Patients with glioblastoma (GBM) are treated with radiotherapy (RT) and temozolomide (TMZ). These treatments may cause prolonged systemic lymphopenia, which itself is associated with poor outcomes. NT-I7 is a long-acting IL7 that expands CD4 and CD8 T-cell numbers in humans and mice. We tested whether NT-I7 prevents systemic lymphopenia and improves survival in mouse models of GBM.
C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day × 5 days), TMZ (33 mg/kg/day × 5 days), and/or NT-I7 (10 mg/kg on the final day of RT). We followed the mice for survival while serially analyzing levels of circulating T lymphocytes. We assessed regulatory T cells (Treg) and cytotoxic T lymphocytes in the tumor microenvironment, cervical lymph nodes, spleen, and thymus, and hematopoietic stem and progenitor cells in the bone marrow.
GBM tumor–bearing mice treated with RT+NT-I7 increased T lymphocytes in the lymph nodes, thymus, and spleen, enhanced IFNγ production, and decreased Tregs in the tumor which was associated with a significant increase in survival. NT-I7 also enhanced central memory and effector memory CD8 T cells in lymphoid organs and tumor. Depleting CD8 T cells abrogated the effects of NT-I7. Furthermore, NT-I7 treatment decreased progenitor cells in the bone marrow.
In orthotopic glioma-bearing mice, NT-I7 mitigates RT-related lymphopenia, increases cytotoxic CD8 T lymphocytes systemically and in the tumor, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).
Radiotherapy (RT) alone or in combination with temozolomide (TMZ) can induce iatrogenic immunosuppression in patients with glioblastoma (GBM), termed treatment-related lymphopenia (TRL). TRL occurs in 40% of patients with GBM and is associated with worse survival. IL7 is required for human T-cell development and homeostasis. In this study, we found that NT-I7, a long-acting recombinant human IL7, combined with RT improved survival in tumor-bearing mice. NT-I7 enhanced CD8 T cells in peripheral blood and secondary lymphoid tissues such as the thymus, spleen, and lymph nodes. NT-I7 improved CD8 T-cell infiltration in the tumor microenvironment and was associated with a relative decrease in the number of regulatory T cells. NT-I7 combined with RT augmented tumor immunity and improved survival. A phase I/II trial evaluating long-acting IL7 in patients with high-grade gliomas is ongoing (NCT03687957).
Glioblastoma (GBM) is the most aggressive primary brain tumor, with a 5-year overall survival of less than 10% (1). Most patients with GBM receive radiotherapy (RT) and temozolomide (TMZ). These treatments can induce severe prolonged systemic lymphopenia. Systemic lymphopenia is associated with shorter survival not only in GBM (2–4), but also sarcomas and cancers of the lung, colon, pancreas, breast, and rectum (2, 5–7). In brain tumors, irradiated brain volume, concurrent TMZ, and overall corticosteroid exposure are all contributing factors to systemic lymphopenia (8). GBM itself can induce T-cell sequestration within the bone marrow which might exacerbate lymphopenia (9). However, the biological mechanisms of lymphopenia in GBM and more importantly its association with poor survival are not clearly understood.
Previously, we have shown that lymphopenia is associated with inappropriately low levels of IL7 (10). IL7 is a 25-kDa glycoprotein that is required for human T-cell development and maintaining the homeostasis of mature T cells (11). IL7 is the main homeostatic driver of T-cell numbers (12). IL7 signals through the IL7 receptor (IL7R) and the downstream JAK-STAT pathway (11). The IL7R consists of a common cytokine receptor γ-chain (γc) and a unique IL7R α-chain (IL7Rα). The IL7R is first expressed on common lymphoid progenitor (CLP) cells in the bone marrow during lymphocyte development (13). IL7 increases T-cell numbers by (i) enhancing their proliferation and maturation in the thymus, (ii) boosting proliferation of precursors/progenitors in the bone marrow, and (iii) preventing T-cell apoptosis (14, 15). The human gene for IL7 resides on chromosome 8q12–13 and shares 81% homology with its murine ortholog.
NT-I7 (efineptakin alfa) is a novel long-acting homodimeric recombinant human IL7, which expands CD8 T cells with enhanced tumor tropism in murine tumor models (16) and has demonstrated a significant increase in T lymphocytes without safety concerns in healthy human subjects (17). Given the critical role of IL7 on lymphocyte expansion, we decided to test whether NT-I7 can correct treatment-related lymphopenia and thereby improve survival in GBM mouse models.
