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Human Immunodeficiency Virus (HIV) Phenotype and Interleukin-2/
Interleukin-10 Ratio Are Associated Markers of Protection
and Progression in HIV Infection
By Mario Clerici, Claudia Balotta, Antonino Salvaggio, Chiara Riva, Daria Trabattoni, Laura Papagno,
Alberto Berlusconi, Stefan0 Rusconi, Maria Luisa Villa, Mauro Moroni, and Massimo Galli
Human immunodeficiency virus (HIV) isolability, rate of viral
replication, HIV phenotype, type 1 and type 2 cytokine production, and CD4 counts were cross-sectionally analyzed in
63 HIV-seropositive (HIV') individuals to establish possible
correlations between virologic and immunologic markers of
protection and progression. We observed that these markers
are tightly correlated. Thus, lack or low prevalence of HIV
isolability and the presence of nonsyncitium inducing strains
are associated with the strongest type 1 cytokine production, the weakest type 2 cytokine production, and highest
CD4 counts. Conversely, the isolation of highly replicating,
syncitium-inducing HIV strains is associated with the weakest type 1 cytokine production, the strongest type 2 cytokine
production, and lowest CD4 counts. Additionally, it was determined that the interleukin (IL)-IO/IL-2 ratio best discriminates among different virologic scenarios. These data s u g
gest that the virologic and immunologic correlates of
disease protection and progression might be associated variables that define two different subsets of HIV' individuals
and lend support to a viro-immunologic hypothesis of HIV
infection.
0 1996 by The American Society of Hematology.
A
antigens precedes and heralds the development of opportunistic infections and
Analogously, defective production of the CMI-inducing cytokines interleukin (1L)-2,
1L-12, and gamma interferon (1FN-y) and augmented production of type 2 cytokines IL-4, IL-5, IL-6, and IL-I0 are
observed in HIV infection and have been proposed as an in
vitro immunologic marker of progression in HIV' individuals, 11)-12
NALYSES OF VIRAL and host factors show that a complex pattern of interactions takes place between intrinsic viral properties and defense mechanisms in human immunodeficiency virus (HIV) infection.'.' Biological and
molecular studies of HIV type 1 demonstrated that HIV is
highly heterogeneous. In particular, HIV primary isolates
display in vitro distinct biological properties, among which
the rate of viral replication (slow/low or rapidhigh),' and
ability to induce a cytopathic effect (nonsyncytium-inducing
or syncytium-inducing) differentiate diverse viral strains.'
The prevalence and the emergence of distinct phenotypic
variants appear to be correlated with different stages of infection. Thus, the asymptomatic phase is characterized by the
inability to isolate HIV in culture or by the presence of
slowly replicating nonsyncytium-inducing (NSI) isolates. In
contrast, highly replicating syncytium-inducing (SI) variants
emerge in 50% to 60% of HIV-seropositive (HIV') individuals in the progression of HIV infection'.'; the emergence of
these viral variants is related to rapid CD4' T-cell depletion
and progression to acquired immune deficiency syndrome
(AIDS).'
Progression of HIV infection is also associated with impairment of cell-mediated immunity (CMI), which has both
in vivo and in vitro correlates. Thus, the inability to develop
delayed type hypersensitivity reactions (DTH) to ubiquitous
From Cuttedru di Immunologiu, Universitu ' degli Studi di Milano.
Padiglione L.I. T.A.. Ospedale Luigi Sacco, Miluno; Clinicu Malarrie
Infettive, Ospedale Luigi Succo, Miluno; and lstituto di Igiene e
Medicina Preventivu, Universitu degli Studi di Miluno, Milano, Italy.
Submitted September 26, 1995; accepted March 2, 1996.
M.C. and C.B. contributed equally to rhis study.
Supported by grunts from Istituto Superiore di Sunitu' "VI1 Progetto AIDS 1994 and VI11 Progetto AIDS 1995" NO. 8204-85, 920489, 9303.13, and 9202-11.
Address reprint requests to Mario Clerici, MD, Cuttedru di Inmunologiu, Universith degli Studi di Milano, Via Veneziun, I , 20133
Miluno, Ituly.
The publication costs of this urticle were defruyed in part by puge
charge payment. This article must therefore he hereby murked
"advertisement" in accordance with 18 U.S.C. section I734 solely t~
indicate this fuct.
