CB T cells mediate enhanced antitumor responses compared with PB T cells in a murine model of B-cell lymphoma.
The antitumor activity correlates with increased tumor-homing of CCR7high CB CD8+ T cells and rapid gain of cytotoxic and Th1 function.
Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rgnull mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7+CD8+ T cells and prompt induction of cytotoxic CD8+ and CD4+ T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.
Unrelated cord blood transplantation (CBT) appears to be more permissive of an HLA mismatch between the recipient and donor, with a lower risk of graft-versus-host disease (GVHD) for a given degree of mismatch compared with hematopoietic cell transplantation from adult donors.1,2 This is likely to reflect the fact that, compared with naïve adult T cells, CB T cells are T-helper (Th)2-Tc2 biased with reduced inducible expression of Th1-Tc1 cytokines, such as interferon γ (IFN-γ).3,4 In addition, the fetal immune system is believed to induce immune tolerance after exposure to foreign antigens.5,6 In vivo T-cell depletion leads to delayed T-cell reconstitution after CBT, resulting in a high morbidity and mortality from infection.7-10 We showed that omission of serotherapy leads to enhanced thymus-independent T-cell expansion of donor CB T cells.11 This enhanced T-cell reconstitution after CBT is CD4+ T-cell biased, a pattern not observed after transplantation using a bone marrow or peripheral blood (PB) stem cell graft, and, intriguingly, occurs despite 1 log lower T cells in the CB graft compared with the bone marrow graft. Furthermore, these rapidly expanding naïve CB T cells differentiate into viral-specific T lymphocytes within 2 months, leading to rapid clearance of viral infections such as cytomegalovirus and adenovirus.11 These observations suggest that, despite an intrinsic bias toward anti-inflammatory Th2-Tc2 immune responses, naive CB T cells can rapidly proliferate and differentiate into antigen-specific T lymphocytes.
In view of clinical data suggesting that CBT appears associated with a significantly lower risk of leukemic relapse, particularly after HLA-mismatched12 and T-cell replete CBT,13-16 we questioned whether the rapidly expanding T-cell compartment after CBT also accounts for enhanced graft-versus-leukemia (GVL) effects. To determine whether CB T cells can mediate potent antitumor effects in vivo and to elucidate the mechanism for this, we compared the ability of HLA-mismatched CB T cells vs adult PB T cells to reject Epstein-Barr virus (EBV)-driven B-cell lymphoma in a xenograft tumor model in the immunodeficient mouse strain NOD/SCID/IL2rγnull (NSG). We demonstrate that CB T cells mediate curative and enhanced antitumor responses compared with PB T cells.
Generation of EBV-driven B-cell lymphoma cell lines
A total of 5 × 106 freshly isolated mononuclear cells from healthy CB or PB donors were cultured with an EBV supernatant produced from the B95-8 cell line in the presence of 1 µg/mL cyclosporin A to inhibit EBV-specific T-cell responses as previously described.17 B-cell lymphoma cell lines (LCLs) were transduced with an SFG retroviral vector encoding green shifted firefly luciferase (F-Luc) and cultured in RPMI medium with 10% fetal calf serum + 1% penicillin/streptomycin (RF10). Expression of F-Luc allows for noninvasive monitoring of xenografted tumors in mice by luminescent imaging in vivo.
Mononuclear cells were separated from CB and PB using Ficoll-Paque PLUS. Negatively selected CD3+ T cells were isolated using Milteyni Pan T-cell Isolation Kit, human (130-096-535) and counted using a Sysmex XE-5000 automated hematology system. The purity of negatively selected T cells was >94% (supplemental Table 2 available on the Blood Web site).
