Donor-derived lymphocytes from allogeneic hematopoietic cell transplantation (allo-HCT) or donor lymphocyte infusion can mediate eradication of host tumor cells in a process labeled the graft-versus-tumor (GVT) effect. Unfortunately, these treatments have produced limited results in various types of leukemia because of an insufficient GVT effect. In this context, molecular engineering of donor lymphocytes to increase the GVT effect may benefit cancer patients. Activating MyD88 signaling in CD8+ T cells via TLR enhances T cell activation and cytotoxicity. However, systemic administration of TLR ligands to stimulate MyD88 could induce hyperinflammation or elicit protumor effects. To circumvent this problem, we devised a synthetic molecule consisting of MyD88 linked to the ectopic domain of CD8a (CD8α:MyD88). We used this construct to test the hypothesis that MyD88 costimulation in donor CD8+ T cells increases tumor control following allo-HCT in mice by increasing T cell activation, function, and direct tumor cytotoxicity. Indeed, an increase in both in vitro and in vivo tumor control was observed with CD8α:MyD88 T cells. This increase in the GVT response was associated with increased T cell expansion, increased functional capacity, and an increase in direct cytotoxic killing of the tumor cells. However, MyD88 costimulation in donor CD8+ T cells was linked to increased yet nonlethal graft-versus-host disease in mice treated with these engineered CD8+ T cells. Given these observations, synthetic CD8α:MyD88 donor T cells may represent a unique and versatile approach to enhance the GVT response that merits further refinement to improve the effectiveness of allo-HCT.

This article is featured in Top Reads, p.665

Allogeneic hematopoietic cell transplantation (allo-HCT) is an established treatment for different blood cancers (1). Over the past several decades, it has become evident that donor-derived lymphocytes increase engraftment and rejection of residual host tumor cells (2, 3). This was initially verified when patients receiving T cell–depleted bone marrow (TCD-BM) grafts experienced an increase in graft failure and tumor relapse (4). The rejection of host tumor cells by donor lymphocytes has been characterized as the unique graft-versus-tumor (GVT) effect (5). Subsequently, donor lymphocyte infusion (DLI) is indicated for high-risk patients undergoing allo-HCT or for the treatment of relapsed disease. This treatment has shown to increase remission and prolong survival in patients with some leukemias; however, efficacy remains poor for treating other cancers, including lymphoma or acute myelogenous leukemia (6, 7). Further manipulation of donor lymphocyte populations and engineering their function could benefit additional patient populations with aggressive or high-risk disease (8). CD8+ T cells or cytotoxic T cells are a population of lymphocytes that target tumor cells to induce a strong GVT effect through different modalities of killing, including cytokine-, death ligand-, and perforin/granzyme–mediated cell death (9). The GVT effect is known to be initiated through T cell activation by residual host dendritic cells, and this is crucial for shaping a response to the host tumor cells (10, 11).

MyD88 activation and costimulation can be achieved through activation of TLRs, IL-1R, IL-18R, and ST2/IL-33R (1214). With the exception of TLR3, MyD88 binds to the Toll/IL-1R (TIR) domain of activated TLRs, initiating the formation of a signaling complex that IRAK-4 binds, leading to recruitment and phosphorylation of IRAK-1 (14). Through a series of steps involving TRAF-6 and TAK1, the NF-κB pathway is initiated, leading to expression of genes involved in cell survival, proliferation, and inflammation (15). Activation of TLRs and the MyD88 signalosome can also function to recruit factors that activate the MAPKs ERK, JNK, and p38. MAPKs recruit transcription factors for growth, proliferation, and survival (14). Activating MyD88 signaling in CD8+ T cells via TLR engagement enhances T cell function and proliferation (12, 13). However, using TLR ligands to stimulate T cells presents major challenges because of ephemeral or low expression of TLRs (16). Additionally, TLR ligand activation of tumor cells has been shown to promote tumor survival and growth (16). To circumvent these challenges, we have developed a construct to produce CD8+ T cell–targeted MyD88 costimulation. This construct consists of MyD88 directly linked to the ectopic domain of CD8α to induce MyD88 activation with TCR engagement. After transduction with this construct, tumor-specific pmel T cells display characteristics of TLR activation and have enhanced efficacy in an experimental melanoma tumor model (17).

The impact of MyD88 in CD8+ T cells in the context of experimental transplantation is still controversial. One study using MyD88 knockout T cells showed that MyD88 was indispensable for an effective GVT effect, whereas another report claimed MyD88 was dispensable for a GVT effect (18, 19). In this study, we sought to define the effect of synthetic MyD88 costimulation in donor CD8+ T cells on tumor control in the setting of allo-HCT. In this study, we find that synthetic MyD88 costimulation in CD8+ T cells significantly improves tumor control in vitro and in vivo. We observed enhanced proliferative capacity and expansion of CD8α:MyD88–transduced donor CD8+ T cells. Furthermore, MyD88 costimulation increased the cytotoxic functions of donor CD8+ T cells. The increase in T cell activation and function from MyD88 costimulus was linked to an increase in graft-versus-host disease (GvHD) but not host death. Ultimately, we conclude that MyD88 costimulation in donor CD8+ T cells increases the GVT response after allo-HCT.

BALB/c, 129 × 1/SvJ, and C57BL/6 male mice were obtained from The Jackson Laboratory. A20 lymphoma, derived from the BALB/c strain, was transduced to express luciferase as previously described (20, 21). All mice were maintained in specific pathogen–free housing, and all experiments were conducted in accordance with the animal care guidelines from the Office of Animal Welfare Assurance at the University of Maryland School of Medicine Veterinary Resources using protocols approved by the Institutional Animal Care and Use Committee.

All TCD-BM cells were isolated from wild-type (WT) C57BL/6 or 129 × 1/SvJ mice. T cell depletion was performed with autoMACS by using anti-CD90.2 MicroBeads (Miltenyi Biotec). Donor CD8+ T cells (purity ∼90–95%) were purified from the spleens of WT C57BL/6 mice by using the CD8+ T Cell (negative) Isolation Kit (STEMCELL Technologies). CD8+ T cells were activated to prepare for retroviral transduction by plating 1 × 106 cells per well in 24-well plates that were coated with 1 μg of anti-CD3 (BD Pharmingen) and supplemented with 2 μg/ml soluble anti-CD28 (BD Pharmingen) and 100 U/ml IL-2 (Novus Biologicals). Plasmids were designed and cloned, as described previously, using the pMIG retroviral vector (modified MSCV-IRES-GFP) (17). Plasmids for viral packaging were isolated from bacteria using an EndoFree Plasmid Maxi Kit (QIAGEN). The retrovirus was prepared by transfecting the Phoenix-ECO (CRL-3214; American Type Culture Collection) packaging cell line with GFP control or CD8α:MyD88 experimental construct plasmids using Lipofectamine 2000 (Invitrogen). The viral supernatant was collected at 48 and 72 h and used for transduction 48 h after T cell activation. The activated CD8+ T cells were transduced on RetroNectin (Takara Bio). The virus was washed off the next day, and cells were given 20 U/ml IL-2 and allowed to propagate before T cell infusion or in vitro use.

