Leukemia & Lymphoma

ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: https://www.tandfonline.com/loi/ilal20

Nivolumab induces dynamic alterations in CD8

T-cell function and TIM-3 expression when used
to treat relapsed acute myeloid leukemia after
allogeneic stem cell transplantation

Eric Wong, Joanne Davis, Rachel Koldej, Jeff Szer, Andrew Grigg & David
Ritchie

To cite this article: Eric Wong, Joanne Davis, Rachel Koldej, Jeff Szer, Andrew Grigg & David
Ritchie (2019): Nivolumab induces dynamic alterations in CD8 T-cell function and TIM-3 expression
when used to treat relapsed acute myeloid leukemia after allogeneic stem cell transplantation,
Leukemia & Lymphoma, DOI: 10.1080/10428194.2019.1648803

To link to this article: https://doi.org/10.1080/10428194.2019.1648803

Published online: 07 Aug 2019.

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LEUKEMIA & LYMPHOMA
https://doi.org/10.1080/10428194.2019.1648803
LETTER TO THE EDITOR
Nivolumab induces dynamic alterations in CD8 T-cell function and TIM-3
expression when used to treat relapsed acute myeloid leukemia after
allogeneic stem cell transplantation
Eric Wonga,b,c,d, Joanne Davisb, Rachel Koldejb, Jeff Szera,d, Andrew Griggc,d and David Ritchiea,b,d
a
Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia;
b
Australian Cancer Research Foundation Translation Research Laboratory, Sydney, Australia; cDepartment of Clinical Oncology and
Olivia Newton-John Cancer Research Institute, Austin Hospital, Heidelberg, Australia; dDepartment of Medicine, Dentistry and Health
Sciences, University of Melbourne, Melbourne, Australia
ARTICLE HISTORY Received 30 April 2019; revised 3 July 2019; accepted 15 July 2019
Relapse of acute myeloid leukemia (AML) after allogeneic
hematopoietic stem cell transplantation (alloSCT) is a
major cause of mortality. Programmed death-1 (PD-1)
and its cognate ligand PD-L1 may contribute to post-
alloSCT immune escape by repressing the CD8 T-cell
mediated graft-versus-tumor (GVT) effect [1]. Blockade of
PD-1/PD-L1 interactions post-alloSCT is being investi-
gated as a therapy for relapse, albeit with caution due to
the risk of graft-versus-host disease (GVHD). Here, we
describe the dynamics of T-cell phenotype and function
in a patient who received nivolumab for AML relapsing
after alloSCT.
A 43-year-old man with myelodysplastic syndrome
(MDS) developed secondary AML with adverse prognosis
features including a complex monosomal karyotype and
three TP53 coding region mutations. Induction chemo-
therapy achieved complete morphologic remission from
AML but persistent dysplasia consistent with the underly-
ing MDS. The patient proceeded to alloSCT from an
HLA-matched unrelated donor with non-myeloablative
conditioning due to severe hepatic steatosis. GVHD
prophylaxis is comprised of in vivo T-cell depletion with
