Transplant Immunology 70 (2022) 101513

Contents lists available at ScienceDirect

Transplant Immunology
journal homepage: www.elsevier.com/locate/trim

Evaluation of cytokine profile in the different phases of the autologous

hematopoietic stem cell transplantation in patients with multiple myeloma
AlexandraFernandes Callera a, Fernando Callera b, Aurileia Aparecida Brito c,
Carlos Rocha Oliveira d, e, Andre Luis Lacerda Bachi f, Rodolfo P. Vieira a, g, h, i, *
a
Department of Pathology, School of Medicine, University of São Paulo, São Paulo, SP, Brazil
b
Centro de Hematologia do Vale, São José dos Campos, São Paulo, SP, Brazil
c
Nove de Julho University, São Paulo, SP, Brazil
d
Anhembi Morumbi University, São José dos Campos, SP, Brazil
e
Post-graduation Program in Biomedical Engineering, Federal University of Sao Paulo, São José dos Campos, SP, Brazil
f
University Santo Amaro, São Paulo, SP, Brazil
g
Brazilian Institute of Teaching and Research in Pulmonary and Exercise Immunology (IBEPIPE), São José dos Campos, São Paulo, SP, Brazil
h
Post-graduation Program in Bioengineering, Universidade Brasil, São Paulo, SP, Brazil
i
Post-graduation Program in Sciences of Human Movement and Rehabilitation, Federal University of Sao Paulo, Santos, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Background: The autologous hematopoietic stem cell transplantation (ASCT) is of fundamental importance in the
Multiple myeloma treatment of patients with multiple myeloma (MM). Nevertheless, due to its toxicity, it decreases the number of
ASCT bone marrow cells available, altering the cell interactions and causing an imbalance between pro- and anti-
Cytokines
inflammatory cytokines.
Methods: Thus, we determined the serum levels of pro- and anti-inflammatory cytokines in samples of patients
with MM obtained from the different phases of ASCT.
Results: In summary, the cytokines levels varied considering the different phases of ASCT. The levels of IL-1ra
tend to increase in the post-apheresis period suggesting an anti-inflammatory role induced by the apheresis
process. A response characterized by the increase in the concentrations of IL-5 and IL-8 was observed in the post-
conditioning bone marrow aplasia phase. The rise in IL-5 levels was not correlated with any clinical or laboratory
event in this framework; IL-8 was associated with positive blood cultures and seems to have an effect against
microbial agents. The increase in the levels of IL-10 and IL-12 suggests a possible regulatory effect of the in­
flammatory response in the period of bone marrow recovery and IL-12 seems to be inversely associated with the
presence of minimal residual disease.
Conclusions: Apheresis process seems to induce an anti-inflammatory response, followed by a pro-inflammatory
response and a stimulus for granulocytes differentiation.

1. Introduction
Multiple myeloma (MM) is a malignant hematological neoplasia
characterized by clonal plasma cell proliferation in the bone marrow.
Despite the advance in current therapies, including chemotherapies and
immunotherapies, MM is still considered an incurable disease, with low
rates of event-free survival and overall survival [1].
High-dose chemotherapy followed by autologous hematopoietic
stem cell transplantation (ASCT) is of fundamental importance in the
treatment of patients with MM and results in higher rates of negative
minimal residual disease, better rates of remission and patient survival
[2–4]. Nevertheless, high doses of chemotherapeutic agents cause toxic
lesions in the bone marrow whose main function is the production of
cells that constitute the blood and the immune system. In this context,
chemotherapeutic agents act against neoplastic cells; however, they
attack normal and habitual cells located in the bone marrow. Conse­
quently, the patient can present anemia (decrease in red blood cells),
serious infectious phenomena resulting from leukopenia (decrease in
white blood cells) and bleeds due to thrombocytopenia (decrease in the
number of platelets). These toxic effects, named bone marrow aplasia,

* Corresponding author at: Federal University of São Paulo (UNIFESP), Rua Talim 330, São José dos Campos, SP 12231-280, Brazil.
E-mail address: rodrelena@yahoo.com.br (R.P. Vieira).

https://doi.org/10.1016/j.trim.2021.101513
Received 18 June 2021; Received in revised form 2 December 2021; Accepted 3 December 2021
Available online 8 December 2021
0966-3274/© 2021 Elsevier B.V. All rights reserved.
A. Callera et al. Transplant Immunology 70 (2022) 101513

can be irreversible and potentially lethal if they remain over long pe­ Table 1
riods. With the aim of circumventing bone marrow toxicity resulting Physical (presented as median and minimum – maximum values) and clinical
from high doses of chemotherapeutic agents, hematopoietic stem cells (presented as number or percentage) characteristics of the patients with MM
(HSC), characteristically called CD34+ cells, are mobilized and and submitted to ASCT participating in this study.
collected from the own patient before the execution of the chemo­ Median age 61 (40–70)
therapy by using a method termed apheresis. After CD34+ cells have Male / Female (%) 19 (63) / 11 (37)
Median weight 70 (57–108)
been collected, they can be preserved or cryopreserved at temperatures
Diagnostic classification n (%)
lower than 86 negative centigrade degrees. Following the high-dose Immunoglobulin G 18 (60)
chemotherapy protocol prescribed (called conditioning), the CD34+ Immunoglobulin A 7 (23)
cells are reinfused into the patient intravenously. Since these cells do not Immunoglobulin light chain 5 (17)
suffer the action of chemotherapeutic agents, they rapidly migrate to the ISS Staging (International Staging System)
patient's bone marrow, proliferate, and differentiate themselves into ISS I 16 (53)
mature cells, recovering the bone marrow functionality in a short period ISS II 9 (30)
[5]. ISS III 5 (17)