Abs, including anti-mouse CD11b (M1/70), B220 (RA3-6B2), CD3 (145-2C11), TCRB (H57-597), TNF-α (MP6-XT22), IFN-γ (XMG1.2), GzmB (GB11), CD107a (1D4B), H-2Kb (AF6-88.5), H-2kd (SF1-1.1), Tim-3 (B8.2C12), and PD-1 (29F.1A12), were ordered from BioLegend and used for spectral flow cytometry. The Ki67 Ab (SolA15) from eBioscience was used for intracellular flow cytometric staining. Briefly, cells were washed using flow buffer (PBS with 2% FBS), and Fc receptors were blocked with the addition of unlabeled anti-CD16/CD32 for 20 min. External markers and fixable LIVE/DEAD Fixable Aqua or Near-IR (Invitrogen) were stained together in PBS for 30 min and washed two times with flow buffer. Intracellular stains were performed using the Intracellular Fixation and Permeabilization Buffer Set by eBioscience. Briefly, cells were fixed for 30 min at room temperature using the Intracellular Fixation Buffer. Cells were resuspended in 1× permeabilization buffer and incubated for 5 min. Samples were split, and test Abs or isotype control Abs were added and incubated for 1 h at room temperature and washed twice in 1× permeabilization buffer before resuspending in flow buffer. Samples were run on the Aurora spectral flow cytometer (Cytek Biosciences) in the Center for Innovative Biomedical Resources at the University of Maryland School of Medicine. Unmixed samples were analyzed using FlowJo software (FlowJo). Background staining was assessed with unstained controls, stained isotype controls, and experimental negative controls when possible. “Spike-in dead” and “all dead” controls of mixed cell populations (T cells and tumor) were used as positive controls to test the staining and gating strategy for annexin V/7-aminoactinomycin D (7-AAD) staining.

T cell killing assays were setup as modified mixed lymphocyte cytotoxicity reactions by adding a variable number of transduced C57BL/6 CD8+ T cells (1 × 104–2 × 105; 1:1–20:1) to a constant 1 × 104 tumor cells (BALB/C origin). Assays were performed in T cell media containing an RPMI 1640 base with 10% FBS, 1% nonessential amino acids, 1% glutamine, and 0.05 mM of 2-ME. Cells were added to a round-bottom plate for flow cytometry assays, and a black flat-bottom plate was used for luciferase measurements (round bottoms are not available in black). Mismatched bone marrow–derived dendritic cells (BMDC) (BALB/C; H-2d) were added to the wells at a ratio metric 1:10 to the variable T cell number. Assays were quickly stained for surface markers (15 min) and washed with calcium buffer followed by the addition of annexin V/7-AAD that was purchased as a kit from BioLegend. These samples were placed on ice and run immediately on the Aurora spectral flow cytometer (Cytek Biosciences) mentioned previously. Live tumor control luciferase assays were setup similarly but cultured in black flat-bottom plates. On the day of acquisition, these assays were supplemented with 0.15 mg/ml luciferin and imaged on the Xenogen IVIS-200 (PerkinElmer) and analyzed using the Living Image Software (PerkinElmer).

Experimental transplant tumor models used male C57BL/6 or 129 × 1/Svj donors and BALB/c host mice, which were irradiated with 9.5 Gy before transplant and T cell treatment. One day later, the hosts were inoculated i.v. with A20 cells (doses indicated in figure legends). After injecting the tumor, mice received 3.5 × 106 TCD-BM cells alone or combined with (C57BL/6) WT naive CD8+ T cells (nCD8), control-transduced CD8+ T cells (pMIG), or CD8α:MyD88 (pCM8)–transduced CD8+ T cells (dose indicated in figure legends). In the experiment in which T cell transfer was delayed, i.v. injection of T cells was performed 7 d after tumor and bone marrow transplant in 200 μl of PBS. Bioluminescence imaging was performed to monitor tumor burden, as previously described (22). In GVT experiments, tumor burden was expressed as photon flux (photons per second), and disease score and weight were also used to assess the health of the animals through treatment. To assess GvHD, we used the groups consisting of TCD-BM alone, TCD-BM plus pMig-GFP–transduced control T cells, and TCD-BM plus pCM8-transduced T cells. To separate effects from tumor versus GvHD, these mice were not injected with tumor. T cell dose is indicated in the figure legends. In the GvHD T cell dose escalation experiment, mice were euthanized at the end of the experiment. Liver and large and small intestines were removed, formalin-fixed, sectioned, and stained with H&E. Blinded assessments of histopathological GvHD were made by an experienced pathologist using an established semiquantitative scoring system (23, 24). Representative pictures were captured at ×100.

All data processing was performed in Prism GraphPad V8. Student t tests were used to compare statistical significance between groups of normally distributed samples, and asterisks (ns = p > 0.05; *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001) indicate the degree and magnitude of significance between two groups. Kaplan–Meier plots were created, and survival analysis was performed using the Gehan–Breslow–Wilcoxon test. To compare significance between survival groups, all combinations of groups were separated and paired, and p values were adjusted for multiple comparisons. In assessing statistical differences of tumor burden over time between in vivo groups, a two-way ANOVA test was used up to before mice began dying. To assess significance at individual time points, a pound sign (#) was used to indicate when the pCM8 group was significantly different (p < 0.05) from all other controls when compared using Student t test. Logistic growth/inhibition curves were made in GraphPad using the transformed data algorithm, in which data are normalized to the control samples (0:1; tumor only) as 100% growth without transforming to log. With this, we could derive IC50 values, which are defined as the number of T cells required to produce 50% growth inhibition of the controls.

