-1hi CD8 TIL subset that retains some functional capacity was reduced in sepsis survivors (Fig. 6A, 6B). Direct ex vivo Ki67 labeling showed CD8 TILs from sepsis survivors had dramatically impaired proliferative capacity compared with sham mice, and, once again, PD-1hi CD8 TILs proved to be highly proliferative/functional (Fig. 6C, 6D). The absence of proliferation in PD-1hi CD8 TILs from sepsis survivors was particularly striking because these cells acquired a phenotype associated with robust cellular activation/function in nonseptic counterparts. The health of CD8 TILs not currently undergoing apoptosis was determined by labeling cells with mitochondrial membrane potential (ΔΨ) dye (49), which showed the health of CD8 TILs from sepsis survivors was diminished compared with nonseptic counterparts (Fig. 6E, 6F). Lastly, an increased rate of apoptosis (activated caspases 3/7) and cell death (propidium iodide) was seen in CD8 TILs from sepsis survivors (Fig. 6G–J). As expected, PD-1hi CD8 TILs had the highest frequency of dead cells as a consequence of activation/inhibitory receptor expression; however, despite examining the same population of PD-1hi CD8 TILs across both groups, sepsis survivors had a dramatically higher frequency of dead cells compared with sham controls. Thus, the data in Fig. 6 suggest the functional/survival capacity of CD8 TILs from sepsis survivors is greatly diminished even across subsets with similar activation/inhibitory receptor expression, clearly indicating the tumor microenvironment in sepsis survivors is not conducive to efficacious CD8 T cell–mediated antitumor immunity.

FIGURE 6.

The chronic immunoparalysis phase of sepsis diminishes proliferative capacity and survival of CD8 TILs. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 10 d later. Tumor samples were analyzed 14 d later. (B) PD-1 expression on CD8 TILs. (C) Representative histograms and (D) summary data of Ki67 expression of CD8 TILs grouped by PD-1 expression. (E) Representative histograms and (F) summary data of ΔΨ+ CD8 TILs grouped by PD-1 expression. (G) Representative histograms and (H) summary data of activated caspase 3/7+ (detected with FLICA) CD8 TILs grouped by PD-1 expression. (I) Representative histograms and (J) summary data of propidium iodide+ CD8 TILs grouped by PD-1 expression. Data are representative of at least two independent experiments. Bar graphs with single or multiple comparisons were analyzed using unpaired t test or two-way ANOVA, respectively. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 6.

The chronic immunoparalysis phase of sepsis diminishes proliferative capacity and survival of CD8 TILs. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 10 d later. Tumor samples were analyzed 14 d later. (B) PD-1 expression on CD8 TILs. (C) Representative histograms and (D) summary data of Ki67 expression of CD8 TILs grouped by PD-1 expression. (E) Representative histograms and (F) summary data of ΔΨ+ CD8 TILs grouped by PD-1 expression. (G) Representative histograms and (H) summary data of activated caspase 3/7+ (detected with FLICA) CD8 TILs grouped by PD-1 expression. (I) Representative histograms and (J) summary data of propidium iodide+ CD8 TILs grouped by PD-1 expression. Data are representative of at least two independent experiments. Bar graphs with single or multiple comparisons were analyzed using unpaired t test or two-way ANOVA, respectively. *p < 0.05, **p < 0.01, ****p < 0.0001.

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Tumor-specific CD8 TILs express the activation/inhibitory receptors PD-1 and LAG-3 (seen in Fig. 3), marking cells, in this model, that retain detectable levels of effector functions but are susceptible to apoptotic death (seen in Fig. 6). Targeted therapeutic strategies to prevent activation/inhibitory receptor ligation have proven successful in clinical and experimental melanoma that reduces cell death of the highly activated PD-1hi CD8 TIL subset (5052). Our data indicated the rapid loss of PD-1hi CD8 TILs and the impaired functional capacity of remaining cells could make sepsis survivors less responsive to this immunotherapy strategy. To confirm this striking difference in phenotype observed in CD8 TILs obtained from tumor-bearing hosts after CLP or sham surgeries, an additional method was used to analyze flow cytometric data. SPADE is a nonbiased approach to analyze flow cytometric data and separate cells with similar characteristics into nodes. In this study, CD8 TILs were separated, based on the intensity of PD-1 expression, into bubbles, and the frequency of cells within each bubble is shown. The SPADE trees show the relative expression of LAG-3 on cells from each node (low [blue] to high [yellow] expression). LAG-3 expression was mainly seen on cells within the PD-1hi bubble (also seen in Fig. 3). Furthermore, the representation of PD-1hi CD8 TILs was dramatically decreased in sepsis survivors, based on node size (cell number) (Fig. 7A). These data suggest checkpoint blockade therapy designed to improve the durability of cells with high expression of activation/inhibitory receptors may not be efficacious if administered at this point of disease progression in tumor-bearing sepsis survivors.