Based on the information mentioned above, ASCT is commonly
divided into four phases named 1) mobilization of CD34+ cells, 2)
collection of CD34+ cells, 3) bone marrow aplasia after conditioning,
and lastly 4) bone marrow recovery. In addition, ASCT enables that the
patients have the benefit of using high doses of chemotherapeutic agents
with a reduction of the period of bone marrow aplasia. On the other
hand, due to its transient toxicity, ASCT drastically alters the cell
interaction dynamics, decreases the number of bone marrow cells
available can cause an imbalance between pro- and anti-inflammatory
cytokines.
In the context of high-dose chemotherapy treatments including he­
matopoietic stem cell transplants and chemoradiation, cytokines seem
to have a role in the control of tumor cells, restoration of the immune
and hematopoietic systems, post-transplant complications and eventu­
ally in the risk of relapse of the underlying disease [6–13]. Wang and
collaborators [10] recently reported the association among inflamma­
tory markers and development of symptom burden in MM patients
through ASCT, however, there is a paucity of data available in this
research field.
2. Objectives
In view of these aspects, we believe that the investigation into the
effects of different phases of the ASCT on the profile of pro- and anti-
inflammatory cytokine secretion in patients with MM could reveal
important elements for the better comprehension of the pathophysio­
logical processes involved and for the eventual development of new
therapeutic strategies.
Remission induction treatments
Single chemotherapy protocol 18 (60)
Two chemotherapy protocols 8 (27)
Associated radiotherapy 4 (13)
Pre-transplantation therapy response
Complete response (CR) 04 (13)
Very good partial response (VGPR) 06 (20)
Partial response (PR) 20 (67)
ISS I = serum beta-2-microglobulin <3,5 mg/L and serum albumin ≥3,5 g/Dl,
ISS II = intermediary between I and III, ISS III = serum beta-2-microglobulin
≥5,5 mg/L. Complete Response (CR) = negative immunofixation (serum and
urine), clonal plasma cells in the bone marrow ≤5%, disappearance of extra­
osseous plasmacytoma and no increase in size or number of lytic lesions. Very
good partial response (VGPR) = detectable monoclonal protein by immuno­
fixation, but not by electrophoresis, reduction in monoclonal protein in serum
or urine ≥90% and proteinuria <100 mg/24 h. Partial response (PR): reduc­
tion in monoclonal protein in serum ≥50%, in urine ≥90% and proteinuria
<200 mg/24 h.
3.2.2. Inclusion and exclusion criteria
Patients who reached at least partial remission with the following
chemotherapy protocols (cyclophosphamide and dexamethasone,
cyclophosphamide and dexamethasone combined with thalidomide, and
bortezomib associated with cyclophosphamide and dexamethasone)
were included in the study. Patients with heart disease and ejection
fraction lower than 50%, with lung or limiting hepatic disease, with
creatinine clearance lower than 40 ml/min, ECOG higher than 1 or
Karnofsky index lower than 70, insufficient collection of HSC or positive
HIV or HCV serologies were not eligible.

3. Material and methods

3.1. Ethical approval
The present study was conducted in partnership with a bone marrow
transplant unit of Hospital PIO XII of São José dos Campos and was
approved by the Research Ethics Committee (CEP) of the Clinics Hos­
pital of the Faculty of Medicine at University of São Paulo. Process
number 2.564.035/2017.
3.2. Study design
3.2.1. Patients
In the period between August of 2017 and August of 2019, thirty (30)
patients with recent diagnosis of multiple myeloma (MM), treated with
ASCT, were included in this prospective study (Table 1). Initially,
samples of twenty (20) patients, obtained from each phase of the
transplantation, were analyzed. Subsequently, 10 more cases were
inserted in each phase of the study in order to confirm the results pre­
viously found; we also included 10 patients in the period prior to
mobilization of CD34+ cells in an effort to demonstrate the possible
influence of the mobilization process on the serum concentration of
cytokines.
3.3. Mobilization and quantification of CD34+ cells
The mobilization of CD34+ cells to the peripheral blood was
executed two weeks before the transplantation by using the cytarabine
protocol (Ara-C) in association with granulocyte growth factor (G-CSF)
as previously reported [14]. Throughout the mobilization period, the
patients were evaluated regarding adverse hematological and non-
hematological reactions, classified according to the Common Termi­
nology Criteria for adverse events [15]. The CD34+ cells were quanti­
fied by flow cytometry according to the protocols described by the
International Society of Hematotherapy and Graft Engineering (ISH­
AGE) [16].
3.4. Collection of cells by apheresis
The HSC mobilized from the bone marrow to the peripheral blood
were collected by a cell separator device of intermittent flow (Haemo­
netics MCS+ 9000E, Haemonetics Corporation, Massachusetts, USA)
according to protocols defined by the manufacturer for the collection of
peripheral blood mononuclear cells. The objective in this phase was to
reach 5,0 × 106 CD34+ cells / kg of the patient in the final product of
apheresis.