We previously found that TLR5 agonist CBLB502 enhanced GVT without increasing GvHD (25). This effect was indirect and mediated through stimulation of accessory innate immune cells, increasing CD8+ T cell killing of tumor cells (25). However, other studies have found that CD8+ T cells express other TLRs and use MyD88 signaling to amplify activation and effector functions (26). To test the direct effect of the MyD88 pathway on T cells without confounding systemic treatment, we developed a synthetic MyD88 construct (Fig. 1A, left) which has since been tested in a solid tumor model and found effective for the treatment of experimental melanoma in mice (17). Our construct contains an ectopic CD8α domain with a MyD88 molecule linked internally but without the TIR domain. This construct uses the pMIG retroviral vector as the backbone, requiring primary T cell activation for transduction. After transduction, secondary activation through the TCR or anti-CD3 Abs induces activation of this novel construct. Transduction efficiencies are typically around 60%, with a range between 55 and 72% observed (Fig. 1A, right). Previous studies show that the effects of this construct are not from ectopic CD8α expression, but from the aggregation and activation of the linked MyD88 in conjunction with TCR stimulus (17). As a proof of concept for MyD88-stimulated CD8+ T cell control of tumor in an allogeneic setting, we designed a modified MLR, in which H-2b CD8+ T cells were sorted from C57BL/6 mice and transduced to express the control GFP vector (pMig) or the CD8α:MyD88 vector (pCM8). These cells were combined with BMDC from BALB/c (H-2d) mice at a 10:1 ratio. A constant of 1 × 104 luciferase-expressing A20 lymphoma cells were used as live reporter cells to assess tumor control over time (Fig. 1B). At 24 and 48 h, d-luciferin was added, and the plates were imaged and analyzed. We found that pCM8-transduced CD8+ T cells were better than pMig control T cells in inhibiting tumor growth at high ratios of 20:1 and 10:1 after 24 h (Fig. 1C). After 48 h of culture, pCM8-transduced CD8+ T cells were better able to control tumor growth, with significant differences with the greatest separation at the 1:1 ratio (Fig. 1D). This experiment provided antecedent evidence that MyD88 costimulation in donor CD8+ T cells could increase tumor control in an allogeneic setting. To determine the impact of MyD88-costimulated CD8+ donor T cells in the GVT response, BALB/c (H-2d) host mice were conditioned with total body irradiation (TBI). One day later, mice were first i.v. injected with 0.5 × 106 A20-luciferase lymphoma cells. This was immediately followed by transplantation with 3.5 × 106 TCD-BM and treatments, including no T cell infusion (TCD-BM only), infusion of 0.5 × 106 CD8+ naive T cells, infusion of pMig-GFP control CD8+ T cells, or infusion of experimental pCM8 CD8+ T cells (Fig. 2A). We performed bioluminescence imaging to measure tumor burden. As expected, there was tumor control in CD8 naive and pMig control groups compared with the TCD-BM only group, yet there was no significant difference between the activated pMig control and the naive CD8+ T cell control. When the pCM8 T cell group was compared with all controls, there was a significant decrease in tumor burden observed over time (p < 0.0001) (Fig. 2B, top). Also apparent in each of the pCM8-treated mice were spontaneous but ephemeral regressions that varied in magnitude and time (Fig. 2B bottom). To further examine the impact of MyD88 costimulation of CD8+ T cells with transplant on the tumor-bearing mice, we monitored survival over 50 d (Fig. 2C). Transplant recipients of the nCD8 or pMig CD8+ T cells had a median survival (MS) of 25 d compared with the TCD-BM only control, which had an MS of 17 d. Recipients of the pCM8-transduced CD8+ T cells had an MS of 47 d, which was significantly longer than all other groups (pMig:pCM8, p = 0.0043; nCD8:pCM8, p = 0.0041; TCD:pCM8, p = 0.0039). Additionally, mice were scored using a disease metric accounting for fur texture, posture, activity, skin condition, weight loss, and diarrhea (Fig. 2D). The disease score in mice treated with pCM8-transduced CD8+ T cells was significantly lower after day 21 and until the end of the study.

FIGURE 1.

Synthetic CD8α:MyD88 coreceptor in donor CD8+ T cells increases killing of allogeneic A20 lymphoma cells in vitro. (A) Left, Schematic representing the synthetic CD8α:MyD88 coreceptor. Magnetically sorted CD8 T cells were polyclonally activated with 1 μg/ml plate-bound anti-CD3 and 2 μg/ml soluble anti-CD28. On day 3 postactivation they were retrovirally transduced to express either CD8α:MyD88 (pCM8) or the empty vector (pMIG) with GFP only. Right, Representative data for typical transduction efficiency for CD8+ T cells. Efficiency is measured 2 d posttransduction by analyzing GFP expression on CD8+ cells via flow cytometry before injection into corresponding treatment group. (B) Schematic describing a cytotoxic MLR with A20-luciferase tumor to test proof of concept of engineered T cells in an allo-HCT tumor model. Transduced T cells (H-2b) were cocultured with constant A20 tumor (H-2d) and activated BMDC (H-2d) 1:10 to T cells. To assess tumor control, luciferin substrate was added at (C) 24 h and (D) 48 h after culture; plates were imaged using the Xenogen IVIS-200 system. Total photon flux was plotted to assess tumor signal in each well. Representative results are shown from one of two independent experiments, and data are presented as mean ± SD from triplicate samples. Student t test was used to determine statistical difference between pMig and pCM8 groups at a given timepoint (*p < 0.05, **p < 0.01).

FIGURE 1.

Synthetic CD8α:MyD88 coreceptor in donor CD8+ T cells increases killing of allogeneic A20 lymphoma cells in vitro. (A) Left, Schematic representing the synthetic CD8α:MyD88 coreceptor. Magnetically sorted CD8 T cells were polyclonally activated with 1 μg/ml plate-bound anti-CD3 and 2 μg/ml soluble anti-CD28. On day 3 postactivation they were retrovirally transduced to express either CD8α:MyD88 (pCM8) or the empty vector (pMIG) with GFP only. Right, Representative data for typical transduction efficiency for CD8+ T cells. Efficiency is measured 2 d posttransduction by analyzing GFP expression on CD8+ cells via flow cytometry before injection into corresponding treatment group. (B) Schematic describing a cytotoxic MLR with A20-luciferase tumor to test proof of concept of engineered T cells in an allo-HCT tumor model. Transduced T cells (H-2b) were cocultured with constant A20 tumor (H-2d) and activated BMDC (H-2d) 1:10 to T cells. To assess tumor control, luciferin substrate was added at (C) 24 h and (D) 48 h after culture; plates were imaged using the Xenogen IVIS-200 system. Total photon flux was plotted to assess tumor signal in each well. Representative results are shown from one of two independent experiments, and data are presented as mean ± SD from triplicate samples. Student t test was used to determine statistical difference between pMig and pCM8 groups at a given timepoint (*p < 0.05, **p < 0.01).

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FIGURE 2.

Synthetic CD8α:MyD88 coreceptor in donor CD8+ T cells increases GVT effect. (A) Schematic describing the experiment in which BALB/c host mice are given TBI, inoculated with 0.5 × 106 A20-luciferase cells, injected with 3.5 × 106 TCD-BM and 0.5 × 106 T cells from C57BL/6 or 129 × 1/SvJ mice for the corresponding adoptive T cell transfer group (n = 5 per group). Using live imaging, tumor measurements were obtained in A20-luciferase tumor-bearing mice. (B) Total photon flux was plotted over time with average and SEM. ANOVA was used to analyze mice during the period in which they were all alive; pound signs (#) indicate significant difference between pCM8 group and all other (remaining) groups at that time point. #p < 0.05 by Student t test between variables. Additionally, (C) survival was measured until the experiment was terminated at 50 d. (D) Disease score was assessed on a 0–12 scale by measuring and adding mouse activity (0–2), fur texture (0–2), posture (0–2), diarrhea (0–2), skin (0–2), and weight (0–4) in each animal at time of image acquisition. Deceased mice maintain a disease score of 10 throughout the study. A similar experiment was performed but instead used 129 × 1/SvJ mice as bone marrow and T cell donors into BALB/c hosts; live imaging was performed as in (A). TCD-BM (n = 4), the other groups (n = 5). (E) Total photon flux was plotted over time with average and ± SEM. Pound signs (#) indicate significance (p < 0.05) between pCM8 group and all other (remaining) groups at that time point. (F) Survival was measured until the experiment was terminated at 60 d. In survival studies, ****p < 0.0001. ANOVA test was used to ascertain differences between groups over time. Representative results are shown from two independent experiments with similar results for both C57BL/6 and 129 × 1/SvJ donors.

