Circulating immune profile can predict survival of metastatic uveal melanoma patients: results of an exploratory study

Abstract

Metastatic uveal melanoma (UM) is a poor prognosis malignancy. Immunotherapy is commonly employed, despite the low activity, considering the lack of other effective systemic treatments. In this study, the prognostic and predictive role of soluble immune checkpoints and inflammatory cytokines/chemokines in 22 metastatic UM patients was evaluated. Baseline levels of these molecules were assessed, as well as their changes during anti-PD-1 therapy. The correlation between soluble immune checkpoints/cytokines/chemokines and survival was analyzed. A comparison between circulating immune profile of metastatic cutaneous melanoma (CM), for which immunotherapy is a mainstay of treatment, and UM during anti-PD-1 therapy was also performed. Three immune molecules resulted significantly higher in metastatic UM patients with survival <6 months versus patients with survival ≥6 months: IL-8, HVEM and IDO activity. Considering these three molecules, we obtained a baseline score able to predict patients' survival. The same three molecules, together with soluble(s) CD137, sGITR and sCD27, resulted significantly lower in patients with survival >30 months. We also observed an increase of sCD137, sCD28, sPD-1, sPD-L2 sLAG3, sCD80 and sTim3 during anti-PD-1 treatment, as well as IDO activity, IP-10 and CCL2. Several of these molecules were significantly higher in UM compared to CM patients during anti-PD-1 therapy. The analysis of circulating immune molecules allows to identify patients with poor prognosis despite immunotherapy and patients with long survival treated with an anti-PD-1 agent. The different serum concentration of these molecules during anti-PD-1 therapy between UM and CM reflects the different efficacy of immune checkpoint inhibitors.

Full text

RESEARCH PAPER
Circulating immune prole can predict survival of metastatic uveal melanoma
patients: results of an exploratory study
Ernesto Rossi
a
*, Ilaria Grazia Zizzari
b
*, Alessandra Di Filippo
b
, Anna Acampora
c
, Monica Maria Pagliara
d
,
Maria Grazia Sammarco
d
, Maurizio Simmaco
e
, Luana Lionetto
e
, Andrea Botticelli
f
, Emilio Bria
a,g
, Paolo Marchetti
f
,
Maria Antonietta Blasi
d
, Giampaolo Tortora
a,g
, Giovanni Schinzari
a,g
*, and Marianna Nuti
b
*
a
Medical Oncology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy;
b
Laboratory of Tumor Immunology and Cell Therapy,
Department of Experimental Medicine, Policlinico Umberto I, Sapienza University, Rome, Italy;
c
Department of Life Sciences and Public Health,
Università Cattolica del Sacro Cuore, Rome, Italy;
d
Ophtalmology, Fondazione Policlinico Universitario Agostino Gemelli, Rome, Italy;
e
Spectrometry-
Clinical Biochemistry Laboratory, Sant’Andrea University Hospital, Rome, Italy;
f
Medical Oncology, Policlinico Umberto I, Sapienza University, Rome,
Italy;
g
Medical Oncology, Università Cattolica del Sacro Cuore, Rome, Italy
ABSTRACT
Metastatic uveal melanoma (UM) is a poor prognosis malignancy. Immunotherapy is commonly
employed, despite the low activity, considering the lack of other eective systemic treatments. In this
study, the prognostic and predictive role of soluble immune checkpoints and inammatory cytokines/
chemokines in 22 metastatic UM patients was evaluated. Baseline levels of these molecules were assessed,
as well as their changes during anti-PD-1 therapy. The correlation between soluble immune checkpoints/
cytokines/chemokines and survival was analyzed. A comparison between circulating immune prole of
metastatic cutaneous melanoma (CM), for which immunotherapy is a mainstay of treatment, and UM
during anti-PD-1 therapy was also performed. Three immune molecules resulted signicantly higher in
metastatic UM patients with survival <6 months versus patients with survival ≥6 months: IL-8, HVEM and
IDO activity. Considering these three molecules, we obtained a baseline score able to predict patients’
survival. The same three molecules, together with soluble(s) CD137, sGITR and sCD27, resulted signi-
cantly lower in patients with survival >30 months. We also observed an increase of sCD137, sCD28, sPD-1,
sPD-L2 sLAG3, sCD80 and sTim3 during anti-PD-1 treatment, as well as IDO activity, IP-10 and CCL2.
Several of these molecules were signicantly higher in UM compared to CM patients during anti-PD-1
therapy. The analysis of circulating immune molecules allows to identify patients with poor prognosis
despite immunotherapy and patients with long survival treated with an anti-PD-1 agent. The dierent
serum concentration of these molecules during anti-PD-1 therapy between UM and CM reects the
dierent ecacy of immune checkpoint inhibitors.
ARTICLE HISTORY
Received 19 September 2021
Revised 27 November 2021
Accepted 14 December 2021
KEYWORDS
Uveal melanoma; PD-1;
soluble checkpoint;
immunotherapy
Introduction
Uveal melanoma (UM) represents the most common tumor
with origin in the eye and is a rare malignancy, with an
incidence of 4.9 cases per million.
1
Despite the radical treat-
ment of primary tumor, metastatic spread often occurs.
2
The
first and most frequent site of metastases is the liver.
3
Hepatic
involvement, which accounts for about 90% of metastatic
disease,
4
is usually characterized by multifocal metastases.
Therefore, surgical resection of hepatic metastases is not pos-
sible for the majority of the patients.
5
Survival for patients with
metastatic disease is limited. The systemic treatments com-
monly used are the same tested in clinical trials for cutaneous
melanoma (CM)
6
despite the different clinical and biological
features of these tumors.
Chemotherapy and target therapies have been employed with
poor results.
7–11
To date, immune checkpoint inhibitors (ICIs) are
used for the treatment of metastatic uveal melanoma (mUM).
6
Ipilimumab showed a modest activity, with a survival ran-
ging from 6.8 to 9 months.
12,13
In pre-treated patients, pem-
brolizumab, nivolumab and atezolizumab demonstrated
a progression-free survival (PFS) of about 3 months.
14,15
Pembrolizumab showed a limited efficacy with a PFS of
3.8 months in first-line setting, as demonstrated by a prospec-
tive observational study.
16
The association of nivolumab and
ipilimumab allowed an overall survival (OS) of 12.7 months
and a PFS of 3 months.
17
In contrast to CM, UM is unresponsive to checkpoint inhi-
bitors in the majority of the cases
18
and the reasons of the poor
response remain speculative. The low activity of the ICIs can be
explained by the ability of UM cells to elude immunity, upre-
gulating and expressing immunosuppressive molecules.