Xenograft model and tumor imaging
NSG mice were obtained from the Jackson Laboratory and raised under pathogen-free conditions. All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the Animals (Scientific Procedures) Act 1986 (Home Office, London, UK). In primary murine modeling experiments performed to study the differential GVL effect between CB and PB T cells, mice were inoculated with 5 × 106 F-Luc expressing EBV LCLs subcutaneously on the nape of the neck on day –2. To evaluate the antitumor activity, 5 × 106 comparably HLA-mismatched CB or PB CD3+ T cells were injected intravenously (by tail vein injection) after 2 days on day +0 (supplemental Figure 1). HLA typing was performed for A, B, C, DRB1, and DQB1 antigens. In the first experiment, 10 of 10, and in the second experiment, 7 of 10, HLA antigens were mismatched between CB T cells vs LCLs and PB T cells vs LCLs (supplemental Table 1). Tumor growth was evaluated using the in vivo imaging system (Xenogen; Caliper Life Sciences, Hopkinton, MA). Animals were imaged on day +10, +20, +25, +30, and +35. Photon emission from FLuc+ LCLs expressed in photon per second per cm2 per steradian (p/s/cm2/sr) was quantified using Living Image software (Xenogen) as previously described.18 In addition to monitoring the tumor bioluminescence, 2-dimensional caliper measurements of the tumor were performed, and tumor volume was derived using the following formula: tumor volume = 1/2 × (length × width2). Mice were euthanized if the tumor growth exceeded the permitted threshold of 10 mm.
Secondary murine modeling experiments to study the mechanisms of the GVL effect between CB and PB T cells are shown in a schematic diagram in supplemental Figure 1.
Tumors were minced and incubated in RF10 medium with 0.1 mg/mL collagenase A (Roche) and 60 U/mL DNase I (Sigma-Aldrich) for 30 minutes at 37°C. Tumors were then homogenized and filtered through a nylon filter (70 µm), and the lymphocytes were isolated in RF-10. Tumor-infiltrating lymphocytes (TILs) were studied for (1) IFN-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-4 responses; (2) perforin expression; and (3) naïve memory effector differentiation and FoxP3 staining,19 as detailed in the supplemental Methods.
Immunohistochemical mouse anti-human CD3 staining of tumor sections was performed to study the extent of T-cell infiltration. Using Image J, 10 RGB images of 40× magnification for each tumor slide were converted to black and white mask output using a calibrated threshold for each stack of images, and average density of T cells per square millimeter of tumor was calculated as shown in supplemental Figure 2.20
Statistical analysis was done using GraphPad Prism software. Tumor volumes of either photons/s/cm2/sr or mm3 are expressed as mean and standard error of mean. A two-tailed Student t test was performed for comparisons of experimental groups. A log-rank (Mantel-Cox) test was used to compare survival between different groups of mice.
CB T cells mediate enhanced antitumor effects compared with PB T cells
We initially compared the antitumor effects of equivalent doses of T cells derived from comparably HLA-mismatched CB vs PB in a NSG mouse model of EBV-driven B-cell lymphoma. In 2 consecutive experiments where mice were treated with 5 × 106 allogeneic T cells derived from different donors 2 days after inoculation with 5 × 106 tumor cells, we observed that control mice receiving B-cell lymphoma without T cells (n = 6) and mice receiving B-cell lymphoma followed by PB T cells (n = 10) exhibited tumor growth exceeding the threshold limit of 10 mm between 20 and 22 days following tumor inoculation, so that these mice had to be euthanized. In contrast, 9 of 10 mice receiving the same dose of CB T cells showed slower tumor growth followed by complete tumor regression (Figure 1A). These findings were confirmed by measuring tumor bioluminescence in photons/s/cm2/sr using a Xenogen-IVIS imaging system. The mean tumor bioluminescence of the CB group on day +20 was significantly lower than that of the PB and control group (9.65 ± 2.65 × 109 vs 3.83 ± 1.08 × 1010 and 3.04 ± 0.84 × 1010, respectively; P < .01; Figure 1B). These IVIS findings were mirrored by caliper-measured tumor volumes. On day +20, the mean tumor volume in the CB group was 204 mm3 (Figure 1C), which was significantly lower than the mean tumor volume of the PB (814 mm3) and control (812 mm3) group (P < .01).
