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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2001 as doi:10.1096/fj.00-0619fje. |
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,
2
Departments of
* Medical Microbiology;
Pediatrics and Child Health,
Immunology, University of Manitoba, Winnipeg, Canada R3E 0W3; and Departments of
Medical Microbiology and
Community Health, University of Nairobi, Kenya
2Correspondence: Department of Immunology, University of Manitoba, 626–730 William Ave. Winnipeg, MB R3E 0W3, Canada. E-mail: hayglass{at}ms.umanitoba.ca
SPECIFIC AIMS
We previously conducted epidemiologic studies identifying a cohort of prostitutes who exhibit profound resistance to HIV-1 infection despite intense, chronic sexual exposure. Many demonstrate CTL responses to HIVIIIB env, gag, and pol gene products and interleukin 2 (IL-2) production to HIV peptides, suggesting that immunological factors are involved in this type of HIV-1 resistance. Here, we hypothesized that differences in virus-specific cytokine responses may underlie resistance vs. susceptibility to HIV infection among Nairobi sex workers and therefore we examined the nature of HIV-1-specific and recall antigen responses in resistant vs. susceptible populations compared with healthy (non-sex worker) controls.
PRINCIPAL FINDINGS
1. Resistant sex workers exhibit significantly enhanced
interferon 
(IFN-) and lower IL-4 HIV-1-specific responses
independent of changes in other type 2 cytokines
Although exposed uninfected individuals clearly possess a variety
of HIV-1-specific, MHC class I- and II-restricted immune effector
responses, how and why these develop is unknown. In particular, the
nature of the immunoregulatory cytokine response in resistant vs.
susceptible populations is unclear. Quantitative analyses of cytokine
responses by fresh peripheral blood mononuclear cells (PBMC) stimulated
in short-term primary culture by intact HIV-1, exhibiting a spectrum of
potential HIV epitopes, have not been performed to date. We examined
antigen-specific cytokine responses in HIV-1-resistant and infected CDC
stage A1 Kenyan sex workers. All participants were asymptomatic,
exhibited CD4 > 500/µl, and had not received anti-retroviral
therapy. All exposed subjects exhibited HIV-dependent responses in
primary culture whereas low-risk, seronegative, presumably unexposed
Kenyan women (not shown) did not. The nature of the cytokine response
was markedly different in resistant and susceptible women. The
frequency of resistant women exhibiting detectable IFN- in culture
with HIV-1 was 86% (12 of 14 subjects) compared to 18% of susceptible
women (2/11, Fisher’s two-tailed, P<0.001). Resistant
women also exhibited more intense IFN- production than did
HIV-1-infected individuals in response to virus-mediated stimulation
(Fig. 1
, Mann-Whitney P<0.005). In marked contrast to IFN-,
HIV-1 stimulated IL-4 responses were substantially less frequent among
resistant women, with 2 of 14 (14%) generating detectable IL-4
responses vs. 10 of 11 susceptible women (91%, Fisher’s
P<0.0002). The median intensity of this HIV-stimulated IL-4
production was at least 20-fold lower among resistant than susceptible
women (<0.45 vs. 9.3 pg/ml; P<0.005, Fig. 1
). When these
data are examined as the ratio of virus-stimulated IL-4: IFN-
production, a commonly used strategy for evaluation of type 1 vs. type
2 dominance of immune responses, resistant subjects exhibit patterns
commonly termed ‘type 1’ dominance, whereas healthy HIV-infected
subjects display ‘type 2’-dominated responses (IL-4:IFN- ratios of
0.27 vs. 46, P<0.0002, Mann-Whitney).
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However, notwithstanding the 
20-fold lower median IL-4 production
seen in the resistant population, IL-13 production was intense and
similar in both groups (P=0.09), and HIV-driven IL-5
responses were marginally higher in the resistant population
(P=0.046). Thus, IL-5:IFN- or IL-13:IFN- ratios were
indistinguishable between resistant and infected groups, arguing
against a classical type 2 immunity association with resistance vs.
susceptibility to HIV-1 infection. IL-10 production, not selectively
associated with human Th1- or Th2-like T cell clones in vitro or with
type 1- or type 2-dominated responses in vivo, was indistinguishable in
resistant and susceptible populations.