19,20
Moreover, the eye is considered an immune-privileged site
with own immunosuppressive mechanisms. UM cells are able
to escape from systemic immune surveillance also in the liver.
21
CONTACT Ernesto Rossi ernesto.rossi@policlinicogemelli.it; ernestorossi.rm@gmail.com Medical Oncology, Fondazione Policlinico Universitario Agostino
Gemelli IRCCS, L.go A. Gemelli 8, Roma 00168, Italy; Ilaria Grazia Zizzari ilaria.zizzari@uniroma1.it Laboratory of Tumor Immunology and Cell Therapy,
Department of Experimental Medicine, Policlinico Umberto I, Sapienza University, V.le Regina Elena 324, Roma 00161, Italy
*These authors contributed equally to this work.
Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2022.2034377.
https://doi.org/10.1080/21645515.2022.2034377
© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
HUMAN VACCINES & IMMUNOTHERAPEUTICS
2022, VOL. 18, NO. 3, e2034377 (10 pages)

In addition, the low mutational burden of this type of mela-
noma can be responsible for the poor results obtained with
immunotherapy until now.
22
Understanding the immune status of UM patients is essen-
tial to identify biomarkers useful for selecting patients who
could benefit more from immunotherapy.
23,24
Interestingly, recent studies indicate that soluble isoforms of
immune checkpoint (IC) receptors, released in the serum of
patients, are centrally involved in immune regulation and asso-
ciated with clinical outcomes.
25,26
The origin of the soluble
receptors has not been completely elucidated. It has been
reported that they can be produced by a proteolytic cleavage
of membrane bound, by an alternative splicing of mRNA, or
released with exosomes or microvesicles.
27,28
It was demon-
strated that high serum levels of several IC molecules correlate
with resistance to immunotherapy in melanoma patients.
29
Cytokines and chemokines also play a key role during
immunotherapy. For example, an upregulation of 11 cytokines
was observed in melanoma patients treated with an anti-PD-1
alone or in combination with an anti-CTLA-4 who developed
high grade immune-related adverse events.
30
Here, we have studied for the first time the circulating
immune profile of UM patients in order to evaluate their
immunological status and investigate the role of soluble
immune molecules in patients during anti-PD-1 treatment.
Patients and methods
Patients enrollments and samples collection
We considered 22 patients with metastatic UM referred to the
Oncology Unit of Fondazione Policlinico Universitario Agostino
Gemelli IRCCS. Patients older than 18 years, with measurable
unresectable metastatic disease, received pembrolizumab as first-
line therapy, administered intravenously at a dose of 2 mg/kg
every 3 weeks or 200 mg flat dose every 3 weeks (when the flat
dose has been introduced) until disease progression, unaccepta-
ble toxicity or consent withdrawn. Toxicity was reported accord-
ing to the Common Terminology Criteria for Adverse Events
(CTCAE v. 5.0). Radiological and clinical assessments were
performed according to good clinical practice. Progression-free
survival, response rate, clinical benefit, OS and tolerability were
evaluated. Responses were assessed in accordance with the
RECIST criteria 1.1. PFS was calculated from the first day of
treatment to progression or death for any reason. Survival was
defined as the interval from the first detection of metastases to
death for any cause.
A group of 11 BRAF wild-type metastatic cutaneous mela-
noma (mCM) patients treated in first-line setting with an anti-
PD-1 agent were also evaluated for the comparison with mUM
patients. Patients with stable brain metastases (no neurological
symptoms, no radiologic evidence of progression, no steroid
requirement) could be considered.
Peripheral blood samples were drawn from all patients into
a tube without anticoagulant and left at room temperature to
allow blood to clot. Later, samples were centrifugated to collect
serum that was stored and frozen at −80°C until use. In a group
of metastatic UM patients, samples were collected before treat-
ment with pembrolizumab (T0) and after three cycles (>T0).
Samples from a group of CM patients, during first-line anti-PD
-1 therapy, were also collected. The study was conducted in
accordance with the Helsinki declaration of 1975 and was
approved by the local ethics committee. Informed consent
was obtained from all the subjects involved in the study.
Detection of soluble molecules in serum
Sera from uveal and CM patients were assayed to evaluate the
levels of cytokines and soluble immune checkpoint molecules
(sICs) by multiplex immunoassay analysis using the
ProcartaPlex Human Inflammation Panel (20 Plex, catalog
number EPX200-12185-901; sE-Selectin; GM-CSF; ICAM-1/
CD54; IFN alpha; IFN gamma; IL-1 alpha; IL-1 beta; IL-4; IL-
6; IL-8; IL-10; IL-12p70; IL-13; IL-17A/CTLA-8; IP-10/
CXCL10; MCP-1/CCL2; MIP-1alpha/CCL3; MIP-1 beta/
CCL4; sP-Selectin; TNF alpha) (eBioscence) and the Human
Immuno-Oncology Checkpoint 14-plex ProcartaPlex Panel 1
(catalog number EPX14A-15803-901; BTLA; GITR; HVEM;
IDO; LAG-3: 47; PD-1; PD-L1; PD-L2; TIM-3; CD28; CD80;
CD137; CD27; CD152) (eBioscence). Assay was conducted
using 50 µl of serum for each sample and adding it in a 96-
well plate with a mixture of color-coded magnetic beads coated
with antibody that recognize specific analytes. Later, biotiny-
lated detection antibodies that bind analytes of interest were
added and then bound to Phycoerythrin-conjugated streptavi-
din that through its signal intensity allow to detect the analyte
concentration. Samples were measured using Luminex 200
platform (BioPlex, Bio-Rad) and data, expressed in pg/ml of
protein, were analyzed using Bio-Plex Manager Software.
Trp/kyn ratio analysis
Serum levels of tryptophan (trp) and kynurenine (kyn) were
evaluated through modified liquid chromatography – tandem
mass spectrometry method.
Samples were deproteinized using 50 μl of TCA 4% aqueous
solution and following centrifuged at 14,000 rpm for 15 min.
Supernatants were injected into chromatographic system to
perform separation using an Agilent Liquid Chromatography
System series 1100 (Agilent Technologies, USA), on a biphenyl
column (100 × 2.1 mm, Kinetex 2.6 μm Biphenyl, 100 Å,
Phenomenex, CA, USA) equipped with a security guard pre-
column (Phenomenex, Torrance, CA, USA). Gradient elution
was performed with a flow rate of 400 μl/min and mobile
phases consisted of 0.1% aqueous formic acid and 100%
acetonitrile.
The mass spectrometry method was performed on a 3200
triple quadrupole system (Applied Biosystems, Foster City, CA,
USA), equipped with a Turbo Ion Spray source. The detector
was set in the positive ion mode. The instrument was set in the
Multiple Reaction Monitoring mode. Data were acquired and
processed by the Analyst 1.5.1 Software.
