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Prepublished online November 14, 2008; doi:10.1182/blood-2008-05-155697

Keratinocyte growth factor enhances DNA plasmid tumor vaccine responses following murine allogeneic bone marrow transplantation Robert R Jenq, Christopher G King, Christine Volk, David Suh, Odette M Smith, Uttam Rao, Nury Yim, Amanda M. Holland, Sydney X. Lu, Johannes L Zakrzewski, Gabrielle L Goldberg, Adi Diab, Onder Alpdogan, Olaf Penack, Il-Kang Na, Lucy W Kappel, Jedd D Wolchok, Alan N Houghton, Miguel-Angel Perales and Marcel R.M. van den Brink

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Blood First Edition Paper, prepublished online November 14, 2008; DOI 10.1182/blood-2008-05-155697

Keratinocyte growth factor enhances DNA plasmid tumor vaccine responses following murine allogeneic bone marrow transplantation

Robert R. Jenq1,2, Christopher G. King1, Christine Volk1,3, David Suh1, Odette M. Smith1, Uttam Rao1, Nury Yim1, Amanda M. Holland1, Sydney X. Lu1, Johannes L. Zakrzewski1, Gabrielle L. Goldberg1, Adi Diab1,3, Onder Alpdogan1, Olaf Penack1, Il-Kang Na1, Lucy W. Kappel1, Jedd D. Wolchok1,2,3, Alan N. Houghton1,2,3, Miguel-Angel Perales1,2,3 & Marcel R. M. van den Brink1,2

1

Department Immunology, 2Department of Medicine, 3Swim Across America Laboratory of

Tumor Immunology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA. Correspondence should be addressed to M.R.M.v.d.B. ([email protected]).

All authors concur with the submission. The material submitted for publication has not been previously reported and is not under consideration for publication elsewhere. The authors report no financial conflicts of interest.

Condensed title: KGF and DNA tumor vaccination after allogeneic BMT

Abbreviations: allogeneic bone marrow transplantation (allo-BMT) graft-versus-host disease (GVHD)

Copyright © 2008 American Society of Hematology

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keratinocyte growth factor (KGF) regulatory T cells (Tregs) tyrosinase-related protein 1 (TRP1)

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Abstract Keratinocyte growth factor (KGF), given exogenously to allogeneic bone marrow transplantation (allo-BMT) recipients, supports thymic epithelial cells and increases thymic output of naïve T cells. Here we demonstrate that this improved T cell reconstitution leads to enhanced responses to DNA plasmid tumor vaccination. Tumor-bearing mice treated with KGF and DNA vaccination have improved long-term survival and decreased tumor burden after allo-BMT. When assayed prior to vaccination, KGF-treated allo-BMT recipients have increased numbers of peripheral T cells, including CD8+ T cells with vaccine-recognition potential. In response to vaccination, KGF-treated allo-BMT recipients, compared to controls, generate increased numbers of tumor-specific CD8+ cells, as well as increased numbers of CD8+ cells producing IFN-γ and TNF-α. We also found unanticipated benefits to anti-tumor immunity with KGF administration. KGF-treated allo-BMT recipients have an improved T effector-to-regulatory T cell ratio, a larger fraction of effector cells that display a central memory phenotype, and effector cells that are derived from a broader T cell receptor repertoire. In conclusion, our data suggest KGF can function as a potent vaccine adjuvant following allo-BMT through its effects on posttransplant T cell reconstitution.