2
A. Callera et al.
3.5. Cell cryopreservation
After the execution of the apheresis procedure, part of the product
was maintained between 2◦ and 8 ◦ C and returned to the patient after
high-dose chemotherapy; the other part was frozen using a cryopro­
tectant mixture containing dimethyl sulfoxide, hydroxyethyl starch and
human albumin. The bags containing the cells were cryopreserved in
freezers with temperatures below minus 84 ◦ C.
3.6. ASCT protocols
For the execution of ASCT, the patients were admitted to the hospital
in individual rooms with HEPA (high-efficiency particulate arrestance)
filters. The conditioning (high-dose chemotherapy) used in all patients
was melphalan at a dose of 200 mg/m2. One day after the end of con­
ditioning, the fresh or thawed CD34+ cells were infused into the pa­
tients. The moment of infusion of CD34+ cells is called day 0 (D 0) and
the patient's daily evolution after the infusion of CD34+ cells is classified
as D + 1, D + 2, and so on until the bone marrow recovery. Generally,
the phase of bone marrow aplasia lasts 3 to 10 days after conditioning
and can be defined by means of the hemogram that is presented with a
hemoglobin level < 10 g/dL, white blood cell counts <0,5 × 109/L and
frequently platelets <20 × 109/L. The clinical protocols after the
transplantation involved the use of specific care such as the adminis­
tration of prophylactic antimicrobials (acyclovir and fluconazole), blood
component transfusion, when necessary, empirical antimicrobial treat­
ment for febrile neutropenia, collection of blood cultures, treatment of
mucositis and parenteral nutrition when low oral calorie intake. The
bone marrow recovery was characterized when neutrophils were higher
than 0,5 × 109/L and platelets higher than 20 × 109/L without need for
transfusion.
3.7. Sample collection and determination of cytokine profile in the
different phases of ASCT
Peripheral blood samples from each patient were collected before the
mobilization of CD34+ cells, before and after the apheresis session (one
day before and one day after) in the period of bone marrow aplasia (on
day +7) and after the bone marrow recovery (on day +12) for the
evaluation of serum levels of pro- and anti-inflammatory cytokines GM-
CSF, IL-1ra, IL1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13. IL-15, IL-
17, INF-gamma (γ), Klotho and TNF-alpha (α). The blood was collected
by venipuncture using steryle material and vacuum tubes containing
anticoagulant EDTA K3 (two tubes of 5 mL); after the blood collection,
the two tubes were centrifuged at 900g, for 7 min, at 4 ◦ C. The plasma
obtained was stored at temperatures lower than − 84 ◦ C for later eval­
uation. The measurements of the above-described cytokines were per­
formed using Duo Set® ELISA kits (R&D Systems, MN, EUA) as
recommended by the manufacturers and the results were expressed in
pg/mL.
3.8. Research on minimal residual disease (MRD)
Bone marrow blood samples obtained by myelogram were collected
from patients between 90 and 100 days after ASCT. The research on
clonal plasma cells in the material obtained (MRD) was conducted by
flow cytometry as previously described [17].
3.9. Statistical analyses
The statistical analyses were conducted considering nonparametric
and paired samples; the statistical relationship between sets of variables
were determined with multiple regression analysis. Categorical vari­
ables were tested with Fisher's exact test. The variance analyses of the
samples were executed with Friedman test (One Way ANOVA), and the
correlations between variables with Spearman test. The comparison
3
Transplant Immunology 70 (2022) 101513
between the pre-mobilization and pre-apheresis periods was made with
Mann-Whitney test, considering, in this situation, nonparametric and
unpaired samples. The results were compared with the pre-apheresis
column. P values lower than 0,05 were considered as indicative of sta­
tistical significance (* for p > 0.05, ** for p < 0.05, *** for p < 0.01, and
**** for p < 0.001). Data were analyzed with the software GraphPad
Prism 6.
4. Results
4.1. Study population characteristics before and after apheresis
As shown in Table 1, 70% of patients were treated with only one
chemotherapeutic protocol and 60% were in partial response at trans­
plantation (explanation note on the legend of the Fig. 1). The mobili­
zation protocol was uneventful; patients did not require hospitalization
nor showed persistente or substantial disabilities or had important
medical consequences (Table 2). Cell elements were not different before
and after the apheresis session; the median found was 8,7 × 106 CD34+
cells/kg, ranging from 4.1 to 38.7 cell/Kg. In the period of post-
conditioning bone marrow aplasia, all patients presented febrile neu­
tropenia with indication of empirical antimicrobial treatment; blood
culture sample were positive in sixteen patients (53%). All needed blood
component transfusion (Table 3). There were no bone marrow recovery
failures and the medians of the times for neutrophils >0.5 × 109/L and
for platelets >20 × 109/L were 10 days (9–12 days) and 11 days (9–13
days) respectively.
4.2. Cytokines analysis before and after apheresis
Cytokine concentrations did not suffer interference from the mobi­
lization process (Table 4). The levels of IL-1ra increased significantly
after the apheresis session (p = 0.0001) and decreased in the phases of
post-conditioning aplasia and bone marrow recovery to values like the
observed in the pre-apheresis phase. There was a significant increase in
levels of IL-5 (p = 0.0003) and IL-8 (p < 0.0001) in the period of post-
conditioning aplasia; however, they decreased in the bone marrow re­
covery. An increase in the levels of IL-10 (p < 0.0001) and IL-12p70 (p
= 0.0001) in the period of bone marrow recovery was observed (Fig. 1).
These findings were not associated with the response to pre-
transplantation treatment or correlated to the quantity of CD34+
transplanted cells or blood component transfusions. The increase in IL-8
was significantly associated with the positivity of blood cultures (IL-8 p
= 0.021, relative risk = 3.68, 95% CI = 1.05 to 12.90); there was no
association between IL-5 and positive blood cultures (p = 0.06); IL-5
levels was not correlated with any clinical or laboratory event in this
framework. Lastly, variations in the serum concentrations of cytokines
GM-CSF, IL-1beta, IL-2, IL-4, IL-6, IL-13, IL-15, IL-17, INF-gamma,
Klotho and TNF-alpha in the different phases of ASCT were not
observed (Table 5). An association between the increase in IL-12 in the
bone marrow recovery and the research on minimal residual disease
(MRD) after ASCT was observed. In patients with positive MRD, the
average of serum concentrations of IL-12 was significantly lower (p =
0.001), and the percentages of clonal bone marrow plasma cells were
inversely proportional to the concentrations of IL-12 (r = − 0.845, p =
0.0001), Fig. 2. In contrast, increased IL-10 was not associated with
MRD.
5. Discussion
Over 80% of the patients in our study came from the Brazilian public
health system, where the available treatments [18] produce low rates of
CR of MM, thus explaining the high proportion of patients in PR. Wang
and collaborators [10] evaluated the profile of several cytokines in 63
patients with MM during ASCT, most of them in CR, and different results
were reported; there was a decrease in IL-1ra on day +5, an increase in
A. Callera et al. Transplant Immunology 70 (2022) 101513