0 1996 by The American Society of Hematology.
0006-4971/96/8802 -002 I$3.00/0
574
Because possible correlations between these virologic and
immunologic parameters of protection and progression have
not been established, we cross-sectionally analyzed HIV isolates and cytokine production in 63 consecutively enrolled
HTV' individuals. Additionally, we analyzed which reciprocal pair of type 1 and type 2 cytokine is better capable to
discriminate among HIV' individuals with different virologic characteristics.
MATERIALS AND METHODS
Purienrs und controls. We analyzed virologic and immunologic
parameters in 63 consecutively enrolled HIV-infected individuals
treated at our outpatient clinic. Active drug users, as well as patients
with overt AIDS according to the 1987 Centers for Disease Control
(CDC) classification were not included in this study. Of the enrolled
individuals, 58 were asymptomatic and five mildly symptomatic with
constitutional symptoms or minor opportunistic infections (Group
IVA or IV C2). Tdbk I shows the clinical and demographic features
of the population studied. A total of 24 H I V ~healthy individuals
were tested in parallel.
Virus culture. Peripheral blood mononuclear cells (PBMC) from
H I V ~healthy individuals were stimulated for 24 to 48 hours with
phytohemagglutinin P (PHA, lrvine Scientific, Irvine, CA). A total
of 2 x IO" cells were cocultured with 2 X 10' cells from HIV'
patients for 3 weeks in RPMI medium supplemented with 20%
fetal calf serum and 10% recombinant IL-2 (Biosource International.
Camarillo, CA). Supernatants were sampled twice a week and tested
for p24 antigen production using a commercial enzyme-linked immunosorbent assay (ELISA). Once a week, fresh 2-day PHA-prestimulated donor cells were added to the cultures, maintaining a I : I
ratio. A culture was considered positive if two serial supernatant
samples were positive and showed a IO-fold increase in p24 antigen.
Syncytiu ussay. Each week, the viral cultures were tested for
syncytia formation using the MT-2 assay, as described." Briefly. 5
x IO' MT-2 cells were cocultured with an equal number of cells
from the PBMC virus cultures in complete RPMI medium in a 96Blood, Vol 88, No 2 (July 15), 1996: pp 574-579
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IMMUNOVIROLOGY OF HIV INFECTION
575
Table 1. Demographic and Clinical Characterization
of the 63 HIV* Patients Studied
N I%)
~
~
Mean age (years t SD)
Malefiemale
Risk group:
Former IVDU3
Homosexual
Heterosexual
CDC Classification*
34.1 t 7.3
35/28
35 (55)
12 (19)
16 (26)
11-111
IV A
IV c2
~
~
~
~
* According to the CDC 1987 criteria.
well flat-bottom plate in triplicate. Syncytia formation was scored
visually after 24 and 48 hours under 40X magnification.
In vitro cytokine production. PBMC were separated on lymphocyte separation medium (Organon Teknika Corp, Durham, NC),
washed twice in phosphate-buffered saline (PBS), and the number
of viable leukocytes was determined by trypan blue exclusion.
PBMC of HIV+ individuals or healthy controls (HC) resuspended
at 3 x 106/nL in RPMI 1640 (GIBCO, Grand Island, NY) were
either unstimulated or stimulated in vitro for 2 days with PHA
(GIBCO) diluted 1:lOO at 37°C in a moist, 7% C 0 2 atmosphere.
Stimulation with a mitogen was chosen because soluble antigens do
not induce the production of quantities of m y , IL-4, and IL-IO
sufficient to be detectable with current ELISA p r o t o c ~ l sand
' ~ to
allow the comparison of these results to those obtained in previous
st~dies.'~,''
Supematants were harvested after 48 hours (kinetic studies indicated that 48 hours was the optimal time for assessing PHAIL-2, IFNy, IL-4, and IL-IO
stimulated lymphokines prod~ction).'~
production was evaluated with commercially available ELISA
assays: IL-2: human IL-2 Quantikine (R&D Systems, Minneapolis,
MN); IFNy: human interferon gamma Intertest y (Genzyme, Cambridge, MA); IL-4: human IL-4 Intertest-4 (Genzyme); IL-IO: human
IL- 10 ELISA (Bender MedSystems, Vienna, Austria) following the
procedures suggested by the manufacturers. Cytokine production
was calculated from a standard curve of the corresponding recombinant human cytokine.
