Am. J. Trop. Med. Hyg., 98(3), 2018, pp.

875–882
doi:10.4269/ajtmh.16-0747
Copyright © 2018 by The American Society of Tropical Medicine and Hygiene

CD45RO+ T Cells and T Cell Activation in the Long-Lasting Immunity after

Leishmania infantum Infection
João F. Rodrigues-Neto,1,2 Gloria R. Monteiro,2 Tatjana S. L. Keesen,1 Henio G. Lacerda,2,3 Edgar M. Carvalho,4,5
and Selma M. B. Jeronimo1,2,4*
1
Department of Biochemistry, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil; 2Institute of Tropical Medicine
of Rio Grande do Norte, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil; 3Department of Infectious
Diseases, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil; 4National Institute of Science and
Technology of Tropical Diseases (INCT-DT/CNPq), Federal University of Bahia, Salvador, Bahia, Brazil; 5Immunology
Service, Federal University of Bahia, Salvador, Bahia, Brazil

Abstract. Manifestations of Leishmania infantum infection range from asymptomatic to symptomatic visceral
leishmaniasis (VL). People with symptomatic VL (sVL) have suppressed immune responses against Leishmania antigens
that are reversed after clinical cure. The intradermal leishmanin skin test (LST) is negative during sVL, but it becomes
positive after treatment. The aim of this study was to compare T cell responses in individuals with sVL, recovered VL
(RecVL), and endemic controls. Endemic controls were household contacts of a VL case and they were grouped by their
LST results, either positive (LST+) or negative (LST−). Mononuclear cells were studied ex vivo or after stimulation with
soluble Leishmania antigens (SLA); cell surface markers and cytokines were determined. T cells, ex vivo, from individuals
with sVL and from LST+ individuals presented a higher activation for CD4+ and CD8+ cells expressing CD69.
However, lymphocytes from sVL stimulated with SLA had lower percentages of CD4+ and CD8+ cells expressing CD69
and CD8+ cells expressing CD25, with no release of interferon-γ or tumor necrosis factor. sVL subjects had lower
percentage of memory cells (CD4+ CD45RO+), ex vivo, without SLA stimulation than RecVL, LST+, or LST− (P = 0.0022).
However, individuals with sVL had fewer regulatory cells after SLA stimulation (CD4+ CD25HIGH, P = 0.04 and CD4+
FOXP3+, P = 0.02) than RecVL. The decrease in specific memory and activated CD4+ and CD8+ cells, as in response to
Leishmania antigens, could explain, in part, the immune impairment during sVL. Finally, protective T cell responses are
long lasting because both RecVL or LST+ individuals maintain a specific protective response to Leishmania years after
the primary infection.