FIGURE 2.

Synthetic CD8α:MyD88 coreceptor in donor CD8+ T cells increases GVT effect. (A) Schematic describing the experiment in which BALB/c host mice are given TBI, inoculated with 0.5 × 106 A20-luciferase cells, injected with 3.5 × 106 TCD-BM and 0.5 × 106 T cells from C57BL/6 or 129 × 1/SvJ mice for the corresponding adoptive T cell transfer group (n = 5 per group). Using live imaging, tumor measurements were obtained in A20-luciferase tumor-bearing mice. (B) Total photon flux was plotted over time with average and SEM. ANOVA was used to analyze mice during the period in which they were all alive; pound signs (#) indicate significant difference between pCM8 group and all other (remaining) groups at that time point. #p < 0.05 by Student t test between variables. Additionally, (C) survival was measured until the experiment was terminated at 50 d. (D) Disease score was assessed on a 0–12 scale by measuring and adding mouse activity (0–2), fur texture (0–2), posture (0–2), diarrhea (0–2), skin (0–2), and weight (0–4) in each animal at time of image acquisition. Deceased mice maintain a disease score of 10 throughout the study. A similar experiment was performed but instead used 129 × 1/SvJ mice as bone marrow and T cell donors into BALB/c hosts; live imaging was performed as in (A). TCD-BM (n = 4), the other groups (n = 5). (E) Total photon flux was plotted over time with average and ± SEM. Pound signs (#) indicate significance (p < 0.05) between pCM8 group and all other (remaining) groups at that time point. (F) Survival was measured until the experiment was terminated at 60 d. In survival studies, ****p < 0.0001. ANOVA test was used to ascertain differences between groups over time. Representative results are shown from two independent experiments with similar results for both C57BL/6 and 129 × 1/SvJ donors.

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To determine whether the observed effects were generalizable across transplant models, we performed a GVT experiment using 129/SvJ donors to the BALB/c hosts. In the first 2 wk, all the CD8+ T cell treatment groups displayed an enhanced control of tumor growth over the TCD-BM only control group. After day 18, the pCM8-transduced T cell group began showing a significant decrease in tumor burden compared with other groups throughout the remainder of the experiment (p < 0.0001) (Fig. 2E). Additionally, the pCM8 group displayed significantly improved survival compared with all other groups (pMig:pCM8, p = 0.0047; nCD8:pCM8, p = 0.0038; TCD:pCM8 p = 0.0047) (Fig. 2F).

In the standard GVT models described above, the bone marrow, tumor cells, and T cells are injected on the same day. This established model (25, 27, 28) for studying GVT in mice mimics a scenario in which a transplant is performed for treatment but residual tumor cells remain. However, second-line treatments, such as DLI, are typically given later, after transplant. To test whether the enhanced efficacy of CD8:MyD88 T cells could occur in the setting of DLI, we performed an experiment in which the injection of T cells was delayed for 7 d (Fig. 3A). Starting on day 18, the group treated with the pCM8-transduced T cells displayed significantly reduced tumor growth compared with all other treated and control groups (p < 0.0001) (Fig. 3B). Also apparent in the pCM8 T cell–treated group were various spontaneous tumor regressions (Fig. 3C), which were also observed in our standard model. The pCM8-transduced T cell group also maintained better survival compared with all other groups (pMig:pCM8, p = 0.0495; nCD8:pCM8, p = 0.0197; TCD:pCM8, p = 0.0039) (Fig. 3D). Although the standard model in Fig. 2 shows greater differences in tumor kinetics and survival, both the standard model and delayed DLI model show a similar pattern of efficacy.

FIGURE 3.

Delayed transfer of CD8α:MyD88 T cells after experimental transplant increases GVT effect. (A) Schematic describing the in vivo experiment in which BALB/c host mice are given TBI, injected with 3.5 × 106 TCD-BM from C57BL/6 mice and inoculated with 0.1 × 106 A20-luciferase. Seven days later, mice were injected with 0.5 × 106 T cells from the corresponding adoptive T cell transfer group. Using live imaging, tumor measurements were obtained in A20-luciferase tumor-bearing mice. (B) Total photon flux was plotted over time with average and SEM. The pound signs (#) indicate significance between pCM8 group and all other (remaining) groups at that time point. #p < 0.05 by Student t test between each group. (C) Individual values were also plotted to show spontaneous regressions in tumor growth. Additionally, (D) survival was measured until the experiment was terminated at 50 d. In survival studies, ***p < 0.001. Representative results are shown as mean ± SEM. Multiple t tests were used to assess significance between groups at a single timepoint, whereas an ANOVA test was used to ascertain differences between groups over time.

FIGURE 3.

Delayed transfer of CD8α:MyD88 T cells after experimental transplant increases GVT effect. (A) Schematic describing the in vivo experiment in which BALB/c host mice are given TBI, injected with 3.5 × 106 TCD-BM from C57BL/6 mice and inoculated with 0.1 × 106 A20-luciferase. Seven days later, mice were injected with 0.5 × 106 T cells from the corresponding adoptive T cell transfer group. Using live imaging, tumor measurements were obtained in A20-luciferase tumor-bearing mice. (B) Total photon flux was plotted over time with average and SEM. The pound signs (#) indicate significance between pCM8 group and all other (remaining) groups at that time point. #p < 0.05 by Student t test between each group. (C) Individual values were also plotted to show spontaneous regressions in tumor growth. Additionally, (D) survival was measured until the experiment was terminated at 50 d. In survival studies, ***p < 0.001. Representative results are shown as mean ± SEM. Multiple t tests were used to assess significance between groups at a single timepoint, whereas an ANOVA test was used to ascertain differences between groups over time.

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Together, results from these experiments indicate that synthetic MyD88 costimulation in donor CD8+ T cells enhances the GVT effect.

GVT and GvHD are linked in that both are mediated by allogeneic T cells, yet GvHD involves migration and damage to susceptible organs (29). This provided the rationale to examine the effect of this strategy on inducing GvHD. To separate the symptoms caused by the tumor and GvHD, allo-HCT was performed with TCD-BM and T cell infusion without tumor inoculation. In Fig. 2, our data indicate there were NS differences between naive and activated CD8+ T cell infusion; thus, nCD8 were excluded from this experiment. Mice were scored for disease over time on the same established metric from Fig. 2. We found that mice transplanted with the pCM8-transduced donor CD8+ T cells displayed a significantly higher disease score starting on day 17 through the remaining days of the study (Fig. 4A). The weight loss metric was the highest fraction of the score, so mouse weight was assessed separately and found significantly lower than either the pMig-activated control T cells or the TCD-BM only control throughout the length of the study (Fig. 4B). Despite the observed weight changes, none of the treatment groups, including pCM8 infused mice, experienced mortalities through the length of the study (Fig. 4C). To determine whether this effect was correlated with T cell expansion, some mice were bled on day 7 to determine their immune profile. In the pCM8 T cell–infused mice, we found that live CD8+ T cells made up a significantly higher proportion of the donor immune cells in the blood, indicating increased expansion of these cells (Fig. 4D). This expansion came at the cost of donor myeloid cells in the blood, which were significantly decreased in mice treated with the CD8+ T cells transduced with pCM8 (Fig. 4E). When comparing the myeloid to T cells fractionally, there were on average three times more myeloid cells than T cells in the mice treated with the pMig control group compared with the pCM8 group (Fig. 4F). We also examined activation status of these cells by staining for the high-affinity IL-2Rα (CD25). We found that mice treated with CD8+ T cells transduced with the pCM8 construct had donor CD8+ T cells with ∼2.5 times more expression of CD25 compared with the pMig-GFP control CD8+ T cells (Fig. 4G). Additionally, mice treated with the pCM8-transduced CD8+ T cells had significantly lower TIM-3+ PD-1+ double-positive donor CD8+ T cells. This was due to a striking decrease in TIM-3 expression in these cells (Fig. 4H). Overall, we conclude that, although MyD88 costimulation in donor CD8+ T cells increases the GVT effect, it also impacts GvHD as correlated with increased T cell expansion and a more activated phenotype.