2. Polyclonal stimuli fail to reveal alterations in HIV-1-specific
type 1 vs. type 2 cytokine responses
Use of polyclonal activators as surrogate antigens elicits
intense, hence readily quantified, cytokine responses, leading to their
widespread usage to evaluate HIV-specific responses. However, these
stimuli initiate distinct intracellular signaling events and frequently
elicit cytokine responses qualitatively different from those seen after
MHC-dependent T cell activation. For comparison, primary cultures were
established using immobilized anti-CD3 monoclonal antibody or
phytohemagglutinin (PHA) as polyclonal stimuli. As anticipated, median
cytokine responses by resistant and HIV-1-infected subjects were 5- to
50-fold stronger than those seen in that individual’s HIV-specific
response. IFN- IL-4, IL-5, or IL-10 production in the study groups
did not differ significantly under any of the conditions tested
(P>0.05) whereas IL-13 was marginally higher
(P=0.04) after anti-CD3, but not PHA stimulation. These
results indicate the necessity of using Ag-specific stimulation.
3. Recall antigen-mediated stimulation reveals a global deficiency
in the capacity of HIV-1-resistant women to mount IL-4 responses
Because resistant women exhibit strongly IFN--dominated
cytokine responses to HIV-1 and resistance to HIV has been associated
with enhanced type 1 responses (IFN- synthesis, CTL activity), we
considered the hypothesis that these women exhibit a generalized
tendency for enhanced IFN- production relative to the general
population. We examined their capacity to respond to a panel of common
recall antigens relative to low-risk, also seronegative, presumably
unexposed Kenyan women.
Primary culture with polyclonal stimuli (PHA, anti-CD3) or
antigens unrelated to HIV-1 (streptokinase, purified protein
derivative, or Candida) revealed that the increased IFN-
response to HIV seen among HIV-1-resistant prostitutes (Fig. 1)
is not
paralleled by general hyper-responsiveness in IFN- production.
Similarly, production of IL-5, IL-10, and IL-13 to these environmental
antigens was comparable in the HIV-1-resistant, HIV-infected, and HIV-1
unexposed groups (data not shown).
In marked contrast, IL-4 responses generated by HIV-1-resistant women
to recall antigens were dramatically lower than those of either
healthy, unexposed Kenyan women or the HIV-infected women examined
above (Summary Fig. 2
). This reduced capacity of HIV-1 exposed but uninfected women to mount
IL-4 responses was evident whether examining the frequency of
individuals in each group capable of mounting Ag-specific IL-4
responses (Fisher’s, P=0.02 to 0.005 for the five different
stimuli examined) or the median intensity of the IL-4 responses
generated (Mann-Whitney P=0.01 to 0.0015). IL-4 responses to
streptokinase, PPD, or Candida were indistinguishable in
HIV-1-infected vs. HIV-1 unexposed populations (Mann-Whitney
P=0.43 to 0.54). Thus, HIV-1-resistant women exhibit global
hyporesponsiveness in their capacity to generate IL-4 responses to HIV
or common recall antigens that distinguishes them from either
HIV-1-infected or unexposed (predominately susceptible) groups.