INTRODUCTION Memory T cells stimulated by specific antigens aid the

differentiation of T cells to effector T cells. Their responses via
Visceral leishmaniasis (VL) in Brazil, Europe, and northern cytokines or chemokines enable CD4+ and CD8+ T cells to
Africa is caused by Leishmania infantum infections.1,2 The migrate to the site of infection and to secrete proinflammatory
natural course of L. infantum infection depends on the host cytokines such as tumor necrosis factor (TNF) and IFN-γ.24,25
immune responses and environmental factors,3,4 and ranges Heterogeneity in CD4+ T cells influences immune responses
from asymptomatic to symptomatic VL (sVL).5–8 VL can be to Leishmania infection.26–28 Regulatory T (Treg) cells are
fatal, even with treatment, in 5% to 10% of the cases.5 How- capable of recognizing self- and non-self-antigens; they can
ever, most of the people infected with L. infantum have downregulate both Th1 and Th2 immune responses,29,30
self-resolution of the infection without presenting clinical and they play a role in both experimental and human VL.29,31
symptoms,9 and usually they can be identified by a positive The molecular mechanisms by which Treg cells suppress
response to Leishmania antigens, in vitro, or by a positive effector T cells are under investigation, but it is believed that
leishmanin skin test (LST+). Both recovered VL (RecVL) indi- Treg cells suppress the effector T cells by releasing sup-
viduals and people with self-resolving Leishmania infections pressive cytokines (interleukin [IL]-10, transforming growth
tend to present long-term protection against disease devel- factor-β) or in a contact-dependent manner or both. Re-
opment, if there is no immunosuppression.10,11 cently, it was observed that CD4+ T cells suppress T cell
Factors involved in susceptibility or resistance to Leishmania activation at the pathologic site of infection in human VL due
infection are due, in part, to the balance between pathogenic and to Leishmania donovani.30 However, in another study, no
protective immune responses.12,13 The latter depends on the increase in Foxp3 + Treg was observed in patients with
genetic background of the host, strain of infecting Leishmania, sVLdue to L. donovani infection.29 In addition, Th17 cells
sand fly factors, and comorbidity.14–16 sVL is characterized by seem to promote a proinflammatory environment by the
impaired Th1 responses, whereas resistance to developing release of cytokines and chemokines, which are key com-
disease is characterized by activation of CD4+ T cells to a Th1 ponents in activating and attracting neutrophils and other
phenotype. However, the decreased ability of peripheral blood cells to sites of inflammation.32,33
mononuclear cells (PBMCs) to proliferate and produce in- The aim of this study was to assess activation in mem-
terferon (IFN)-γ upon Leishmania antigen stimulation17–19 ory and Treg cells during sVL and after successful clinical
contrasts with the detection of IFN-γ in sera of VL patients20–22 recovery (RecVL), and in controls from the VL endemic
or its release and detection in whole blood assays.23 area that present signs of Leishmania infection (LST+) or
not (LST−). The overall goal was to assess whether the
* Address correspondence to Selma M. B. Jeronimo, Institute of
presence of long-term memory may explain the long-term
Tropical Medicine of Rio Grande do Norte, UFRN, Natal, RN 59078- immunity in RecVL individuals or among individuals who
970, Brazil. E-mail: smbj@cb.ufrn.br are LST+.

