Recipient T Cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells

Accepted Manuscript Title: Recipient T Cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells Author: Robin L. Williams, Sarah Cooley, Veronika Bachanova, Bruce R. Blazar, Daniel J. Weisdorf, Jeffrey S. Miller, Michael R. Verneris PII: DOI: Reference:

S1083-8791(17)30869-8 https://doi.org/10.1016/j.bbmt.2017.11.022 YBBMT 54879

To appear in:

Biology of Blood and Marrow Transplantation

Received date: Accepted date:

16-6-2017 12-11-2017

Please cite this article as: Robin L. Williams, Sarah Cooley, Veronika Bachanova, Bruce R. Blazar, Daniel J. Weisdorf, Jeffrey S. Miller, Michael R. Verneris, Recipient T Cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells, Biology of Blood and Marrow Transplantation (2017), https://doi.org/10.1016/j.bbmt.2017.11.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Recipient T cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells Authors: Robin L Williams, MD1, Sarah Cooley, MD2, Veronika Bachanova, MD2, Bruce R Blazar, MD3, Daniel J Weisdorf, MD2, Jeffrey S Miller, MD2, and Michael R Verneris, MD4 1

Division of Pediatric Hematology/Oncology, University of Minnesota Masonic Children’s Hospital, Minneapolis, MN 2

Division of Hematology, Oncology and Transplantation, University of Minnesota Medical Center, Minneapolis, MN 3

Division of Pediatric Blood and Marrow Transplantation, University of Minnesota Masonic Children’s Hospital, Minneapolis, MN 4

Pediatric Hematology/Oncology/Bone Marrow Transplant, University of Colorado Anschutz Medical Campus, RC1 North, MS 8302, Aurora, CO 80045 Word Count Abstract: 194 Body: 1390 Figures: 2 Tables: 0

Short Title: Recipient T cell exhaustion and donor NK expansion

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Key Point: 

Prior to chemotherapy, AML patients with absolute CD3+ T cell count > 500/µL are less likely to have donor haplo-NK cell expansion.

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Recipient T cell exhaustion at D+14 after chemotherapy and haplo-NK infusion is associated with in vivo donor NK cell expansion.

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Abstract: NK cells mediate surveillance for malignancy. In some chemotherapy refractory myeloid leukemia patients, adoptive transfer of NK cells from haploidentical donors can induce remission. We have previously shown that remission induction is linked to NK persistence at D+14, but the factors influencing NK cell persistence are unknown. To address this question, patient samples from a phase I trial of NCI IL-15 in whom either did or did not show NK cell expansion were compared to healthy donor controls. Prior to lymphodepleting chemotherapy, high absolute CD3+ count was predictive of patients who failed to expand their haplo-NK cell graft. Interestingly, both groups had elevated expression of inhibitory receptors and decreased cytokine production compared to controls, suggestive of T cell exhaustion among all patients prior to haplo-NK cell infusion. At D+14, however, haplo-NK expanders had persistence of recipient CD8+ T cells with the most exhausted inhibitory phenotype (either PD-1high or dual PD1+Tim-3+) and elevated expression of T-bet and Eomes compared to NK cell non-expanders and controls. This suggested that maintenance of an exhausted T cell state at D+14 permits haploNK cell expansion and supports further efforts to selectively deplete recipient T cells or modulate their dysfunction.

Keywords: NK cells T cell exhaustion AML

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Introduction: Natural killer (NK) cells are cytotoxic innate lymphoid cells, which play a role in cancer surveillance.1 Haploidentical NK cells (haplo-NK) can be safely infused following lymphodepleting chemotherapy and successful in vivo expansion 14 days after infusion (D+14) is associated with remission induction in chemotherapy-refractory acute myeloid leukemia (AML) patients.2,3 The factors that modulate haplo-NK cell expansion are unknown and understanding the regulators could impact future adoptive haplo-NK cell therapeutics. We hypothesized that recipient T cells dampen proliferation of adoptively transferred, MHCmismatched haplo-NK cells and that patients with T cell dysfunction are more likely to have haplo-NK persistence. T cell exhaustion is an established state of T cell dysfunction occurring after chronic and continuous antigen stimulation and is well-documented in human cancer.4,5 It is characterized by progressive loss of effector functions, such as loss of cytokine production in response to stimulation, co-expression of multiple inhibitory receptors, including PD-1 and Tim-3 and the altered use of key transcription factors, Eomes and T-bet.4 To test our hypothesis, we used samples collected before and after haplo-NK cell adoptive therapy to investigate recipient T cell exhaustion in patients with or without successful in vivo haplo-NK expansion.