Surviving mice in the CB group were monitored for 60 days after T-cell injection without recurrence of tumor or xenogeneic GVHD. This resulted in significantly improved survival in the CB group compared with the PB group (P < .0003; Figure 1D). In addition to these primary in vivo imaging experiments, the superior antitumor effect of CB T cells was also observed in 2 additional functional experiments with different CB and PB T-cell donors, where tumor volumes were measured using a caliper (supplemental Figure 3). This observation suggests that CB T cells can mediate potent and enhanced graft-versus-tumor effects compared with PB T cells in this model.
CB T cells are not xenoreactive, whereas PB T-cell antitumor effects correlate with xenoreactivity
It is well established that human PB T cells can mediate xenoreactivity.21-23 We next compared the xenoreactive capacity of 5 × 106 CB vs PB T cells in tumor-free NSG mice. In mice receiving PB T cells, we observed the onset of xenoreactivity 3 weeks following T-cell injection: all mice developed ruffled fur, lethargy, and weight loss 3 weeks after T-cell injections (supplemental Figure 4A). In contrast, mice receiving CB T cells did not develop any symptoms of xenoreactivity, despite the presence of circulating human T cells up to 60 days after injection. Xenogeneic GVHD in the PB T-cell group was confirmed histologically in the liver and skin with T-cell infiltration, distortion of normal architecture, and apoptotic changes around the bile ducts and in the intradermal region, but this was not seen in CB T-cell recipients (supplemental Figure 4B).
To model the antitumor effects of PB T cells and how this correlates with xenoreactivity, we performed a similar tumor modeling experiment with a lower burden of B-cell lymphoma. We injected 2.5 × 106 LCLs subcutaneously on day –2, followed by the tail vein injection of 5 × 106 PB T cells in the treatment but not the control group. We observed that the control mice had tumor growth >10 mm on day +27 after tumor inoculation, requiring these mice to be euthanized. In mice receiving PB T cells, tumors grew for the first 3 weeks with no significant difference in mean tumor bioluminescence between the tumors in the control and PB T-cell groups (3.89 ± 2.15 × 108 vs 5.69 ± 1.69 × 108; P = not significant) but subsequently started regressing. This tumor regression correlated with the onset of signs of xenoreactivity such as ruffled fur, lethargy, and weight loss (supplemental Figure 5A-B).
Thus, the antitumor effect of CB T cells appears independent of xenoreactivity. PB T cells can also mediate an antitumor effect in this model, but this was attenuated compared with CB T cells and correlated with xenoreactivity.
Antitumor effects of CB T cells are mediated through alloreactivity
Because the B-cell lymphoma used in this model is an EBV-driven tumor expressing viral antigens,24,25 we next determined whether the tumor regression observed in the CB group was due to an antiviral or an alloreactive effect. To model this, we compared the ability of 5 × 106 autologous and allogeneic CB T cells to reject LCL tumors. We observed that control mice receiving LCLs without T cells (n = 3) and mice receiving LCLs followed by autologous CB T cells (n = 5) had tumor growth exceeding the threshold limit of 10 mm on day +22 after tumor inoculation and were therefore euthanized. In contrast, mice injected with LCLs and allogeneic CB T cells (n = 5) had slower tumor growth followed by complete tumor regression. On day +22, the autologous CB T-cell group had a mean tumor volume (mm3) and mean tumor bioluminescence (photons/s/cm2/sr) of 1223 ± 72 and 1.32 ± 0.25 × 1011, respectively. In contrast to these large tumors, the mean tumor volume (mm3) and mean tumor bioluminescence (photons/s/cm2/sr) in the allogeneic CB T-cell group were significantly lower: 12 ± 6 and 1.80 ± 1.09 × 109, respectively (P < .01; Figure 2A-C). These results indicate that the tumor regression observed with CB T cells appears to be mediated through an alloreactive rather than an EBV-specific T-cell response.