Statistical analyses. To analyze differences in the HIV replication rate, we used a two-tailed t test for two samples, with equal
variance assumed. Differences in immunologic variables between
HC and HIV' individuals classified according to virus isolation and
phenotype were analyzed using a distribution-free Kruskal-Wallis
test (non-parametric one-way analysis of variance).I6Possible relationships between immunologic parameters and the increasing severity of virologic results (considering the isolation negative situation
the most benign one, and the one in which SI virus is isolated the
least favorable one) were evaluated in HIV+ individuals by using a
non-parametric Kendall's rank correlation."^" Because the infonna-
NSI Primary Isolates
SI Primary Isolates
l-T
t-
lwk
lwk
3wk
2wk
3wk
lime (weeks)
Fig 1. Kinetics of replication of primary isolates in PBMC of 47 of 63 HIV' individuals (74.6%) in whom HIV was isolated. Twenty-six HIV+
individuals in whom nonsyncytium-inducingvirus was isolated fNSl primary isolates) and 19 HlV+ individuals in whom syncytium-inducing
virus was isolated (SI primary isolates) are shown.
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576
CLERIC1 ET AL
tion provided by different immunologic variables was likely to be
interdependent, we performed a discriminant analysis lookmg for
a simple combination of immunologic variables able to separate
individuals grouped according to their virologic characteristics.
Thus, we searched for the most important variables among 1L-2,
IFNy, IL-4,and IL-IO capable to discriminate subjects with different
virologic characteristics. A linear discriminant analysis” was performed. IL-2, IFNy, IL-4, and 1L-10 were square-root transformed
to stabilize their covariance matrices: Box’s M test, P > . I ; composition and size of the best subset was established according to Wilks’
criterion (Wilks’), F-to-enter 2.0; discriminant functions have been
expressed as functions of the original variables, possibly with integer
coefficients, and without unrelevant constant terms.
RESULTS
Virologic analyses. The rate of viral isolation and the
cytopathic effect of primary isolates was determined in a
consecutive series of 63 HIV’ patients. HIV could not be
isolated in 16 of 63 patients (25.4%), and viral isolation was
thus possible in 47 of 63 H I V individuals (74.6%). SI HIV
isolates were present in 19 of 47 (40.4%) individuals in
whom viral isolation was possible. HIV-1 primary isolates
showed different patterns of viral replication in regard to lag
phase of the first positive detection of p24 antigen, and viral
titers of the cultures. Thus, NSI isolates showed uniformly
lower p24 antigen titers in cultures compared with SI variants at all the points tested. The mean amount of p24 antigen
in the cultures was as follows: week 1: NSI = 404 pg/mL;
SI = 7,045 pg/mL (P = .03); week 2: NSI = 14,808 pg/
mL; SI = 72,114 pg/mL (P = .0005);week 3: NSI = 16,034
pg/mL; SI = 60,413 pg/mL (P = .00002).These results are
shown in Fig 1 .
Immunologic and statistical analyses. Mitogen-stimulated and unstimulated type 1 and type 2 cytokine production
by PBMC was examined in the same patients and in 24
healthy controls. Unstimulated cytokine production was constantly less that 5% of the mitogen-stimulated value for each
cytokine and was subtracted by all the results shown. IL-2,
IFNy, IL-4, and IL-10 production is shown as box plots in
Fig 2. The results of the analyses and summary statistics are
shown in Table 2. According to the results of Kruskal-Wallis
tests, important differences between the individuals classified according to HIV positivity, isolation of HIV, and identification of SI variants were observed for all immunologic
variables with the exception of p2. Also, a relationship between the severity of the virologic status in HIV’ subjects,
as inferred by the isolation of the virus and the presence of
SI variants, was evident for all immunologic variables, again
with the exception of p2. Thus, mitogen-stimulated production of the type 1 cytokines IL-2 and IFNy was significantly
reduced in parallel with the increasing severity of the viral
results, whereas mitogen-stimulated production of the type
2 cytokines IL-4 and IL-10 was significantly increased in
parallel with the increasing severity of the viral results and
the reduction of CD4 counts (Kendall’s rank correlation).