Statistical analysis
PFS and OS were calculated using Kaplan–Meier method.
Summary data were expressed as average and standard error
of mean.
-2
e2034377 E. ROSSI ET AL.

Two-tailed nonparametric statistical tests were used to com-
pare different groups. Wilcoxon matched-pairs signed rank test
was used to analyze mUM group and compare T0 to >T0.
mUM patients were divided into three groups according to
survival: fast progressors (FP), slow progressors (SP) and long
survivors (LS); one-way Anova test was used to compare these
populations. Comparison between mUM and mCM was per-
formed using unpaired Mann–Whitney test. All the results
were considered significant when p value was <0.05.
The level of all the studied molecules was described by calcu-
lating mean and standard deviation (SD), and median and
interquartile range (IQR). The values were compared between
patients with a survival <6 months or ≥6 months using the
Mann–Whitney test because the data were not normally distrib-
uted. The molecules for which the comparison showed a p value
< .05 or for which there was a particular clinical interest (con-
sidering a p value not higher than 0.2) were investigated with the
objective to identify the better cutoff for distinguishing long
survivors or no long survivors and a Receiver Operating
Characteristic (ROC) analysis was performed. A score was finally
defined by assigning a value of 1 to each molecule associated
with the worst prognosis cased at the cut-off. Consequently, the
overall score was obtained by summing the single molecule score
with the higher value corresponding to the worst prognosis.
Finally, a Spearman correlation was performed to correlate
the value of the score with the survival time in months.
Results
Patients’ characteristics
A total of 22 UM patients were evaluated. Patients’ character-
istics are summarized in Table 1. A group of 11 advanced
BRAF wild-type CM patients treated with an anti-PD-1 agent
(3 patients treated with pembrolizumab, 8 patients with nivo-
lumab) in first-line setting was also considered.
Among the metastatic UM patients, the median age was
67.9 years (range 54–87). Eleven subjects were male and 11
female. Liver metastases were found in 21 patients. None of the
patients had BRAF mutation. Ocular enucleation was pre-
viously performed for the treatment of primary tumor in 16
patients, while 6 patients never underwent enucleation. Local
treatment of liver metastases (metastasectomy) was previously
carried out in two patients. One of them developed a non-
resectable metastatic disease, while the other patient was free
from disease recurrence at the time of data analysis.
All 20 patients with non-resectable metastatic disease under-
went anti-PD-1 treatment with pembrolizumab. A median of 8.8
cycles for patients were administered (range 1–52)
A score based on immune molecules can select UM
patients with better survival
Twelve mUM patients were divided into two groups according
to OS from the time of starting pembrolizumab: patients with
survival <6 months and patients with survival ≥6 months. The
concentration of circulating immune checkpoints and cyto-
kines/chemokines released in serum before starting
pembrolizumab (T0) was analyzed. Indoleamine 2,3-dioxygen-
ase (IDO) activity, evaluated as Kynurenine/tryptophan ratio
was also measured.
Serum level of two molecules resulted significantly higher in
patients with survival <6 months: HVEM with a median value
of 47 pg/ml (IQR 9–111,75) for those with survival <6 months
and 6 pg/ml (IQR 6–35) for those with survival ≥6 months
(p = .045). IDO activity showed a median value of 0.038 (IQR
0.024–0.043) for patients with survival <6 months and 0.019
(IQR 0.017–0.024) for those with survival ≥6 months (p= .035).
A third molecule, IL-8, was considered for the ROC analysis
because of clinical interest. Indeed, it has been previously
demonstrated that increased levels of this cytokine are predic-
tive of poor efficacy in patients treated with an ICI.
31
In our
study, IL-8 showed a median value of 289.92 pg/ml (IQR
48.30–314.38) for patients with survival <6 months and of
7 pg/ml (IQR 2.70–57.15) for those with survival ≥6 months.
ROC curve analysis identified the better cut-off for the three
selected molecules (Figure 1a-c): HVEM ≥50 pg/ml (sensibility
60%; specificity 83.3%; Accuracy 72.7%; AUC 0.817); IDO
activity ≥0.024 (sensibility 100%; specificity 66.7%; Accuracy
81.8%; AUC 0.883); IL-8 ≥ 50 pg/ml (sensibility 80%; specifi-
city 66.7%; Accuracy 72.7%; AUC 0.743).
Assigning a score of 1 to values higher than the cut-off for
each of the three identified molecules and by summing single
scores, an overall score ranging from 0 to 3 was obtained,
where 3 corresponds to the worst prognosis.
The overall score showed significantly higher value in
patients with survival <6 months (p= .028) (Figure 2a). The
ROC analysis for the overall score (Figure 2b) identified the
better cutoff for worst survival prediction, which was 2 (overall
score ≥2; sensibility 60%; specificity 100%; Accuracy 81.8%;
AUC 0.867). These results suggest that the presence of two or
more of the identified molecules with values higher than the
critical level is associated with a worst prognosis. Furthermore,
higher values of the overall score correlate with lower survival
(in months) with a coefficient rho = −0.490. Figure 2c shows
the survival of mUM patients according to the score (p= .007).
Table 1. Patients’ characteristics.
Metastatic uveal melanoma 22
Median age (range) 67.9 y (54–87)
M/F 11/11
Enucleation for primary tumor 16
Previous local treatment for liver metastases 2
Site of metastases
Liver 21
Lung 5
Bone 6
Brain 0
Other 7
Hepatic and extra-hepatic metastases 9
Extra-hepatic metastases only 1
BRAF mutation 0
Metastatic cutaneous melanoma 11
Median age (range) 67.8 (42–84)
M/F 8/3
Site of metastases
Liver 2
Lung 4
Bone 2
Brain 2
Other 10
BRAF mutation 0
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e2034377 3

Figure 1. Baseline levels of cytokines and soluble immune checkpoint inhibitors can select patients with better survival. A-C. ROC curve analysis for the identified
molecules: A: HVEM; B IDO ratio; C: IL-8. The values were compared between patients with a survival <6 months or ≥6 months using the Mann–Whitney test.
Figure 2. A score based on cytokines and soluble immune checkpoint inhibitors can predict patients’ survival. A. Overall score for total sample and by patients’ survival.
B. ROC curve analysis for the overall score. A score of 1 was assigned to values higher than the cutoff for each of the three identified molecules; an overall score ranging
from 0 to 3 was obtained by summing single scores. C. Patients survival based on the score: solid line: patients with score 2–3; dotted line: patients with score 0–1. (OS:
overall survival. SD: standard deviation. IQR: interquartile range).
