Archives of Medical Research 34 (2003) 184193

ORIGINAL ARTICLE
Activation and Proliferation of T Lymphocyte Subpopulations
in Patients with Brucellosis
Martha Cecilia Moreno-Lafont,a,c Ruben Lopez-Santiago,a Vladimir Paredes-Cervantes,a
Ariel Estrada-Aguilerab and Leopoldo Santos-Argumedoc
a
Departamento de Inmunologa, Escuela Nacional de Ciencias Biologicas (ENCB), Instituto Politecnico Nacional (IPN), Mexico City, Mexico
b
Hospital de Infectologa, Centro Medico Nacional La Raza, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
c
Departamento de Biomedicina Molecular, Centro de Investigaciones y de Estudios Avanzados (CINVESTAV), Mexico City, Mexico

Received for publication October 26, 2001; accepted July 12, 2002 (01/192).

Background. T-cell proliferation is a standard method to evaluate cellular immune

responses against intracellular infectious agents. The present study was undertaken to
look for expression of an early activation marker (CD69) and proliferation using a
nonradioactive method to evaluate cellular immune response against a salt-extractable
antigen from Brucella melitensis 16M (RCM-BM) in patients suffering from brucellosis.
Methods. Expression of CD69 on membrane of CD4 and CD8 T-cells was determined
by flow cytometry. Lymphoproliferation was determined by tritiated thymidine and 5-
bromo-2-deoxyuridine (BrdU) incorporation using liquid scintillation counter or flow
cytometry, respectively, to evaluate DNA synthesis.
Results. Thirty healthy donors and 24 patients suffering from brucellosis were included in
this study. In all cases, incubation with mitogen induced expression of CD69 and
proliferation of both CD4 and CD8 T-cells. In contrast, only brucellosis patients
responded with expression of CD69 and proliferation against RCM-BM antigen from
Brucella melitensis ( p 0.001).
Conclusions. Methods used in this study were useful to evaluate immune response against
specific antigen or polyclonal stimulation. CD4 and CD8 T cells from patients became
equally activated and proliferated in response to RCM-BM antigen. Our data suggest that
both T-cell subpopulations play an important role in immune response against Brucella.
2003 IMSS. Published by Elsevier Science Inc.
Key Words: 5-Bromo-2-deoxyuridine, CD69, Human brucellosis, Cellular immune response,
[3H] thymidine uptake.

Introduction undulant fever, sweats, anorexia, fatigue, weight loss, and

Human brucellosis is a systemic infection that involves any
organ or organ systems of the body. Symptoms are nonspe-
cific and generally occur within 46 weeks of incubation.
The majority of symptoms are somatic complaints such as
Address reprint requests to: Dr. Leopoldo Santos-Argumedo, De-
partamento de Biomedicina Molecular, CINVESTAV, Av. IPN #2508, Col.
Zacatenco, 07360 Mexico, D.F., Mexico. Phone: (+52) (55) 5747-3800,
exts. 3323 and 5020; FAX: (+52) (55) 5747-7134; E-mail: lesantos@
mail.cinvestav.mx
depression (1).
Previous studies in murine models have shown that cyto-
kine-secreting CD4 T cells are fundamental components
of immunity against Brucella (2,3). On the other hand, CD8
T lymphocytes also are very important in protective immu-
nity (4) in that they possess cytotoxic activity against macro-
phages infected with Brucella (57).
Studies looking for antigen-specific T-cell activation have
typically measured either proliferative responses as a func-
tion of tritiated thymidine ([3H]TdR) incorporation or cyto-
kine secretion from activated cells following long-term
culture of peripheral blood mononuclear cells (PBMN).