Methods: Clinical trial, NK cell expansion and patient samples: This research was approved by the IRB and all human participants gave written informed consent. NK cell persistence was prospectively determined at D+14 after adoptive transfer using short tandem repeat analysis performed on peripheral blood (PB) and correlated with NK cell absolute number.3 Patients who had donor DNA and NK cells present at this time point had at least 75 donor NK cells/µl and were defined as “haplo-NK expanders” and those with no donor DNA and T cells present were defined as haplo-NK non-expanders. Using this definition, we compared PB samples from 4 haplo-NK expanders, 6 haplo-NK non-expanders and 5 healthy donor controls. These patients 4 Page 4 of 15

were enrolled on University of Minnesota protocol (MT2010-10) which is a phase I dose escalation trial of NCI monomeric IL-15 administered IV after lymphodepleting chemotherapy (cyclophosphamide 50 mg/kg and fludarabine 25 mg/m2) and NK cell infusion. Donor apheresis products were enriched for NK cells through T cell- and B cell- (CD3/CD19) depletion and then activated overnight in the presence of NCI IL-15 (10 mg/mL). On D+1 through D+12, patients received daily IV infusions of NCI IL-15.

In Vitro Assays: Samples were thawed and incubated for 12-18 hours in RPMI media. For stimulation assays, CD3/CD28 beads (Life Technologies) were added 1:1 to the media during this incubation. Antibodies: L/D aqua (ThermoFisher), CD3-PreCP-Cy5.5, CD8-APC-C7, CD56BV605 (BD), PD-1-FITC, Tim-3-BV711, TIGIT-APC (R&D Systems), CD45RA-AF700 (BD), CD27-PB, T-bet-PE-Cy7, Eomes-PE (eBioscience), IL-2-PE-Cy7, TNF-α-PE, and IFN-γ-PB. (BioLegend unless otherwise specified.) Cells were evaluated on BD LSR II flow cytometer and analyzed using FlowJo V10. Fluorescence minus one (FMOs) samples determined positive antibody expression.

Statistical Analysis – GraphPad software of one way ANOVA with Sidak’s multiple comparisons test was used to compare groups.

Results and Discussion: Recipient absolute CD3+ T cell Count Predicts Success of Haploidentical NK Cell Expansion at D+14. We first examined the T cell compartment of haplo-NK cell expanders and compared them to that of haplo-NK non-expanders to determine whether there were predictable differences before and after NK cell infusion. Prior to chemotherapy, there was a general trend toward higher absolute counts including ALC, CD3+ and CD8+ in the haplo-NK non-expanders compared to 5 Page 5 of 15

the expanders although only the absolute CD3+ T cell count reached statistical significance between the two groups (Figure 1A-C). There was also a trend toward a higher percentage of CD3+ or CD8+ T cells in the haplo-NK non-expanders compared to the expanders but this difference did not reach statistical significance (Figure 1D, E). Interesting, the percentage of CD3+ T cells among haplo-NK expander group was significantly less than controls and this finding persisted at D+14 (Figure 1D). Additionally, at D+14, the percentage of CD3+ T cells in the haplo-NK non-expander group was significantly increased compared to the haplo-NK expanders and the percentage of CD8+ T cells in this group was significantly higher than both the expander group and controls (Figure 1D, E). There was no difference in T cell naïve or memory subsets between groups (Figure 1F).

Haploidentical NK cell Expanders Maintained a Highly-Exhausted T cell Phenotype at D+14. Next, we evaluated the expression of PD-1 and Tim-3 individually on CD8+ T cells, as well as states most suggestive of T cell exhaustion, including high expression of PD-1 and dual PD1+Tim-3+ cells.6 Prior to chemotherapy, the percent expression and median fluorescent intensity (MFI) of these two inhibitory receptors were increased but not significantly on CD8+ T cells in both haplo-NK expander and non-expander groups compared to controls (Figures 1G-J). At D+14, however, higher percentages and MFI of PD-1 and Tim-3 were maintained on the CD8+ T cells of haplo-NK cell expanders, while non-expanders became indistinguishable from controls (Figures 1G-J). There was a similar pattern of inhibitory receptor expression among PD-1hi cells (Figure 1K) and dual PD-1+Tim-3+ cells (Figure 1L) such that both patient groups had a relative increase in the percent expression prechemotherapy, but at D+14, this exhausted recipient T cell phenotype only persisted in patients with haplo-NK cell expansion.