Materials and Methods
Cell culture and reagents
GL261 (NCI) and CT2A murine glioma cells (18), transduced with luciferase, were confirmed mycoplasma-free by PCR. Cells were maintained in DMEM (Gibco), supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). NT-I7 is a long-acting Ig fusion protein composed of a recombinant form of the endogenous human protein IL7 (rhIL7), fused to a hybrid Fc region of a human antibody (NeoImmuneTech, Inc.).
Animals, intracranial glioma implantation, and bioluminescence imaging
Six- to 8-week-old female C57BL/6 mice were obtained from Charles River and maintained under pathogen-free conditions at Washington University School of Medicine. All studies involving mice were performed per the guidelines of the Institutional Animal Care and Use Committee. Protocols were approved by the Washington University Division of Comparative Medicine.
Prior to implantation, 70% confluent cells were trypsinized, washed, and prepared as a single cell suspension. After mice were anesthetized with an intraperitoneal injection of xylazine (80 mg/kg) and ketamine (10 mg/kg), 4 × 104 cells in 3 μL were stereotactically injected into the right frontal cortex/striatum over 5 minutes. To confirm tumor engraftment and growth, approximately 1 week after tumor implantation, bioluminescence imaging (BLI) was performed using a Xenogen IVIS-100 system. Animals were injected with D-luciferin (150 mg/kg) in 100 μL of phosphate buffer saline intraperitoneally and kept in a light-tight imaging chamber under 2% isoflurane anesthesia after 10 minutes of injection. Images were acquired and analyzed using Living Image software (Perkin Elmer). Following BLI, mice were serpentine sorted such that each treatment group had similar averages of the tumor.
RT and treatments
Mice were irradiated with 1.8 Gy/day to the head by shielding the rest of the body with a lead shield. TMZ (Sigma-Aldrich) powder was reconstituted in dimethyl sulfoxide at a concentration of 50 mg/mL, aliquoted, and stored at −20°C. Mice were treated with an intraperitoneal injection of TMZ (34 mg/kg) for 5 consecutive days, 45 minutes prior to RT. Recombinant human IL7 (NT-I7; 10 mg/kg) was injected subcutaenously within 30 minutes after the final RT administration.
CD8 T-cell depletion
Anti-CD8 (clone 2.43, cat no. BE0061, Bio X Cell) and corresponding isotype control (clone LTF-2, cat no. BE0090, Bio X Cell) were injected intraperitoneally in tumor-bearing mice (±RT). The anti–CD8-treated doses were 500 μg/mice on the first day and following 250 μg/mice every 4 days (total five doses). The antibody was injected after 2 hours of RT. Depletion was confirmed by flow cytometry to quantify CD8 populations in peripheral blood. All mice in survival studies were followed until the development of severe neurologic morbidity or death.
Organ processing for flow cytometry
Three hundred microliter of blood was withdrawn from the jugular vein and collected into an EDTA-coated tube prior to euthanasia. Peripheral blood mononuclear cells were isolated by density centrifugation (400 × g for 30 minutes) using histopaque-1083 (Sigma). Single cells were resuspended into freshly prepared FACS buffer (1X PBS and 0.5% BSA) for further antibody staining.
Brain, spleen, lymph nodes, thymus, and bone marrow
Mouse brains were harvested and prepared as single-cell suspension as described previously (19). Spleen, lymph nodes, and thymus were crushed on a 70 μm strainer with a syringe plunger into DMEM. Cells were pelleted (400 × g, 10 minutes, 4C), lysed with ACK buffer (Thermo-Fisher), washed twice, and finally resuspended in FACS buffer. Similarly, bone marrow cells were isolated by flushing the femur and tibia in DMEM, followed by treatment with ACK buffer, washed twice, and resuspended in FACS buffer for further antibody staining.
Flow cytometry and antibodies
For extracellular surface staining, dead cells were depleted using the Dead Cell Removal Kit (Miltenyi Biotec) and washed with FACS buffer. Cells were incubated with antibodies for 1 hour at 4°C and washed twice with FACS buffer. The following antibodies were used for T-cell subpopulation staining: phycoerythrin (PE)-conjugated anti-mouse CD3 (145-2C11; BD Biosciences), APC-Cy7–conjugated anti-mouse CD4 (RM5-5; BD Biosciences), Brilliant violet 421–conjugated anti-mouse CD4 (GK1.5, BioLegend), PerCP-conjugated anti-mouse CD8 (53-6.7; BD Biosciences), PE-Cy7–conjugated anti-mouse CD62 L (MEL-14; BD Biosciences), and APC-conjugated anti-mouse CD44 (IM7; BD Biosciences). After surface staining, cells were fixed and permeabilized using Fixation/Permeabilization (BD) solution and stained with Alexa-fluor488–conjugated anti-mouse FoxP3 (R16-715; BD Biosciences). For exhausted and functional T-cell profiling, FITC-conjugated anti-mouse CD279/PD-1 (29F.1A12, BioLegend) was used along with the above-mentioned CD3, CD4, and CD8 antibodies. Then, APC-conjugated anti-mouse IFNγ (XMG1.2, BioLegend) was used for intracellular staining.