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Introduction KGF, also called fibroblast growth factor 7, is indicated and widely used for preventing mucositis associated with high-dose cytotoxic therapy1,2. Interestingly, its receptor, FgfR2-IIIb, is expressed by thymic epithelium as well as gastrointestinal mucosa. A role for KGF in supporting thymic epithelial cells was first suggested by the observation that FgfR2-IIIbdeficient mice show severely hypoplastic thymi at birth3. We have previously shown that KGFdeficient mice develop normal thymi but display increased sensitivity to thymic damage and impaired thymic reconstitution, suggesting a role for KGF in supporting thymic epithelium during regeneration4. The endogenous sources of KGF in the thymus include mesenchymal cells5 as well as αβlineage thymocytes6. When given exogenously, KGF restores the hypoplastic medullary thymic epithelial compartment in Rag-deficient mice6. In mice with thymic injury secondary to radiation, cyclophosphamide or dexamethasone, or thymic involution due to age, KGF restores thymic architecture and improves thymocyte generation4, translating into improved humoral immune responses7. KGF directly increases proliferation of both mature and immature thymic epithelial cells, activating the p53, NF-κB, BMP, and Wnt pathways, resulting in enhanced T cell development8. KGF has been shown to increase intrathymic levels of IL-7 and appears to act at least partially via IL-7 signaling9,10. When given to mice undergoing allo-BMT, KGF preserves thymic architecture and improves thymocyte counts10. This benefit has also been demonstrated in aged mice4 and mice with active graft-versus-host disease (GVHD)11. KGF also increases numbers of naïve T cells in the periphery post-allo-BMT4,10, with further enhanced T cell reconstitution when combined with androgen blockade12. Similar benefits of KGF have recently been reported in rhesus macaques

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which had received autologous BMT13. Clinical studies on the effects of KGF on immune function in allo-BMT patients are ongoing at our center. One potential clinical benefit of accelerated T cell reconstitution after allo-BMT is improved immunity against pathogens and residual malignancy. In this study, we asked if KGF leads to improved anti-tumor immunity in response to vaccination. We and others have reported on the utility of tumor vaccines as a means of enhancing the graft-versus-tumor effect in alloBMT recipients14-22, and this strategy was also reported in a clinical trial23. We previously showed in murine models that DNA tumor vaccination after allo-BMT is quite effective, capable of producing anti-tumor T cell responses, rejection of subsequent tumor challenges, and markedly improving the long-term survival of tumor-bearing mice in treatment models14. Thus far strategies to enhance CD8+ T cell responses to vaccination have not focused on agents with a predominant effect on thymopoiesis. Therefore we assessed the effects of administrating KGF to allo-BMT recipients receiving DNA tumor vaccination. In this study we utilized the murine melanoma model B16, as well as a DNA plasmid vaccine targeting the melanoma antigen tyrosinase-related protein 1 (TRP1) to examine the effects of KGF on responses to tumor vaccination after allo-BMT.

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Materials and Methods Mice and stem cell transplantation. Female C57BL/6 (B6, H-2b) and LP (H-2b) mice 8-10 weeks of age were obtained from the Jackson Laboratory. On day 0, bone marrow cells were removed aseptically from femurs and tibias of LP mice. Donor bone marrow was T cell-depleted by incubation with anti-Thy-1.2 for 40 min at 4°C followed by incubation with Low-TOX-M rabbit complement (Cedarlane Laboratories) for 40 min at 37°C. Five million cells were transplanted by tail vein injection into lethally irradiated B6 recipients (11 Gy total body irradiation from a 137Cs source as a split dose with a 2.5- to 3-hour interval between doses). Mice were housed in sterilized microisolator cages and received normal chow and autoclaved hyperchlorinated drinking water (pH 3.0). BMT protocols were approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee.

KGF administration. Recombinant human KGF was administered daily at 5 mg/kg/day subcutaneously on days -6, -5, and -4 prior to allo-BMT as previously described4. Recombinant human KGF was kindly provided by Amgen (Thousand Oaks, CA). Control mice received PBS instead of KGF.

DNA plasmid vaccine construct. VP22-opt-TRP1 is a DNA plasmid vaccine based on a tyrosinase-related protein 1 (TRP1) vaccine described previously24, designed using software based on the concept of heteroclitic peptides25. The coding sequence of mouse TRP1 was modified to optimize MHC class I binding to both Kb and Db, and subsequently fused to VP22, an HSV1 protein that has been shown to enhance vaccine potency26. We have previously shown

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that immunization with the plasmid vector alone did not induce tumor rejection or antigenspecific responses27.