The median production of 1L-2 and IFNy was higher in the
healthy controls than in all groups of HIV+ subjects, whereas
the median production of IL-4 and IL-10 was lower in HIV+
isolation-negative subjects compared with all other HIV’
individuals.
1500
1 IL-2
1000
1
G
E
\
CT
500
0
a
0
0
I
T
v
c
.-*
1000
U
500
0
V
3
IFNy
0
0
I
I
2
a
0)
C
.Y
0
4
>r
0
1000
750
1
1
IL-4
I
0
‘0
Q)
4
-0
3
.-E
I
3;
I
a
1000
a
750
500
I
1
1
I
I
IL-10
A
250t & &
0
CONTR
ISOL-
0Q 8 A
€+I-8‘
SI-
SI+
Fig 2. Immunologic variables in HIV- healthy controls and HlV+
individualsgrouped according to the isolabili of HIV and the identification of SI variants. Designations are: HIV- controls (CONTR); no
virus isolationIISOL-); nonsyncytia-inducingvirus isolated (SI-); syncytia-inducing virus isolated (SI+l.Each data set is displayed as a box
plot: the box covers the range between the lower and the upper
quartile, and the central line is at the median. The ’whiskers’ extend
to the 10th and the 90th percentile; more extreme data are plotted
as separate points.
Discriminating value of tjpe I and tjpe 2 cytokines. IL2 and IL-IO (square-root transformed) resulted in the best
discriminating variables. The best discrimination resulted in
the direction of the dotted line in Fig 3A, where values of
IL-2 were weighted about two times the values of IL-IO
(score = JIL-10 - 1.9.dIL-2= JIL-10 - 2.dIL-2; note the
opposite sign assigned to IL-2 and IL-IO). However, the best
discriminant function in HIV+ individuals is given by simply
subtracting JIL-2 from hL-10 (score = JIL-10 - JIL-2;
dashed line in Fig 3A). According to the computed scores,
only 56.3% of all subjects and 58.7% of HIV’ subjects were
correctly classified. However, ordering the virologic classes
from the healthy controls to HlV+ individuals in whom SI
HIV variants are isolated (individuals with the most severe
virologic status), only two of 87 individuals were misplaced
by more than one class. Moreover, both the scores appeared
to separate completely HIV+ individuals in whom SI HIV
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IMMUNOVIROLOGY OF
HIV INFECTION
577
Table 2. Statistical Analysis of the Association Between Virologic and Immunologic Parameters Obsewed in 63 HlV+ Individuals
Cases (HIV')
lsol'
P
IL-2
Median
1.q. range*
Range
IFNy
Median
1.q. range
Range
IL-4
Median
1.q. range
Range
IL-10
Median
1.q. range
Range
CD48
Median
1.q. range
Range
8211
Median
1.q. range
Range
IL-1O/l L-21
Median
1.q. range
Range
Controls
ISOl~
SI'
(24)
(16)
1191
300.0
220.0-767.5
130-1,400
150.0
77.5-200.0
0-400
60.0
23.8- 100.0
0-350
5.0
0.5-50.0
0-250
<0.0001
<0.0001
(-0.41)
100.0
45.0-21 2.5
10-800
75.0
20.0-177.5
1-800
75.0
35.0-150.0
1-300
10.0
0.5-40.0
0-330
0.0035
0.0002
(-0.30)
15.0
10.0-150.0
5-220
1.5
0.8-85.0
0-250
15.0
0.0-127.5
0-400
80.0
50.0-225.0
0-1100
0.0250
0.0016
(0.25)
100.0
13.8-135.0
10-400
40.0
11.5-96.3
0-330
94.0
50.0-200.0
0-700
280.0
195.0-512.5
60-800
<0.0001
<0.0001
(0.47)
626.0
545.3-700.0
208- 1451
394.0
292.8-561.3
120-1224
319.0
192.0-413.0
2-1039
<0.0001
<0.0001
(-0.39)
2.790
1.91 5-3.318
1.63-3.87
2.780
2.350-3.850
1.53-5.20
2.530
2.300-2.890
1.90-4.28
20.1
(>0.1)
(0.01)
0.37
0.06-0.94
0.0-2.3
1.53
0.80-5.96
0.0-125.0
0.21
0.05-0.46
0.0-1.0
The immunologic parameters are compared with those of 24 HIV- HC.