The following antibodies were used to stain bone marrow hematopoietic stem and progenitor cells: APC-Cy7–conjugated anti-mouse CD11b, CD45R/B220, Ly6G/Ly6C, CD3e, TER-119/Erythroid cells (M1/70, RA3-6B2, RB6-8C5145-2C11, and TER-119; BioLegend), PE-conjugated anti-mouse CD117 (2B8; BD Biosciences), PE-Cy7–conjugated anti-mouse Ly6A/E (D7; BD Biosciences), FITC-conjugated anti-mouse CD34 (RAM34; BD Biosciences), APC-conjugated anti-mouse CD135 (A2F10.1; BD Biosciences), BV421-conjugated anti-mouse CD16/32 (2.4G2; BD Biosciences), and FITC-conjugated anti-mouse CD127 (A7R34, BioLegend). Data were acquired using a MACSQuant Analyzer 10 instrument (Miltenyi Biotec) and analyzed with FlowJo (v10.6.1) software.
For all the studies, 5 to 15 mice were used per treatment group. The data were expressed as mean ± SD. Statistical differences between more than two groups were determined by ANOVA with Tukey multiple comparisons test. Statistics were performed in GraphPad Prism version 8 (GraphPad Software, Inc.). A P value less than 0.05 was considered significant and indicated in each graph where applicable or otherwise mentioned in Supplementary Tables.
NT-I7 treatment is associated with enhanced survival and partially rescues T cells after chemoradiation
To determine the effects of NT-I7 on survival and treatment-induced lymphopenia in glioma-bearing mice, we used a syngeneic murine glioma model (GL261). After confirmation of tumor engraftment and growth with BLI, mice with orthotopic GL261 tumors were treated with NT-I7 (10 mg/kg) alone; RT combined with TMZ (RT+TMZ; TMZ 33 mg/kg, 45 minutes before each irradiation treatment); triple combination therapy with RT, TMZ and NT-I7 (RT+TMZ+NT-I7; NT-I7 at 10 mg/kg, 45 minutes after final irradiation); or no treatment as control (Fig. 1A). We monitored survival for up to 90 days. RT+NT-I7+TMZ had significantly better median survival (86 days) when compared with NT-I7 alone (29 days) or untreated (20 days). NT-I7+RT+TMZ had better survival (86 days) when compared with RT+TMZ (56 days), although not statistically significant (Fig. 1B). Because IL7 is a cytokine required for T-cell maturation, development, and homeostasis, we evaluated its effects on T lymphocytes after RT+TMZ. Baseline blood was drawn on day 8 prior to initiation of RT+TMZ, and serial blood was drawn weekly on days 15, 18, 25, and 32 (Fig. 1A). GL261 tumor-bearing mice treated with NT-I7 had significant increases in CD3, CD4, and CD8 T cells on day 15 compared with control (Fig. 1C–E). Mice treated with NT-I7+RT+TMZ significantly increased the number of CD3 T cells at day 15 (Fig. 1C), with CD4 and CD8 T cells following a similar trend (Fig. 1D and E). These results suggest that NT-I7 may rescue lymphopenia induced by chemoradiation.
NT-I7 combined with RT significantly improves survival
To determine whether NT-I7 could improve the survival of glioma-bearing mice receiving chemoradiation, we tested RT+NT-I7 and RT+TMZ+NT-I7 in two mouse glioma models, GL261 and CT2A. Tumors were implanted intracranially on day 0, treated with daily RT and TMZ starting at Day 8, and treated with a single injection of NT-I7 on Day 12, which was the final day of RT (Fig. 2A). In the GL261 model, median survival was significantly longer in mice treated with combined treatment of RT+TMZ+NT-I7 (47 days) when compared with RT alone (34 days) and NT-I7 alone (24 days). The mice treated with RT+NT-I7 and the triple combination of RT+TMZ+NT-I7 had similarly improved survival when compared with the untreated control, NT-I7 alone, or RT alone (Fig. 2B). Remarkably, the percentage of long-term surviving mice bearing the CT2A model after the triple combination of RT+TMZ+NT-I7 was 80%. Median survival in this group (undefined; greater than 90 days) was significantly better than that for RT alone (40 days; P = 0.0088), or NT-I7 alone (32 days; P = 0.01). There was no significant difference between the triple combination of RT+TMZ+NT-I7 to RT+NT-I7 in both GBM models (Fig. 2B and C). In both models, RT+NT-I7 significantly improved survival compared with RT alone (Fig. 2B and C).