Enhanced recruitment of CB T cells to the tumor may mediate a more potent antitumor effect
To study the immunologic basis of this differential antitumor effect following the injection of CB and PB T cells, we first studied the differences in the recruitment of TILs. In a separate allogeneic LCL tumor experiment, mice receiving CB (n = 5) and PB (n = 5) T cells were euthanized on day +15 following T-cell injections, and tumor sections were studied. Immunohistochemistry staining of tumor sections with CD3 antibody showed higher numbers of TILs in the CB group compared with the PB group (Figure 3A). A significantly higher number of TILs per square millimeter were observed in the CB group compared with the PB group (4702 ± 426 vs 1275 ± 330; P < .01; Figure 3B). Further, flow cytometric analysis of isolated TILs showed that a majority of TILs in the CB group were CD8+ T cells, with a median CD4:CD8 ratio of 0.49 (range, 0.47–0.58; Figure 3C). In contrast, CD4+ T cells were predominant in the PB group, with a median CD4:CD8 ratio of 1.72 (range, 1.60-1.88; P < .01). This inversion of the CD4:CD8 ratio in the CB TILs was observed despite a higher initial CD4:CD8 ratio in the injected CB T cells compared with the injected PB T cells (4.9 vs 2.9). This suggests that CB CD8+ T cells are preferentially recruited to the tumor and may play a key role in mediating antitumor effects in this model.
CB TILs show a CD8+ T-cell bias and higher CD8:Treg ratio than PB TILs
To further gain insight into the kinetics of infiltration of CB and PB T cells in this B-cell tumor and the role of regulatory T cells in this, we compared the infiltration of CD4+, CD8+, and CD4+CD25+FoxP3+ T-regulatory cells in the tumor on day +10 and day +20 after T-cell injections. NSG mice were injected with 5 × 106 lymphoma cells subcutaneously followed 2 days later by 5 × 106 CB (n = 8) or PB T cells (n = 8). Ten and 20 days after T-cell injections, 4 mice from each group were euthanized, and isolated TILs were studied flow cytometrically for the CD4:CD8 and CD8:CD4+ T-regulatory cells ratio (Figure 4A-C).
At day +10 and day +20 after T-cell injection, the median CD4:CD8 ratio in the CB TILs was 0.58 (range, 0.51-0.69) and 0.03 (range, 0.03-0.06), respectively, compared with a median ratio of 1.54 (range, 1.3-1.9) and 0.26 (range, 0.14-0.33) in the PB TILs (P < .001 and P < .001, respectively; Figure 4D). Thus, as previously observed, the CD4:CD8 ratio in the CB TILs was significantly reversed compared with PB TILs, despite a higher initial CD4:CD8 ratio in the injected CB T cells compared with the injected PB T cells (3.4 vs 2.7).
Similarly, at day +10 and day +20 after T-cell injection, the median CD8:CD4+CD25+FoxP3+ ratio in the CB TILs was 29 (range, 25-52) and 2043 (range, 950-3288), respectively, compared with a median ratio of 11.9 (range, 7.8-15.5) and 132 (range, 72-166) in the PB TILs (P < .01 and P < .01, respectively; Figure 4D). This observation was complemented by the observed persistence of FoxP3 positivity in 3-dimensional color inspector images of 10× tumor sections in the PB group but not in the CB group (supplemental Figure 6). This differential pattern of T-cell infiltration indicate that, although CB CD8+ T cells may be the primary mediators of the enhanced antitumor effect in the CB group, a relatively increased recruitment of T-regulatory cells compared with cytolytic effectors may also attenuate the antitumor effect in the PB group.
CCR7-enriched CB CD8+ T cells have enhanced tumor-homing ability
Because the vast majority of CB CD8+ T cells are CCR7high (Figure 5A) and CCR7 is a homing receptor for naïve T cells,26-28 we sought to identify whether CCR7+CD8+ T cells predominantly infiltrate the tumor.