875
876 RODRIGUES-NETO AND OTHERS

METHODS supplemented with glutamine and were incubated at a concen-

tration of 107 cells/mL for 18 hours at 37°C, in 5% CO2 atmosphere,
Study population. A total of 55 people were recruited from either with or without stimulation by SLA at a concentration of
a cohort residing in an endemic area for VL in the state of Rio 10 μg/mL. SLA was prepared from promastigotes grown in culture
Grande Norte, northeast Brazil, as previously described.34,35 derived from a local Leishmania isolate that was typed by a ref-
Table 1 shows the characteristics of the studied subjects. erence laboratory (IOC 3071). The cells were stained ex vivo for
Majority of the population were adults (mean age 29.3 years). CD25, CD69, CD45RO, CD4, and CD8 and after SLA stimulation
Patients with sVL had symptoms of disease plus parasito- for CD25, CD69, FOXP3, CD4, and CD8 markers. CD4 and CD8
logical confirmation (presence of Leishmania in the bone memory cells were identified by the presence of the CD45RO
marrow) and/or positive anti-Leishmania antibodies.36 VL marker, whereas Treg cells were identified by the presence of
cases were under treatment (sVL) or post-treatment (RecVL), FOXP3 or CD25HIGH. The cells were also stained with annexin/
whereas the controls had no history of VL or symptoms sug- propidium iodide (PI) and programed cell death 1 (PD-). For each
gestive of VL. RecVL were individuals within 1 year or more sample, 30,000 events were analyzed using the FlowJo software
than 10 years post-treatment. All VL subjects were treated (version 7.6.4; Treestar, Ashland, Oregon). Data acquisition was
with antimony and had no relapse of disease. The control obtained by using FACSCanto II (BD Biosciences, San Jose, CA).
subjects were household contacts of VL cases. Antibodies used in the experiments were acquired from
Blood was collected from all subjects and the LST was used eBiosciences or BD Pharmingen (San Diego, CA).
as a measure of the delayed-type hypersensitivity response. Cytokine analysis. Levels of cytokines in cell culture
Controls were examined for Leishmania infection (anti- supernatants were determined by using a cytometric bead
Leishmania antibodies, by polymerase chain reaction and array (CBA; IL-17, IL-4, IL-2, IL-10, IL-6, TNF, and IFN-γ,
LST), and they were grouped in accordance to their LST, either Becton Dickinson [BD], San Jose, CA), following the instruc-
positive (LST+) or negative (LST−). None of the controls had a tions provided by the manufacturer. The supernatants were
history of VL. The clinical evaluations were performed on at evaluated after 18 hours at 37°C, in 5% CO2 atmosphere,
least two occasions, with an interval of at least 1 year between either with or without stimulation by SLA at a concentration of
assessments, to avoid boosting. In this way, the controls were 10 μg/mL. SLA was prepared from a local L. infantum isolate
grouped as either those who remained with a positive LST (IOC 3071). Cytokine concentrations were calculated based
response over time (LST+/LST+), or those who were LST+ but on fluorescence intensity measurements with the use of
lost this response over time (LST+/LST−) or remained LST software designed for the CBA analysis (FCAP Array™ v3.0
negative (endemic control), (LST−/LST−). All study partici- Software; BD Biosciences).
pants remained residing in the VL endemic area. Statistical analysis. Kruskal–Wallis or Wilcoxon tests were
Assessment of Leishmania infection. Anti-Leishmania used for comparison between groups (sVL, RecVL, and con-
antibodies were determined by enzyme-linked immunosor- trols) using data obtained from samples ex vivo or subjected
bent assay using soluble Leishmania antigen (SLA) lysate from to SLA stimulation. Analyses were performed using Graph-
a local Leishmania isolate (IOC 3071) and rK39 (Infectious Pad Prism (Graph Pad Software, San Diego, CA) and a
Disease Research Laboratory, Seattle, WA), as previously P value < 0.05 was considered significant.
reported.36 We assessed the delayed-type hypersensitivity Ethical considerations. This protocol and informed con-
responses using LST, which was performed by injecting sent were reviewed and approved by the Federal University
0.1 mL (25 μg) of the antigens at 3 cm from the cubital fold on Ethical Committee. The certificate of ethical approval is CAAE
the forearm of the study participants. The responses were 12584513.1.0000.5537. All participants or their legal guard-
read between 48 and 72 hours after injection of the antigens, ians signed the informed consent.
by the ballpoint pen technique.37,38 An induration greater than
5 mm was considered positive. The antigens were kindly do- RESULTS
nated by Centro de Pesquisa e Produção de Imunobiológicos
(Curitiba, Paraná, Brazil). T cell activation markers in L. infantum infection. Non-
Isolation of cells, culture, and typing. The PBMCs were stimulated PBMCs from sVL and LST+/LST+, ex vivo,
isolated by using a Ficoll-HyPaque gradient (GE Health Care, had higher expression of CD69 on CD4+ and CD8+ T cells
Freiburg, Germany). Cells were suspended in RPMI 1640 when compared with RecVL < 1 year post-treatment, or