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e2034377 E. ROSSI ET AL.
4

Levels of soluble immune molecules are associated with
prognosis in mUM patients
Based on the course of the metastatic disease, we identified
three different groups of UM patients associated with OS
(Figure 3a):
(1) Patients defined as “fast progressors” (FP) who rapidly
progressed with a median survival <6 months despite
immunotherapy;
(2) Patients defined as “slow progressors” (SP) who
remained alive despite disease progression but with
a median survival shorter than 30 months;
(3) Patients defined as “long survivors” (LS) with a median
survival >30 months. Among them, three patients were
undergoing treatment with pembrolizumab at the time
of inclusion in the study.
As expected, among the molecules analyzed, IL-8 resulted
higher in serum of FP patients compared to LP (p= .01)
and LS (p= .04) patients. Similarly, high levels of IDO
activity were detected in fast progressive patients (p= .02),
whereas LS and SP patients showed comparable amounts.
Serum HVEM also highlighted a major concentration in FP
vs LS (p= .07) and a significantly higher value in SP
patients vs LS (p= .04) (Figure 3b).
Other immune checkpoint molecules such as sCD137,
sGITR and sCD27 resulted significantly changed among the
three groups of patients (p< .05) (Figure 3c). Patients with
fast-progressive disease had higher concentration of sICs
compared to SP and LS patients (sCD137: FP vs SP
p = .03; FP vs LS p = .02; sGITR: FP vs SP p = .04;
sCD27: FP vs LS p = .05). Similarly, patients with slow
progressive disease showed higher levels of sCD137
(p = .01), sGITR (p = .01) and sCD27 (p = .01) compared
to long survivors. These results suggest that the release in
serum of these immune molecules could be related to the
severity of the disease.
Modulation of immune molecules during anti-PD-1
treatment in metastatic uveal melanoma patients
In order to evaluate the impact of anti-PD-1 treatment on
the release of immune molecules, the serum of UM
patients was also collected and analyzed after three cycles
of therapy (>T0) (except for three patients who rapidly
progressed). The concentration of numerous sICs resulted
significantly modulated during anti-PD-1 treatment
(Figure 4a-e). In particular, analyzing the levels of
sCD137 and sCD28 at T0 and >T0 (Figure 4a), the mole-
cules resulted significantly enhanced by 3.25-fold
(p = .007) and 2.4-fold (p = .01), respectively. Similarly,
the concentration of the soluble form of inhibitory recep-
tors belonging to the immunoglobulin family significantly
increased during anti-PD-1 therapy, including sPD-1
(1.96-fold, p = .01), sLAG3 (1.6-fold, p = .007) and
sTim3 (1.54-fold, p = .03) (Figure 4b). In addition, also
the levels of sPD-L2 (1.38-fold, p = .04) and sCD80
(1.3-fold, p = .01), the soluble forms of PD-1 and CTLA-
4 ligands, respectively, resulted augmented at >T0 com-
pared to baseline (T0) (Figure 4c).
Pro and anti-inflammatory cytokines and chemokines
were also evaluated. The most remarkable results were the
significant increase of IP-10 (2.16-fold, p = .007) and CCL2
(1.29-fold, p = .004) after the beginning of anti-PD-1 ther-
apy (>T0) (Figure 4d). Furthermore, also IDO activity
resulted increased (p = .046) during anti-PD-1 treatment
(Figure 4e).
Figure 3. Profiling of soluble immune molecules in UM patients stratified according to the course of metastatic disease. A. UM patients were classified in Fast progressor
(FP), slow progressor (LP) and long survivor (LS). In the histograms, the serum levels of each protein were reported as average value ± SEM: B. Levels of IL-8 (FP:
212.31 pg/ml ± 114.3; SP: 12.33 ± 4.017; LS: 6.333 ± 2.348), sHVEM (FP: 325.6 pg/ml ± 282; SP: 94.91 pg/ml ± 41.98; LS: 6.8 ± 1.562) and IDO activity (FP: 0.047 ± 0.021;
SP: 0.02 ± 0.007; LS: 0.023 ± 0.006). C. sCD137 (FP: 361.8 pg/ml ± 123.1; SP: 140.1 pg/ml ± 16.77; LS: 65.5 pg/ml ± 20.75), sGITR (FP: 70.2 pg/ml ± 29.81; SP:
25.33 ± 6.644; LS: 11.4 ± 0.4) and sCD27 (FP: 10258 ± 3758; SP: 6027 ± 870.5; LS: 2610 ± 460.5). ANOVA test was used to compare three groups. Student’s unpaired t-test
for two groups. p < .05 was considered statistically significant. (OS: overall survival. FP: fast progressors, with a survival <6 months. SP: slow progressors, with a survival
>6 months and <30 months. LS: long survivors, with a survival >30 months).
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e2034377 5

Figure 4. Modulation of soluble immune molecules in mUM patients during anti-PD-1 treatment. Levels of soluble immune checkpoint proteins and inflammatory
cytokines/chemokines were measured in sera of metastatic uveal melanoma patients at baseline (T0) and during anti-PD-1 treatment (>T0). Proteins were analyzed by
Luminex multiplex beads and results are reported as concentration (pg/ml). A-C. sCD137, sCD28, sPD-1, sLAG3, sTim3, sPD-L2 and sCD80; D. chemokines IP-10 and CCL2.
E. IDO activity measured as KYN/trp ratio. The kyn/trp ratio was reported as average value ± SEM (T0: 0.033 ± 0.015; >T0: 0.063 ± 0.032). Wilcoxon matched-pairs signed
rank test was used and a p value <.05 was considered statistically significant.
Figure 5. Immune profile of mUM patients compared to mCM. A. PFS and OS of patients treated with an anti-PD-1 as first line treatment for metastatic disease. Solid
line: uveal melanoma. Dotted line: cutaneous melanoma. B-E. mUM patients were compared to CM patients, both treated with anti-PD-1 therapy. In the scatter plot the
longest bars represent the average value, the shortest ones indicate the error bar (± SEM). B. sGITR (mUM: 52.49 pg/ml ± 20.56; mCM: 12.88 ± 2.065) and sCD27 (mUM:
8204 ± 2190, mCM: 3025 ± 641.4); C. sPD-1 (mUM: 129.6 ± 34.79; mCM: 40.18 ± 13.84; D. levels of sCD80 (mUM: 630.9 ± 185.6; mCM: 240.1 ± 113.1); E. IFNγ (mUM:
258.1 ± 92.4; mCM: 4.778 ± 1.665). F. The histogram shows the IDO activity (Kyn/trp ratio) in mUM and CM (UM: 0.069 ± 0.01; CM: 0.035 ± 0.0036). (PFS: progression-free
survival. OS: overall survival. UM: uveal melanoma. CM: cutaneous melanoma. NR: not reached).