FIGURE 4.

MyD88-costimulated expansion of donor CD8+ T cells correlates with GvHD-related weight loss. BALB/c host mice were subjected to 9.5-Gy TBI, injected with 3.5 × 106 TCD-BM from C57BL/6 mice, and injected with 1 × 106 of indicated CD8+ T cell group in the absence of tumor (n = 5 per group). (A) Disease score was assessed on a scale by measuring and adding mouse activity (0–2), fur texture (0–2), posture (0–2), diarrhea (0–2), skin (0–2), and weight (0–4) in each animal. (B) Percentage weight change was assessed separately, and (C) survival was tracked during the length of the experiment. Furthermore, live retro-orbital bleeds were used on day 7 to assess (D) donor CD8+ T cells, (E) donor myeloid cells, and (F) their ratios. Donor (H-2kb) CD8+ T cells were analyzed for (G) CD25 expression by measuring mean fluorescence intensity (MFI). (H) PD-1 and TIM-3 positivity was also assessed on donor CD8+ T cells. This experiment is representative of two independently replicated experiments with similar results. Representative results are shown as mean ± SEM and significant differences of disease score and weight between groups over time was ascertained using ANOVA test. The p values and significance indications were acquired by Student t tests (ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 4.

MyD88-costimulated expansion of donor CD8+ T cells correlates with GvHD-related weight loss. BALB/c host mice were subjected to 9.5-Gy TBI, injected with 3.5 × 106 TCD-BM from C57BL/6 mice, and injected with 1 × 106 of indicated CD8+ T cell group in the absence of tumor (n = 5 per group). (A) Disease score was assessed on a scale by measuring and adding mouse activity (0–2), fur texture (0–2), posture (0–2), diarrhea (0–2), skin (0–2), and weight (0–4) in each animal. (B) Percentage weight change was assessed separately, and (C) survival was tracked during the length of the experiment. Furthermore, live retro-orbital bleeds were used on day 7 to assess (D) donor CD8+ T cells, (E) donor myeloid cells, and (F) their ratios. Donor (H-2kb) CD8+ T cells were analyzed for (G) CD25 expression by measuring mean fluorescence intensity (MFI). (H) PD-1 and TIM-3 positivity was also assessed on donor CD8+ T cells. This experiment is representative of two independently replicated experiments with similar results. Representative results are shown as mean ± SEM and significant differences of disease score and weight between groups over time was ascertained using ANOVA test. The p values and significance indications were acquired by Student t tests (ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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We next analyzed the effect of increasing doses of CD8:MyD88 T cells on GvHD, coupled with histopathological analysis, to determine the source of GvHD-related weight loss. Like the previous experiment, both pCM8-transduced T cell doses trended toward higher disease scores than either doses of the pMig control T cells or the TCD-BM only control (Fig. 5A). These mice also trended toward lower body weight, starting on day 22 through the remaining days of the study (Fig. 5B). However, the effects of the pCM8-transduced T cells did not show a dose-dependent increase in disease score or weight loss. Despite the observed weight changes, none of the treatment groups experienced mortalities through the length of the study (Fig. 5C). On day 55, the mice were euthanized, the tissues were fixed in formalin and sent for processing into H&E slides. Tissue slides were blind scored for 17 metrics in the intestinal tissues and 19 metrics in the liver tissues. Minor increases in score were noted in the large intestine of both the 2 × 106 and 5 × 106 doses of the pCM8 T cell infusion (Fig. 5D). The metrics with the greatest change in the pCM8 groups included increased lamina propria inflammation, increased lymphocyte infiltration, and increased apoptosis (Fig. 5E). Together, these results suggest that pCM8-transduced T cells can cause increased but nonlethal GvHD.

FIGURE 5.

MyD88-costimulated donor CD8+ T cells induce clinical, histopathological but nonlethal GvHD. BALB/c host mice were subjected to 9.5-Gy TBI injected with 3.5 × 106 TCD-BM from C57BL/6 mice and injected with 2 × 106 or 5 × 106 of the indicated CD8+ T cell group in the absence of tumor (n = 4 per group). (A) Disease score was assessed as described in Fig. 3A. (B) Percentage weight change was assessed separately as the strongest indicator of systemic GvHD. Disease score and weight are shown as mean ± SEM, and significance between groups over time was ascertained using ANOVA test. (C) Survival was tracked until experiment termination on day 55. Furthermore, the mice were euthanized, and liver and large and small intestines were fixed in 10% formalin for processing into H&E-stained slides. Histopathological GvHD was scored blind by a trained pathologist and (D) summarized. The mean summarized score ± SD is indicated on the graph with score maximums represented by a dashed line and Student t test used for statistical comparisons. (E) Representative images are displayed for the large intestine. Colored arrows indicate the score indicators of GvHD, including apoptosis (red arrow), intraepithelial lymphocytic infiltrates (blue arrow), and lamina propria inflammation (green arrow).

FIGURE 5.

MyD88-costimulated donor CD8+ T cells induce clinical, histopathological but nonlethal GvHD. BALB/c host mice were subjected to 9.5-Gy TBI injected with 3.5 × 106 TCD-BM from C57BL/6 mice and injected with 2 × 106 or 5 × 106 of the indicated CD8+ T cell group in the absence of tumor (n = 4 per group). (A) Disease score was assessed as described in Fig. 3A. (B) Percentage weight change was assessed separately as the strongest indicator of systemic GvHD. Disease score and weight are shown as mean ± SEM, and significance between groups over time was ascertained using ANOVA test. (C) Survival was tracked until experiment termination on day 55. Furthermore, the mice were euthanized, and liver and large and small intestines were fixed in 10% formalin for processing into H&E-stained slides. Histopathological GvHD was scored blind by a trained pathologist and (D) summarized. The mean summarized score ± SD is indicated on the graph with score maximums represented by a dashed line and Student t test used for statistical comparisons. (E) Representative images are displayed for the large intestine. Colored arrows indicate the score indicators of GvHD, including apoptosis (red arrow), intraepithelial lymphocytic infiltrates (blue arrow), and lamina propria inflammation (green arrow).