Fig. 1. Variation in the serum levels of IL-1ra, IL-5, IL-8, IL-10 and IL-12 (mean and standard error of the mean) in the phases of ASCT.

4
A. Callera et al. Transplant Immunology 70 (2022) 101513

Table 2 Table 4
Adverse events observed during the stem cell mobilization Comparison between the serum concentrations of cytokines evaluated in the
protocol. phases of pre-mobilization and pre-apheresis of ASCT.
Hematological n (%) Cytokine Pre-mobilization (n = 10) Pre-apheresis (n = 30) p

Neutropenia IL-1ra 25.1 ± 6.2 23.3 ± 6.7 0.883

Degree 1 24 (80) IL-1b 4.0 ± 0.6 4.2 ± 0.8 0.890
Degree 2 4 (13) IL-2 134.1 ± 29.4 129.0 ± 30.4 0.928
Degree 3 2 (7) IL-4 6.5 ± 1.3 6.2 ± 1.0 0.875
Degree 4 0 IL-5 11.5 ± 2.2 9.4 ± 1.5 0.473
IL-6 11.6 ± 6.7 13.9 ± 4.4 0.789
Thrombocytopaenia
IL-8 32.1 ± 3.6 31.8 ± 6.5 0.979
Degree 1 0
IL-10 10.9 ± 3.1 8.5 ± 2.1 0.558
Degree 2 20 (67)
IL-12p70 74.3 ± 9.9 77.7 ± 16.1 0.909
Degree 3 8 (27)
IL-13 3.7 ± 0.4 3.2 ± 0.4 0.500
Degree 4 2 (6)
IL-15 5.3 ± 0.6 4.3 ± 0.7 0.435
Transfusions IL-17 46.7 ± 35.5 49.5 ± 31.2 0.961
Packed red blood cells 8 (27) INF-γ 258.7 ± 26.2 292.4 ± 58.1 0.744
Platelet concentrate 11 (37) GM-CSF 10.1 ± 3.4 8.8 ± 4.3 0.867
Klotho 59.2 ± 18.9 59.8 ± 30.7 0.991
Non-hematological TNF-α 7.5 ± 0.9 7.8 ± 2.7 0.949
Bone pain resulting from G-CSF 5 (17)
Weakness 3 (10) Data expressed as mean and standard error of the mean.
Allergy 1 (3)
Diarrhea 1 (3)
Dyspepsia 1 (6) Table 5
Elevation of creatininea 1 (3)
Comparison between the serum concentrations of cytokines evaluated in the
Neutropenia degree 0: without alterations, degree 1: ≥1,5 to <2,0 different phases of ASCT.
× 109/L, degree 2: ≥1,0 to <1,5 × 109/L, degree 3: ≥0,5 to <1,0 Cytokine Pre- Post- Bone Bone p
× 109/L and degree 4: <0,5 × 109/L. Thrombocytopaenia degree apheresis apheresis marrow marrow
0: without alterations, degree 1: 150 to 75 × 109/L, degree 2: ≥50 (n = 30) (n = 30) aplasia (n recovery (n
to <75 × 109/L, degree 3: ≥10 to <50 × 109/L and degree 4: <10 = 30) = 30)
× 109/L. IL-1ra 23.3 ± 6.7 30.6 ± 9.6 10.3 ± 3.1 20.2 ± 4.2 0.0001
a
Variation of 1,3 to 1,5 mg/dL. IL-1b 4.2 ± 0.8 9.4 ± 5.4 27.5 ± 6.9 ± 2.3 0.903
23.1
IL-2 129.0 ± 106.0 ± 128.0 ± 142.0 ± 0.327
Table 3 30.4 24.2 23.6 23.2
Main clinical and laboratory adverse events in the period of post-conditioning IL-4 6.2 ± 1.0 9.5 ± 3.1 11.4 ± 3.8 6.8 ± 1.2 0.696
bone marrow aplasia. IL-5 9.4 ± 1.5 9.2 ± 1.4 38.7 ± 9.1 ± 1.4 0.0003
11.4
Hematimetric values Median (minimum - maximum) IL-6 13.9 ± 4.4 72.5 ± 59.1 78.7 ± 15.9 ± 4.3 0.443
37.5
Hemoglobin (g/dL) 7.2 (6.5–7.9)
IL-8 31.8 ± 6.5 72.7 ± 57.3 395.6 ± 25.4 ± 5.4 <0.0001