* Kruskal-Wallis test, Pvalue.
t Kendall's rank correlation between each immunologic variable and the virologic status of
P value, Pi, and correlation coefficient ( i ) .
lnterquartile range.
§ Only HIV' subjects: 16 IsoI-, 26 Isol+,17 Isol'/SI'.
11 Only HIV' subjects: 14 IsoI-, 25 Isol+/SI-, 9 lsol+/Sl'
1 IL-lOAl + IL-2), to avoid 1/0 ratios.
50.0
4.40-137.50
1.2-350.0
P?t
IP)
<0.0001
<0.0001
(0.57)
HIV' subjects (ordered as follows: isol-, SI-, SI+):
*
variants were isolated from HC (Fig 3B). According to a
nonrigorous approach, the simple ratio IL-lO/IL-2 (or ILlo/( 1 IL-2), to avoid l/O's) appears to separate completely
individuals in whom SI HIV variants were isolated (ratios
1.2) from controls (ratios 1 .O). The second best combination
of two variables was IL-2/IL-4 and IFN-y/IL-lO, among all
individuals and HIV+ individuals, respectively.
Thus, we suggest that immunological characterization of
HIV+ individuals with respect to virologic status should include the evaluation of at least one type 1 cytokine (IL-2
andor IFN-y) and at least one type 2 cytokine (IL-4 and/or
IL-10).
+
DISCUSSION
Because HIV infection was suggested to be an immunovirologic disease, we analyzed possible correlations between
HIV intrinsic biological properties, such as the rate and the
extent of viral replication and viral phenotype and the
strength and the quality of the immune response. Our hypotheses were two: (1) a tight correlation exists between the
immunologic and the virologic aspects of HIV infection; and
(2) the virologically most favorable outcome (inability to
isolate HIV from PBMC) is associated with the strongest
production of type 1 cytokines, the weakest production of
type 2 cytokines, and the highest CD4 counts, whereas the
virologically least favorable outcome (isolation of fast-replicating SI HIV variants) is associated with a specular immunologic situation (ie, defective production of type 1 cytokines, strong production of type 2 cytokines, and lowest CD4
counts). Thus, we cross-sectionally analyzed the rate and
extent of HIV replication, HIV phenotype, cytokine production, and CD4 counts in 63 consecutively enrolled HIV+
individuals, and we examined cytokine production in 24
HIV- HC. The results suggest that the virologic and immunologic aspects of HIV infection are likely to be correlated.
Therefore, the most dramatic reduction of type 1 cytokine
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578
CLERIC1 E T AL
r
uniformly reduced in LTNP.zo-2' Additionally, a correlation
exists between the emergence of HIV SI HIV strains and
disease progression. Thus, ( 1 ) HIV SI variants are isolated
in SO% to 60% of HIV' individuals with AIDS, but in less
than 10% of HIV' asymptomatic individuals'; (2) 71% of
HIV' individuals in whom SI HIV variants could be isolated,
but only 16% of HIV' individuals in whom SI variants could
not be isolated progressed to AIDS in a 30-month followup period'; and (3) the isolation of HIV SI variants is associi
I *
ated with increased decline in CD4+ T lymphocytes.24
Immunological correlates of disease progression include
abnormalities affecting both humoral and cell-mediated imI
I
I
I
I
munity. Thus, it was shown that antibodies with better neu0
100
400
900
1600
tralizing ability for laboratory and primary isolates are present in LTNP and that the antibody repertoire of these
individuals is directed toward multiple HIV epitopes." We
have proposed progressive impairment of type 1 cytokinen
I
driven cell-mediated immunity to be the main immunologic
30
B
8
correlate of disease progression.""' This hypothesis is based
I
on different independent observations: (1) the gradual reducI
W
tion of the ability of PBMC to produce IL-2, IL-12, and
0
IFNy (type 1 cytokines) is accompanied in the progression
-15
of HIV infection by an increased generation of IL-4, IL-6,
0 -30
0
" . ~cytokine
~;
production
and IL-10 (type 2 ~ y t o k i n e s ) ~ " ~(2)
v)
profile of HIV+ LTNP individuals is characterized as high
CONTR ISOLSISI+
IL-2, high IFNyllow IL-4, low IL-10, whereas the cytokine
0
0
e
production profile of HIV+ individuals with progressive HIV
Fig 3. (A) IL-2 and IL-10 production in HIV- healthy controls (0); infection is exactly specular (low IL-2, low IFNylhigh ILHIV'llsol- individuals r),HIV'/lsol'/SI- individuals P); and HlV+/
4, high IL-10)"; and (3) better preserved generation of HIVIsol'/SI+ individuals (0).The dotted and the dashed lines indicate
