Differential cytokine responsiveness to toll-like receptor (TLR) ligand stimulation in HIV-1 resistant sex-workers from Nairobi, Kenya T. Blake Ball Ph.D. Department of Medical Microbiology University of Manitoba

HIV resistance in the Pumwani cohort: Epidemiological definition of resistance: active sex worker >3 years of follow-up HIV negative by PCR and serology Clinically no evidence of disease Reduced incidence of HIV seroconversion with increasing duration of follow-up Modified from Fowke et al.Lancet 1996

Why Does Resistance Develop? Resistance to infection by HIV-1 associates with: HIV specific cell mediated immune responses, Compartmentalized HIV specific cell mediated and antibody responses Multifactoral immunogenetic factors: specific HLA alleles (Lacap, Turk, Hardie MOPE0017, MOPE0020, MOPE0024), polymorphisms in IRF-1 genes (Ji MOAX 0103), site specific HIV inhibitory factors (Iqbal WEAA 0504) undescribed factors

Toll-like receptors (TLR) play a critical role in innate immunity, recognizing conserved Pathogen-Associated Molecular Patterns

TLRs link innate and adaptive immunity: Regulation of CD4 T cell development through TLRs NEJM 2000

We hypothesize that: HIV resistant women have altered innate responses such that they are more likely to develop cell-mediated immune responses to HIV-1.

Study design: Cross sectional comparison of cytokines known to drive cell mediated (Th1) responses in HIV resistant sex workers, newly enrolled HIV negative sex workers and HIV non-SW controls. Measure cytokines (IL-12, IFN- , IFN-  and IL-10) after short-term PBMC culture (18 hr) in response to ligands for TLR-2 (Staphylococcus aureus), TLR-4 (LPS) and TLR-7 (Imiquamode anolog-3M)

HIV resistant women have elevated IL-10, but not IL-12p40, IFN- , nor IFN-  responses to TLR2 stimulation Media SAC (0.001%) Media SAC (0.004%) HIV resistant women HIV negative SW LR controls

HIV resistant women have elevated IL-10, depressed IFN-  to TLR4 stimulation HIV resistant women HIV negative SW LR controls

HIV resistant women have elevated IL-10, depressed IFN-  to TLR7 stimulation HIV resistant women HIV negative SW LR controls

Results Resistant women have depressed responses in cytokines responsible for cell mediated immune responses (IFN-  ) and elevated immunosuppressive (IL-10) responses. Resistant women have depressed responses in cytokines responsible for cell mediated immune responses (IFN-  ) and elevated immunosuppressive (IL-10) responses. Is this a deficit of IFN-  production or are the depressed IFN-  responses simply due to immunoregulation by excess IL-10. Is this a deficit of IFN-  production or are the depressed IFN-  responses simply due to immunoregulation by excess IL-10. We tested the IFN-  responses to TLR stimulation in the absence of IL-10 (by blocking IL-10). We tested the IFN-  responses to TLR stimulation in the absence of IL-10 (by blocking IL-10).

Does blocking of immunosuppressive IL-10 recover responsiveness of IFN-  Removal of “higher” levels of IL-10 does not restore IFN-  levels to those observed in controls. LPS SAC HIV resistant women LR controls

Conclusions Resistant women have significantly altered innate immune responses to TLR stimulation. Resistant women have significantly altered innate immune responses to TLR stimulation. Altered innate and therefore adaptive immunity has implications for resistance/susceptibility to other infectious and immune mediated diseases. Altered innate and therefore adaptive immunity has implications for resistance/susceptibility to other infectious and immune mediated diseases. No apparent bias towards generation of cell mediated responses. Overall, appears to be a suppressed immune response. No apparent bias towards generation of cell mediated responses. Overall, appears to be a suppressed immune response. How could they lead to resistance ? HIV resistance may be due to the LACK of a response, rather than the induction of a strong response. How could they lead to resistance ? HIV resistance may be due to the LACK of a response, rather than the induction of a strong response.

Further Questions…. Why are these women different? Why are these women different? Altered TLR expression, signaling? Altered TLR expression, signaling? Known TLR 2+4 polymorphisms do not account for this (Marlin TUPDA07) Known TLR 2+4 polymorphisms do not account for this (Marlin TUPDA07) Several correlates of HIV resistance have been identified Several correlates of HIV resistance have been identified Demonstrates that this group is biologically distinct Demonstrates that this group is biologically distinct How may these associations explain resistance either alone or in concert? How may these associations explain resistance either alone or in concert? Resistance is likely multifactoral and complex. Replication in other HEPS groups critical as is multivariate analysis Resistance is likely multifactoral and complex. Replication in other HEPS groups critical as is multivariate analysis Altered TLR signaling relevant to other infectious/immune diseases? Altered TLR signaling relevant to other infectious/immune diseases?

Correlates of resistance HIV systemic compartment genital tract Trappin-2 RANTES IgA  IRF-1  CTL CTL HIV stim  IL-10 TLR stim CTL  CD3

Thanks to: The women of the Majengo and Pumwani cohorts University of ManitobaFunding Dr. Kent HayGlass Dr Frank Plummer Crystal Marlin Rongrong Mao University of Nairobi Dr Charles Wachihi Dr Joshua Kimani Colleagues and staff of the WHO collaborative center for STI Research