0188-4409/03 $see front matter. Copyright 2003 IMSS. Published by Elsevier Science Inc.
d o i : 10 .1 0 1 6/ S0 1 88 - 4 40 9 ( 0 3) 0 0 02 0 - 1
 T-Cell Activation and Proliferation in Human Brucellosis
CD69 molecule is one of the earliest surface markers ex-
pressed on activated T, B, and NK cells following stimula-
tion. Its expression can be detected 23 h post-stimulation,
peaking at 1824 h and declining at 24 h if stimulus is no
longer present (812). CD69 function has yet to be defined
but is expressed in a variety of hematopoietic cells including
activated platelets (13), eosinophils (14), neutrophils (15),
and monocytes (1618) in addition to activated lympho-
cytes (9,11). In several of these cells, CD69 expression
is constitutive (monocytes and platelets). In lymphoid cells,
CD69 is not expressed in resting populations but is rapidly
induced following activation (11,12). CD69 expression in
lymphocyte membranes appears to have an important func-
tional role in T-cell activation cycle (19,20); thus, it is possi-
ble to identify responder population to mitogen or antigen
stimulation by determining CD69 expression simultaneously
with T-cell surface phenotype.
5-Bromo-2-deoxyuridine (BrdU), a thymidine analog,
was also incorporated into DNA during S-phase of cell cycle;
therefore, BrdU-labeled cells can be identified by using fluo-
rescein-conjugated antibody (anti-BrdU-FITC). The main
advantage of this method was the use of a nonradioactive
label. Anti-BrdU-FITC reacted only with single-stranded
DNA; thus, native double-stranded conformation must be
denatured (2123).
In the present work, we evaluated response of individual T
lymphocytes against salt-extractable antigen from Brucella
melitensis 16M (RCM-BM) from patients with brucellosis.
At the same time, responses against phytohemagglutinin
(PHA) or CD2/CD2R were used as positive controls. Results
clearly showed that both CD4 and CD8 T cells from
patients became equally activated and induced to proliferate
by specific stimuli given by RCM-BM antigen.
Materials and Methods
Study population. Blood samples from 16 female and 8 male
patients 2564 years of age with brucellosis from 3 months
to several years were obtained from the Centro Medico
Nacional La Raza, IMSS and from the Mexican Health
Secretariat Hospital Juarez, all in Mexico City. Diagnosis
of positive brucellosis was established according to the fol-
lowing: isolation of Brucella spp. from blood; presence of
specific antibodies using Rose Bengal test (standard aggluti-
nation test) and 2-mercaptoethanol test (24), and certain
symptoms such as fever, chills, generalized aches and pains,
anorexia, and general weakness. Patients participating in
this study did not consume alcohol. Eight females and 22
males, 25- to 35-year-old healthy donors with neither sero-
logic nor epidemiologic evidence of brucellosis who were
not pregnant and not infected with other bacteria were re-
cruited. Both patients and healthy donors gave informed
consent.
185
Mitogen and bacterial antigens. Phytohemagglutinin
(PHA) or CD2/CD2R were used as positive controls and
Brucella melitensis antigen was used as specific antigen. B.
melitensis strain 16M was kindly provided by M. Corbel
(National Institute for Biological Standards and Control
[NIBSC], Hertfordshire, UK). It was used as the source for
salt-extractable antigen RCM-BM, obtained as described by
Moreno-Lafont et al. (25). Briefly, frozen stored cultures
were grown on trypticase soy agar and then transferred to
trypticase soy broth (Becton Dickinson, Mexico) supple-
mented with 12% yeast extract (Oxoid, Unipath, Ltd., Basing-
stoke, UK) and 30% dextrose. Cultures were incubated at
37C for 48 h with continuous shaking (New Brunswick
rotary shaker). Formaldehyde-inactivated bacteria were
washed and resuspended in 0.85% saline and finally stirred
magnetically with sterile 0.1-mm glass beads at 4C for
18 h. After centrifugation at 20,000 g, supernatant was
recovered, filtered, aliquoted, and stored at 20C. RCM-
BM antigen was assayed for protein (26), total carbohydrates
(27), nucleic acids (28), and KDO (29) contents.
Determination of CD69 on membrane of lymphocytes. To
assess early T-cell activation, CD69 expression on cell mem-
brane was determined as described by Maino et al. (30).
Briefly, 500 L of heparinized venous blood was placed
into each of three Eppendorf tubes and PHA, CD2/CD2R
(positive controls of activation), RCM-BM antigen, or PBS
(negative control) was added. Stimulated and unstimulated
samples were incubated in a water bath at 37C for 8 h.