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e2034377 E. ROSSI ET AL.
6

Metastatic uveal melanoma vs metastatic cutaneous
melanoma: dierences in the release of soluble immune
molecules during anti-PD-1 treatment
Since UM shares the same treatment with metastatic CM but
shows differences in clinical benefit, we compared these set-
tings of patients during first-line anti-PD-1 treatment in order
to evaluate possible discrepancies in the regulation of the
immune system.
As expected, first-line anti-PD-1 therapy allowed different
clinical outcomes in mUM and mCM. Median PFS for UM
melanoma patients was 3.8 months, while median PFS for CM
patients was not reached at the time of data analysis
(Figure 5a). Median OS for UM patients was 15.3 months,
not reached for CM (Figure 5a). Although all patients were
treated with an anti-PD-1 agent, the levels of several soluble
immune molecules resulted remarkably different between the
two groups during the treatment. The concentration of sGITR
and sCD27 (Figure 5b) were significantly higher in UM
patients (p = .02 and p = .03, respectively). sPD-1 was the
only soluble form of immune checkpoint receptors whose
concentration was statistically different between the two types
of cancer: higher in UM patients compared to CM (p = .01)
(Figure 5c). Among the soluble form of ligands, also the levels
of sCD80 resulted increased in UM (p = .03) (Figure 5d).
Moreover, we observed that UM and CM release differently
IFNγ, whose concentration was higher in the serum UM
patients (p < .001) (Figure 5e). As IFNγ is a potent inducer of
IDO expression, we also compared IDO activity between the
two tumors and the results confirmed the higher presence of
this enzyme in UM patients (p = .04) (Figure 5f).
No significant modulation was observed for the other mole-
cules tested (data not shown).
Discussion
UM and CM share systemic treatments despite their different
clinical and biological behaviors. Indeed, anti-PD-1 therapy is
largely used also for metastatic UM but it allows a limited
benefit in this disease.
16
The poor efficacy can be related to
the low mutational burden with few nonsynonymous muta-
tions and no ultraviolet-induced mutational damage.
6,22
Therefore, a current issue is to understand if there is
a rationale for immunotherapy in metastatic UM.
24
A deeper
knowledge of immunological features of this disease can help
to answer the questions: 1) if alternative strategies involving
pathways different from PD-1 and CTLA-4 can be more pro-
mising; 2) if a selection of the patients based on immunological
factors can allow a better outcome also for patients treated with
anti-PD-1 agents; 3) if a combination therapy including radio-
therapy + immunotherapy can have a role for the treatment of
UM through the immunogenicity induced by radiation
therapy.
32
Our study aimed to investigate the circulating immune
profile of metastatic UM in order to find an answer to these
issues. Our data demonstrated that after three cycles of pem-
brolizumab, the concentration of several soluble immune
checkpoint molecules is significantly changed compared to
T0. In particular, the levels of sCD137 and sCD28 during anti-
PD-1 treatment resulted significantly enhanced. CD137 and
CD28 are known as costimulatory receptors but previous stu-
dies have shown that their soluble forms have an inhibitory
role in immune response.
33,34
Similarly, the concentration of
the soluble form of inhibitory receptors including sPD-1,
sLAG3 and sTim3 significantly increased during anti-PD-1
therapy. Trials investigating the addition of an anti-LAG3 to
an anti-PD-1 agent as first-line therapy for mUM are
ongoing.
35
The chemokines IP-10 and CCL2 also increased after the
beginning of anti-PD-1 therapy (>T0). Considering that these
chemokines, as well as the soluble immune checkpoint mole-
cules, are associated with poor prognosis in many tumors,
36,37
these results suggest that immunosuppression appears to be
predominant in this setting of patients despite anti-PD-1
therapy.
Furthermore, the IDO activity increased during the sys-
temic treatment. IDO acts as immune checkpoint involved in
peripheral immune tolerance due to its ability to inhibit T-cell
proliferation by depleting them from tryptophan to sensitize
T-cells to apoptosis,
38–43
reducing immune activation.
Moreover, it was demonstrated that IDO can predict primary
resistance to anti-PD-1 treatment in solid tumors, such as non-
small cell lung cancer.
44,45
This data supports the observation
that an immunosuppressive profile can be found in patients
with metastatic UM under anti-PD-1 treatment.
In this study, we also investigated the predictive role of
survival of sICs, cytokines and chemokines at baseline in
patients treated with pembrolizumab.
Among all the factors evaluated, we found that HVEM, IDO
activity and IL-8 were correlated with survival, with higher
values in patients with poor survival (<6 months). Similar to
IDO, HVEM seems to promote Treg functions.
46
Thus, in
mUM an immunosuppressive environment detectable in
patients’ blood samples is associated with poor prognosis
despite anti-PD-1 treatment. IL-8 is a proinflammatory cyto-
kine: high plasmatic levels of IL-8 are associated with decreased
efficacy of checkpoint inhibitors in an inflamed tumor.
31,47
Considering IDO, HVEM, and IL-8, we obtained a score
based on their serum basal levels, able to predict patients’
survival. A prospective validation in a larger patients’ popula-
tion is advisable to avoid ineffective treatments.
Considering all the mUM patients included in the study, we
found three different course of disease with specific profiles of
circulating immune checkpoints and cytokines. All the patients
with mUM were divided into three groups considering the
survival: fast progressors (FP), slow progressors (SP) and long
survivors (LS). IL-8, IDO activity and HVEM are also able to
distinguish the different course of the disease. Indeed, their
concentration was higher in patients with dismal survival.
Moreover, serum levels of CD137, GITR, and CD27 resulted
significantly different among the three groups of the patients,
with fast-progressive disease group that had higher concentra-
tion of sICs compared to SP and LS.
In contrast to CM, UM is usually poorly responsive to
checkpoint inhibitors.
18
In order to define immunological dif-
ferences between metastatic UM and mCM during anti-PD-1
therapy, we evaluated the release of sICs, cytokines/chemokines
and IDO activity in CM patients and the results were compared
HUMAN VACCINES & IMMUNOTHERAPEUTICS -
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with UM patients. Although all the patients were treated with an
anti-PD-1 agent, the levels of several soluble immune molecules
were remarkably different between the two groups during the
treatment. In fact, the concentration of sGITR and sCD27 were
significantly higher in UM patients. It has been demonstrated
that sGITR promotes Helios expression and enhances the func-
tion of regulatory T cells.
