Activated MHC-Mismatched T helper-1 Lymphocyte Infusion Enhances Graft-versus-Leukemia with Limited Graft-versus-Host Disease

Jessica Stokes1,2,3, Emely Hoffman3, Seongmin Hahn3, Yi Zeng3, Emmanuel Katsanis3.1Department of Molecular and Cellular Biology. 2Department of Chemistry and Biochemistry. 3Department of Pediatrics, Steele Children’s Research Center. The University of Arizona, Tucson, AZ.

ABSTRACTDonor lymphocyte infusion (DLI) is used to provide graft-versus-leukemia (GvL) effects when given to patients relapsing post-hematopoietic stem cell transplantation (HSCT), but is associated with significant graft-versus-host disease (GvHD). Novel cellular therapies are needed. These cell therapies should be optimized to induce GvL with an acceptable degree of GvHD. Activated allogeneic T helper-1 (aTh-1) cells are CD4+ CD25+ CD40L+ CD62Llo effector memory cells that produce large amounts of IFN-γ and TNF-α. We have shown that when aTh-1 cells are combined with tumor vaccination, they augment long-lasting protection against murine leukemia. In this study, we demonstrate that post-transplant adoptive aTh-1 cell therapy enhances GvL with limited GvHD in a major histocompatibility complex (MHC)-mismatched murine bone marrow transplantation (BMT) model. aTh-1 cell infusions result in superior leukemia-free survival when compared to splenocytes (SC) and persist in vivo for at least two months following adoptive transfer. In contrast to SC infusion, aTh-1 cell therapy is associated with only transient, mild suppression of donor-derived hematopoiesis. aTh-1 cell therapy may, therefore, provide an effective alternative to DLI.

SupportFunding provided by NIH grant R01 CA104926, Tee Up for Tots, and The Hyundai

Foundation. Thanks to Alexis Lorenz, Martin Asimis, Neale Hanke, Darya Alizadeh, and Dr. Nicolas Larmonier for their support and contribution.

THERAPEUTIC METHOD

CONCLUSIONS AND APPLICATION

RESULTS

• aTh-1 therapy improves the survival of mice bearing A20 leukemia following an MHC-mismatched BMT.

• aTh-1 cells are associated with reduced graft-versus-host disease toxicity compared to SC.

• aTh-1 cells are positive for Th-1 and activation markers and produce type-1 cytokines.

• aTh-1 cells persist in vivo without suppressing or skewing hematopoietic reconstitution.

• aTh-1 cells may be an effective alternative to DLI.

aTh-1 therapy following allogeneic BMT is associated with minimal GvHD

when compared to total SC

PBS aTh-1 SC

Figure 1. BALB/c recipient mice received 1x106 A20 cells intravenously (i.v.) on day -5. On day -1, the mice received 750 cGy of total body irradiation (TBI) (Cesium-137 Irradiator). On day 0, the femurs, tibiae, humeri, and iliac bones of C57BL/6 donor mice were harvested, and bone marrow (BM) was flushed from the bones. After red blood cell lysis, T-cells were depleted (CD3e MicroBeads; Miltenyi Biotec). The recipient mice received 107 BM cells i.v. aTh-1 cells were generated from the spleens of C57BL/6 mice. Red blood cells were lysed (BD Pharm Lyse), and a positive selection for CD4+ cells was performed using CD4 (L3T4) MicroBeads and an LS column (Miltenyi Biotec). Cells were cultured in 24-well plates (0.5x106/mL/well) with 20 IU/mL IL-2, 20 ng/mL IL-7, 10 ng/mL IL-12 (Peprotech), 5 μg/mL anti-IL-4 (eBioscience), and anti-CD3/anti-CD28 T-cell expander beads (Invitrogen) at a ratio of 3 beads:1 cell. On days 12 and 17, the recipient mice were injected with 3x106 aTh-1 or 107 total SC i.v.