We observed that CCR7 expression was similar in CB and PB CD8+ TILs (median, fluorescence intensity [MFI] = 6854 ± 1542 vs 6737 ± 1626; P = not significant). CCR7 MFI expression was significantly higher in TILs compared with circulating PB CD8+ T cells (1378 ± 10; P < 0.01), whereas circulating CB CD8+ T cells remained CCR7high (MFI = 21 128 ± 534; Figure 5B-C). This indicates that CCR7high CD8+ T cells were primarily recruited to the tumor, and higher expression of CCR7 may endow CB CD8+ T cells with the enhanced tumor-homing ability.
Tumor-infiltrating naïve CB T cells rapidly differentiate into memory/effector cells and gain IFN-γ and TNF-α functions
For naïve CB T cells infiltrating the tumor to exert antitumor effects, they must undergo memory effector differentiation. Therefore, we studied the memory phenotype of TILs and circulating lymphocytes (CLs). We observed that TILs in the CB group rapidly underwent differentiation to central memory (CM) and effector memory (EM) T cells, whereas circulating CB T cells remained naïve (Figure 6A-B). A similar percentage of CD8+ TILs were observed to have a CM and EM phenotype in the groups receiving CB and PB T cells (CM = 77.8 ± 1.4 vs 78.0 ± 1.8; EM = 1.48 ± 0.3 vs 2.1 ± 0.5; P = not significant).
Finally, mice in the PB (n = 5) and CB (n = 5) groups were euthanized after the onset of tumor regression in both the groups (day +26 after the injection of T cells), and isolated TILs were studied for differences in effector functions.
CD8+ TILs in both the groups demonstrated IFN-γ responses, and there was no difference in the percentage of CD8+ TILs expressing IFN-γ responses in the 2 groups (3.5 ± 0.14 vs 3.4 ± 0.15; P = not significant; Figure 7A). In contrast, a significantly higher percentage of CB CD8+ TILs acquired TNF-α responses (1.2 ± 0.07 vs 0.48 ± 0.06; P < .0001; Figure 7B). Likewise, both the percentage of CD8+ T cells expressing perforin and the MFI were significantly higher in CB CD8+ TILs compared with PB CD8+ TILs (MFI = 443 ± 16 vs 365 ± 13; percentage of CD8+ T cells = 75 ± 3.0 vs 58 ± 5.0; P < .01; Figure 7C). Thus, CB CD8+ TILs rapidly acquire higher TNF-α and perforin effector functions than PB T cells in the tumor microenvironment.
We also studied the effector functions of CD4+ TILs to gain insight into the role of intratumoral Th1/Th2 balance on differential antitumor effects. A significantly higher percentage of CD4+ TILs were identified as Th2 TILs in the PB group compared with the CB group (16 ± 2.0 vs 6.2 ± 1.0; P < .001; supplemental Figure 7A-B). Th1/Th2 balance measured as the IFN-γ/IL-4 and TNF-α/IL-4 ratios was significantly biased toward Th1 in the CB group compared with the PB group (0.98 ± 0.2 vs 0.3 ± 0.06 and 0.88 ± 0.05 vs 0.39 ± 0.07; P < .05 and P < .0001, respectively; supplemental Figure 6C-F). Thus, CB CD4+ TILs gained Th1-biased responses in the tumor microenvironment.
Taken together, these observations indicate that naïve CB T cells can swiftly undergo memory effector differentiation and gain cytotoxic effector functions in the tumor microenvironment.