48
On the other hand, the immunolo-
gical function of sCD27 has not yet been clarified. Among the
soluble forms of inhibitory receptors, only sPD-1 concentration
was statistically different between the two types of cancer:
higher in UM patients compared to CM. Regarding the soluble
form of ligands, the levels of sCD80 resulted increased in UM. It
is well known that CD80 on APC cells is required for rejection
of immunogenic tumor in animal models.
49
Recently, it has
been demonstrated that CD80 on APC can bind PD-L1 avoid-
ing PD-1/PD-L1 interaction and consequently blocking an inhi-
bitory signal for T cell activation.
50
High serum level of CD80
could reflect the shedding of CD80 and contribute to maintain
an immunosuppressive microenvironment.
Furthermore, we observed a higher concentration of both
IFNγ in the serum of UM patients. IFNγ is a potent inducer of
IDO expression, and indeed a higher presence of this enzyme
in UM patients was confirmed.
The different expression of circulating factors in the serum
of patients with metastatic UM and mCM during anti-PD-1
therapy can offer a possible explanation of the different clinical
efficacy obtained with checkpoint inhibitors in these mela-
noma subtypes. Indeed, our findings show immunosuppressive
features of UM compared to CM.
The rarity of the disease influenced the number of patients
enrolled. The small sample size and the unavailability of
a baseline sample for all the patients with UM represent limita-
tions of the study. Further analyses with the largest number of
patients are necessary to confirm our observations.
Conclusions
This study provides preliminary data on a limited population
of patients with a rare disease as UM. This is the first study to
evaluate the correlation between sICs, cytokines and chemo-
kines with clinical outcomes in metastatic UM.
The unsatisfactory response to anti-PD-1 therapy in UM
may be justified by poor activation of the immune system.
However, some patients with metastatic UM had a long survi-
val. These patients could be identified by a score based on the
circulating immune molecules such as HVEM, IDO, and IL-8.
The immune response could influence the course of advanced
disease and some of the studied immunological molecules
could also offer new therapeutic targets.
Moreover, the comparison of circulating immune profile
during anti-PD-1 therapy between UM and CM could reflect
the different efficacy of ICIs in these diseases.
Acknowledgments
EB is currently supported by the Associazione Italiana per la Ricerca sul
Cancro (AIRC) under In-vestigator Grant (IG) No. IG20583 and by
Institutional funds of Università Cattolica del Sacro Cuore (UCSC-
project D1-2018/2019). GT is supported by AIRC, IG18599, AIRC
5 × 1000 21052. GS is currently supported by Institutional funds of
Università Cattolica del Sacro Cuore (UCSC-project D1 2018/2019). MN
reports research grant from Incyte and IPSEN.
Disclosure statement
The authors declare that they have no competing interests. ER had a role as
consultant for MSD and Novartis. EB reported speakers’ and travel fees from
MSD, Astra-Zeneca, Celgene, Pfizer, Hel-sinn, Eli-Lilly, BMS, Novartis, and
Roche; consultant’s fees from Roche and Pfizer; and institution-al research
grants from AstraZeneca and Roche. PM has/had a role as consultant/advi-
sory for BMS, Roche Genentech, MSD, Novartis, Amgen, Merk Serono,
Pierre Fabre and Incyte. GT has a role as consultant for BMS and MSD.
Funding
The study was funded by institutional resources by Università Cattolica
del Sacro Cuore.
ORCID
Ernesto Rossi http://orcid.org/0000-0002-6442-1707
References
1. McLaughlin CC, Wu XC, Jemal A, Martin HJ, Roche LM,
Chen VW. Incidence of noncutaneous melanomas in the US.
Cancer. 2005;103:1000–07. doi:10.1002/cncr.20866.
2. Kujala E, Mäkitie T, Kivelä T. Very long-term prognosis of patients
with malignant uveal melanoma. Invest Ophthalmol Vis Sci.
2003;44:4651–59. doi:10.1167/iovs.03-0538.
3. Rietschel P, Panageas KS, Hanlon C, Patel A, Abramson DH,
Chapman PB. Variates of survival in metastatic uveal melanoma.
J Clin Oncol. 2005;23:8076–80. doi:10.1200/JCO.2005.02.6534.
4. Collaborative Ocular Melanoma Study Group, Diener-West M,
Reynolds SM, Agugliaro DJ, Caldwell R, Cumming K, Earle JD,
Hawkins BS, Hayman JA, Jaiyesimi I, Jampol LM, et al.
Development of metastatic disease after enrollment in the COMS
trials for treatment of choroidal melanoma: collaborative ocular
melanoma study group report no. 26. Arch Ophthalmol.
2005;123:1639–43. doi:10.1001/archopht.123.12.1639.
5. Agarwala SS, Eggermont AM, O’Day S, Zager JS. Metastatic mel-
anoma to the liver: a contemporary and comprehensive review of
surgical, systemic, and regional therapeutic options. Cancer.
2014;120:781–89. doi:10.1002/cncr.28480.
6. Rossi E, Schinzari G, Maiorano BA, Indellicati G, Di Stefani A,
Pagliara MM, Fragomeni SM, De Luca EV, Sammarco MG,
Garganese G, et al. Efficacy of immune checkpoint inhibitors in
different types of melanoma. Hum Vaccin Immunother.
2020;1–10. doi:10.1080/21645515.2020.1771986.
7. Flaherty LE, Unger JM, Liu PY, Mertens WC, Sondak VK.
Metastatic melanoma from intraocular primary tumors: the
Southwest oncology group experience in phase II advanced mela-
noma clinical trials. Am J Clin Oncol. 1998;21:568–72. doi:10.1097/
00000421-199812000-00008.
8. Nathan F, Sato T, and Hart E. Response to combination che-
motherapy of liver metastasis from choroidal melanoma compared
with cutaneous melanoma (abstract). Proc Am Soc Clin Onc .
1994;13.
9. Bedikian AY, Legha SS, Mavligit G, Carrasco CH, Khorana S,
Plager C, Papadopoulos N, Benjamin RS. Treatment of uveal
melanoma metastatic to the liver; a review of the MD Anderson
cancer center experience and prognostic factors. Cancer.
1995;76:1665–70. doi:10.1002/1097-0142(19951101)76:9<1665::
aid-cncr2820760925>3.0.co;2-j.
-
e2034377 E. ROSSI ET AL.
8

10. Schinzari G, Rossi E, Cassano A, Dadduzio V, Quirino M, Pagliara M,
Blasi MA, Barone C. Cisplatin, dacarbazine and vinblastine as first line
chemotherapy for liver metastatic uveal melanoma in the era of
immunotherapy: a single institution phase II study. Melanoma Res.
2017;27:591–95. doi:10.1097/CMR.0000000000000401.