FIGURE 7.

The chronic immunoparalysis phase of sepsis diminishes the therapeutic window of checkpoint blockade because of the rapid loss of PD-1hi CD8 TILs. (A) Representative SPADE trees of CD8 TILs in mice that received 2 × 104 B16 cells 14 d after sham (top) or CLP (bottom) surgery. Expression of PD-1 is shown in bubbles with frequency of population shown. In a nonbiased approach, cells sharing similar characteristics are grouped into nodes. Relative expression of LAG-3 is shown, based on node color (blue–yellow). Node size indicates the number of cells that are associated with each node. (B) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 14 d later. Mice that received treatment were injected i.p. with 200 μg of anti–PD-L1 and anti–LAG-3 mAb or received equal amounts of control rat IgG at indicated time points (every 3 d). (C) Tumor size at indicated time point after B16 injection in mice that received treatment (square) or control IgG (circle). Treatment regimen is shown in red below x-axis. (D) Frequency of CD8+ cells among TILs in tumor samples. (E) Representative histograms and (F) summary data showing frequency of PD-1hi CD8 TILs in tumor samples. Tumor size ± SEM are shown. Data are representative of >3 mice per group. Bar graphs and tumor growth analyzed using unpaired t test and two-way ANOVA, respectively. *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant.

FIGURE 7.

The chronic immunoparalysis phase of sepsis diminishes the therapeutic window of checkpoint blockade because of the rapid loss of PD-1hi CD8 TILs. (A) Representative SPADE trees of CD8 TILs in mice that received 2 × 104 B16 cells 14 d after sham (top) or CLP (bottom) surgery. Expression of PD-1 is shown in bubbles with frequency of population shown. In a nonbiased approach, cells sharing similar characteristics are grouped into nodes. Relative expression of LAG-3 is shown, based on node color (blue–yellow). Node size indicates the number of cells that are associated with each node. (B) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 14 d later. Mice that received treatment were injected i.p. with 200 μg of anti–PD-L1 and anti–LAG-3 mAb or received equal amounts of control rat IgG at indicated time points (every 3 d). (C) Tumor size at indicated time point after B16 injection in mice that received treatment (square) or control IgG (circle). Treatment regimen is shown in red below x-axis. (D) Frequency of CD8+ cells among TILs in tumor samples. (E) Representative histograms and (F) summary data showing frequency of PD-1hi CD8 TILs in tumor samples. Tumor size ± SEM are shown. Data are representative of >3 mice per group. Bar graphs and tumor growth analyzed using unpaired t test and two-way ANOVA, respectively. *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant.

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To formally test this hypothesis, mice received sham or CLP surgery and 14 d later were implanted with B16 cells. Anti–PD-L1/anti–LAG-3 or control IgG mAb was administered 13 d after tumor implantation when loss of PD-1hi CD8 TILs has been shown to occur in sepsis survivors (Fig. 7B). Consistent with previously discussed data, sepsis survivors that did not receive therapy had increased tumor growth compared with untreated nonseptic mice. As predicted from the SPADE, nonseptic hosts responded to the checkpoint blockade therapy with rapidly diminishing tumor progression. However, checkpoint blockade that proved efficacious in nonseptic hosts did not have therapeutic efficacy in CLP-treated mice (Fig. 7C). CD8 TIL responses that underlie these tumor growth curves further indicated sepsis survivors were not amenable to checkpoint blockade and did not increase the frequency of PD-1hi CD8 TIL to levels seen in nonseptic counterparts (Fig. 7D–F). Next, to determine if tumor-bearing sepsis survivors could be amenable to checkpoint blockade therapy when administered earlier during tumor progression and before lesions in CD8 TIL responses were observed, the similar experimental approach was used with mAb treatments starting 7 d after tumor challenge (Fig. 8A). First, sepsis survivors that did not receive therapy had increased tumor growth compared with untreated sham mice. Importantly, initiating checkpoint blockade at day 7 in tumor-bearing sepsis hosts led to a significant increase in the tumor control of sepsis survivors that resulted in tumor growth that was indistinguishable to nonseptic hosts receiving therapy (Fig. 8B). The underlying antitumor response support these conclusions with a high amount of PD-1hi CD8 TILs now observed in sepsis survivors that received therapy (Fig. 8C–E). Histological analysis of tumor samples from this experiment confirmed that sepsis survivors who did not receive therapy had low levels of CD8 T cells (marked with asterisk) that poorly penetrated the tumor (Fig. 8F). Checkpoint blockade therapy promoted tumor penetration of CD8 TILs, resulting in a diffuse cellular infiltration because of the inhibition of PD-1 and LAG-3 activation/inhibitory receptors. These histological data further confirm timely checkpoint blockade therapy can dramatically extend the durability of CD8 TIL responses in sepsis survivors. In total, the rapid loss of PD-1hi CD8 TILs during the chronic immunoparalysis phase of sepsis diminishes the therapeutic window of checkpoint blockade that has significant implications in future attempts to manage cancer-associated mortality of sepsis survivors.