NT-I7 increased cytotoxic CD8 T cells and decreased regulatory T cells within the tumor
There is emerging evidence that although cytotoxic CD8 T cells recognize and target brain tumor cells, regulatory T cells (Treg) play an immunosuppressive role in the brain tumor microenvironment (TME; ref. 20). One reason for the continued failure of immunotherapy in brain tumors may be that there is an inadequate number of tumor-specific CD8 T cells and the existence of a preponderance of Tregs. To determine whether NT-I7 treatment shifts the balance of lymphocytes in the TME, we analyzed T lymphocyte subsets after NT-I7 treatment in GL261-bearing mice (Fig. 3A). Blood and tumors were collected 6 days after NT-I7 treatment (Day 18). In the blood of NT-I7–treated mice, we found significant increases in CD3 and CD8 T cells (Fig. 3C; Supplementary Fig. S1A). We also observed a significant increase in CD4 T cells in mice treated with RT in combination with TMZ and NT-I7, when compared with NT-I7 alone (Fig. 3B). Interestingly, combining NT-I7 with RT rescued the CD8 T-cell levels when compared with RT alone, whereas CD8 T cells remained low following triple therapy (Fig. 3C).
In the tumor, similar to our previous observation (21), CD3 T-cell numbers were significantly reduced following treatment with RT compared with untreated controls. Significant decreases in CD3 T cells (Supplementary Fig. S1B) and CD4 T cells (Fig. 3D) were seen in all treatment groups when compared with tumor-bearing, untreated controls. In contrast, we observed a significant increase in CD8 T cells in mice treated with NT-I7 alone. However, despite the trend observed at day 15 (Fig. 1E) adding NT-I7 to RT or RT+TMZ did not significantly increase the number of CD8 T cells at day 18 (Fig. 3E), suggesting that the effect of NT-I7 when combined with RT+TMZ may be transient and several doses may be necessary to achieve long-lasting effects.
Next, we evaluated CD4 and FoxP3 immunosuppressive regulatory T cells, referred to as Treg, in the tumor (representative gating strategy showed in Supplementary Fig. S2). When compared with untreated controls, we observed significant decreases in Treg in all treatment groups including RT alone, NT-I7 alone, RT+TMZ, RT+NT-I7, and RT+TMZ+NT-I7 groups (Fig. 3F). Treg suppress cytotoxic CD8 T-cell responses in the TME. The CD8 to Treg ratio was significantly higher in mice treated with NT-I7, compared with untreated control (P < 0.0001). However, when RT was administered, NT-I7 failed to increase the CD8 to Treg ratio in the tumor (Fig. 3G). Similarly, adding NT-I7 to RT+TMZ did not significantly change the ratio compared with the RT+TMZ group (Fig. 3G). This suggests that although NT-I7 as a single agent increases CD8 T cells in the tumor, it does not completely rescue the immunosuppressive effects of RT or RT+TMZ.
To further characterize the exhaustion and effects of CD4 and CD8 T cells in the tumor, we evaluated the expression of programmed cell death-1 (PD-1) and intracellular IFNγ production, respectively (Supplementary Fig. S2B). PD-1 is the major inhibitory receptor regulating T-cell exhaustion; T cells with high expression of PD-1 limit its ability to eliminate cancer (22). IFNγ plays a vital role in the activation of T cells and subsequently evoking an antitumor immune response (23). NT-I7 treatment alone significantly decreased the expression of PD-1 on both CD4 and CD8 T cells compared with untreated controls. When NT-I7 was added to either RT or RT+TMZ, there was no significant change in PD-1 expression compared with their respective controls (Fig. 3H and I). NT-I7 significantly increased IFNγ production in both CD4 and CD8 T cells compared with untreated controls (Fig. 3J and K). Interestingly, when NT-I7 was added to the RT+TMZ combination, IFNγ production was increased when compared with RT+TMZ treatment alone (Fig. 3J and K). This indicates that although the addition of NT-I7 to RT+TMZ does not affect T-cell exhaustion, it does increase T-cell function within the tumor.