TABLE 1
Clinical and demographic data of the study groups
Phenotypic groups

VL Asymptomatic Leishmania infection Potentially exposed

Parameters sVL RecVL* < 1 year RecVL* > 10 years LST+/LST+ LST+/LST− LST−/LST−

n 11 11 9 9 6 9
Age (years) 32 ± 14.2 27 ± 14.9 17 ± 12.1 50 ± 17.6 33 ± 18.2 17 ± 3.5
Sex % (M/F) 66/34 80/20 44/56 55/45 16/84 29/71
Leukocytes† 2,525 ± 660.1 6,120 ± 286.3 7,814 ± 2,830.8 6,500 ± 1,831.6 9,766 ± 2,313.5 7,883 ± 2,885.6
(mm3)
Platelets (mm3) 138,400 ± 79,682.5 261,000 ± 62,382.7 355,428 ± 74,631.8 277,000 ± 34,100.7 300,833 ± 64,436.2 340,333 ± 74,783.6
Treatment (days) 3 ± 2.6 193 ± 97.8 4,457 ± 728.1 N/A N/A N/A
N/A = not applicable; RecVL = recovered VL; sVL = symptomatic VL; VL = visceral leishmaniasis.
* RecVLs are not from the same donor.
† Leukocytes in sVL presented a statistical difference when compared with the other groups (P < 0.05). Values shown in absolute numbers or as mean ± SD.
 LONG-TERM IMMUNE RESPONSES AFTER LEISHMANIA INFECTION 877

FIGURE 1. Increased lymphocyte activation ex vivo in symptomatic VL (sVL) and LST+/LST+. Peripheral blood mononuclear cells from
individuals with sVL, recovered VL (RecVL) (< 1 year or > 10 years), and endemic controls (LST+/LST+, LST+/LST−, or LST/LST) were stained for
CD69 and CD25 in CD4+ and CD8+ T cells. The graphs represent the percentage of CD69 in CD4+ T cells (A), CD69 in CD8+ T cells (B), and CD25
in CD8+ T cells (C). Medians were compared using Mann–Whitney. A P value of < 0.05 was considered significant. (*P < 0.05, **P < 0.01; LST =
leishmanin skin test).

RecVL > 10 years or LST+/LST− (Figure 1A and B). CD25 ex- A representation of the dot plot graphs of cellular activation
pression was higher in CD8+ T cells from sVL subjects, when profiles in ex vivo condition or after SLA stimulation is shown in
compared with RecVL subjects < 1 year (Figure 1C). However, Supplemental Fig. 2. There was no increase in annexin/PI cell
T cells from sVL individuals stimulated with SLA had a lower death in cells from sVL individuals after SLA stimulation, P >
percentage of CD69 or CD25 when compared with RecVL in- 0.05 (data not shown). CD4 and CD8 T cells from sVL indi-
dividuals (P = 0.008 for CD4+ CD69+, P = 0.0006 for CD8+ CD69+ viduals were stained for PD-1 ex vivo and after SLA stimula-
and P = 0.01 for CD8+ CD25+), or LST+/+ (Figure 2A–C). tion. However, no difference was observed between the
The LST+/LST+ individuals presented a median of 15% of groups (P > 0.05) (Supplemental Fig. 3).
CD69 in CD4 T cells, and the LST+/LST− individuals had a sVL has reduced levels of memory T cells. The percent-
median of 8% of CD69 in CD4 T cells, whereas the LST−/LST− age of memory CD4+ T cells in PBMCs without SLA stimula-
individuals had a median of 4% of CD69 in CD4+ T cells tion was reduced in sVL subjects compared with that in the
(Figure 2A). Similar findings were observed for CD8+ T cells other groups (P < 0.05). Lymphocytes from sVL ex vivo pre-
expressing CD69, but individuals who were LST+/LST− had sented 24% of CD45RO expression in CD4+ T cells, whereas
the highest percentages of CD8+ T cells expressing CD69 RecVL presented 54% CD45RO expression in this population
(about 15%). However, for CD8+ T cells, only RecVL patients < 1 (Figure 3A). There was a difference in the percentage of CD4+
year posttreatment presented a higher percentage of cells T cells expressing CD45RO between sVL and RecVL individ-
expressing CD25 (Figure 2C). RecVL subjects > 10 years post- uals 1 year post-treatment (P = 0.002) and between sVL indi-
treatment still responded to SLA stimulation, with increased viduals and the other groups (P < 0.05). There was no
CD4+ and CD8+ T cells expressing CD69 (P = 0.01 and P = difference in CD8+ CD45RO+ in sVL or RecVL individuals
0.03, respectively; Supplemental Fig. 1). The percentage of (Figure 3B), respectively, 22% and 29%. There was also no
cells expressing activation markers in the presence or ab- difference in CD45RO expression in both CD4 and CD8 T cells
sence of SLA, for all groups, is shown in Supplemental Fig. 1. between RecVL groups and endemic controls, (P > 0.05),