Haematocrit (%) 21.3 (19.1–27.6)
120.8
Leukocytes (×109/L) 0.1 (0.04–0.1)
IL-10 8.5 ± 2.1 8.6 ± 2.7 10.8 ± 2.1 20.4 ± 3.3 <0.0001
Neutrophils (×109/L) 0
IL- 77.7 ± 16.1 99.2 ± 23.7 86.7 ± 182.2 ± 0.0001
Eosinophils (×109/L) 0
12p70 16.4 29.1
Lymphocytes (×109/L) 0
IL-13 3.2 ± 0.4 6.5 ± 2.1 4.5 ± 1.1 3.5 ± 0.5 0.629
Monocytes (×109/L) 0
IL-15 4.3 ± 0.7 8.5 ± 4.0 9.2 ± 3.6 6.1 ± 1.0 0.111
Platelets (×109/L) 19 (3–55)
IL-17 49.5 ± 31.2 113.1 ± 65.2 ± 71.6 ± 35.3 0.434
Blood components used 85.1 39.3
Packed red blood cells 2 (0–4) INF-γ 292.4 ± 347.6 ± 308.1 ± 313.3 ± 0.123
Platelet concentrate 14 (7–35) 58.1 102.6 70.45 44.5
Positive blood cultures n (%) GM-CSF 8.8 ± 4.3 11.5 ± 6.1 6.2 ± 3.2 4.7 ± 2.4 0.150
Staphylococcus epidermidis 7 (23) Klotho 59.8 ± 30.7 38.8 ± 26.9 85.3 ± 28.8 ± 11.8 0.675
Klebisella pneumoniae 3 (10) 33.3
Escherichia coli 5 (17) TNF-α 7.8 ± 2.7 17.6 ± 11.2 33.7 ± 8.5 ± 2.3 0.122
Morganella morgana 1 (3) 19.7

Antimicrobials Data expressed as mean and standard error of the mean.

Cefepime alone 4 (13)
Cefepime associated with vancomycin 21 (70)
Substitution of cefepime for meropenem 5 (17) IL-6 on day +9, maintenance of IL-8 in values close to zero during all
phases of the transplantation and absence of alterations in others
Digestive tract lesion
including IL-12. As in our study, the mentioned author found an increase
Diarrhea 5 (17)
Mucositis 21 (70) in IL-10 in the phase of bone marrow recovery. The authors did not carry
Degree 1 7 (23) out evaluations related to the apheresis process or adverse events during
Degree 2 5 (17) the aplasia phase. Based on the elements described, it is speculated
Degree 3 5 (17)
about the role of the type of therapeutic response (CR, VGPR or PR) in
Degree 4 4 (13)
Parenteral nutrition 4 (13) the profile of pro- and anti-inflammatory markers along the various
phases of ASCT.
Mucositis degree 0: without alterations, degree 1: painless ulcers, erythema or Apheresis procedures were recently associated with the production
mild irritability in the absence of lesions, degree 2: painful erythema, edema or
of IL-1ra [19]. Devices for selective apheresis that contain cell adsorp­
ulcers, but able to eat and swallow, degree 3: painful erythema, edema or ulcers
tion columns remove granulocytes, monocytes, and lymphocytes from
that require intravenous hydration, degree 4; serious ulcers that require enteral
or parenteral nutritional support or prophylactic intubation. peripheral blood and can be part of therapeutic planning for some
autoimmune or inflammatory diseases [20]. Sakimura and collaborators