NT-I7 increases T lymphocytes in the lymph nodes, thymus, and spleen
To determine the systemic effects of NT-I7 treatment, we analyzed CD3, CD4, and CD8 T-cell populations in the lymph nodes, thymus, and spleen of GL261 glioma-bearing mice (Fig. 3A). In the cervical lymph nodes, NT-I7 added to RT or RT+TMZ treatments significantly increased the levels of CD3 T cells (P < 0.0001) mainly due to an increase in the CD8 T-cell subset (Fig. 4A–C). In the thymus, we found significantly higher CD3 and CD8 T cells in mice treated with NT-I7 when compared with untreated mice. Similar to other lymphoid organs, RT or RT+TMZ greatly reduced CD3, CD4, and CD8 T cells (1.6 × 104 to 0.86 × 104; P = 0.1035) in the thymus. Adding NT-I7 to RT or RT+TMZ did not reverse the reduction of these T cells (Fig. 4D–F). T-cell progenitors evolve into thymocytes in the thymus. The earliest thymocytes are double-negative (DN) for CD4 and CD8. As thymocytes mature, they express both CD4 and CD8 and are termed double-positive (DP) cells. The thymocytes then undergo thymic selection to commit to either the CD4 or CD8 lineage (24). The mice treated with NT-I7 had significantly higher DN cells and DP cells. RT was associated with a reduction of both DN cells and DP cells, although this was not statistically significant. Adding NT-I7 after RT increased DN cell numbers but not DP cells when compared with RT alone (Supplementary Fig. S3A and S3B). RT or RT+TMZ led to a significant reduction in the size of the thymus when compared with untreated mice (70.5 mg to 48.1 mg; P = 0.0157; 70.5 mg to 33.5 mg; P < 0.0001, respectively). Adding NT-I7 to RT resulted in slightly larger thymuses; however, this was not significant (Supplementary Fig. S3C and S3D).
RT resulted in a reduction in the size and weight of the spleen. Notably, mice treated with NT-I7 had significantly larger spleens (P < 0.0001). Interestingly, adding NT-I7 to RT or RT+TMZ resulted in a significantly enlarged spleen, suggesting NT-I7 might rescue the spleen from RT-induced damage (Supplementary Fig. S4A and S4B). Spleens from mice treated with NT-I7 had significantly greater numbers of CD3 and CD8 T cells (P = 0.0017 and P < 0.0001, respectively) compared with untreated controls (Fig. 4G and I). However, we did not see the same effect in CD4 T cells in the spleen for this Day 18 time point (Fig. 4H). These results indicate that NT-I7 monotherapy increases CD8 T cells in the lymph nodes, thymus, and spleen. In addition, combining NT-I7 with RT and TMZ rescues treatment-induced CD4 and CD8 T-cell loss in the lymph nodes.
NT-I7 enhances central memory and effector memory CD8 T cells in lymphoid organs and tumor
To determine whether IL7 treatment alters the number of naïve, central memory (CM), and effector memory (EM) CD8 T cells, we assessed blood, lymph nodes, tumor, spleen, and thymus. The gating strategy is shown in Supplementary Fig. S2A. In the blood, on day 18 after tumor implantation, we did not observe significant differences in the number of naïve CD8 T cells between any treatment groups (Fig. 5A). We found a significant increase in CM (8.8 × 104 to 0.4 × 104; P < 0.0001; Fig. 5B) and EM (13.05× 104 to 0.66 × 104; P < 0.0001; Fig. 5C) CD8 T cells in the mice treated with NT-I7 when compared with untreated mice. Treating the mice with NT-I7 in combination with RT significantly increased EM (Fig. 5C) and not CM (Fig. 5B) CD8 T cells when compared with RT alone. The addition of NT-I7 to RT+TMZ increased EM and CM CD8 T cells, despite not reaching statistical significance (Fig. 5B and C). In the draining lymph nodes, NT-I7 was able to significantly increase naïve T-cell numbers when added to RT or RT+TMZ treatments, successfully recovering, and even increasing, the cell numbers when compared with untreated control (Fig. 5D). A similar trend was observed for CM and EM CD8 T cells in the lymph nodes (Fig. 5E and F). In the tumor, we found significantly more CM and EM CD8 T cells in mice treated with NT-I7 when compared with untreated control (Fig. 5H and I). However, adding NT-I7 to RT or RT+TMZ did not have a significant effect on either naïve, CM, or EM CD8 T cells in the tumor (Fig. 5G–I). Interestingly, in the spleen, we found that the addition of NT-I7 significantly increased naïve, EM, and CM CD8 T cells (Fig. 5J–L) when compared with non–NT-I7-treated mice. In the thymus, NT-I7 treatment did not affect the levels of naïve CD8 T cells compared with untreated controls (Fig. 5M) but significantly increased CM and EM CD8 T cells (Fig. 5N and O). However, when NT-I7 was added to RT+TMZ, we observed a significant increase in naïve CD8 T-cell levels but not EM or CM CD8 T cells (Fig. 5M–O). In summary, NT-I7 monotherapy increased the number of CM and EM CD8 T cells in the blood, lymphoid organs, and tumor. The combination of NT-I7 to RT+TMZ enhanced the number of naïve T cells in lymph nodes, spleen, and thymus.