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To produce a GVT response, lymphocytes must be activated and expand in response to host APC (30). To investigate whether MyD88-costimulated donor CD8+ T cells expand in vivo after allo-HCT, T cell proliferative capacity was assessed on day 4 posttransplant. T cell transduction was assessed before injection (Fig. 6A). The spleens of the mice were dissociated and stained for flow cytometric analysis (Fig. 6B). Donor CD8+ T cells were analyzed for Ki67, a graded marker indicative of cell proliferation that increases as the cells move toward mitosis (31). Residual host CD8+ cells were also analyzed as an internal control and displayed low Ki67 expression (Fig. 6C). The pCM8-transduced donor CD8+ T cells had significantly higher Ki67 expression compared with either the pMig control or the naive CD8+ control (Fig. 6D). When comparing the transduced GFP+ CD8+ donor T cells between the pMig control T cell and pCM8 T cell groups, the differences were more apparent (Fig. 6E). When analyzing the frequency of donor CD8+ T cells in the spleens of these mice over time, the pCM8 group remained higher at the earlier timepoints, indicating early expansion of these cells (Fig. 6F). We also noted that on day 12 there was a contraction of donor T cells from both groups (Fig. 6F). To see whether these populations were maintained in the circulation, blood samples from these mice were analyzed. We found that mice treated with pCM8-transduced CD8+ T cells had significantly more donor T cells within the blood at day 12 compared with the pMig control (Fig. 6G). In contrast, the inguinal lymph nodes collected from these mice also displayed a drop-off in donor CD8+ T cell populations, mimicking what we saw in the spleen (Fig. 6H).

FIGURE 6.

MyD88 costimulation enhances proliferation of donor CD8+ T cells in vivo. (A) CD8+ T cells were sorted and transduced with pMig control or pCM8 experimental constructs, and (right) efficiency was measured. BALB/c host mice underwent TBI and then were injected with 3.5 × 106 TCD-BM from C57BL/6 mice, 0.5 × 106 A20-luciferase, and 0.5 × 106 of the indicated CD8+ T cells. On day 4 postinjection, mice were euthanized, and their spleens were harvested for cell isolation, staining, and analysis. (B) Donor and residual host CD8+ T cells were gated as shown. (C) Quiescent residual host CD8+ cells were analyzed for Ki67 as a baseline intrinsic control. (D) Ki67 was measured in all donor CD8+ T cells and (E) between GFP-positive (transduction) cells only. (F) Donor CD8+ T cell expansion was measured in the spleen of these mice at multiple timepoints. (G) Blood and (H) inguinal lymph nodes were taken at day 6 and 12 posttransplant in the presence of A20 tumor. Donor CD8+ populations were assessed to determine whether these cells enter circulation or are retained in the secondary lymphatics. This experiment is representative of two independently replicated experiments with similar results shown using mean ± SD. The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01).

FIGURE 6.

MyD88 costimulation enhances proliferation of donor CD8+ T cells in vivo. (A) CD8+ T cells were sorted and transduced with pMig control or pCM8 experimental constructs, and (right) efficiency was measured. BALB/c host mice underwent TBI and then were injected with 3.5 × 106 TCD-BM from C57BL/6 mice, 0.5 × 106 A20-luciferase, and 0.5 × 106 of the indicated CD8+ T cells. On day 4 postinjection, mice were euthanized, and their spleens were harvested for cell isolation, staining, and analysis. (B) Donor and residual host CD8+ T cells were gated as shown. (C) Quiescent residual host CD8+ cells were analyzed for Ki67 as a baseline intrinsic control. (D) Ki67 was measured in all donor CD8+ T cells and (E) between GFP-positive (transduction) cells only. (F) Donor CD8+ T cell expansion was measured in the spleen of these mice at multiple timepoints. (G) Blood and (H) inguinal lymph nodes were taken at day 6 and 12 posttransplant in the presence of A20 tumor. Donor CD8+ populations were assessed to determine whether these cells enter circulation or are retained in the secondary lymphatics. This experiment is representative of two independently replicated experiments with similar results shown using mean ± SD. The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01).

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To test survival of donor CD8+ T cells, a modified MLR was used, combining variable ratios of transduced donor CD8+ T cells (H-2b), activated BMDC 1:10 (H-2d), and constant A20 tumor (H-2d). This culture models the allogeneic host cell types in vivo and allows us to analyze how these populations impact the survival of CD8+ T cells with and without MyD88 costimulus. At each time point we used annexin V/7-AAD to see the fraction of T cells dead (7-AAD+/annexin V+) or initiating apoptosis (7-AAD/annexin V+) at 24 or 48 h (Fig. 7A). Twenty-four hours after culture, we found that the pMig control T cells had a significantly increased frequency of dead cells at every ratio compared with the pCM8-transduced CD8+ T cells (Fig. 7B). Additionally, at 24 h, there were significantly more early apoptotic cells in the pMig control cultures at the 10:1 and 1:1 ratios, indicating better survival of the pCM8-transduced CD8+ T cells, even when tumor is increased (Fig. 7C). At 48 h the results were similar with increased death of pMig controls compared with the transduced pCM8 CD8+ T cell cultures (Fig. 7D). Early apoptosis of the pMig control was also significantly higher than the transduced pCM8 CD8+ T cells after 48 h (Fig. 7E).

FIGURE 7.

MyD88 costimulation enhances survival of allogeneic CD8+ T cells. (A) Top, Transduced T cells (H-2b) were cocultured with constant A20 tumor (H-2d) and activated BMDC (H-2d) 1:10 to T cells. Bottom, These cells were stained with phenotypic markers, and annexin V/7-AAD was added to assess death and survival of these cells. Dead cells (annexin V+ 7-AAD+) at (B) 24 h and (D) 48 h and apoptotic cells (annexin V+ 7-AAD) at (C) 24 h and (E) 48 h were analyzed on CD8+ T cells. This experiment is representative of one of two independently replicated experiments with similar results shown using mean ± SD (in triplicate). The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 7.

MyD88 costimulation enhances survival of allogeneic CD8+ T cells. (A) Top, Transduced T cells (H-2b) were cocultured with constant A20 tumor (H-2d) and activated BMDC (H-2d) 1:10 to T cells. Bottom, These cells were stained with phenotypic markers, and annexin V/7-AAD was added to assess death and survival of these cells. Dead cells (annexin V+ 7-AAD+) at (B) 24 h and (D) 48 h and apoptotic cells (annexin V+ 7-AAD) at (C) 24 h and (E) 48 h were analyzed on CD8+ T cells. This experiment is representative of one of two independently replicated experiments with similar results shown using mean ± SD (in triplicate). The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01, ***p < 0.001).

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Together, these data indicate that the MyD88-costimulated donor CD8+ T cells have an increased capacity for proliferation and survival in the allogeneic setting.