DNA vaccine administration. Mice were immunized by helium-driven particle bombardment, as previously reported28. Briefly, plasmid DNA was purified, coated onto 1-µm diameter gold particles (Alfa Aesar), and allowed to settle on bullets of Teflon tubing. Gold particles containing 1 µg of DNA were delivered to each abdominal quadrant using a helium-driven gene gun (Accell; PowderMed), for a total of 4 µg of DNA per mouse. Mice were immunized every five days starting day 14 for a total of either 3 or 5 immunizations and harvested 6 days after the last immunization for cell counts and flow cytometry studies. For survival studies, tumor-bearing mice were vaccinated every 5 days starting day 14.

Mouse tumor studies and in vivo bioluminescence. Tumor challenge experiments were conducted with melanoma B16 cells. Briefly, 5×104 B16F10 melanoma cells (gift from I. Fidler, M.D. Anderson Cancer Center, Houston, TX) were injected intravenously by tail vein. Mice were monitored daily and were euthanized when suffering from respiratory distress or found to have progressive large bulky tumors > 1cm in diameter. Survival was assessed from the day of tumor challenge. Kaplan-Meier survival curves were generated and compared using the log-rank test. For quantitating tumor burden, B16-TGL cells (B16 retrovirally transduced to express a triple fusion protein consisting of Herpes simplex virus thymidine kinase, enhanced green fluorescent protein and firefly luciferase)29 were administered intravenously at a dose of 10×105. Bioluminescent signal intensity of tumor-bearing mice was determined weekly starting 2 weeks after BMT. Ten minutes after intra-peritoneal injection of 3 mg/mouse D-Luciferin (Caliper LS),

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mice were anesthetized and placed into the light tight chamber of an IVIS 200 bioluminescence imaging system (Xenogen). Gray-scale photographic images of the mice were acquired first and then a low-level bioluminescent signal was recorded. Pseudo-color images showing the whole body distribution of bioluminescent signal intensity were superimposed on the gray-scale photographs and total flux (photons/sec) was determined for individual mice.

Cells. Single-cell suspensions were prepared from spleen, thymus, and lymph nodes (LNs) according to standard protocols and analyzed by flow cytometry.

Antibodies and flow cytometry. Anti-murine CD16/CD32 Fc receptor block (2.4G2) and all of the following fluorochrome-labeled antibodies against antigens (all murine unless otherwise indicated) were obtained from BD Pharmingen: CD3ε (500A2), CD4 (RM4-5), CD45 (30-F11), CD8α (53-6.7), cleaved caspase-3 (C92-605), human KI-67 (B56), Ly 9.1 (30C7), IFN-γ (XMG1.2), TNF-α (MP6-XT22) and Vβ TCR screening panel. Antibodies against CD44 (IM7), CD62L (MEL-14), and Foxp3 (FJK-16s) were obtained from BioLegend, Caltag, and eBioscience, respectively. iTAg MHC tetramer with peptide TAPDNLGYM (amino acids 455463 from murine TRP1, optimized for H-2Db-binding) was custom ordered from Beckman Coulter. Intracellular cytokine production was assayed with overnight stimulation of whole splenocytes with peptide TAPDNLGYA (amino acids 455-463 from murine TRP1, wild-type sequence) at a concentration of 1 μg/mL in the presence of Golgiplug from BD Pharmingen per the manufacturer's directions. Cells were acquired on a LSRII cytometer (BD Biosciences) with FACSDiva software. Data were analyzed with FlowJo software (Tree Star).

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Statistics. Statistical comparisons of experimental data were performed with the nonparametric unpaired Mann-Whitney t test. A p value less than 0.05 was considered statistically significant. For Vβ experiments, background usage for each Vβ was determined by the 20% trimmed-mean percentage of Vβ+ tetramer-negative cells from the same experiments.