Unrelated CBT is increasingly used to treat high-risk hematologic malignancies.12,13 It is therefore critical to determine whether the rapidly expanding donor T cells following T-replete CBT can confer differential GVL effects compared with donor T cells transferred with adult stem cell sources, despite an intrinsic Tc2-Th2 bias in the CB T cells.3,4 We therefore compared the ability of CB vs adult PB T cells to mediate an antitumor effect against the EBV-driven B-cell lymphoma in NSG mice. In this model, we observed that CB T cells mediate enhanced antitumor effects compared with PB T cells, resulting in improved survival of mice receiving CB T cells. As previously described by other groups,29 CB T cells were not xenoreactive, and tumor regression was associated with alloreactivity rather than recognition of EBV antigens. In the primary in vivo tumor imaging experiments, the superior antitumor effect observed with CB T cells is unlikely to reflect differences in HLA mismatch between the PB and CB T cells, as T cells from both sources were comparably mismatched with the LCLs, and the effect was observed with 2 different donor-recipient pairs. One limitation of our model is that both CB and PB T cells were highly (7-10 of 10) mismatched with the LCL tumor, and ideally these experiments should be repeated with a more clinically relevant degree of HLA matching (ie, 0-2 mismatches) but we were unable to source CB units closely matched to the LCL tumor cells. However, we plan to perform these experiment if such CB units become available in future. The difference in antitumor effect mediated by CB and PB T cells correlated with enhanced tumoral recruitment of CB T cells compared with PB T cells. Analysis of TILs demonstrated that, after adoptive transfer of CB T cells, CD8+ T cells rapidly infiltrate the tumor with induction of cytotoxic CD8+ and Th1 CD4+ responses in the tumor microenvironment. In contrast, in the PB group, this antitumor effect appears attenuated because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells.
Despite the strong expression of both major histocompatibility complex class I and II in this lymphoma and the fact that both CB CD4+ and CD8+ T cells proliferate robustly in response to stimulation with allogeneic LCL in vitro, the majority of T cells recruited to the tumor after transfer of CB T cells were from the CD8+ subset. The role of CD8+ TILs in tumor immunology has been demonstrated in numerous murine models and also in cancer patients, including those with leukemia.30-40 In a murine model of allogeneic bone marrow transplantation for acute T-cell leukemia39 and chronic myeloid leukemia,40 infusion of mixed donor CD4+ and CD8+ T cells or donor CD8+ T cells alone mediated a potent GVL effect, whereas GVL was not observed with donor CD4+ T cells alone. It is therefore apparent that CD8+ T cells are critical as antileukemic effectors at the tumor site. Hence, in our model, the observation of the preferential infiltration of CB CD8+ TILs compared with PB CD8+ TILs suggests that CB CD8+ T cells mediate the observed enhanced antitumor responses.
Despite the naïve phenotype of CB T cells, we observed a similar percentage of CM and EM CD8+ T cells in the CB and PB TILs. Interestingly, circulating CB T cells did not undergo memory effector differentiation, in contrast to circulating PB T cells, which showed an increased percentage of EM T cells compared with PB TILs. These findings suggest that naïve CB T cells rapidly undergo memory effector differentiation within the tumor microenvironment, whereas circulating CB T cells retain a naïve phenotype.
Naïve CB CD8+ T cells rapidly gained effector activity including expression of IFN-γ, TNF-α, and perforin in the tumor microenvironment. We observed similar percentages of IFN-γ–secreting CD8+ TILs in the CB and PB groups, whereas the percentage of TNF-α–secreting and perforin-expressing CD8+ TILs was significantly higher in the CB group. In contrast to the cytotoxic CD8+ T-cell bias in the CB TILs, a preponderance of CD4+ TILs was observed in the PB group with a relative bias toward suppressive Th2 and T-regulatory cells. Although CD8+ TILs may primarily mediate cytotoxic antitumor effects, the Th1/Th2 balance of CD4+ TILs is also important in mediating tumor-specific immune responses.41-45 In general, immune deviation toward Th1 effectors mediates an antitumor response, whereas Th2 responses prevent tumor rejection. The relative skewing of Th1/Th2 intratumoral balance toward Th1 in the CB TILs is noteworthy, because this occurred despite the fact that naïve CB CD4+ T cells are reported to be physiologically biased toward Th2, produce lower levels of IFN-γ than adult naïve T cells in vitro, and are hypermethylated at cytosine guanine dinucleotide and non-cytosine guanine dinucleotide sites within the IFN-γ promoter.4 This therefore suggests that CB CD4+ T cells can rapidly mature into Th1 effectors in the tumor microenvironment. Similarly, despite the immune-tolerant ontogeny of fetus,5,6 the CD8+ T cell/CD4+ T-regulatory balance was significantly skewed toward CD8+ T cells in the CB TILs. The immunosuppressive role of FoxP3+ regulatory T cells as a crucial tumor immune-evading mechanism is well established.46-49 In a meta-analysis of TILs in cancer sections, although CD3+ and CD8+ TILs were associated with good prognosis and improved survival, the CD8+/FoxP3+ ratio produced a more impressive hazard ratio compared with CD3+ or CD8+ TILs alone.50 The enhanced GVL effect after infusion of CD4+CD25+ regulatory T cell-depleted donor lymphocytes has been demonstrated further, indicating immune-evading role of T-regulatory cells following allogeneic transplantation.51 Thus, Th1 CD4+ T cells may synergize with tumor infiltrating CD8+ cytotoxic effectors to enhance antitumor effects after transfer of CB T cells, whereas the relative Th2 bias and increased number of tumor-infiltrating T-regulatory cells in the PB group may impair tumor rejection in this model.