After incubation, 50 L aliquots were stained with 20 L
of a mixture of three monoclonal antibodies for 20 min at
room temperature in the dark. For analysis of T-cell activa-
tion, antibody combination included the following: CD69
conjugated with phycoerythrin (CD69-PE); CD4 or CD8
conjugated with fluorescein isothiocyanate (CD4-FITC or
CD8-FITC), and CD3 conjugated with peridinin chlorophyll
protein (CD3-PerCP). IgG1 isotype control antibody conju-
gated with FITC and PE in addition to CD3-PerCP (1-FITC/
1-PE/CD3-PerCP) were included to establish background
fluorescence and to gate in CD3 lymphocytes. All anti-
bodies used in this staining protocol were obtained from
Becton Dickinson Immunocytometry System (BDIS) (San
Jose, CA, USA). After incubation, red blood cells were lysed
with FACS lysing solution (BDIS). Samples were analyzed by
three-color analysis using FACSCalibur (BDIS), and data
were acquired with CellQuest software package (BDIS) by
triggering in FL3 channel (CD3-PerCP) to gate in T-lympho-
cyte populations. Data were displayed as two-color dot plots
(FL1 vs. FL2) to measure proportion of activated lymphocyte
subsets expressing CD69 and analyzed using CellQuest soft-
ware on a Quadra 650 Macintosh computer.
[3H]-thymidine uptake. Peripheral blood mononuclear
(PBMN) cells were isolated from 40 mL of heparinized ven-
ous blood by centrifugation on Histopaque (d 1.077
0.001 g/L, Sigma Chemical Co., St. Louis, MO, USA) at
186 Moreno-Lafont et al. / Archives of Medical Research 34 (2003) 184193
room temperature for 30 min at 400 g. PBMN cells were
recovered from interface and washed three times in minimal
essential medium (MEM) by centrifugation. Viable PBMN
cells were counted by Trypan blue dye exclusion, and cell
suspension was adjusted to 5 107 PBMN/mL in AIM-V
medium (Gibco BRL, Grand Island, NY, USA). To determine
lymphocyte response to antigen, 2.5 105 PBMN cells in a
200-L final volume per well were added to each of two
separate 96 flat bottom-well microtiter plates in AIM-V
medium. Optimal concentrations of RCM-BM antigen (20
g protein/mL) were standardized in a previous report (25).
This was added in triplicate and three wells without antigen
were used as controls. In the second plate, 5 g/mL PHA
(Sigma Chemical) was added in triplicate, and three wells
without stimulus were used as controls. Plates were cultured
in a 37C, 5% CO2 humidified incubator. After 3-day incuba-
tion for mitogen and 5-day incubation for antigen, cultures
were pulsed for 6 h with 0.5 Ci of [3H]TdR (specific activity
6.7 Ci/mmol, Amersham Life Science, Buckinghamshire,
UK) per well. Cells were harvested onto glass fiber mats
and counts per minute (cpm) were measured in a liquid
scintillation counter (Beckman Instruments Inc., Fullerton,
CA, USA). Cell proliferation results were expressed as cpm
(stimuli triplicate cultures minus control cpm) standard
deviation (SD).
5-Bromo-2-deoxyuridine (BrdU) incorporation. To assess
lymphoproliferative cells, BrdU incorporation was deter-
mined as described by Dolbeare et al. (21). Briefly, PBMN
cells from the same healthy donors and patients were ad-
justed to 4 106 PBMN/mL in AIM-V medium, and 1 mL
per well was added to a 24 flat-bottomed well multidish.