To determine the tumor control mechanisms of MyD88-costimulated donor CD8+ T cells, CD8+ T cells from mice were characterized 4 d after allo-HCT. Mouse spleens were extracted and stained for the cytokines IFN-γ and TNF-α (Fig. 8A). We were able to detect significantly increased double-positive donor CD8+ T cells in mice treated with CD8α:MyD88 donor T cells (Fig. 8B). This increase in multifunctional CD8+ T cells was weighted by a significant increase in TNF-α–positive cells (Fig. 8C) and a trending increase in IFN-γ (Fig. 8D). We also stained the cells for granzyme B and CD107a to determine their potential for killing with granzyme B and assess their secretory capacity (Fig. 8E). Donor CD8+ T cells transduced with CD8α:MyD88 had three times more granzyme B–positive cells than pMig control or naive CD8 control T cells (Fig. 8F). This group also displayed two times more granzyme B+ CD107a+ cells than controls (Fig. 8G) and had twice the total granzyme B expression of control T cell groups (Fig. 8H). Together, these data indicate that MyD88-costimulated donor CD8+ T cells have enhanced functions for cytotoxicity.

FIGURE 8.

MyD88 costimulation enhances multimodal function of donor CD8+ T cells. BALB/c host mice were conditioned with TBI (9.5 Gy), then they were injected with 3.5 × 106 TCD-BM from C57BL/6 mice, 0.5 × 106 A20-luciferase, and 1 × 106 of the indicated T cell infusion (TCD-BM, n = 3; pMig, n = 4; pCM8, n = 4). On day 4 postinjection, mice were euthanized, and their spleens were harvested for cell isolation, staining, and analysis. Donor CD8+ T cells were gated on from live → singlet → H-2kb+ populations. (A) These cells were analyzed for IFN-γ and TNF-α (B) together as double-positive cells and individual (C) TNF-α and (D) IFN-γ single-positive populations. (E) Additionally, donor CD8+ T cells were examined for expression of granzyme B and CD107a and assessed for (F) granzyme B positivity, (G) secretory CD107a+ granzyme B+, and (H) granzyme B mean fluorescence intensity (MFI). Results shown are mean ± SD and representative from one of two independently replicated experiments. The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 8.

MyD88 costimulation enhances multimodal function of donor CD8+ T cells. BALB/c host mice were conditioned with TBI (9.5 Gy), then they were injected with 3.5 × 106 TCD-BM from C57BL/6 mice, 0.5 × 106 A20-luciferase, and 1 × 106 of the indicated T cell infusion (TCD-BM, n = 3; pMig, n = 4; pCM8, n = 4). On day 4 postinjection, mice were euthanized, and their spleens were harvested for cell isolation, staining, and analysis. Donor CD8+ T cells were gated on from live → singlet → H-2kb+ populations. (A) These cells were analyzed for IFN-γ and TNF-α (B) together as double-positive cells and individual (C) TNF-α and (D) IFN-γ single-positive populations. (E) Additionally, donor CD8+ T cells were examined for expression of granzyme B and CD107a and assessed for (F) granzyme B positivity, (G) secretory CD107a+ granzyme B+, and (H) granzyme B mean fluorescence intensity (MFI). Results shown are mean ± SD and representative from one of two independently replicated experiments. The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01, ***p < 0.001).

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To determine whether MyD88-stimulated control of tumor is through a direct killing mechanism, we used our modified MLR system. Again, CD8+ T cells were transduced with equivalent efficiency (Fig. 9A). Transduced CD8+ T cells (H-2b) were cocultured with 1:10 activated BMDC (H-2d) and a constant 1 × 104 A20 lymphoma (H-2d). Different ratios of transduced CD8+ T cells were added to the cocultures to assess killing and tumor control caused by the T cells. At each timepoint, cells were stained for surface markers, annexin V, and 7-AAD, and data were immediately acquired. Tumor cells were analyzed for death and early apoptosis within the population. Additionally, live tumor cells of the total cell population were normalized to the control and used to assess percentage tumor growth and obtain an IC50 to measure and compare T cell efficacy. At 24 h, we observed a significant increase in dead tumor cells (Fig. 9B) and early apoptotic tumor cells (Fig. 9C) in samples treated with CD8α:MyD88 T cells. At 24 h, significant differences were observed at higher T cell/tumor ratios of 20:1 and 10:1. When assessing how CD8α:MyD88 T cells impacted tumor growth, we found a significant decrease in growth at each ratio (Fig. 9D). After 48 h of coculture, we found that the samples containing the pCM8-transduced CD8+ T cells had significantly increased dead tumor cells across all ratios (Fig. 9E), although the early apoptotic percentages were overall decreased compared with 24 h (Fig. 9F). When analyzing the impact of CD8α:MyD88–transduced CD8+ T cells on percentage tumor growth after 48 h, we found the IC50 was still under one T cell per tumor cell (0.49), whereas the pMig IC50 was 2.77 T cells per tumor cell for 50% control of tumor growth (Fig. 9G). Tumor growth across all ratios was significantly less in the CD8α:MyD88 T cell group (Fig. 9G). Most striking was the 1:1 ratio that was under 50% tumor cell growth in the CD8α:MyD88 T cell group, whereas the GFP control group had tumor growth almost equal to that of the tumor-only control (e.g., 100%) at the same ratio (Fig. 9G). Together, these data indicate that MyD88-costimulated donor CD8+ T cells directly kill tumor cells and do so more efficiently than the transduced GFP control T cells.

FIGURE 9.

Tumor control by MyD88-costimulation donor CD8+ T cells is the result of direct killing. (A) Left, Donor CD8+ T cells were sorted, activated, and transduced with either pMig-GFP control vector (pMIG) or pMIG-CD8α:MyD88 vector (pCM8). Various T cell concentrations were than cocultured with a constant number (1 × 104) of A20 lymphoma+/− H2kd BMDCs kept at 1:10 ratio (BMDC/T cells). Right, Tumor cells were analyzed by gating on singlet B220+ cells and further on 7-AAD viability dye by annexin V apoptosis indicator. Tumor populations were separated by dead tumor cells at (B) 24 h and (E) 48 h, early apoptotic tumor cells at (C) 24 h and (F) 48 h, and logistic tumor growth curves at (D) 24 h and (G) 48 h. Experiments were performed in triplicate and independently replicated two times with results as mean ± SD. IC50 values were obtained from logistic growth curve analysis in GraphPad Prism software. The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 9.

Tumor control by MyD88-costimulation donor CD8+ T cells is the result of direct killing. (A) Left, Donor CD8+ T cells were sorted, activated, and transduced with either pMig-GFP control vector (pMIG) or pMIG-CD8α:MyD88 vector (pCM8). Various T cell concentrations were than cocultured with a constant number (1 × 104) of A20 lymphoma+/− H2kd BMDCs kept at 1:10 ratio (BMDC/T cells). Right, Tumor cells were analyzed by gating on singlet B220+ cells and further on 7-AAD viability dye by annexin V apoptosis indicator. Tumor populations were separated by dead tumor cells at (B) 24 h and (E) 48 h, early apoptotic tumor cells at (C) 24 h and (F) 48 h, and logistic tumor growth curves at (D) 24 h and (G) 48 h. Experiments were performed in triplicate and independently replicated two times with results as mean ± SD. IC50 values were obtained from logistic growth curve analysis in GraphPad Prism software. The p values and significance indications were acquired by Student t tests (*p < 0.05, **p < 0.01, ***p < 0.001).