FIGURE 2. T lymphocyte activation after Leishmania antigen stimulation. Peripheral blood mononuclear cells from symptomatic VL (sVL),
recovered VL (RecVL) (< 1 year or > 10 years) and endemic controls (LST+/LST+, LST+/LST−, or LST−/LST−) were cultured in the presence of
soluble Leishmania antigens. The graphs represent the percentage of CD69 in CD4+ T cells (A), CD69 in CD8+ T cells (B), and CD25 in CD8+
T cells (C). Medians were compared using Mann–Whitney. A P value of < 0.05 was considered significant. (*P < 0.05, **P < 0.01; LST = leishmanin
skin test).
878 RODRIGUES-NETO AND OTHERS

FIGURE 3. CD45RO expression in CD4 and CD8 T, in ex vivo condition. Peripheral blood mononuclear cells from symptomatic VL (sVL),
recovered VL (RecVL) (< 1 year or > 10 years) and endemic controls (LST+/LST+, LST+/LST−, LST−/LST−) were stained for CD45RO in CD4 and
CD8 T cells. The graphs represent the percentage of CD45RO in CD4+ T cells (A) and CD45RO in CD8+ T cells (B). (**P < 0.01; LST = leishmanin
skin test).

either LST+/LST+, LST+/LST−, or LST−/LST−. There was no Increased IFN-γ, TNF, and IL-6 release after VL clinical
difference in CD45RO expression in either CD4 and CD8 recovery. There was no difference in IL-17, IL-4, IL-2, IL-10,
T cells after SLA stimulation (data not shown). IL-6, TNF, and IFN-γ in the supernatants of cells isolated from
Decreased Treg cells during sVL. The percentage of individuals with sVL after SLA stimulation (P > 0.05) when
T cells expressing CD25High and FOXP3 is shown in Figure 4. compared with that in cells stimulated with media, but an in-
There was no difference in the percentage of CD4+ CD25High crease in IFN-γ, TNF, and IL-6 was observed for RecVL < 1
between the sVL group (median of 0.6%) and the RecVL < 1 year (P = 0.01) (Figure 5A–C). Likewise, an increase in the ratio
year post-treatment (median of 0.8%), ex vivo, but there was a of IFN-γ/IL-10 after SLA stimulation in RecVL < 1 year was
difference when compared with cells from RecVL > 10 years detected, showing a predominance of IFN-γ production after
post-treatment (median of 1.5%, P = 0.009) (Figure 4A). Cells clinical cure (Figure 5D). There was no production of IL-17,
from sVL subjects had a median of 0.7% for CD4+ CD25High IL-10, IL-4, and IL-2 in cells isolated from individuals with sVL
when compared with RecVL < 1 year (1.3%) (P = 0.04), or > 10 or RecVL < 1 year after SLA stimulation (data not shown).
years (2.8%) after SLA stimulation (Figure 4B). Moreover, the
group that was LST+/LST− had a mean of 2.2% of CD4+ DISCUSSION
CD25High (Figure 4B). In addition, the expression of FOXP3 in
the CD4+ T cells was increased in RecVL but not in sVL (me- VL is a severe disease that is usually associated with de-
dian of 1.8% in sVL and 3.7% in RecVL < 1 year, P = 0.02) after creased ability of the host to kill Leishmania, and there is about
SLA stimulation (Figure 4C). CD4+ T cells expressing FOXP3 5% to 10% mortality, even with treatment.39,40 This disease is
were increased in subjects who were LST+/LST+ (median of the result of an interplay between the host defense and the
6.0%). The LST+/LST− group had a median of 1.3% of FOXP3 parasite survival strategies.12 Studies have demonstrated
expression in CD4+ T cells, which was similar to the sVL group the importance of cytokines, such as TNF and IFN-γ, and
(median of 1.9%) (Figure 4C). T lymphocytes in Leishmania infection.41–43 The activation of