5
A. Callera et al.
Fig. 2. Serum concentration of IL-12 in the bone marrow recovery phase according to the occurrence of MRD (A) and correlation between IL-12 and the percentage
of clonal plasma cells in the bone marrow (B).
[21] observed that selective apheresis produces an anti-inflammatory
effect possibly mediated by IL-1ra on patients with ulcerative colitis.
The collection of peripheral stem cells by apheresis follows similar
principles; it does not use adsorption columns, however. Mosevoll and
collaborators [22] studied fifteen patients with MM treated with ASCT
and observed that apheresis for the collection of stem cells increased the
plasmatic level of some mediators, among them IL-1ra, ligand to CD40,
CCL5 and CXCL 5, 8, 10 and 11; the authors suggested that the profile of
cytokines can be altered by the apheresis process in patients with MM.
Our results suggest that the patients' clinical and laboratory conditions
were similar in the pre-apheresis period, therefore elements contained in
the apheresis process might explain the increase in IL-1ra in the post-
apheresis period.
The increased IL-5 in the phase of bone marrow aplasia remains
6
Transplant Immunology 70 (2022) 101513
unclear. IL-5 seems to have an important role in the pathogenesis of
allergic process and inflammatory diseases mediated by eosinophils
[23–25]. IL-5 seems to play a role in the differentiation of B cells in
antibody-secreting plasma cells, including IgA in mucous membranes,
and may also act as an angiogenic factor [26]. As demonstrated in
Table 3, most patients presented mucositis in the phase of bone marrow
aplasia after the conditioning chemotherapy; we speculated a possible
association between IL-5 and the need for production of antibodies,
mainly IgA in mucous membranes as a humoral immune response
against mucositis. We also hypothesized that the angiogenic action of IL-
5 in the bone marrow microenvironment might be important in the
reparation process of the bone marrow lesion induced by the cytotoxic
action of the high-dose chemotherapy. Obviously, further experiments
will be required to share some light on these questions.
A. Callera et al.
Regarding IL-8 [27–29], Tromp and collaborators [30] studied 73
patients with post-chemotherapy febrile neutropenia and found that
values of IL-8 under 60 pg/mL were related to fever of undetermined
origin and values above with infectious processes. In a different manner,
Engel and collaborators [31] demonstrated in 147 episodes of post-
chemotherapy febrile neutropenia that values of IL-8 above 1000 pg/
mL were associated with a worse prognosis of patients including septic
shock, respiratory insufficiency, or death. Our study demonstrated a
significant increase in the serum concentrations of IL-8 in the period of
post-conditioning bone marrow aplasia; however, we did not observe
concomitance with serious infections or reserved prognoses in our
sampling. On the other hand, we observed a significant association be­
tween IL-8 levels and the presence of positive blood cultures, suggesting
a possible organization of the inflammatory response against microbial
agents even in the absence of a defense system cells.
IL-10 is an anti-inflammatory cytokine, produced by monocytes, T
lymphocytes, regulatory B lymphocytes, macrophages and dendritic
cells that presents an important role in the negative regulation of the
immune response to microbial agents [32]. It also acts in the prevention
of excessive inflammatory responses providing a proper balance be­
tween the immune response and the tissue protection [33]. In turn, the
IL-12 family has a fundamental role in the regulation of the inflamma­
tory response of T cells [34–36]. Considering these aspects, it is
reasonable to imagine that the increases in IL-10 and IL-12 observed in
our work might represent the resumption of a homeostatic balance of the
immune system in the period of bone marrow recovery.
An interesting finding was the association between the concentra­
tions of IL-12 in the phase of bone marrow recovery and the research on
MRD. In the last two decades, several studies have demonstrated the
antitumor activity of IL-12 [37] and safety studies have been reported in
patients with metastatic renal-cell carcinoma, melanoma, colon carci­
noma, multiple myeloma, Hodgkin's lymphoma, ovarian tumors and
head and neck carcinomas [38]. In our study, half of all patients had
negative MRD, a result that compares favorably with previous studies
that reported absence of MRD in 42% to 58% of patients with MM
treated with ASCT [17]. Based on the above mentioned arguments, the
antitumor effect of IL-12 might explain, at least partly, the high con­
centration of IL-12 in patients with negative MRD and the inverse cor­
relation between IL-12 levels and the number of clonal plasma cells
obtained from the bone marrow of patients who maintained positive
MRD even after treatment with ASCT.
Lastly, the present study has several limitations. Although the dif­
ferences observed have been significant, the results found must be
confirmed with a higher number of patients. The data were based on
comparisons between different times of the same procedure; however,