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This study presents a strategy of donor T cell engineering and infusion for allo-HCT, in which fusion protein CD8α:MyD88 activates MyD88 signaling in donor CD8+ T cells without the need for systemic administration of a ligand or cytokine. This approach resulted in an enhanced GVT effect. To the best of our knowledge, this is the first strategy designed to boost the GVT response through molecular engineering of MyD88 for donor T cell infusion. In some leukemias, DLIs often fail to clear tumor, and patients often succumb to their disease. For example, one study found that patients with adult lymphoid malignancies had an overall survival of only 13% in the 3 years after receiving a DLI (32). Our results provide proof of concept that synthetic engineering of CD8α:MyD88 may improve the effectiveness of DLI in the setting of allo-HCT. However, because of the link between GVT and GvHD, it was rational to predict that enhanced MyD88 in donor CD8+ T cells may elicit some GvHD symptoms in these mice. Our results indicated that mice treated with CD8α:MyD88 T cells showed significantly increased weight loss, yet these mice did not display other severe GvHD-related side effects and survived throughout the course of the study. When we escalated the dose of administered T cells, we found there was no dose-dependent increase in GvHD-related symptoms. Upon histopathological analysis, we found that weight loss was associated with minor increases in lamina propria inflammation, intraepithelial lymphocyte infiltration, and apoptosis occurring in the large intestine. However, these mice survived throughout the course of the study, even at the highest dose, indicating MyD88 costimulation induces increased but nonlethal GvHD in mice.

From our previous study, we can conclude that the CD8α domain of the construct enhances activation of MyD88 in conjunction with activation of TCR (17). Only when these cells underwent Ag-specific activation was MyD88 found aggregating at or proximal to the immunological synapse, indicating that the CD8α domain of the construct promotes MyD88 aggregation during TCR activation (17). In this study, the TCR activation signal would come from allogeneic MHC stimulation by host dendritic cells and tumor cells. Our CD8α:MyD88 and GFP control T cells are already activated going into the host mouse; however, not all T cells respond to allogeneic stimulation. Host dendritic cells and tumor are likely to play a role in further stimulating and shaping the responding population of T cells. We found that CD8α:MyD88 T cells displayed enhanced capacity to proliferate in the allo-HCT system. Ki67 is a graded marker that increases as cells move toward S phase, G2 phase, and peaks at M phase (31). In addition to showing an increased proliferative capacity, CD8α:MyD88 donor CD8+ T cells expanded early in secondary lymphatics, where they would see residual host APCs. This was followed by a contraction of all donor T cell populations by day 12. Systemically, MyD88-costimulated CD8+ T cells maintained a larger presence in the blood through day 12 posttransplant, indicating systemic retention of these cells. The contraction of these cells in secondary lymphatics is important to make room for reconstitution of other immune cells, whereas retention in the blood shows these cells could still be active in eliminating tumor cells. Ultimately, the proliferation profile, expansion, and notable increase in survival of the CD8α:MyD88 T cells further mimic treatment of T cells with TLR ligand but without systemic off-target effects (33).

TLR2 agonists in CD8+ T cells increase T-bet and its binding to granzyme B, perforin, and IFN-γ promoters (34). In previous studies using our CD8α:MyD88 construct in pmel-specific CD8+ T cells, we also found a notable increase in IFN-γ and other cytokines (17). In the current study, we measured several functional cytokines in the donor CD8+ T cells after isolating them from the spleens of these mice on day 4 posttransplant. We were able to detect increased multiple cytokine-producing donor CD8+ T cells. There were also more granzyme B–positive donor CD8+ T cells, and these cells coexpressed the secretory marker CD107a. The increase in granzyme B and CD107a double-positive donor T cells in the mice treated with CD8α:MyD88 T cells could indicate an increased expansion of allogeneic responders. We know that this construct can produce a more sensitive MHC and TCR interaction (17); thus, it is likely that synthetic CD8α:MyD88 increases the reactivity of donor CD8+ T cells to host Ags and tumor Ags in this allo-HCT system. The observed increase in T cell functions led us to test whether MyD88 costimulation in T cells elicits direct killing in target tumor cells. We found that lymphoma cells cocultured with CD8α:MyD88 T cells had significantly more apoptotic and dead cells down to the 1:1 T cell/tumor ratio and at each timepoint. Conversely, pMig control T cells could not control tumor growth. This occurred in a T cell dose-dependent manner, indicating the T cells were the dominant parameter modulating tumor death.

The biological role of MyD88 in T cells in the context of experimental transplant has been controversial. Most studies analyzing MyD88 use a knockout approach. One study showed that MyD88 is indispensable for effective GVT (18), complementing other studies that found that Ag-specific CD8+ T cells lacking MyD88 are partially defective and have impaired responses to lymphocytic choriomeningitis virus (35, 36). In contrast, another study concluded that MyD88 ablation reduces GvHD while maintaining GVT effects (37). In both studies, pan T cells, including CD4+ T cells, were used. Our previous studies (38, 39) have shown that although CD4+ T cells contribute to the GVT effect, a small number (5 × 104) of CD4+ T cells could cause hyperacute lethal GvHD between 1 and 2 wk after transplant in this model system. Because of this concern, we have devised this CD8α:MyD88 construct to selectively enhance CD8+ T cell function as a strategy to improve the GVT effect without causing severe and lethal GvHD. Although we did see an increase in GvHD symptoms of the gut with our CD8α:MyD88-modified CD8+ T cells, we did not observe any lethality, even at a 5 × 106 CD8+ T cell dose. Therefore, our study would support a new strategy of engineering human CD8+ T cells for DLI. Currently, DLI in the clinic does not separate CD4+ and CD8+ lymphocytes, so the use of this construct would require a different protocol from the current clinical procedure. Further modifications to this construct (e.g., inducible suicide genes, tumor-specific TCRs) may increase safety, specificity, and efficacy in the clinical setting.

To summarize, synthetic engineering of MyD88 costimulation into donor CD8+ T cells represents a unique and versatile approach to enhance the GVT response. We found these cells were more activated and primed to kill tumor cells in the setting of allo-HCT. Overall, our approach provides proof of concept for engineering donor T cell infusions to improve their efficacy.

We thank the University of Maryland School of Medicine Center for Innovative Biomedical Resources, Flow Cytometry Core and Translational Research in Imaging at Maryland, University of Maryland School of Medicine, Baltimore, MD.

This work was supported by grants from the National Cancer Institute (R01CA184728 to X.C. and R01CA207913 to E.D.).

Abbreviations used in this article:

7-AAD

7-aminoactinomycin D

allo-HCT

allogeneic hematopoietic cell transplantation

BMDC

bone marrow–derived dendritic cell

DLI

donor lymphocyte infusion

GvHD

graft-versus-host disease

GVT

graft-versus-tumor

MS

median survival

nCD8

naive CD8+ T cell

TBI

total body irradiation

TCD-BM

T cell–depleted bone marrow

WT

wild-type.

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E.D. has shares in a company that is developing products related to the research being reported. The other authors have no financial conflicts of interest.