FIGURE 4. Profile of Regulatory T (Treg) cells in Leishmania infection. Peripheral blood mononuclear cells from symptomatic VL (sVL), recovered
VL (RecVL) (< 1 year or > 10 years), and controls (LST+/LST+, LST+/LST− or LST−/LST−) were evaluated ex vivo and cultured in the presence or
absence of Leishmania antigen. The graphs represent the ex vivo percentage of CD4CD25High T cells (A), the percentage after antigen stimulation of
CD4+ CD25High T cells (B), and FOXP3 in CD4+ T cells (C). Medians were compared using Mann–Whitney with a P value of < 0.05 considered
significant. (*P < 0.05, **P < 0.01; LST = leishmanin skin test).
 LONG-TERM IMMUNE RESPONSES AFTER LEISHMANIA INFECTION 879

FIGURE 5. Cytokine evaluation in symptomatic VL (sVL) and recovered VL (RecVL) < 1 year after soluble Leishmania antigen (SLA) stimulation.
Plots show the quantity of interferon (IFN)-γ (A), tumor necrosis factor (B), and interleukin (IL)-6 (C), and the ratio of IFN-γ/IL-10 (D) in sVL and
RecVL< 1 year. Data are represented in pg/mL, *P < 0.05.

CD4 and CD8 T cells is known to have an important role in and IFN-γ is detected in the sera and whole blood cell culture
controlling infection by intracellular pathogens, by eliciting of sVL patients.21 In this study, we found that cells from sVL
protective immunity through the proinflammatory cytokines. individuals displayed, ex vivo, a greater activation pattern, but
In this study, we observed a decreased frequency in activated T cell activation was absent in response to SLA stimulation.
T cells from sVL patients after SLA stimulation, which was Whereas cytokines such as IFN-γ are found in sera from sVL
accompanied by a lack of IFN-γ and TNF production after patients, PBMCs from these patients do not proliferate in vitro
specific antigen stimulation and decreased memory T cells. and do not produce IFN-γ after SLA stimulation. However, this
The increase in the ratio of IFN-γ/IL-10 after the clinical cure reduction in activated T cells (in vitro culture) in sVL could be
was in accordance with the restoration of the specific immune due to cell exhaustion, because of the overstimulation during
response. Moreover, as no increased frequency in Treg cells chronic Leishmania infection leading to the inability of T cells
was found in sVL, the impairment in the immune response to respond to an additional load of antigen stimulation. How-
might be due to the lack of memory T cells, rather than an ever, as annexin and PD-1 expression was not enhanced, it is
increase in Treg cells, as seen in other diseases.44,45 likely that the increase in costimulatory molecules, such as
sVL patients are anergic to Leishmania antigen cytotoxic T-lymphocyte–associated protein-4, and regulatory
stimulation,17,46 which is reversed after clinical cure. This can cytokines, such as IL-10, could be involved in promoting a
be estimated by a positive LST response, by the proliferation negative regulation. Studies of tegumentary leishmaniasis
of T lymphocytes, or by the amount of IFN-γ released by showed that subjects with diffuse cutaneous leishmaniasis
PBMCs in response to parasite antigen stimulation. However, have an anergic response to Leishmania antigens similar to
mRNA expression for IFN-γ has been documented in T cells,47 VL.48 In a murine model of schistosomiasis, decreased T cell
880 RODRIGUES-NETO AND OTHERS