they do not draw comparisons with control groups. The collection of
samples was carried out in the morning in accordance with the routines
established for transplanted patients limiting their number. Possibly,
more samples from the different phases provide more detailed
information.
Finally, in accordance with the objectives proposed, this studied
determined the serum levels of pro- and anti-inflammatory cytokines in
samples of patients with MM obtained from the different phases of the
ASCT. Our findings can serve as a basis for further investigations into the
mechanisms involved in the production of IL-1ra after the apheresis
procedure, the angiogenic role of IL-5 in the period of bone marrow
aplasia and their possible influence on the bone marrow recovery, the
association between MRD and IL-12 in the bone marrow recovery, and
the possible use of IL-12 as complementary therapy after the ASCT of
patients with MM.
6. Conclusions
The results suggest that cytokines with varied expression levels can
be found in the different phases of the ASCT. The levels of IL-1ra tend to
increase in the post-apheresis period. The rise in IL-5 levels was not
7
Transplant Immunology 70 (2022) 101513
associated with any clinical or laboratory event. The response charac­
terized by the increase in the concentrations of IL-8 in the period of bone
marrow aplasia seems to be associated with microbial agents in the
bloodstream. After bone marrow recovery there is an increase in IL-10
and IL-12 levels. Additionally, IL-12 was inversely associated with the
presence of MRD in MM patients treated with the ASCT.
Funding
The authors thank all the staff members of the Hospital PIO XII of São
José dos Campos and São Paulo Research Foundation (FAPESP) for the
financial support for this study [grant #2012/15165-2].
Author's contribution
Author's specific contributions to the work: A.F.C., F.C., A.A.B., C.R.
O., A.L.L.B., and R.P⋅V participated in conceptualization, investigation,
performance of the research, data analysis and writing of the article.
Declaration of Competing Interest
The results presented in this paper have not been published previ­
ously in whole or part, even as an abstract or poster form. The authors
received no personal financial support for the research, authorship, and/
or publication of this article. No conflict of interest relevant to this
article was reported.
References
[1] A. Palumbo, K. Anderson, Multiple Myeloma, N. Engl. J. Med. 364 (11) (2011)
1046–1060.
[2] F.R. Appelbaum, The current status of hematopoietic cell transplantation, Annu.
Rev. Med. 54 (2003) 491–512.
[3] M. Attal, J.L. Harousseau, A.M. Stoppa, A prospective, randomized trial of
autologous bone marrow transplantation and chemotherapy in multiple myeloma,
N. Engl. J. Med. 335 (1996) 91–95.
[4] J.L. Harousseau, P. Moreau, Autologous hematopoietic stem-cell transplantantion
for multiple myeloma, N. Engl. J. Med. 360 (25) (2009) 2645–2654.
[5] E.A. Copelan, Hematopoietic stem-cell transplantation, N. Engl. J. Med. 354 (2006)
1813–1826.
[6] O. Bruserud, A.O. Kittang, The chemokine system in experimental and clinical
hematology, Curr. Top. Microbiol. Immunol. 341 (2010) 3–12.
[7] X.S. Wang, Q. Shi, L.A. Williams, C.S. Cleeland, G.M. Mobley, J.M. Reuben, et al.,
Serum interleukin-6 predicts the development of multiple symptoms at nadir of
allogeneic hematopoietic stem cell transplantation, Cancer. 113 (8) (2008)
2102–2109.
[8] X.S. Wang, Q. Shi, L.A. Williams, L. Mao, C.S. Cleeland, R.R. Komari, et al.,
Inflammatory cytokines are associated with the development of symptom burden
in patients with NSCLC undergoing concurrent chemoradiation therapy, Brain
Behav. Immun. 24 (6) (2010) 968–974.
[9] X.S. Wang, L.A. Williams, S. Krishnan, Z. Liao, P. Liu, L. Mao, et al., Serum sTNF-
R1, IL-6, and the development of fatigue in patients with gastrointestinal cancer
undergoing chemoradiation therapy, Brain Behav. Immun. 26 (5) (2012) 699–705.
[10] X.S. Wang, Q. Shi, N.D. Shah, C.J. Heijnen, E.N. Cohen, J.M. Reuben, et al.,
Inflammatory markers and development of symptom burden in patients with
multiple myeloma during autologous stem cell transplantation, Clin. Cancer Res.
20 (5) (2014) 1366–1374.
[11] H. Reikvam, K.A. Mosevoll, G.K. Melve, C.C. Gunther, M. Sjo, P.T. Bentsen, et al.,
The pretransplantation serum cytokine profile in allogeneic stem cell recipients
differs from healthy individuals, and various profiles are associated with different
risks of posttransplantation complications, Biol. Blood Marrow Transplant. 18 (2)
(2012) 190–199.
[12] O. Bruserud, A. Ryningen, A.M. Olsnes, L. Stordrange, A.M. Oyan, K.H. Kalland, et
al., Subclassification of patients with acute myelogenous leukemia based on
chemokine responsiveness and constitutive chemokine release by their leukemic
cells, Haematologica. 92 (2007) 332–341.
[13] S.M. Kornblau, D. Mccue, N. Singh, W. Chen, Z. Estrov, K.R. Coombes, et al.,
Recurrent expression signatures of cytokines and chemokines are present and are
independently prognostic in acute myelogenous leukemia and myelodysplasia,
Blood. 116 (2010) 4251–4261.