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Results KGF administration increases thymic cellularity and peripheral T cell numbers in alloBMT recipients. A previous study of KGF in mice undergoing T cell-depleted MHC-disparate allo-BMT utilized the BALB/c→B6 (H2d→H2b) model and found enhanced thymopoiesis and increased T cell numbers in the periphery as early as day 28 after BMT10. In the current study, we examined the effects of KGF in a model with minor histocompatibility antigen disparities, LP→B6 (H2b→H2b), which we used previously in our studies of DNA tumor vaccination14. On day 0, B6 mice were lethally irradiated and intravenously injected with T cell-depleted LP bone marrow. KGF was given subcutaneously prior to BMT on days -6, -5, and -4. We found in this model that KGF produces a two-fold increase in thymic cellularity as early as day 19 (Figure 1A), similar to the results in the MHC-disparate model. Splenic and inguinal lymph node (LN) cellularities were unchanged in KGF-treated recipients (data not shown), but the composition of cells in these peripheral lymphoid organs were markedly changed, with increased total numbers of CD4+ and CD8+ cells in both spleens (Figures 1B-C) and inguinal LNs (Figures 1D-E), as well as increased percentages of CD4+ and CD8+ cells (data not shown). KGF increases the overall survival of tumor-bearing mice treated with allo-BMT with and without DNA tumor vaccination. We hypothesized that the accelerated T cell reconstitution seen with KGF administration would lead to more robust T cell responses to tumor vaccination. To test this, we utilized a DNA plasmid vaccine targeting the melanoma differentiation antigen TRP1. Mice were inoculated intravenously with B16 melanoma 7 days prior to allo-BMT, received DNA immunizations starting on day 14 after allo-BMT delivered by gene gun every 5 days, and were monitored for survival. We found that BMT alone had a significant impact on survival (Figure 2A). While adding DNA vaccination to BMT did not

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produce an appreciable benefit, mice treated with both KGF and DNA vaccination showed marked improvement in median survival and 37% 90-day survival (Figure 2B). The combination of KGF and vaccination trended towards a benefit compared with KGF alone (p=0.08). We found a similar benefit when we replaced the DNA vaccine with a GM-CSF-secreting B16 cell vaccine (data not shown). To evaluate the effect of our treatments on tumor burden, we utilized an in vivo biolumenscence technique using luciferase-positive B16-TGL cells. We found that again, DNA vaccination did not add significantly to BMT alone, but treatment with KGF+DNA resulted in improved control of tumor burden (Figures 2C, D). With the more immunogenic B16-TGL cell line, which expresses a triple fusion protein consisting of Herpes simplex virus thymidine kinase, enhanced green fluorescent protein and firefly luciferase, we found that KGF+DNA was no longer trending towards a benefit compared to KGF alone. This raised the possibility that KGF may act independently of vaccination, and we thus performed a series of experiments to assess if KGF was improving the efficacy of DNA vaccination by augmenting immune responses. KGF administration increases the number of T cells with vaccine-recognizing potential in unvaccinated allo-BMT recipients, as well as the number of CD4+Foxp3+ regulatory T cells (Tregs). We first evaluated the frequency of CD8+ T cells that could potentially respond to vaccination. CD8+ T cells from spleens and inguinal LNs were purified with magnetic beads on day 19 after BMT and stained with 455-opt-TRP1 tetramer to identify CD8+ cells that, in the absence of vaccination, recognize the immunodominant heteroclitic epitope of our DNA vaccine. We found that while the tetramer+ percentage of CD8+ cells does not change with KGF, the total number of tetramer+ cells in each individual mouse is increased in the KGF-treated group (Figures 3A-B). Thus the overall increase in CD8+ cells seen in KGF-