The mechanisms by which CB T cells are recruited to the tumor, proliferate preferentially in the tumor microenvironment, and mediate enhanced antitumor effects in this model remain to be elucidated, and this will require further work. CB CD8+ T cells are predominantly CCR7high compared with a mix of CCR7high and CCR7lowCD8+ T cells in the PB. The CD8+ T cells homing into the tumor in both the CB and PB groups were CCR7high, whereas CCR7lowCD8+ T cells from the PB group remained in circulation. It is thus possible that high expression of chemokine receptor CCR7 in CB CD8+ T cells may facilitate enhanced tumor homing. We therefore plan to optimize disruption of CCR7 receptor in the CB T cells using the CRISPR/Cas9 system to more closely define the role of CCR7 receptor in tumor homing.
More broadly, there is growing evidence that fetal and adult T cells are functionally distinct populations with different gene expression profiles arising from different ontogenic populations of hematopoietic stem cells.52,53 Our as yet unpublished results indicate that the early adaptive immune system derived after T-replete CBT recapitulates a distinct molecular program of fetal ontogeny. Thus, these ontogenic differences between CB and PB T cells may not only endow CB T cells with the ability to rapidly reconstitute in the lymphopenic host following CBT,11 but may also confer CB T cells with the ability to rapidly infiltrate the tumor and activate and differentiate in the tumor microenvironment.
In summary, CB T cells mediate more potent antitumor effects than adult T cells in this B-cell lymphoma model through enhanced recruitment of naïve CB T cells (particularly CB CD8+ T cells), prompt induction of memory effector differentiation, and gain of cytotoxic effector functions in the tumor microenvironment. The findings that CB T cells mediated an enhanced GVL effect without exhibiting xenoreactivity parallel the clinical situation in humans where CBT may exert a powerful GVL effect yet with reduced rates of GVHD. Our work thus supports omission or minimization of serotherapy from the preparative regimens of CBT recipients for high-risk hematologic malignancies to enhance T cell-mediated GVL. Although we recognize the limitations in extrapolating from this model system to the clinical setting and that other immune cells such as natural killer cells following CB grafts may also mediate a GVL effect,54,55 these findings nonetheless provide a mechanistic insight into how CB T cells may mediate the potent GVL effects observed after T-replete CBT.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
The authors thank Nick Davies, Mike Blundell, and the staff at University College London Biological Services for assistance with animal handling and Dale Moulding for assistance with Image J analysis.
This work was supported by Bloodwise (Leukaemia and Lymphoma Research), the Olivia Hodson Cancer fund, Great Ormond Street Hospital Children's Charity, and National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital.
Contribution: P.H., W.Q., I.R., K.G., S.Q., A.S., P.A., and P.V. designed research; P.H. and I.R. performed experiments; P.H. analyzed the data and performed statistical analysis; and P.H., W.Q., P.A., and P.V. wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Prashant Hiwarkar, Department of Haematology and Bone Marrow transplantation, Birmingham Children’s Hospital, Steelhouse Lane, Birmingham B4 6NH, United Kingdom; e-mail: firstname.lastname@example.org.
P.A. and P.V. contributed equally to this work.