Optimal concentrations of PHA (10 g/mL) and RCM-BM
antigen (20 g protein/mL) were added in duplicate. Plates
were incubated at 37C under 5% CO2. After 3-day incuba-
tion for mitogen and 4-day incubation for antigen, BrdU
(Aldrich Chemical Co., Inc., Milwaukee, WI, USA) was ad-
ded to achieve a final concentration of 10 M, and cells
were incubated for 30 min. Cells were washed twice with 1%
bovine serum albumin in phosphate-balanced saline (BSA/
PBS) and centrifuged at 500 g for 5 min at room tempera-
ture. Cells were resuspended in cold saline and fixed with
70% cold ethanol for 30 min. Fixed cells were centrifuged
and resuspended in 2 N HCl/0.5% Triton X-100 (Sigma
Chemical), then incubated for 30 min at room temperature,
centrifuged at 500 g for 10 min, and resuspended in 1 mL
of 0.1 M Na2B4O7 (pH 8.5) to neutralize the acid. Cells
were counted and 106 cells in 50 L of 0.5% Tween 20/
BSA/PBS solution were placed in polystyrene tubes (Becton
Dickinson Labware, Lincoln Park, NJ, USA), and 20 L of
anti-BrdU-FITC (BDIS) was added. Tubes were incubated
for 30 min at room temperature in the dark. Cells were
centrifuged, washed with BSA/PBS, resuspended in 1 mL
of 1% p-formaldehyde (Sigma Chemical), and analyzed in
a flow cytometer (FACSCalibur, BDIS). Ten thousand events
were acquired with CellQuest software package by using
lymphocyte region on forward- vs. side-scatter dot plot and
displayed as histograms to measure percentage of lympho-
cyte that incorporated BrdU.
BrdU incorporation combined with surface staining. To
identify specific proliferating T-cell subsets, CD3, CD4 or
CD8 cells plus BrdU incorporation were determined as
described by Carayon and Bord (31). Briefly, PBMN cells
from both patients and healthy donors were adjusted to
3 106 PBMN/mL in AIM-V medium, and 1 mL per well
was added to a 24 flat-bottomed well multidish. Optimal con-
centration of RCM-BM antigen (20 g protein/mL) or 10
g/mL PHA (as positive control) was added. Cells without
stimuli were used as negative control. Cell cultures were
incubated at 37C in a 5% CO2 atmosphere. After 3-day
incubation for mitogen and 4-day incubation for antigen,
BrdU (Aldrich Chemical Co.) was added to achieve a final
concentration of 60 M for the final 56 h of activation.
One hundred microliters of 20 mM EDTA in sterile PBS
was added to avoid cell clumping at end of activation time and
incubated for 15 min at room temperature. Cells were
washed with cold phosphate-balanced saline (PBS) and cen-
trifuged at 500 g for 10 min at room temperature. Superna-
tant was decanted, leaving approximately 100 L of fluid,
and cell suspensions were treated with FACS lysing solution
(BDIS) to lyse residual red blood cells and fix surface epi-
topes. Cells were incubated for 10 min at room temperature,
centrifuged at 500 g, washed once with PBS with 0.5%
bovine serum albumin and 0.1% sodium azide (PBS/BSA),
and resuspended in 1 mL of PBS/BSA. One hundred-microli-
ter aliquots were placed into 12 75-mm tubes and 500 L
of FACS permeabilizing solution (BDIS) was added; this
was incubated for 10 min and washed with PBS/BSA.
Twenty microliters of anti-BrdU-FITC plus DNAse (BDIS)
and CD3-, CD4-, or CD8-PE were added. Isotype control
was included (mouse IgG1-FITC/IgG2a-PE) as negative con-
trol. Tubes were incubated for 30 min at room temperature
in the dark. Cells were centrifuged, washed with BSA/PBS,
resuspended in 500 L of 1% p-formaldehyde solution
(Sigma Chemical), and analyzed by two-color analysis using
FACSCalibur (BDIS) after counting 40,000 total events.
Data were acquired with CellQuest software package by
using a gate on lymphocyte population on forward- vs. side-
scatter dot plot and displayed as two-color dot plot (FL1
vs. FL2) to measure percentage of lymphocyte subsets that
incorporated BrdU.
Statistical methods. Statistical analyses for three indepen-
dent experiments were performed by Mann-Whitney rank
sum test; comparisons among studied groups were per-
formed also by Mann-Whitney rank sum test. Correlation
among alternative technologies was determined by Pear-
son correlation coefficient (r) using SigmaStat software pro-
gram v2.03 (SPSS, Inc., Chicago, IL, USA).