11. Carvajal RD, Piperno-Neumann S, Kapiteijn E, Chapman PB,
Frank S, Joshua AM, Piulats JM, Wolter P, Cocquyt V,
Chmielowski B, et al. Selumetinib in combination with dacarbazine
in patients with metastatic uveal melanoma: a phase III, multi-
center, randomized trial (SUMIT). J Clin Oncol. 2018;36:1232–39.
doi:10.1200/JCO.2017.74.1090.
12. Danielli R, Ridolfi R, Chiarion-Sileni V, Queirolo P, Testori A,
Plummer R, Boitano M, Calabrò L, Rossi CD, Giacomo AM, et al.
Ipilimumab in pretreated patients with metastatic uveal melanoma:
safety and clinical efficacy. Cancer Immunol Immunother. 2012;61
(1):41–48. doi:10.1007/s00262-011-1089-0.
13. Zimmer L, Vaubel J, Mohr P, Hauschild A, Utikal J, Simon J,
Garbe C, Herbst R, Enk A, Kämpgen E, et al. Phase II
DeCOG-study of ipilimumab in pretreated and treatment-naive
patients with metastatic uveal melanoma. PLoS One. 2015;10:
e0118564. doi:10.1371/journal.pone.0118564.
14. Karydis I, Chan PY, Wheater M, Arriola E, Szlosarek PW,
Ottensmeier CH. Clinical activity and safety of pembrolizumab in
ipilimumab pre-treated patients with uveal melanoma.
Oncoimmunology. 2016;5:e1143997. doi:10.1080/2162402X.20
16.1143997.
15. Algazi PA, Tsai KK, Shoushtari AN, Munhoz RR, Eroglu Z,
Piulats JP. Clinical outcomes in metastatic uveal melanoma treated
with PD-1 and PD-L1 antibodies. Cancer. 2016;122:3344–53.
doi:10.1002/cncr.30258.
16. Rossi E, Pagliara MM, Orteschi D, Dosa T, Sammarco MG,
Caputo CG, Petrone G, Rindi G, Zollino M, Blasi MA, et al.
Pembrolizumab as first-line treatment for metastatic uveal
melanoma. Cancer Immunol Immunother. 2019;68:1179–85.
doi:10.1007/s00262-019-02352-6.
17. Piulats JM, Espinosa E, de la Cruz Merino L, Varela M, Alonso
Carrión L, Martín-Algarra S, López Castro R, Curiel T, Rodríguez-
Abreu D, Redrado M, et al. Nivolumab plus ipilimumab for treatment-
naïve metastatic uveal melanoma: an open-label, multicenter, phase II
trial by the Spanish Multidisciplinary Melanoma Group (GEM-1402).
J Clin Oncol. 2021;39:586–98. doi:10.1200/JCO.20.00550.
18. Durante MA, Rodriguez DA, Kurtenbach S, Kuznetsov JN,
Sanchez MI, Decatur CL, Snyder H, Feun LG, Livingstone AS,
Harbour JW. Single-cell analysis reveals new evolutionary complexity
in uveal melanoma. Nat Commun. 2020;11:496. doi:10.1038/s41467-
019-14256-1.
19. Niederkorn JY. Immune escape mechanisms of intraocular
tumors. Progr Retin Eye Res. 2009;28:329–47. doi:10.1016/j.
preteyeres.2009.06.002.
20. Terai M, Londin E, Rochani A, Link E, Lam B, Kaushal G,
Bhushan A, Orloff M, Sato T. Expression of tryptophan
2,3-Dioxygenase in metastatic uveal melanoma. Cancers (Basel).
2020;12:405. doi:10.3390/cancers12020405.
21. Jenne CN, Kubes P. Immune surveillance by the liver. Nat
Immunol. 2013;14:996–1006. doi:10.1038/ni.2691.
22. Furney SJ, Pedersen M, Gentien D, Dumont AG, Rapinat A,
Desjardins L, Turajlic S, Piperno-Neumann S, de la Grange P,
Roman-Roman, et al. SF3B1 mutations are associated with alter-
native splicing in uveal melanoma. Cancer Discov. 2013;3:1122–29.
doi:10.1158/2159-8290.CD-13-0330.
23. Rodrigues M, Mobuchon L, Houy A, Fiévet A, Gardrat S,
Barnhill RL, Popova T, Servois V, Rampanou A, Mouton A, et al.
Outlier response to anti-PD1 in uveal melanoma reveals germline
MBD4 mutations in hypermutated tumors. Nat. Commun.
2018;9:1866. doi:10.1038/s41467-018-04322-5.
24. Rossi E, Schinzari G, Zizzari IG, Maiorano BA, Pagliara MM,
Sammarco MG, Fiorentino V, Petrone G, Cassano A, Rindi G,
et al. Immunological backbone of uveal melanoma: is there
a rationale for immunotherapy? Cancers (Basel). 2019;11:1055.
doi:10.3390/cancers11081055.
25. Zizzari IG, Napoletano C, Di Filippo A, Botticelli A, Gelibter A,
Calabrò F, Rossi E, Schinzari G, Urbano F, Pomati G, et al.
Exploratory pilot study of circulating biomarkers in metastatic
renal cell carcinoma. Cancers (Basel). 2020;12:2620. doi:10.3390/
cancers12092620.
26. Zizzari IG, Di Filippo A, Scirocchi F, Di Pietro FR, Rahimi H,
Ugolini A, Scagnoli S, Vernocchi P, Del Chierico F, Putignani L,
et al. Soluble immune checkpoints, gut metabolites and perfor-
mance status as parameters of response to nivolumab treatment in
NSCLC patients. J Pers Med. 2020;10:208. doi:10.3390/
jpm10040208.
27. Daassi D, Mahoney KM, Freeman GJ. The importance of exosomal
PDL1 in tumour immune evasion. Nat Rev Immunol.
2020;20:209–15. doi:10.1038/s41577-019-0264-y.
28. Gu D, Ao X, Yang Y, Chen Z, Xu X. Soluble immune checkpoints
in cancer: production, function and biological significance.
J Immunother Cancer. 2018;6:132. doi:10.1186/s40425-018-0449-
0.
29. Machiraju D, Wiecken M, Lang N, Hülsmeyer I, Roth J,
Schank TE, Eurich R, Halama N, Enk A, Hassel JC. Soluble
immune checkpoints and T-cell subsets in blood as biomarkers
for resistance to immunotherapy in melanoma patients.
Oncoimmunology. 2021 May 25;10(1):1926762. doi:10.1080/
2162402X.2021.1926762.