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CONCLUSIONS
There is considerable heterogeneity in the results of HIV-1 infection,
ranging from rapid progression to AIDS to survival for many years with
little evidence of disease. The mechanisms that underlie resistance in
exposed/uninfected individuals are heterogeneous and may include
defective expression of the CCR5 coreceptor for HIV-1 internalization,
elevated production of CC chemokines RANTES, MIP-1
, and MIP-1ß,
HIV-specific T helper and CTL responses, and secretory IgA responses in
the genital tract. The nature of the immunoregulatory differences in
cytokine synthesis that underlie such responses is unresolved and
controversial. Nairobi prostitutes provide one of the most clear-cut
examples of resistance to HIV-1 infection in the face of ongoing HIV-1
exposure. We report our discovery that these HIV-1-resistant subjects
exhibit cytokine responses to HIV or common environmental antigens
profoundly different from those of infected or healthy controls. Thus,
resistance to HIV-1 infection among African sex workers is strongly
associated with global hyporesponsiveness in IL-4
production, independent of changes in other type 2 cytokines (IL-5,
IL-13, and sometimes incorrectly used as a prototypical Th2 cytokine,
IL-10). As such, it is not readily interpretable in terms of classical
type 1 vs. type 2 skewing of the HIV-specific cytokine response as
previously used in discussion of HIV disease progression and a wide
variety of other infectious diseases.
Whether globally decreased capacity to mount IL-4 responses is a
prerequisite for, or occurs as a consequence of, resistance in the
5% of the population exhibiting long-term HIV-1 resistance despite
ongoing exposure to infectious virus will require prospective studies.
If resistance reflects acquired immunity, individuals with reduced
capacity to mount IL-4 responses to HIV-1 may preferentially develop
resistance. Alternatively, the reduced IL-4 responsiveness seen in
these women may be secondary to induction of resistance. HIV-1 has been
reported to replicate more efficiently in IL-4-producing T cell clones
(Th2/Th0) than in Th1 clones. Certainly, IL-4 is a pivotal cytokine in
promoting commitment of naive T cells to IL-4-dominated response
patterns and is capable in vitro of overriding potent IFN-/Th1
promoting stimuli such as IL-12. We also note that IL-4 substantially
up-regulates in vitro expression of CXCR4, a coreceptor for T cell
tropic HIV-1, making those cells more susceptible to HIV-1 infection
and resulting in higher levels of HIV-1 production, whereas IFN-
down-regulates expression of this receptor. Collectively these
observations raise the possibility that resistant individuals who
exhibit markedly lower HIV-1 and environmental Ag-driven IL-4 responses
may be characterized by lower levels of infectious HIV production at
initial exposure, combined with enhanced clearance of HIV-1,
attributable in part to intense IFN--dominated responses and CTL
generation.
Finally, antigen-specific activation in this study was carried out in vitro using HIV-1IIIB, a widely available clade B virus. Currently dominant Kenyan isolates are from clades A, C, D. The finding that HIV-1IIIB readily evokes cytokine responses in subjects with minimal exposure to this specific clade is consistent with epidemiologic findings that these women, who have exposure to a broad variety of HIV variants within and between clades, are resistant to a broad range of HIV-1 subtypes. Thus, resistance to HIV-1 in these women is broadly cross-protective. Similar cross-reactivity in CTL activity and ß chemokine production between HIV-1 and HIV-2 responses has recently been observed. This finding may have significant implications for design of HIV vaccines.
In summary, the data suggest that a key differentiator of resistant
subjects from the population as a whole is the nature of the cytokine
response elicited upon HIV exposure. Specifically, resistance vs.
susceptibility to HIV-1 infection can be distinguished by reciprocal
differences in the intensity of HIV-1-driven IFN- and IL-4 cytokine
production independent of substantial alterations in IL-5, IL-10, and
IL-13 responses in these women.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0619fje ; to cite this
article, use FASEB J. (June 18, 2001)
10.1096/fj.00-0619fje 
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500/µl, 
). Cells were cultured for 2–8 days with intact
inactivated HIV IIIB and in parallel wells (not shown),
anti-CD3 mAb, or PHA as polyclonal stimuli. Cytokine responses in
culture supernatants were determined by ELISA against WHO or commercial
standards.
) after primary culture. The median
response of resistant women was significantly lower
(P<0.005) than that of healthy HIV-1-infected sex
workers or low-risk controls whereas responses of the latter two
controls were not significantly different (P>0.05).