 T-Cell Activation and Proliferation in Human Brucellosis
Results
Expression of CD69 determines activation of lympho-
cytes. To identify phenotype of cell population activated
by PHA and RCM-BM antigen, CD69 expression on cell
surface of CD4 and CD8 T-cell subsets was determined.
CD69 was detected on CD3CD8 and CD3CD4 cells
after 4 h of stimulation with PHA. Number of cells ex-
pressing CD69 remained steady for at least 24 h. CD69
expression on antigen-activated T-cells was achieved 4 h
post-stimulation, peaking at 8 h for both CD4 and CD8
lymphocytes (data not shown); with regard to PHA-activated
cells, CD69 remained detectable until 24 h post-stimulation
(data not shown). Figure 1 shows a typical cytometry image
from a healthy donor and from a patient with brucellosis.
RCM-BM antigen was able to induce specific CD69 ex-
pression in cells from patients with brucellosis (panels I and
L). Less than 1% of antigen-stimulated cells from healthy
donors expressed CD69 (panels C and F). A higher number
of CD4 than CD8 T cells expressing CD69 induced by
both PHA (panels A, D, G, and J) and CD2/CD2R (panels
B, E, H, and K) mitogens were found.
CD3, CD4 and CD8 cells proliferated in response to
stimuli. To evaluate individual T-lymphocyte proliferation,
BrdU incorporation was determined by flow cytometry. To
determine method specificity, it was established that nonspe-
cific staining due to anti BrdU-FITC was 0.3% as indicated
by the reaction of nonstimulated PBMN cells pulsed with
BrdU for 30 min and stained with anti-BrdU-FITC (Figure
2A). Moreover, PHA-stimulated PBMN cells without
BrdU pulse and stained with anti BrdU-FITC (Figure 2B)
showed 1% positive cells. Specificity of anti BrdU-FITC
staining was verified by stimulating PBMN cells with PHA
and then pulsing them with BrdU. Prior to adding anti BrdU-
FITC conjugate, cell suspension was pre-incubated with
0.01, 0.1, and 1.0 mM BrdU. Anti BrdU-FITC reactivity
diminished as free BrdU concentration increased (Figure
2C). The same treatment for BrdU incorporation as for BrdU
incorporation combined with surface staining (described in
Materials and Methods) was performed. Figure 2D shows
a dot plot from stimulated PBMN cells with PHA pulsed with
BrdU and stained with anti BrdU-FITC and anti CD3 PE.
Once methods were optimized, healthy donor and patient
cells were assayed. Figure 3 shows a typical cytometry image
for a healthy donor and a patient with brucellosis by BrdU
incorporation method. Panels AC represent histograms
from BrdU incorporation without surface staining measuring
percentage of BrdU cells from lymphocyte gate. Panels E
H represent histograms from BrdU incorporation combined
with surface staining, measuring percentage of BrdU cells
from a T cell gate (CD3, CD4, or CD8 cells). In all
cases, response to PHA was obtained (panels A, C, E, and
G). Response to RCM-BM antigen (panels D and H) in
patients with brucellosis showed displacement to the right,
which indicated cells had BrdU in their DNA, i.e., they were
187
proliferating. BrdU incorporation was not observed in cells
from healthy donors when stimulated with antigen (panels
B and F).
Analysis of healthy donors and patients with brucellosis by
CD69 expression and BrdU incorporation in comparison
with thymidine uptake as lymphoproliferation gold
standard. As can be seen in Figure 4A, healthy donors
and patients responded to PHA stimulation; both CD4 and
CD8 T cells showed expression of CD69. Comparison
between patients and healthy controls showed no significant
difference ( p 0.05). In contrast, only patient group re-
sponded to RCM-BM antigen; CD69 expression was found
on both CD4 and CD8 T cells ( p 0.05) (Figure 4A).