specific cytotoxic T lymphocyte and HIV-specific T-cell prothe direction of the best discriminant function and that of the best
liferation are observed in HIV' LTNP
discriminant function among HIV' individuals, respectively. (Notethe
square-root scales on the X- and Y-axis.) (8) Box plots relative to the
Immunologic surrogate markers of disease progression inscores computed according the best discriminant function among
clude defective antigen- and mitogen-stimulated IL-2 proHIV' individuals. For each individual, the score was computed acduction, which is predictive for subsequent decrease in CD4
cording to the function, score = JIL-10- dlL-2, obtained by discrimicounts, time to AIDS, and time to death" and is correlated
nant analysis. The scores correspond to measures computed in the
in pediatric HIV infection with increased incidence of oppordirection of the dashed line shown in (A), which in this panel is
represented by the axis entitled "score (---I." The box plot resume
tunistic infections.** Other markers of progression are the
the score data as already described in Fig 2; the box covers the range
loss of the ability to elicit a DTH reaction to ubiquitous
between the lower and the upper quartile, and the central line is at
antigens (secondary to defective IL-2 production)x,'; hyperthe median. The 'whiskers' extend to the 10th and the 90th percenIgE (secondary to increased IL-4 production)*'^'"; and hypertile; more extreme data are plotted as separate points.
eosynophilia (secondary to increased IL-S production).3'.''
Thus, numerous virologic and immunologic markers of
disease progression have been reported. Nevertheless, no
production, as well as the greatest increment in type 2 cytocomplete analysis of the possible correlations between these
kine production and the lowest CD4 counts are observed in
two groups of correlates has been performed. We report here
those HIV+ individuals in whom fast-replicating SI HIV
that
the immunology and the virology of HIV infection are
variants are isolated. Additionally, the IL-lO/IL-2 ratio is
likely to be connected. Therefore, the worst possible outcapable of the best correlation with viral parameters, ie, in
come of virus analysis (isolation of fast-replicating SI HIV
vitro viral replication of fully infectious virions and ability
variants) is correlated with the least favorable immunologic
to induce a cytopathic effect (SI variants).
results (defective type 1 cytokines/increased type 2 cytoVirologic and immunologic correlates of disease activity
kines; reduced CD4 counts). Additionally, we show that the
have been proposed in HIV infection. Analyses of virologic
simple evaluation of the ratio between two cytokines (IL- 10
correlates have indicated that the rate and the extent of HIV
and IL-2) is capable of completely discriminating among
isolation in in vitro cultures of lymphocytes increase in the
HIV' individuals with different virologic profiles and who
progression of the d i ~ e a s e . ' ' ~Thus,
' ~ it was recently observed
are likely to have different prognoses.
that HIV isolation by PBMC of long-term nonprogressing
Because these results stem from a cross-sectional analysis,
individuals (LTNP) is difficult, and is in some cases, possible
we cannot evaluate whether the emergence of SI HIV varionly following depletion of CD8 lymphocytes."-" Plasma
ants is subsequent to the qualitative impairment of the imlevels of HIV RNA, specific intracellular transcripts, and
mune response, or conversely, if the appearance of SI HIV
the number of PBMC containing integrated viral DNA are
E
A
E 36
e
.
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IMMUNOVIROLOGY OF HIV INFECTION
variants is the cause of the decreased production of type 1
cytokines, in favor of an increased production of type 2
cytokines, which may b e secondary to the different replication requirements of NSI and SI variants. All the patients
are enrolled in a longitudinal study that could answer this
question.
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1996 88: 574-579
Human immunodeficiency virus (HIV) phenotype and interleukin-2/
interleukin-10 ratio are associated markers of protection and
progression in HIV infection
M Clerici, C Balotta, A Salvaggio, C Riva, D Trabattoni, L Papagno, A Berlusconi, S Rusconi, ML
Villa, M Moroni and M Galli
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