thymoglobulin in combination with ciclosporin and short
course methotrexate. Bone marrow biopsy at day 30
demonstrated complete morphologic remission. Relapse
of AML occurred at 97 days post-alloSCT with 23% bone
marrow blasts. Immunosuppression was ceased without
any ensuing GVHD. T-cell chimerism at relapse was 69%
donor origin. He was enrolled on a clinical trial of nivolu-
mab for relapsed hematological malignancies after
alloSCT (ClinicalTrials.gov identifier: NCT03146468) and
received nivolumab 3 mg/kg every 2 weeks commencing
120 days post-alloSCT. Disease re-assessment after four
doses demonstrated reduction in blast count to 12%.
There was no evidence of classical acute or chronic
GVHD; however, he developed postural orthostatic
CONTACT Eric Wong Eric.wong@mh.org.au Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Grattan Street,
Parkville, Victoria 3050, Australia
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
tachycardia syndrome (POTS) which was presumed to be
immune-mediated. After eight doses of nivolumab,
repeat bone marrow biopsy demonstrated an increase in
blast percentage to 17% and treatment was
discontinued.
Peripheral blood samples were obtained prior to nivo-
lumab and at regular intervals while on treatment. To
examine immune cell phenotypic and functional altera-
tions following nivolumab treatment, mononuclear cells
were isolated by density gradient centrifugation and
stained with monoclonal antibodies (BD Biosciences,
Franklin Lakes, NJ) including BD Horizon BUV395 conju-
gated anti-CD3, BV510 anti-CD4, BUV805 anti-CD8, BB515
anti-CD45RA, PE anti-CCR7, PE-Cy7 anti-HLA-DR, BUV496
anti-CD48, BV786 anti-PD1, BV421 anti-TIM3, Alexa Fluor
647 anti-LAG3, and fixable viability stain 780. Intracellular
cytokine production was ascertained after four hours of
in-vitro stimulation at 37
C with anti-CD2/3/28 microbe-
ads (Miltenyi Biotec, Bergisch Gladbach, Germany) in the
presence of protein transport inhibitors (BD GolgiStop
and GolgiPlug, BD Biosciences, Franklin Lakes, NJ) fol-
lowed by staining with BV650 anti-IL2, APC anti-TNFa,
and BV786 anti-IFNc. Samples were analyzed using an
LSRFortessa flow cytometer (BD Biosciences, Franklin
Lakes, NJ) and FlowJo software (Tree Star Inc., Ashland,
OR). Comparisons were made with healthy
patient controls.
At post-transplant relapse prior to treatment with
nivolumab, CD8 T-cells is comprised of CD45RA-CCR7-T
effector memory cells (Tem; 69.3%) and CD45RA þ CCR7-
T effector cells (Teff; 24.9%) with a paucity of T naïve
cells (Tn; 0.8%) as expected at this early time point post-
alloSCT. CD8 T-cells demonstrated increased expression
of activation markers HLA-DR and CD38 compared with
controls (HLA-DR 7.9% vs. 0.5%, p< .01; CD38 1.9% vs.
0.5%, p< .01). There was greater expression of PD-1, TIM-
2 E. WONG ET AL.