Tritiated thymidine uptake in PBMN cells from healthy
donors and patients with brucellosis was evaluated. PHA
response of both groups, used as positive control, did not
show differences. However, only patients with brucellosis
responded to bacterial antigen RCM-BM, showing a signifi-
cant difference ( p 0.05) (Figure 4B).
Although BrdU incorporation without surface staining
can determine number of proliferating cells, it is not possible
to determine which cell subpopulation is responding. To
identify proliferating T-cell subsets, staining with anti-BrdU
plus anti-CD3, anti-CD4, or anti-CD8 was performed. Figure
4C shows results for BrdU incorporation combined with
surface staining. Results are presented as percentage of BrdU
positive cells in CD3, CD4, or CD8 gate. Cells activated
with PHA showed proliferative response in both groups
(p 0.08 for CD3, p 0.12 for CD4, and p 0.09 for
CD8 T-cells); in contrast, only patients responded to spe-
cific antigen RCM-BM in CD3, CD4, and CD8 T-cells
(p 0.001 in all cases). Comparison among numbers of
CD3, BrdU cells, and [3H]-thymidine incorporation
showed significant correlation (r 0.89).
Discussion
In this paper, in vitro T-cell response to RCM-BM antigen
from B. abortus stimuli is described by measuring expres-
sion of T-cell early activation marker CD69, and by prolifera-
tion measuring BrdU incorporation into newly generated
DNA in patients with brucellosis. One of the earliest markers
correlating with cellular activation is expression of CD69 on
cell surface (812,19). By simultaneously staining CD69
and cell subset markers, it is possible to identify phenotype of
mitogen- or antigen-activated T lymphocytes in cell cultures.
However, it should be noted that events in T-cell activation
measured by both BrdU or [3H]TdR incorporation are not
equivalent to CD69 expression. Expression of CD69 does
not reflect some downstream events involved in signals for
proliferation. However, Rouleau et al. (32,33) and Caruso
et al. (34) suggested that even within the first hours of
activation, a number of committed differentiation pathways
188 Moreno-Lafont et al. / Archives of Medical Research 34 (2003) 184193

Figure 1. Representative dot plots of CD69 expression on CD4 and CD8 T-cells from a healthy donor (AF) or a patient with brucellosis (GL) activated
with PHA (A, D, G, and J), CD2/CD2R (B, E, H, and K), or RCM-BM antigen (C, F, I, and L). Values represent percentage of double positive cells
gated into CD3 T lymphocyte population (FL3 channel).

became evident before DNA synthesis and cell division CD8 T lymphocytes were actively involved in immunity
occur. We demonstrated activation of both CD4 and CD8 to Brucella infection in humans, as has been shown in mice
T-cells in response to RCM-BM antigen only in patients with (5). Availability of new markers to be used in combination
brucellosis (Figures 1 and 4A). These findings suggested that with CD69 should provide tools for identifying, at an early
 T-Cell Activation and Proliferation in Human Brucellosis 189

Figure 2. Representative histograms of proliferation measured by BrdU incorporation. Panel A shows non-activated PBMN cells pulsed with BrdU and
stained with anti-BrdU FITC conjugate. Panel B shows PBMN cells activated for 3 days with PHA without BrdU pulse and then stained with FITC. Panel
C shows PBMN cells activated for 3 days with PHA, pulsed with BrdU, and then stained with anti-BrdU FITC. For inhibition, conjugated anti-BrdU
antibody was previously incubated with PBS (a), or 0.01 mM (b), or 0.1 mM (c), or 1.0 mM (d), or free BrdU. Histogram (e) represents autofluorescence
background. Panel D shows BrdU incorporation combined with surface staining of PBMN cells activated for 3 days with PHA pulsed with BrdU and then
stained with anti-BrdU FITC/anti CD3 PE (D).

stage, committed cells that progress to either effector or
anergic pathways of T-cell differentiation.