30. Lim SY, Lee JH, Gide TN, Menzies AM, Guminski A, Carlino MS,
Breen EJ, Yang JYH, Ghazanfar S, Kefford RF, et al. Circulating
cytokines predict immune-related toxicity in melanoma patients
receiving Anti-PD-1-based immunotherapy. Clin Cancer Res.
2019;25:1557–63. doi:10.1158/1078-0432.CCR-18-2795.
31. Bakouny Z, Choueiri TK. IL-8 and cancer prognosis on
immunotherapy. Nat Med. 2020 May;26:650–51. doi:10.1038/
s41591-020-0873-9.
32. Tagliaferri L, Lancellotta V, Fionda B, Mangoni M, Casà C, Di
Stefani A, Pagliara MM, D’Aviero A, Schinzari G, Chiesa S,
et al. Immunotherapy and radiotherapy in melanoma:
a multidisciplinary comprehensive review. Hum Vaccin
Immunother. 2021:1–8. doi:10.1080/21645515.2021.1903827.
33. Luu K, Shao Z, Schwarz H. The relevance of soluble CD137 in the
regulation of immune responses and for immunotherapeutic
intervention. J Leukoc Biol. 2020;107:731–38. doi:10.1002/
JLB.2MR1119-224R.
34. Hebbar M, Jeannin P, Magistrelli G, Hatron PY, Hachulla E,
Devulder B, Bonnefoy JY, Delneste Y. Detection of circulating
soluble CD28 in patients with systemic lupus erythematosus,
primary Sjogren’s syndrome and systemic sclerosis. Clin Exp
Immunol. 2004;136:388–92. doi:10.1111/j.1365-2249.2004.
02427.x.
35. Lutzky J, Feun LG, Magallanes N, Kwon D, Harbour JW.
NCT04552223: a phase II study of nivolumab plus BMS-986016
(relatlimab) in patients with metastatic uveal melanoma (UM)
(CA224-094). Abs TPS9590 2021 ASCO Annual Meeting. Vol.
JCO 30, 15 suppl. doi: 10.1200/JCO.2021.39.15_suppl.TPS9590
36. Lunardi S, Lim SY, Muschel RJ, Brunner TB. IP-10/
CXCL10 attracts regulatory T cells: implication for pancreatic
cancer. Oncoimmunology. 2015;4:e1027473. doi:10.1080/
2162402X.2015.1027473.
37. Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer
microenvironment and their relevance in cancer immunotherapy.
Nat Rev Immunol. 2017;17:559–72. doi:10.1038/nri.2017.49.
38. Mellor AL, Munn DH. Tryptophan catabolism and T-cell toler-
ance: immunosuppression by starvation? Immunol Today.
1999;20:469–73. doi:10.1016/s0167-5699(99)01520-0.
39. Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A,
Mellor AL. Inhibition of T cell proliferation by macrophage tryp-
tophan catabolism. J Exp Med. 1999;189:1363–72. doi:10.1084/
jem.189.9.1363.
40. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA.
Indoleamine 2,3-dioxygenase production by human dendritic
cells results in the inhibition of T cell proliferation. J Immunol.
2000;164:3596–99. doi:10.4049/jimmunol.164.7.3596.
HUMAN VACCINES & IMMUNOTHERAPEUTICS -
e2034377
9

41. Curti A, Pandolfi S, Valzasina B, Aluigi M, Isidori A, Ferri E,
Salvestrini V, Bonanno G, Rutella S, Durelli I, et al. Modulation
of tryptophan catabolism by human leukemic cells results in the
conversion of CD25- into CD25+ T regulatory cells. Blood.
2007;109:2871–77. doi:10.1182/blood-2006-07-036863.
42. Chen W, Liang X, Peterson AJ, Munn DH, Blazar BR. The indo-
leamine 2,3-dioxygenase pathway is essential for human plasma-
cytoid dendritic cell-induced adaptive T regulatory cell generation.
J Immunol. 2008;181:5396–404. doi:10.4049/jimmunol.181.8.5396.
43. Chung DJ, Rossi M, Romano E, Ghith J, Yuan J, Munn DH,
Young JW. Indoleamine 2,3-dioxygenase-expressing mature
human monocyte-derived dendritic cells expand potent autolo-
gous regulatory T cells. Blood. 2009;114:555–63. doi:10.1182/
blood-2008-11-191197.
44. Botticelli A, Cerbelli B, Lionetto L, Zizzari I, Salati M, Pisano A,
Federica M, Simmaco M, Nuti M, Marchetti P. Can IDO activity
predict primary resistance to anti-PD-1 treatment in NSCLC?
J Transl Med. 2018;16:219. doi:10.1186/s12967-018-1595-3.
45. Botticelli A, Mezi S, Pomati G, Cerbelli B, Cerbelli E, Roberto M,
Giusti R, Cortellini A, Lionetto L, Scagnoli S, et al. Tryptophan
catabolism as immune mechanism of primary resistance to
anti-PD-1. Front Immunol. 2020;11:1243. doi:10.3389/
fimmu.2020.01243.
46. Jones A, Bourque J, Kuehm L, Opejin A, Teague RM, Gross C,
Hawiger D. Immunomodulatory functions of BTLA and HVEM
govern induction of extrathymic regulatory T cells and tolerance by
dendritic cells. Immunity. 2016;45:1066–77. doi:10.1016/j.
immuni.2016.10.008.
47. Yuen KC, Liu LF, Gupta V, Madireddi S, Keerthivasan S, Li C,
Rishipathak D, Williams P, Kadel EE 3rd, Koeppen H, et al. High
systemic and tumor-associated IL-8 correlates with reduced clin-
ical benefit of PD-L1 blockade. Nat Med. 2020;26:693–98.
doi:10.1038/s41591-020-0860-1.
48. Li Y, Yang S, Li Z, Meng H, Jin W, Yang H, Yin W. Soluble
glucocorticoid-induced tumor necrosis factor receptor regulates
Helios expression in myasthenia gravis. J Transl Med.
2019;17:168. doi:10.1186/s12967-019-1916-1.
49. McHugh RS, Ahmed SN, Wang YC, Sell KW, Selvaraj P.
Construction, purification, and functional incorporation on
tumor cells of glycolipid-anchored human B7-1 (CD80). Proc
Natl Acad Sci U S A. 1995;92:8059–63. doi:10.1073/
pnas.92.17.8059.
50. Sugiura D, Maruhashi T, Okazaki IM, Shimizu K, Maeda TK,
Takemoto T, Okazaki T. Restriction of PD-1 function by cis-PD-
L1/CD80 interactions is required for optimal T cell responses.
Science. 2019;364:558–66. doi:10.1126/science.aav7062.
-
e2034377 E. ROSSI ET AL.
10