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treated mice results in a concurrent increase in the number of CD8+ T cells recognizing our DNA vaccine. In a similar manner, we found that while KGF-treated mice had percentages of CD4+ cells that expressed Foxp3 similar to mice not treated with KGF, an overall increase in CD4 + T cells led to an increased total number of Tregs (Figures 3C-D). In non-transplanted mice, KGF was reported previously to increase both the frequency and total numbers of Tregs30. After vaccination KGF-treated allo-BMT recipients have increased numbers of tumor-specific T cells without increased Tregs. We then performed vaccination experiments, administering DNA immunizations every 5 days starting on day 14 after BMT. After the third vaccination, we found a marked increase in tetramer+ cells in KGF-treated vaccinated mice compared with untreated vaccinated mice (Figure 4A). We also again assessed the numbers of Tregs in our KGF-treated versus untreated allo-BMT recipients, now both vaccinated, and found no significant difference (Figure 4B). The ratio of T effector cells to Tregs has been suggested to predict effective immune responses31. In our model, we found that KGF-treated allo-BMT recipients indeed display an increased tumor-specific T cells to Treg ratio compared with control allo-BMT recipients (Figure 4C). Our data demonstrate that post-transplant vaccination of KGFtreated allo-BMT recipients produces improved T cell tumor immunity through an increase in tumor-specific T cells and T effector to Treg ratio. We also evaluated the functionality of CD8+ T cells in vaccinated allo-BMT recipients by assaying for production of IFN-γ and TNF-α in response to stimulation with wild-type TRP1 peptide. We found that KGF-treated mice had increased numbers of CD8+ T cells producing either IFN-γ, TNF-α, either, or both cytokines. Polyfunctionality has been identified as an indicator of superior CD8+ T cell responses in terms of control of HIV replication32.

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KGF does not affect the proliferation, survival or chimerism of vaccine-generated tumor-specific T cells, but does increase the proportion with a central memory phenotype. We then asked if the increased T cell vaccine responses seen in KGF-treated mice was due to changes in the proliferation or survival of CD8+ cells responding to the vaccine. For these studies, we found that assaying after 5 immunizations allowed for adequate numbers of tetramer+ cells in both groups for analysis. Using flow cytometry, we found no significant changes in expression of either Ki-67 or cleaved caspase-3 in tetramer+ cells from mice treated with KGF, suggesting that KGF administration has no effect on the proliferation or apoptosis of tetramer+ cells (Figures 5A-B). We also assayed for donor or host origin at this time point and found no changes in chimerism of tetramer+ cells due to KGF (data not shown). A growing body of literature has demonstrated that central memory CD8+ cells, described as CD44highCD62Lhigh, compared with effector memory cells (CD44highCD62Llow)33, are longerlived, have improved proliferative potential, and produce more effective immune responses34. Using flow cytometry, we found that while the majority of tetramer+ cells in both groups displayed an effector memory phenotype, KGF-treated allo-BMT recipients had a greater than 2fold increase in the percentage of cells with a central memory phenotype (Figure 5C). Vaccine responses in KGF-treated allo-BMT recipients are derived from a more varied T cell receptor repertoire. Since KGF administration enhances post-transplant thymopoiesis, we hypothesized that CD8+ cells responding to vaccine should be derived from a broader T cell receptor (TCR) repertoire. The superiority of a diverse TCR repertoire in controlling viral infections has been suggested by others35,36. We assayed for TCR repertoire breadth by flow cytometry with Vβ family-specific antibodies. We compared Vβ+ percentages of tetramer+ cells with background Vβ usage, determined by Vβ+ percentages of tetramer─ cells

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from the same experiments (Figure 6A). We defined tetramer+ usage of a particular Vβ as having a Vβ+ percentage greater than background. With this analysis, we found that in the KGF-treated group, tetramer+ cells in a given individual are derived from a median of five Vβ families, compared to 3.5 Vβ families in controls (Figure 6B). This suggests that mice which received KGF prior to allo-BMT have vaccine-responsive cells that are derived from a broader TCR repertoire.