CD8 T cells are required for NT-I7 effects on survival
To determine the role of CD8 T cells in survival after treatment with NT-I7 and chemoradiation, GL261-bearing mice were administered CD8 blocking antibody or an isotype control immediately after RT (Fig. 6A). Blood analysis on day 15 confirmed that all mice receiving αCD8 had significant depletion of CD8 T cells compared with isotype controls (Fig. 6B). CD8 depletion reduced the median survival of mice treated with RT (38 days to 26 days), RT+NT-I7 (52 days to 38 days), RT+TMZ (40 days to 34 days), and RT+NT-I7+TMZ (90 days to 40 days). Although the combination of NT-I7 with either RT or RT+TMZ prolongs survival, this survival advantage was lost in the absence of CD8 cells, indicating the effect of NT-I7 is mediated by or requires T cells (Fig. 6C).
NT-I7 decreases hematopoietic stem and progenitor cells from the bone marrow
IL7 acts as a signal for hematopoietic stem and progenitor cell (HSPC) differentiation, especially for common lymphoid progenitor cells in the bone marrow, and increases the mobilization to peripheral sites (15, 25–27). To evaluate the effects of NT-I7 alone or in combination with RT or RT + TMZ on tumor-bearing mice, we analyzed HSPCs in the bone marrow and found significantly lower numbers of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitor cells (MPP), CLP, common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) cells, and megakaryocyte erythroid progenitor (MEP) cells in mice treated with NT-I7 when compared with controls. The gating strategy for each cell population is shown in Supplementary Fig. S5. The mice treated with NT-I7 in combination with RT had significantly lower LT-HSC, ST-HSC, MPP, CLP, CMP, GMP, and MEP when compared with mice treated with RT alone (Fig. 7A–E). The same effect was seen when adding NT-I7 to RT + TMZ (Fig. 7A–E). These data indicate that mice treated with NT-I7 alone and in combination with other treatments resulted in a decrease of hematopoietic and progenitor cells in the bone marrow. Further experiments are needed to determine whether this decrease is due to the migration of these cells from the bone marrow to other immune organs or other underlying mechanisms.
The standard of care for patients with GBM is RT and TMZ. This is even the case for nearly half of patients with MGMT promoter methylation, who are known to poorly respond to TMZ and will only have a slight improvement in survival (28). These treatments can induce severe prolonged lymphopenia (4). This treatment-related lymphopenia is associated with reduced survival. Here, we have followed up on our previous work that demonstrated CD4 T-cell lymphopenia is associated with inappropriately low IL7 levels (10). IL7 is a critical cytokine for T-cell production, maturation, expansion, proliferation, survival, and antigen-specific responses. CYT107 is a short-acting recombinant human IL7 that has been studied in HIV and several solid tumors (29, 30), but a short half-life and instability have limited its use. Here, we have used a newly available, long-acting, recombinant human IL7, NT-I7, which significantly increases T lymphocytes in both healthy human subjects and murine cancer models (16, 17). Given the critical role of IL7 on T-cell homeostasis, we tested whether NT-I7 can correct treatment-related lymphopenia and thereby improve survival in glioma murine models. We observed improved survival in mice treated with NT-I7 in combination with RT or RT+TMZ when compared with RT alone in both GL261 and CT2A orthotopic tumor-bearing mice (Fig. 2B and C). Furthermore, RT+NT-I7 was as effective as RT+TMZ+NT-I7 in both models. The possible removal of TMZ, especially in cases without MGMT promoter methylation, and replacing it with NT-I7, is intriguing given the contribution of TMZ to significant fatigue and hematologic toxicities (31).