FIGURE 8.

Timely checkpoint blockade therapy greatly improves tumor control of sepsis survivors during the chronic immunoparalysis phase of sepsis. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 14 d later. Mice that received treatment were injected i.p. with 200 μg of anti–PD-L1 and anti–LAG-3 mAb or received equal amounts of control rat IgG at indicated time points (every 3 d). (B) Tumor size at indicated time point after B16 injection in mice that received treatment (square) or control IgG (circle). Treatment regimen is shown in red below x-axis. (C) Frequency of CD8+ cells among TILs in tumor samples. (D) Representative histograms and (E) summary data showing frequency of PD-1hi CD8 TILs in tumor samples. (F) Representative confocal microscopic images of CD8+ (green; asterisk symbol) and nucleated cells (blue) in tumors of mice that received CLP (top) or CLP with treatment (bottom). Tumor periphery is marked on the left side of each image. Increased image magnification (×6) is seen in the right column. DAPI staining was used. Tumor size ± SEM are shown. Data are representative of >3 mice per group. Bar graphs and tumor growth analyzed using unpaired t test and two-way ANOVA, respectively. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 8.

Timely checkpoint blockade therapy greatly improves tumor control of sepsis survivors during the chronic immunoparalysis phase of sepsis. (A) Experimental design. Mice received sham or CLP surgery followed by s.c. injection of 2 × 104 B16 cells in the hind flank 14 d later. Mice that received treatment were injected i.p. with 200 μg of anti–PD-L1 and anti–LAG-3 mAb or received equal amounts of control rat IgG at indicated time points (every 3 d). (B) Tumor size at indicated time point after B16 injection in mice that received treatment (square) or control IgG (circle). Treatment regimen is shown in red below x-axis. (C) Frequency of CD8+ cells among TILs in tumor samples. (D) Representative histograms and (E) summary data showing frequency of PD-1hi CD8 TILs in tumor samples. (F) Representative confocal microscopic images of CD8+ (green; asterisk symbol) and nucleated cells (blue) in tumors of mice that received CLP (top) or CLP with treatment (bottom). Tumor periphery is marked on the left side of each image. Increased image magnification (×6) is seen in the right column. DAPI staining was used. Tumor size ± SEM are shown. Data are representative of >3 mice per group. Bar graphs and tumor growth analyzed using unpaired t test and two-way ANOVA, respectively. *p < 0.05, **p < 0.01, ****p < 0.0001.

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The transient lymphopenia after sepsis is followed by chronic immunoparalysis of the remaining immune cells that contributes to the chronic critical illness of sepsis survivors. This long-lasting immunoparalysis increases the host’s susceptibility to secondary complications/diseases that increases the risk for mortality of these patients years after the septic event. Thus, studies elucidating sepsis-induced immunological lesions that contribute to the increased disease burden of sepsis survivors is an important research pursuit. Secondary infections have been the primary method to assess the functional capacity of CD8 T cell–mediated immunity in sepsis survivors (19), but additional secondary complications dramatically affect the quality of life/survival of sepsis patients during the chronic immunoparalysis phase and could be the result of unique immunological lesions that occur in certain disease contexts. Cancer is a common and lethal secondary complication in sepsis survivors, accounting for a high frequency of mortality (21); despite this, sepsis-induced immunological lesions that contribute to this phenotype are largely unknown.

This report revealed that a common immunoparalysis signature occurs after sepsis in pathogen- and cancer-specific CD8 T cell responses upon Ag recognition, including diminished proliferative capacity (Ki67) and effector cytokine production (IFN-γ) that contribute to increased disease burden (31). Use of the B16 tumor model disclosed unique immunological lesions specific to malignancy-bearing hosts, including the rapid loss of highly activated PD-1hi CD8 TILs that occurred exclusively in the tumor environment. Because sepsis globally alters immune cell subsets, it could be predicted that the increased tumor progression seen after sepsis is attributed to altered functionality of several cell subsets in the tumor, some of which have been explored previously (24, 25). Despite this, it seems remarkable that a targeted immunotherapy that inhibits ligation of activation/inhibitory receptors (PD-1 and LAG-3) on CD8 T cells appeared to completely negate the sepsis-induced impairments in tumor control when administered before the loss of PD-1hi CD8 TILs was observed. Therapeutic efficacy was lost after the absence of PD-1hi CD8 TILs in sepsis survivors, resulting in a diminished window of therapeutic opportunity that further indicated the importance of this subset in tumor control. These data suggest that increased monitoring and early detection of malignancy in sepsis patients will be critical in the management of cancer-associated survival during the chronic immunoparalysis phase of sepsis.