To obtain additional information concerning the role of
T cell in immunity against Brucella, we sought capacity
of lymphocytes from patients with brucellosis to proliferate
in response to RCM-BM antigen. By [3H]TdR incorporation
assay, which provides information on overall proliferative
responses, we found that only patient mononuclear cells
proliferated, not healthy donor cells (Figure 4B). Neverthe-
less, BrdU incorporation alone did not render information
concerning the manner in which lymphocyte population was
activated by specific stimulus but helped to detect percentage
of proliferating cells at moment of pulse (21). If BrdU incor-
poration is combined with cell surface staining of cell
190 Moreno-Lafont et al. / Archives of Medical Research 34 (2003) 184193

Figure 3. Representative histograms of BrdU incorporation of PBMN cells from a healthy donor activated with PHA (A and E) or RCM-BM antigen (B
and F), and from a patient with brucellosis activated with PHA (C and G) or RCM-BM antigen (D and H). Values represent percentage of BrdU positive
cells in lymphocyte population (gated into FSC vs. SSC) (panels AD) and for CD3 T-cells (gated into CD3 vs. SSC) (panels EH).
T-Cell Activation and Proliferation in Human Brucellosis 191
192 Moreno-Lafont et al. / Archives of Medical Research 34 (2003) 184193
subset-specific markers (31), assessment of cells under pro-
liferation is then feasible. Both CD4+ and CD8+ T cells from
patients proliferated to RCM-BM antigen, as can be seen in
Figures 3 and 4C. However, not all patients responded at
the same intensity. When clinical history was checked, we
observed that higher numbers of CD4 and CD8 cells
in peripheral blood from some patients (data not shown)
correlated with high proliferative percentages of these T-cell
subsets. Moreno-Lafont et al. (25) demonstrated an increase
in percentage of CD8 T cells in peripheral blood of patients
chronically infected with Brucella spp. Such an increase
correlated with Brucella antigen-specific CD8 T cells. Thus,
CD8 T cells may play a role in evolution of the disease
or may participate in memory immune response to Brucella,
as appears to be the case for Mycobacterium tuberculosis
(3539) and influenza infection (40).
In conclusion, data presented in this paper show that T
cells play an important role in Brucella infection in human
beings. Recent studies showed that CD8 cytotoxic T lym-
phocytes are important in brucellosis murine model (7) and
in patients with tuberculosis (41). It would be interesting to
demonstrate the specific role of CD8 T cells in human
brucellosis because their exact role is not yet fully under-
stood. We can, however, speculate that their participation is
crucial for protective immunity, as has been shown in a
murine model (42). Further work is needed to understand
the role of CD8 T cells during brucellosis in humans.
Acknowledgments
This work was supported in part by a grant from Consejo Nacional
de Ciencia y Tecnologa (CONACyT) Mexico (grant 411300-5-
32678-M), a grant from the United Nations University (Tokyo,
Japan), and grants from the Coordinacion de Estudios de Posgrado e
Investigacion, I.P.N., Mexico (grants 200390 and 200399). MCM-
L and RL-S are EDD and COFAA fellows, while LS-A is a SNI
fellow.
The authors are grateful to Drs. Javier Sanchez-Garca and
Leopoldo Flores-Romo for critical review of the manuscript.
Figure 4. Activation and proliferation of healthy donors and patients with
brucellosis. Panel A. Expression of CD69 on CD4 and CD8 T cells in
healthy donors (n 11 circles) and patients with brucellosis (n 16,
squares) in cultures stimulated with PHA or RCM-BM antigen. Data are
expressed as percentage of CD4CD69 or CD8CD69 from CD3
gated cells. Bars indicate media SD. Panel B. [3H]TdR incorporation
method. Lymphoproliferative response to PHA and to RCM-BM antigen of
PBMN cells from healthy donors (n 30, circles) and patients with brucel-
losis (n 24, squares). Data represent cpm (cpm with stimulicpm
without stimuli, from triplicate cultures). Bars indicate media SD. Panel
C. Lymphoproliferative response by BrdU incorporation of CD3, CD4,
and CD8 cells to PHA or RCM-BM antigen from PMBN cultures prepared
from healthy donors (n 30, circles) or patients with brucellosis (n 15,
squares). Data are expressed as percentage of CD3BrdU, CD4BrdU, or
CD8BrdU from lymphocyte gated cells. Bars indicate media SD.
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