IL7 is a limiting factor for T-cell proliferation and its level is usually increased in lymphopenic conditions (32). Previous studies have shown that IL7 in combination with GM-CSF improves the immune response and survival of mice engrafted with melanoma or colon cancer cell lines (33). The survival advantage with NT-I7 may be due to an enhanced immune response against the tumor. As expected, treatment with NT-I7 had significantly increased CD3, CD4, and CD8 T cells in the blood (Fig. 1). We noted that RT or RT+TMZ significantly decreased lymphocyte counts in the blood, consistent with previous findings (21), and adding NT-I7 to RT+TMZ improved the levels of CD3, CD4, and CD8 T cells (Fig. 1). Notably, the peak of the increase for these cells was on day 15, 3 days after NT-I7 treatment.
Within the tumor, we found significantly increased CD8 T cells and decreased Treg with NT-I7 as a single agent. However, we did not see significant changes in CD8+ T cells or Treg when NT-I7 was added to RT±TMZ (Fig. 3). Notably, the tissues were collected on day 18 after tumor implantation and 6 days after NT-I7 treatment. Our results from the peripheral blood demonstrated the maximum effect of NT-I7 on T cells was on day 15 (Fig. 1). Therefore, optimizing the time of tissue collection after NT-I7 with RT±TMZ treatment is needed. One important effect of NT-I7 within the TME was the increase of IFNγ-producing T lymphocytes. NT-I7–treated mice had a significantly higher frequency of functional CD4 and CD8 T lymphocytes when compared with the control. Similarly, the addition of NT-I7 increased the frequency of functional T lymphocytes within the tumor of RT and RT+TMZ-treated mice. A more immune-reactive TME provides an insight into the potential mechanism of action associated with the increased survival observed in mice treated with NT-I7–containing regimens.
IL7 is required for thymocyte expansion during the early phases of thymic development and maturation (34). We found that NT-I7 was able to increase the DN and DP cells in the thymus (Supplementary Fig. S3A and S3B). NT-I7 was able to increase the number of CD3, CD4, and CD8 T cells in the thymus but numbers were not significantly improved when NT-I7 was combined with RT or RT+TMZ (Fig. 4D–F). It is not clear whether these findings were related to the time of tissue collection after NT-I7 treatment.
In other lymphoid organs, we found the groups treated with NT-I7 had increased CD3 T cells, partially restoring the severe RT-induced lymphopenia in the spleen and lymph nodes (Fig. 4). Despite the deep lymphopenic state induced by RT+TMZ, the addition of NT-I7 to RT+TMZ significantly increased the number of CD8 T cells within the lymph nodes (Fig. 4). As previously reported, RT leads to a reduction in the spleen size (35). We found that mice treated with NT-I7 had dramatically larger spleens than RT-treated mice or tumor-bearing untreated controls. NT-I7 treatment was associated with a normalization of spleen size after RT (Supplementary Fig. S4A and S4B).
To further prove that the enhanced numbers and functionality of CD8 T lymphocytes are part of the underlying mechanisms associated with the clinical benefit of NT-I7 treatment, we performed a survival experiment in mice after total CD8 T lymphocyte depletion (Fig. 6). As expected, depletion of CD8 T lymphocytes shortened the survival in all tested groups. Although the mice treated with NT-I7 had longer survival, depletion of CD8 T cells abrogated this effect and NT-I7 was no longer able to improve survival. This demonstrates that the improved survival of mice treated with NT-I7 requires CD8 T cells.
The effects we observed after NT-I7 treatment are consistent with previous observations that mice over-expressing IL7 or that are administered IL7 have increased numbers of lymphoid, myeloid, and dendritic cells (36, 37). Further experiments are required to understand the reduction in hematopoietic cells and progenitor cells in the bone marrow of mice treated with NT-I7 alone or in combination with RT or RT+TMZ (Fig. 7). However, we hypothesize the reduction is secondary to the mobilization of these cells from the bone marrow to other immune organs such as peripheral blood, spleen, thymus, and lymph nodes.
IL7 is responsible for naïve T-cell survival and homeostasis. IL7 receptor (IL7R) is present on most T cells and IL7 serves as a critical signal to resting T cells. In humans, IL7 levels are known to increase in response to lymphopenic conditions, as there is less consumption by lymphocytes (38). We found that mice treated with NT-I7+RT+TMZ had increased naïve cells in the spleen, thymus, and lymph nodes (Fig. 5). This suggests that NT-I7 rescued T-cell numbers following radiological insult. In addition, we found that adding NT-I7 to RT significantly increased CM and EM CD8 T cells in the blood, lymph nodes, and spleen (Fig. 5). These findings suggest that NT-I7 may be a useful clinical strategy to increase naïve, CM, and EM T cells in lymphoid organs and rescue these lymphocytes from chemoradiation-induced lymphopenic conditions. Although it is possible that priming with NT-I7 prior to RT would have similar effects, we did not test this. Because lymphocytes are sensitive to RT, clinically, we do not want to increase this pool of lymphocytes just before we administer cytotoxic therapies as this would be counter-effective.