The diminished capacity to mount efficacious CD8 T cell responses during the immunoparalysis phase of sepsis can be attributed to both T cell–intrinsic and –extrinsic mechanisms (19). For example, the reduced capacity of pathogen-specific memory CD8 T cells to gain effector functions in the presence of limiting levels of cognate Ag (functional avidity) is diminished on a per-cell basis after sepsis because of a T cell–intrinsic defect (31). In addition, the inability of dendritic cells from sepsis hosts to respond to subsequent TLR stimulation results in reduced levels of “signal 3” cytokines (e.g., IL-12) after secondary infections that can diminish the magnitude of CD8 T cell responses in a T cell–extrinsic mechanism (33). It is currently unknown if the diminished capacity of sepsis survivors to mount efficacious CD8 TIL responses is attributed to CD8 T cell–intrinsic or – extrinsic mechanisms. Previously, the transfer of protumorigenic immune cell subsets, including regulatory CD4 T cells or bone marrow–derived macrophages from the postseptic environment, was sufficient to increase tumor progression, but it is unclear if these cells drive increased tumor progression in physiological settings without cell transfer (24, 25). The striking efficacy of checkpoint inhibitors in tumor-bearing sepsis survivors (Fig. 8) suggests ligation of PD-1 and LAG-3 receptors is a potential T cell­–extrinsic mechanism by which immunosuppressive cell subsets weaken CD8 TIL responses that ultimately increases cancer-associated mortality after sepsis. The myeloid compartment represents a likely population of immune cells affected by checkpoint blockade because of their increased expression of PD-1 and PD-L1 after sepsis, but the cell population responsible for immune suppression of CD8 TIL remains speculative (53). Ultimately, determining the mechanisms that drive impaired CD8 TIL responses in sepsis survivors will be critical in controlling the chronic disease burden of these patients. Therapeutic strategies (e.g., checkpoint blockade) that improve tumor control during the period of immunoparalysis could still prove useful in the short-term management of malignancy burden in sepsis survivors until these precise mechanisms are revealed. The appeal of this therapy is further increased with reports showing checkpoint inhibitors improve sepsis-associated survival in mice (5456) and is showing promise in the clinic (57, 58), suggesting a single therapy could be used to improve both short- and long-term survival of sepsis patients.

This report stresses the importance of modeling secondary diseases/complications that can detrimentally affect the overall health and survival of sepsis survivors because of unique lesions in CD8 T cell responses that arise in a disease-specific context. It is becoming clear that the current notion of how sepsis affects CD8 T cell–mediated immunity modeled in hosts lacking comorbidities will not be sufficient to understand alterations in CD8 T cell responses and, ultimately, the outcome of all sepsis patients. This point is further emphasized by our recent work revealing that in some instances cancer bearing hosts could even benefit from sepsis induction (59). In the scenarios in which sepsis occurs in hosts with smaller, less developed tumors, sepsis has the capacity to reinvigorate otherwise dormant CD8 TILs, resulting in prolonged survival (59). Thus, research that reveals sepsis-induced lesions in CD8 T cell responses that occur universally among all secondary diseases as well as disease-specific lesions will be critical in establishing personalized therapy to address the specific needs of each sepsis patient to begin controlling the increased rate of mortality seen in this population.

We thank members of the Badovinac laboratory for helpful discussion, Lecia Epping (University of Iowa) for reagents, and Dr. Steve Varga and Stacey Hartwig (University of Iowa) for training and access to the Buxco whole-body plethysmography machine.

This work was supported by National Institutes of Health Grants GM113961 (to V.P.B.), GM115462 (to T.S.G.), T32AI007485 (to D.B.D. and I.J.J.), and T32AI007511 (to I.J.J.), the Holden Comprehensive Cancer Center at the University of Iowa and its National Cancer Institute Award P30CA086862 (to V.P.B.), and a Veterans Administration Merit Review Award I01BX001324 (to T.S.G.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

BPM

breaths per minute

CLP

cecal ligation and puncture

MHC I

MHC class I

Penh

enhanced pause

SPADE

Spanning-Tree Progression Analysis of Density-Normalized Events

TIL

tumor-infiltrating lymphocyte.

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The authors have no financial conflicts of interest.

Supplementary data