function due to general T cell overstimulation or “T cell ex- data do not support an increase in the percent of Treg cells
haustion” was also shown.49 In addition, the role of antibodies in sVL.
in the pathogenesis of VL is not entirely understood, but an Finally, in this study, we documented that memory to
effective T cell response is usually observed after a decrease Leishmania antigens is long lasting, both in RecVL people and
in anti-Leishmania antibodies in the blood when there is a in people with asymptomatic Leishmania infection. Whether
development of a positive LST response.50 long-term memory is due to new infection or due to parasite
Interestingly, the sVL and LST+/LST+ groups showed an persistence is not known.57 Finally, a decrease in activated
increase in CD4+ and CD8+ T cell activation markers ex vivo. T cells after restimulation in vitro with SLA and a reduction in
However, a decrease in cells expressing CD69 in the sVL the percentage of memory T cells may explain the impairment
was observed after SLA stimulation, whereas an increase in of T cell responses to Leishmania antigens and the inability of
activated cells was seen in LST+/LST+. RecVL individuals patients who develop VL to control parasite growth.
had an increase in the percent of CD4+ and CD8+ T cells
expressing CD69 and CD8+ T cells expressing CD25, in- Received September 12, 2016. Accepted for publication August 21,
dicating long-lasting immunity to Leishmania. These findings 2017.
may explain the resistance to relapse once a protective Published online December 26, 2017.
T cell response is mounted. Moreover, one potential expla- Note: Supplemental figures appear at www.ajtmh.org.
nation for the higher activation observed for both sVL and
Acknowledgments: We thank the staff of Hospital Giselda Trigueiro
LST+/LST+ groups could be the presence of Leishmania. It (Secretaria de Saúde do Estado do Rio Grande do Norte) and Hospital
could be that individuals who had recovered from VL either Infantil Varella Santiago, in Natal, Brazil, for aiding the recruitment of
within 1 year or more than 10 years ago had successfully individuals with symptomatic visceral leishmaniasis; John Donelson,
responded to the specific Leishmania therapy and might (University of Iowa) for helping the revision of this manuscript and
have better controlled parasite replication. Persistence of Manoel Gomes for his help with the field studies.
Leishmania parasites has been shown to occur in leish- Financial support: This work was supported by the National Institutes
maniasis.51 The sVL group had reduced levels of circulating of Health (AI-30639). J. F. R-N. received a fellowship from CAPES.
memory T cells, similar to those observed for sVL because of Authors’ addresses: João F. Rodrigues-Neto and Tatjana S. L. Keesen,
L. donovani.52 This could reflect the migration to and re- Department of Biochemistry, Universidade Federal do Rio Grande do
tention of cells in infected organs, such as bone marrow.53 Norte, Natal, Rio Grande do Norte, Brazil, E-mails: joao_rneto@yahoo.
com.br and tat.keesen@gmail.com. Gloria R. Monteiro, Instituto de
The level of CD45RO ex vivo has been used to predict the Medicina Tropical do Rio Grande do Norte, Universidade Federal do Rio
intensity of T cell response in cutaneous leishmaniasis.54 In Grande do Norte, Natal, Rio Grande do Norte, Brazil, E-mail: gloriag74@
our study, RecVL individuals, either < 1 year or > 10 years, hotmail.com. Henio G. Lacerda, Department of Infectious Diseases,
had an increase in the percent of cells expressing activation Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do
Norte, Brazil, E-mail: heniolacerda@ufrnet.br. Edgar M. Carvalho, Ser-
markers after antigen stimulation and an increase in memory viço de Imunologia, Universidade Federal da Bahia, Salvador, Bahia,
cells (CD45RO+) in ex vivo conditions when compared with Brazil, E-mail: edgar@ufba.br. Selma M. B. Jeronimo, Department of
individuals with sVL. Although peripheral memory cells were Biochemistry, Universidad Federal do Rio do Norte, Natal, Rio Grande
low in sVL patients, we cannot exclude the possibility that do Norte, Brazil and Universidade Federal do Rio Grand do Norte, Natal,
these individuals may have memory cells in tissues (central Rio Grande do Norte, Brazil, E-mail: smbj@cb.ufrn.br.
memory cells) and that, after cure, memory cells are found in
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