Figure 1. PD-1, TIM-3, and LAG-3 expression on CD8 T-cells in a healthy donor (A–C) and the patient case at the time of AML
relapse prior to treatment with nivolumab (D–F). There was a progressive increase in TIM-3 expression over time, with greatest
expression at the time of disease progression despite nivolumab (H–J). There was an initial increase in TNFa production with nivo-
lumab treatment however this was transient (K). At disease progression despite nivolumab treatment, there was upregulation of
TIM-3 expression and repression of TNFa production to baseline levels.

3, and LAG-3 by CD8 T-cells at baseline compared with
controls (PD-1 48.8% vs. 5.9%; TIM-3 19% vs. 5.5%; LAG-3
7.4% vs. 0.1%, all p< .01) (Figure 1(A–F)), and this was
seen in both CD8 Teff and Tem. Despite the increased
expression of co-inhibitory receptors, CD8 T-cells
remained functionally competent and demonstrated
increased IFNc (3.2% vs. 0.83%, p¼ .03) and IL-2 produc-
tion (5.6% vs. 0.8%, p¼ .004) and similar TNFa production
(2.6% vs. 1.4%, p¼ .3) compared with controls.
Following nivolumab treatment, there was a shift in
CD8 T-cell memory phenotype from Tem to Teff by day 7
together with an increase in TNFa production (5.7% vs.
2.6%). There was no similar pattern in IFNc or IL2 produc-
tion over time. This shift in T-cell memory phenotype
and increased cytokine production mirrored the onset of
the patient’s POTS symptoms, suggesting that this phe-
nomenon may have been immunologically driven. The
increase in T-cell function was associated with a reduc-
tion in marrow blasts. At subsequent leukemia progres-
sion despite continued nivolumab therapy, CD8 T-cells
demonstrated increased expression of TIM-3 compared
with baseline (27% vs. 20%) (Figure 1(G–J)) which was
accompanied by a decline in TNFa production to baseline
levels (Figure 1(K)), and a fall in IL-2 production at pro-
gression to below baseline (3.5% vs. 5.6%), suggesting
that upregulation of TIM3 and repression of T-cell func-
tion may contribute to acquired resistance to PD-1 block-
ade after alloSCT.
This case report highlights several interesting findings.
We demonstrate that nivolumab therapy post-alloSCT is
able to achieve leukemia response, albeit transient, in a
patient with aggressive AML for which a non-augmented
GVT was insufficient. The use of checkpoint inhibitors to
treat relapsed AML after alloSCT has been described in
case series. In a phase I trial of CTLA4 inhibition post-
alloSCT, there were five complete responses out of 18
patients with AML or MDS relapsing after alloSCT [2].
Albring et al. described three patients with relapsed AML
after alloSCT who received treatment with low dose nivo-
lumab, of which two patients achieved partial or com-
plete responses [3].
The proportion of PD-1 expressing CD8þ T-cells was
increased at the time of AML relapse compared with
healthy controls. Despite this phenotype, CD8 T-cells
remained functionally competent with preserved cytokine
production. Schnorfeil et al. reported preserved T-cell
function despite upregulation of PD-1 at the time of AML
relapse, suggesting that CD8 T-cell upregulation of PD-1
at relapse is not sufficient to indicate T-cell exhaustion
[4]. PD-1 is upregulated by CD8 T-cells in response to
activation and may not indicate exhaustion per se [5].
Despite this, the biological rationale to pursue checkpoint
blockade in AML and other hematological malignancies
relapsing post-alloSCT remains, and response to this ther-
apy may be more closely linked to tumor expression of
PD-L1 rather than a PD-1 positive T-cell phenotype. PD-
L1 has been described to be upregulated by AML blasts
NIVOLUMAB AFTER ALLOGENEIC TRANSPLANT 3
at the time of relapse post-alloSCT [1]. Furthermore, the
most impressive responses to PD-1 inhibitors either pre-
transplant or post-alloSCT are seen in patients with
Hodgkin lymphoma where upregulation of PD-L1 is an
intrinsic part of the tumor biology mediated by amplifica-
tion of chromosome 9p24.1 [6].
Consistent with the early onset of clinical responses to
nivolumab post-alloSCT, there was an early increase in
CD8 T-cell TNFa production at seven days following nivo-
lumab. Following initial response, leukemia progression
was accompanied by an increase in TIM-3 expression and
a decline in cytokine production, likely representing a
‘true’ exhausted state. The upregulation of TIM-3 as an
alternative inhibitory pathway may represent a mechan-
ism of acquired resistance to PD-1 blockade, and has
been reported in solid tumors [7]. In a murine model of
lung carcinoma, TIM-3 expression was upregulated with
increasing duration of PD-1 blockade. Therapeutic block-
ade of TIM-3 at the time of acquired PD-1 resistance
achieved tumor responses indicating that loss of T-cell
efficacy may be regained by sequential PD-1 followed by
TIM3 inhibition [7]. Interestingly, there was no observed
change in IFNc or IL-2 production with nivolumab treat-
ment, suggesting that checkpoint blockade post-alloSCT
may not universally enhance T-cell functionality in a man-
ner analogous to that observed in healthy donor T-cells
in vitro [8]. Further analysis in a larger number of patients
will be required to examine this hypothesis.
In conclusion, we demonstrate that nivolumab
induced a potent, if transient, GVT response in highly
aggressive AML. Although PD-1 was upregulated prior to
nivolumab therapy, CD8 T-cells remained functionally
intact and this was augmented by nivolumab therapy.
Upregulation of TIM-3 was accompanied by reduced T-
cell function at leukemia progression, and may represent
a mechanism of acquired resistance to PD-1 blockade
post-alloSCT.
Disclosure statement
The clinical trial was designed solely by the investigators
without input from Bristol-Myers Squibb, who also did not
have any input into the analysis of results or preparation of
the final manuscript. Bristol-Myers Squibb did not provide
any other financial support to any of the authors.
Funding
The work described in this manuscript pertains to a clinical
trial which was funded by Bristol-Myers Squibb.
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