In summary, RT+NT-I7 was as effective as RT+TMZ+NT-I7 in both murine glioma models. There is evidence that NT-I7 helps reshape the TME and its efficacy requires CD8 T cells (16). This raises the possibility of substituting TMZ in patients with GBM whose tumors do not have MGMT promoter methylation with NT-I7. In such cases, TMZ and its consequential fatigue and hematologic toxicities (31), could be avoided. Given the beneficial effects of NT-I7, it may also boost the antitumor response to immune checkpoint blockade, which as a single agent has been a disappointment in GBM. Although NT-I7 alone does not significantly prolong survival, it does have beneficial effects when combined with other treatments. Further studies with more tumor models and a larger sample size are warranted. Overall, our findings suggest NT-I7 may be a new tool in the neuro-oncology treatment armamentarium.
Treatment-related lymphopenia is a significant problem in patients with cancer and is associated with poor survival outcomes in patients with brain tumors. NT-I7 boosts T-cell numbers in lymphoid organs and improves survival in orthotopic glioma mouse models treated with RT or RT+TMZ. Our data suggest that NT-I7 treatment augments the immune response by increasing the ratio of cytotoxic CD8 T cells to immune-suppressive regulatory T cells within the TME; however, further experiments are needed to confirm this hypothesis. Altogether, our results suggest reversing lymphopenia with NT-I7 is a new strategy that could help improve our current treatment armamentarium against brain tumors. A phase I/II trial to evaluate the effect of NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).
J.L. Campian reports grants and other support from NeoImmuneTech during the conduct of the study, as well as other support from Incyte, Merck, Ipsen, and NeoImmuneTech outside the submitted work. B.H. Lee, S. Ferrando-Martinez, and A.A. Wolfarth report other support from NeoImmuneTech, Inc., during the conduct of the study and outside the submitted work. M.G. Chheda reports grants from NeoImmuneTech, National Institutes of Health, Alvin J. Siteman Cancer Center, and The Foundation for Barnes-Jewish Hospital and the Barnard Trust during the conduct of the study, as well as other support from Orbus Therapeutics, Incyte, Merck, and UpToDate outside the submitted work; in addition, M.G. Chheda has a patent for Zika virus strains for the treatment of GBM pending. No disclosures were reported by the other authors.
J.L. Campian: Conceptualization, resources, formal analysis, supervision, funding acquisition, validation, methodology, writing–original draft, writing–review and editing. S. Ghosh: Investigation, methodology, writing–original draft, writing–review and editing. V. Kapoor: Data curation, validation, methodology. R. Yan: Data curation, investigation, methodology. S. Thotala: Data curation. A. Jash: Data curation, investigation. T. Hu: Data curation, investigation. A. Mahadevan: Investigation. K. Rifai: Data curation, investigation. L. Page: Investigation. B.H. Lee: Conceptualization, writing–review and editing. S. Ferrando-Martinez: Writing–review and editing. A.A. Wolfarth: Writing–review and editing. S.H. Yang: Resources, funding acquisition. D. Hallahan: Writing–review and editing. M.G. Chheda: Conceptualization, resources, formal analysis, supervision, validation, investigation, methodology, writing–original draft, project administration, writing–review and editing. D. Thotala: Conceptualization, resources, formal analysis, supervision, validation, investigation, methodology, writing–original draft, project administration, writing–review and editing.
This work was supported by NeoImmuneTech, Inc., Department of Radiation Oncology Startup Funds (D. Thotala), National Institute of Neurological Disorders and Stroke of the National Institutes of Health (NIH) under award number R01 NS117149 (to M.G. Chheda), Alvin J. Siteman Cancer Research Fund (M.G. Chheda), and the Alvin J. Siteman Cancer Center Siteman Investment Program through funding from The Foundation for Barnes-Jewish Hospital and the Barnard Trust (J.L. Campian and M.G. Chheda). The authors are grateful to Sachendra S. Bais for assisting with cell culture and mouse implantations. They thank the Department of Radiation Oncology for shared resources and animal facilities. They also thank Katie Duncan and Julie Prior at Small-Animal Cancer Imaging—Siteman Cancer Center Washington University School of Medicine for help with BLI.
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