[14] A.F. Callera, E.S. Rosa, F. Callera, Intermediate-dose cytarabine plus G-CSF as
mobilization regimen for newly diagnosed multiple myeloma and heavily pre-
treated patients with hematological and non-hematological malignancies, Transf.
Apher. Sci. 58 (2019) 318–322.
[15] E.D. Saad, P.M. Hoff, R.P. Carnelos, A. Katz, Y. Novis, M. Pietrocola, et al., Critérios
comuns de toxicidade do Instituto Nacional de Câncer dos Estados Unidos, Rev.
Bras Cancerologia. 48 (2002) 63–96.
A. Callera et al.
[16] J.W. Gratama, D.R. Sutherland, M. Keeney, Flow cytometric enumeration and
immunophenotyping of hematopoietic stem and progenitor cells, Semin. Hematol.
38 (2001) 139–147.
[17] A.C. Rawstron, J.A. Child, R.M. De Tute, E. Faith, F.E. Davies, W.M. Gregory, et al.,
Minimal residual disease assessed by multiparameter flow cytometry in multiple
myeloma: impact on outcome in the medical research council myeloma IX study,
J. Clin. Oncol. 31 (2013) 2540–2547.
[18] S. Vigolo, J. Zuckerman, R.I. Bittencourt, L. Silla, D.A. Pilger, R.I. Ittencourt,
L. Silla, D.A. Pilger, Comparison of cyclophosphamide-thalidomide-dexamethasone
to bortezomib-cyclophosphamide-dexamethasone as induction therapy for
multiple myeloma patients in Brazil, Hematol. Oncol. Stem Cell Ther. 10 (3) (2017)
135–142.
[19] M. Akdis, S. Burgler, R. Crameri, T. Eiwgger, H. Fugita, E. Gomez, et al.,
Interleukins, from 1 to 37, and interferon-γ: receptors, functions, and roles in
diseases, J. Allergy Clin. Immunol. 127 (3) (2011) 701–721.
[20] A. Lindberg, M. Eberhardson, M. Karlsson, P. Karlen, Long-term follow-up with
granulocyte and monocyte apheresis re-treatment in patients with chronically
active inflammatory bowel disease, BMC Gastroenterol. 10 (2010) 73–82.
[21] K. Sakimura, T. Omori, E. Iwashita, T. Yoshida, Y. Tsuzuki, K. Fujimori, et al.,
Clinical response is associated with elevated plasma interleukin-1 receptor
antagonist during selective granulocyte and monocyte apheresis in patients with
ulcerative colitis, Dig. Dis. Sci. 51 (2006) 1525–1531.
[22] K.A. Mosevoll, Ç.A. Akkok, T. Hervig, G.K. Melve, O. Bruserud, H. Reikvam, Stem
cell mobilization and harvesting by leukapheresis alters systemic cytokine levels in
patients with multiple myeloma, Cytotherapy. 15 (7) (2013) 850–860.
[23] K. Takatsu, T. Kouro, Y. Nagai, Interleukin 5 in the link between the innate and
acquired immune response, Adv. Immunol. 101 (2009) 191–236.
[24] M. Ikutani, T. Yanagibashi, M. Ogasara, K. Tsuneyama, S. Yamamoto, Y. Hattori, et
al., Identification of innate IL-5–producing cells and their role in lung eosinophil
regulation and antitumor immunity, J. Immunol. 188 (2) (2012) 703–713.
[25] G.M. Gauvreau, A.K. Ellis, J.A. Denburg, Haemopoietic processes in allergic
disease: eosinophil/basophil development, Clin. Exp. Allergy 39 (9) (2009)
1297–1306.
[26] S.L. Park, T.W. Chung, S. Kim, B. Hwang, J.M. Kim, H.M. Lee, et al., HSP70-1 is
required for interleukin-5-induced angiogenic responses through eNOS pathway,
Sci. Rep. 7 (2017) 44687, https://doi.org/10.1038/srep44687.
Transplant Immunology 70 (2022) 101513
[27] D.J. Waugh, C. Wilson, The interleukin-8 pathway in cancer, Clin. Cancer Res. 14
(21) (2008) 6735–6741.
[28] N. Mukaida, Interleukin-8: an expanding universe beyond neutrophil chemotaxis
and activation, Int. J. Hematol. 72 (4) (2000) 391–398.
[29] M. Seitz, B. Dewald, N. Gerber, M. Baggiolini, Enhanced production of neutrophil-
activating peptide-1/interleukin-8 in rheumatoid arthritis, J. Clin. Invest. 87
(1991) 463–469.
[30] Y.H. Tromp, S.M. Daenen, W.J. Sluiter, E. Vellenga, The predictive value of
interleukin-8 (IL-8) in hospitalized patients with fever and chemotherapy-induced
neutropenia, Eur. J. Cancer 45 (4) (2009) 596–600.
[31] A. Engel, S. Knoll, P. Kern, W.V. Kern, Interleukin-8 serum levels at fever onset in
patients with neutropenia predict early medical complications, Infection. 33 (5–6)
(2005) 380–382.
[32] A. D’andrea, M. Aste-Amezaga, N.M. Valiante, X. Ma, M. Kubin, G. Trinchieri,
Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by
suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells,
J. Exp. Med. 178 (1993) 1041–1048.
[33] S. Rutz, W. Ouyang, Regulation of interleukin-10 expression, Adv. Exp. Med. Biol.
941 (2016) 89–116.
[34] K. Gee, C. Guzzo, N.F. Che, W. Ma, A. Kumar, The IL-12 family of cytokines in
infection, inflammation and autoimmune disorders, Inflamm. Allergy Drug Targets
8 (1) (2009) 40–52.
[35] M.K. Gately, B.B. Desai, A.G. Wolitzky, P.M. Quinn, C.M. Dwyer, F.J. Podlaski, et
al., Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-
12 (cytotoxic lymphocyte maturation factor), J. Immunol. 147 (1991) 874–882.
[36] R. Medzhitov, Toll-like receptors and innate immunity, Nat. Rev. Immunol. 1
(2001) 135–145.
[37] S. Tugues, S.H. Burkhard, I. Ohs, M. Vrohlings, K. Nussbaum, J.V. Berg, et al., New
insights into IL-12-mediated tumor suppression, Cell Death Differ. 22 (2) (2015)
237–246.
[38] A. Younes, B. Pro, M.J. Robertson, I.W. Fiinn, J.E. Romaguera, F. Hagemeister, et
al., Phase II clinical trial of interleukin-12 in patients with relapsed and refractory
non-Hodgkin’s lymphoma and Hodgkin’s disease, Clin. Cancer Res. 10 (16) (2004)
5432–5438.