Colon

Liver

aTh-1 cell therapy improves survival of leukemia-bearing mice over SC

Figure 2. BALB/c mice received A20 tumor and subsequent treatments, as shown in Figure 1. Mice received phosphate buffered saline (PBS) or aTh-1 treatment, with and without BMT or SC post-BMT. Survival was monitored, and moribund mice were euthanized. Kaplan-Meier survival curves are shown, and the log-rank test was used to determine PBS or aTh-1 vs. BMT + PBS, BMT + PBS vs. BMT + aTh-1, and BMT + aTh-1 vs. BMT + SC are significant. Representative data from four independent experiments is shown.

A

B

Day 0 Day 3 Day 5

CD4

CD44

CD25

CD45RB

Day 4

CD62L

CD40L

CD69

Tbet

IFN-γ

Figure 3. Mice received tumor and subsequent treatment as described in Figure 1. A. At various time points, mice were euthanized and the colons and livers were collected and analyzed histologically by a veterinary pathologist. Representative H&E staining (50X magnification) are shown, collected five days after adoptive cell infusion. The SC colon and liver show inflammation. The PBS liver shows leukemic infiltrates. The graph shows colon scores of individual mice, higher numbers indicating more abnormailty. Representative data from three independent experiments. B. Mice were weighed every three to four days. The weight before adoptive transfer was considered their starting weight (100%). The graph shows the lowest percent weight achieved by individual mice. Representative data from three independent experiments is shown. p<0.05 considered significant.

aTh-1 cells persist in vivo and are less proliferative than SC

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Figure 4. BALB/c mice received TBI, a BMT, an infusion of 3x106 allogeneic aTh-1 cells or SC (generated from CD45.1+ BoyJ mice to allow us to distinguish between donor BM and adoptively-transferred cells) on day 12 for in vivo tracking. On days 3, 7, 10, 17, 24, and 31 after adoptive cell transfer, peripheral blood was collected by tail vein bleeding. Complete blood counts (CBC) were obtained using a Coulter AcT counter (Beckman Coulter). The presence and phenotype of adoptively-transferred aTh-1 cells and SC were determined by flow cytometry. A. The absolute number of adoptively-transferred cells was determined by multiplying the CBC by the percentage of CD45.1+ cells. SC vs. aTh-1: p<0.05 on days 7, 10, and 31. B. Ki-67 flow staining was done separately on days 4, 8, 11, 18, and 25. The graph shows the percentage of CD45.1+ cells (adoptively-transferred cells) that were Ki-67+. SC vs. aTh-1: p<0.05 at all time points except for day 18. N=3 to 4 mice per group. Representative data from three independent experiments is shown.

A B

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aTh-1 cells do not suppress or skew hematopoiesis A

B

C

Figure 5. As described in Figure 4, blood was collected from mice on days 3, 7, 10, 17, 24, and 31 and analyzed by flow cytometry. A. The absolute number of donor-derived BM cells was determined by multiplying the CBC by the percentage of H-2kb+ CD45.2+ cells. SC vs. PBS: p<0.05 on days 10, 24, and 31; aTh-1 vs. PBS: p<0.05 on day 24. B. Of the CD3e+ donor-derived BM T-cells, the percentage of CD4 versus CD8 cells was determined. SC vs. PBS: p<0.05 at all time points. aTh-1 vs. PBS: n.s. C. The percentage of polymorphonuclear neutrophils (PMNs), monocytes, B-cells, and T-cells in the donor BM-derived population. For B-cells, SC vs. PBS: p<0.05 at all time points. For PMNs, SC vs. PBS: p<0.05 on days 3, 7, 10, and 17. For monocytes, SC vs. PBS: p<0.05 on days 7, 17, 24, and 31. For T-cells, SC vs. PBS: n.s. For monocytes, aTh-1 vs. PBS: p<0.05 on days 7 and 10. For everything else, aTh-1 vs. PBS: n.s.

aTh-1 cells produce type-1 cytokines and express markers consistent with activated Th-1

lymphocytesA

B

Figure 6. aTh-1 cells were cultured for five days. On each day, cells were harvested, de-beaded, and re-plated for 4 hours with activation beads and IL-2. Supernatant was used for ELISA assays (A), and cells were used for flow cytometric analysis (B). Representative data of three independent experiments.