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Discussion KGF administration to patients receiving high-dose cytotoxic therapy can reduce the duration and severity of mucositis1,2. Preclinical studies also suggest that KGF may ameliorate GVHD37-39 and enhance post-BMT thymopoiesis4,10-13. In this study, we demonstrate an additional potential benefit of KGF: the enhancement of responses to vaccination after T cell-depleted allo-BMT. Our data suggest that KGF exerts its effects primarily by accelerating T cell reconstitution, thus increasing the availability of naïve T cells capable of responding to vaccination. With vaccination, this leads to increased numbers of tumor-specific T cells, which then translates into improved survival in tumor-bearing mice. To our knowledge, this report is the first demonstration of a thymopoietic agent acting as a CD8+ T cell vaccine adjuvant. We also found unanticipated benefits to anti-tumor immunity with KGF administration, including an improved T effector-to-Treg ratio. Several studies have demonstrated that T cell responses in the lymphopenic setting are markedly enhanced40. Several mechanisms for this phenomenon have been identified, including the relative absence or dysfunction of immune regulatory cells such as Tregs and the increased availability of pro-survival T cell cytokines including IL-7 and IL-15. Our data suggest that administering KGF in the setting of allo-BMT allows generation of more vaccine-responsive precursors. Upon vaccination, KGF-treated alloBMT recipients generate more tumor-specific T cells with an improved T effector-to-Treg ratio. These T effector cells can then benefit from the increased availability of IL-7 and IL-15 in the lymphopenic setting. We found that KGF-treated allo-BMT recipients, when vaccinated, generate a larger fraction of T effector cells that display a central memory, rather than effector memory phenotype. The superiority of central memory T cells over effector memory T cells in

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maintaining immune responses has been demonstrated by others34. This decrease in effector differentiation may be related to the increase in numbers of vaccine-specific T cells that we observed both prior to and after vaccination. Others have reported that in situations of increased antigen availability and decreased clonal competition, full terminal differentiation to an effector phenotype is accelerated41. It is possible that in KGF-treated individuals, the increase in antigenspecific clones leads to less available antigen an a per-cell-basis, resulting in increased numbers of tumor-specific T cells with a central memory phenotype. We also found that T effector cells from KGF-treated allo-BMT recipients are more likely to produce both IFN-γ and TNF-α, and are derived from a broader TCR repertoire. Studies in various viral models suggest that T cells exhibiting polyfunctionality and a diverse TCR repertoire may be more effective at controlling viral replication32 and preventing development of escape variants35,36, respectively. In our survival experiments, a surprising finding was that the KGF+BMT group showed a survival benefit compared with BMT alone, the reasons for which are unclear. The effects of KGF on in vivo proliferation and as a radiosensitizer of B16 were studied previously and found to be negligible42. In our experiments, KGF produced more dramatic benefits with B16-TGL tumors, which are potentially more immunogenic. This suggests that KGF in the setting of BMT could be supporting immune-mediated suppression of tumor growth by accelerating T cell reconstitution even in the absence of tumor vaccine. In retrospective clinical studies, early lymphocyte recovery has been associated with decreased relapse and improved overall survival after autologous BMT for various malignancies43,44. It is possible that the benefits derived from KGF may also be associated with other strategies to improve T cell generation, such as sexsteroid blockade45, growth hormone46, leptin47, IL-748 and insulin-like growth factor I48.

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In summary, KGF administration to allo-BMT recipients produces improved thymopoiesis, resulting in improved T cell reconstitution. This leads to enhanced responses to DNA tumor vaccination and improved survival in tumor-bearing allo-BMT recipients. Interestingly, KGF produced additional benefits to anti-tumor immunity, including an improved T effector-to-regulatory T cell ratio, a larger fraction of effector cells that display a central memory phenotype, more effectors producing both IFN-γ and TNF-α, and effector cells that are derived from a broader T cell receptor repertoire.