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Increased Inhibitory Receptor Expression Correlates with Impaired CD8+ T cell Cytokine Production Prechemotherapy and Elevated Transcription Factor Expression at D+14 in Haploidentical NK cell Expanders. T cells exhaustion is not defined by sustained up-regulation and co-expression of inhibitory receptors alone, but in combination with progressive loss of function and altered transcription factor programs.4 Therefore, lastly, we assessed CD8+ T cells with the most exhausted inhibitory receptor phenotypes, PD-1hi and dual expression of PD-1+Tim-3+, for altered cytokine expression and transcription factor expression. Prior to chemotherapy, the percent expression of IL-2, TNF-α, and IFN-γ was examined after overnight stimulation with CD3/CD28 beads. Among PD-1hi (Figures 2A-C) and dual PD-1+Tim-3+ CD8+ T cells (Figures 2D-F), both haplo-NK cell expanders and non-expanders had decreased expression of cytokines compared to controls with IL-2 reaching significance in the former group and both IL-2 and IFN-α being significant in the latter group. Due to limited sample availability, a similar comparison could not be performed at the D+14. Alternatively, we evaluated PD-1hi and dual PD-1+Tim-3+ CD8+ T cells at D+14 for altered expression of key transcription factors, T-bet and Eomes.7 Both transcription factors were increased among PD-1hi CD8+ T cells in the haplo-NK expander group, compared to the non-expanders and controls (Figure 2G,H). Similarly, among dual PD-1+Tim-3+ CD8+ T cells, Eomes expression was significantly increased in the haplo-NK expander group (Figure 2J), while T-bet was increased in both patient groups compared to controls, albeit to a lesser extent in the haplo-NK non-expanders compared to the expanders (Figure 2I).

Alloreactivity, Haplo-NK Expansion and Recipient T cell Exhaustion: Potential for AML Remission We have previously shown that haplo-NK cell infusions are safe and can induce remission in chemotherapy refractory AML patients, but the factors that dictate haplo-NK expansion are 7 Page 7 of 15

unknown. Depending on the experimental approach, the frequency of alloreactive T cells in humans ranges in frequency from 1:5,000 to 1:50,000, although in some cases, it is not detectable.8,9 Based on the assumption that alloreactivity is relatively frequent, we hypothesized that host alloreactive T cells play a key role in the loss of donor haplo-NK cells. In support of this, we show that higher absolute T cell counts in the recipient prior to haplo-NK infusion are associated with failure to expand the haplo-NK cell graft at D+14. Additionally, we found that recipient T cell dysfunction (i.e., exhaustion) correlates with haplo-NK expansion. Our findings strongly support this assertion since at D+14, patients with haplo-NK cell expansion had fewer T cells, which were mainly exhausted based on inhibitory receptor phenotype and transcription factor expression. In contrast, the NK non-expanders showed high percentages of CD8+ T cells and a paucity of exhausted T cells. Collectively, these studies expose the impact of alloreactivity on donor NK cell expansion and suggest that recipient CD8+ T cell exhaustion is permissive for haplo-NK expansion. Therefore, further efforts to delete, suppress or transiently modulate the function of recipient T cells may improve the efficacy of haplo-NK therapy by increasing engraftment and persistence. One approach might be to use serotherapy, such as ATG, in the conditioning regimen as is used for allogeneic transplantation. However, careful dosing and pharmacokinetic monitoring, as has been recently described, will likely be needed so as to not negatively impact the activity of the adoptively transferred cells.10–13

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Figure 1: Absolute CD3+ T cell Count Predicts Likelihood of NK Cell Expansion at D+14. A) Absolute lymphocyte count, B) absolute CD3 count, and C) absolute CD8 count prior to lymphodepleting chemotherapy in both patient groups. D,E) Percent expression of CD3 and CD8 in controls compared to both patient groups prior to chemotherapy and at D+14 following haplo-NK cell infusion. F) Percent distribution of T cell subsets in controls and both patient groups prechemotherapy. G,H) Percent expression and MFI analysis of PD-1 and I,J) Tim-3 within CD8+ T cells in controls compared to both patient groups prechemotherapy and at D+14. K,L) Percent expression of PD-1hi and dual PD-1+Tim-3+ expression within CD8+ T cells in donor controls compared to both patient groups prior to chemotherapy and at D+14 following haploNK cell infusion. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001

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Figure 2: Impaired Cytokine Production Prechemotherapy and Elevated Percent Expression of Key Transcription Factors in CD8+ T cells with Most Exhausted Receptor Profiles. A) Percent IL-2, B) TNF-α, and C) IFN-γ expression in PD-1hi CD8+ T cells in controls compared to haplo-NK expanders and non-expanders prechemotherapy. D) Percent IL-2, E) TNF-α, and F) IFN-γ expression in dual PD-1+Tim-3+ CD8+ T cells in controls compared to haplo-NK expanders and non-expanders prechemotherapy. G,H) Percent T-bet and Eomes expression in PD-1hi CD8+ T cells in controls compared to haplo-NK expanders and nonexpanders at D+14 following haplo-NK cell infusion. I,J) Percent T-bet and Eomes expression in dual PD-1+Tim-3+ CD8+ T cells in donors compared to haplo-NK expanders and non-expanders at D+14 following haplo-NK infusion. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001

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