Acknowledgements This research was supported by National Institutes of Health grants RO1-HL069929 (MvdB), RO1-CA107096 (MvdB), RO1-AI080455 (MvdB) and PO1-CA33049 (MvdB). Support was also received from the Ryan Gibson Foundation, the Elsa U. Pardee Foundation, the Byrne Fund, the Emerald Foundation, and The Experimental Therapeutics Center of Memorial SloanKettering Cancer Center funded by Mr. William H. Goodwin and Mrs. Alice Goodwin, the Commonwealth Foundation for Cancer Research, The Bobby Zucker Memorial Fund (MvdB) and The Lymphoma Foundation. R.R.J. is the recipient of grants from the American Association for Cancer Research - MedImmune Fellowship for Research on Biologics-Based Therapies for Cancer and the Leukemia & Lymphoma Society Special Fellowship in Clinical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Authorship R.R. Jenq designed and performed research, analyzed data, and wrote the paper. C.G. King performed research and contributed to writing the paper. C. Volk performed research and

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contributed to writing the paper. D. Suh performed research and contributed to writing the paper. O.M. Smith performed research and contributed to writing the paper. U.R. performed research and contributed to writing the paper. N.Y. performed research and contributed to writing the paper. A.M. Holland contributed to writing the paper. S.X. Lu contributed to writing the paper. J.L. Zakrzewski contributed to writing the paper. G.L. Goldberg contributed to writing the paper. A. Diab designed research and analyzed data. O. Alpdogan designed research and analyzed data. O. Penack designed research and analyzed data. I. Na designed research and analyzed data. L.W. Kappel designed research and analyzed data. J.D. Wolchok analyzed data and contributed vital reagents. A.N. Houghton analyzed data and contributed vital reagents. M. Perales designed research and analyzed data. M.R.M. van den Brink designed research, analyzed data, and wrote the paper. The authors have no conflicts of interest to declare.

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Figure legends

Figure 1. KGF increases thymic cellularity and numbers of peripheral T cells on Day 19 after allogeneic BMT. Eight- to 10-week old B6 mice were lethally irradiated (11 Gy) and transplanted with T cell-depleted LP BM (5×106). Some recipients received KGF (5 mg/kg/day) or PBS subcutaneously pre-transplant on days -6 to -4. On day 19, thymic cellularity (A*, p=0.002), CD4+ and CD8+ populations from spleens (B*, p=0.02, and C*, p=0.003) and inguinal LN (D*, p=0.02, and E*, p=0.02) were determined by flow cytometry. Values from individual mice are shown with medians. Data are pooled from 2 experiments with similar results.

Figure 2. KGF improves overall survival and tumor burden of tumor-bearing mice undergoing allogeneic BMT and tumor vaccination. B6 mice were inoculated intravenously with syngeneic B16 melanoma (5×104 cells) 7 days prior to T cell-depleted BMT. BMT alone had a significant effect on overall survival (A*, p 1cm in diameter. Data shown are pooled from 4 experiments with similar results (B*, p=0.003, **, p=0.01, KGF+BMT vs. KGF+BMT+DNA not significant, p=0.08). In a similar experiment, B16-TGL (1×105 cells) expressing firefly luciferase was substituted for B16. Weekly, starting day 21 after tumor challenge, mice were evaluated with in vivo bioluminescence imaging, with median values of luminescence shown in C, pooled from 2 experiments with similar results. Individual data from day 35 are shown in D (*, p=0.047, **, p=0.008).

Figure 3. On day 19 after BMT without vaccination, KGF does not change the percentage of CD8+tetramer+ cells which could respond to vaccination, or the percentage of CD4+ cells which express Foxp3 , but does increase the overall number CD8+tetramer+ and CD4+Foxp3+ cells in each mouse. B6 mice underwent T cell-depleted BMT and were treated with KGF as described in Figure 1. On day 19, frequency and total number of CD8+tetramer+ cells were determined by purifying peripheral CD8+ cells (combined spleens and inguinal LNs) using magnetic beads, and staining with opt-TRP1-455 tetramer. Purified CD8+ cells staining for tetramer (A and B*, p=0.004) and splenocytes staining for Foxp3 (C and D*, p