The IL-7 Receptor A Key Factor in HIV Pathogenesis

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1 The IL-7 Receptor A Key Factor in HIV Pathogenesis Paul MacPherson PhD, MD, FRCPC Associate Professor Division of Infectious Diseases Ottawa Hospital, General Campus Ottawa Hospital Research Institute University of Ottawa

2 Interleukin-7 25 kdal glycoprotein Produced by stromal epithelial cells, dendritic cells, intestinal epithelium, vascular endothelium, hepatocytes Provides communication to T-cell Sasson et al. Curr Drug Targets, 2006

3 Interleukin-7 Receptor IL-7 Receptor: - -chain (CD127) - -chain (CD132) Common to receptors for IL-2, IL-4, IL-7, IL-9, IL-15, IL-21 Schluns et al., Nature Reviews-Immunology Expressed on common lymphoid progenitors, thymocytes, naïve and memory T-cells

4 IL-7 and T-Lymphocytes IL-7 is essential to T-cell development and function and plays important roles throughout the life-span of a T-cell: Differentiation from stem cells in the bone marrow Differentiation and selection of thymocytes T-cell survival and homeostasis Establishment of memory T-cells

5 IL-7 and CD8 T-Cell Activation IL-7 Proliferation Telomerase Glucose metabolism Bcl-2 Establishment of memory Perforin CD25 CD69 CD45RA adapted from Schluns and Lefrançois. Nature Reviews Immunology. 2003

6 CD8 T-cells and HIV

7 Cell Mediated Immunity and HIV Infection CD8 T-cells remain in the circulation even in patients with advanced disease. CD8 T-cells from HIV-infected patients respond poorly to antigens. Do not proliferate or make perforin in response to antigen and demonstrate weak cytolytic activity.

8 Interleukin-7 and HIV Infection Interleukin-7 signaling is essential for optimal CD8 T-cell proliferation and cytotoxic activity Diminished cytotoxic CD8 T-cell activity is a central feature in HIV disease

9 Does interleukin-7 play a role in immune dysregulation during HIV infection?

10 Hypothesis: HIV infection is associated with a down regulation of the IL-7 receptor -chain (CD127) on CD8 T-cells

11 Hypothesis: HIV infection is associated with a down regulation of the IL-7 receptor -chain (CD127) on CD8 T-cells Corollary: Recovery of CD127 is associated with immune recovery following suppression of HIV replication with antiretroviral therapy.

12 Representative FACS analysis from three individuals gating on total lymphocytes. (A) Healthy seronegative control (B) HIV+ patient naïve to antiretroviral therapy with a CD4 count of 278 and a viral load of 6,028 copies/ml (C) HIV+ patient on antiretroviral therapy. Baseline CD4 count was 108. At the time of analysis, the viral load had been maintained at <50 copies/ml for 41 months and the CD4 counts was 694. CD8 CD127

13 IL-7R and HIV Infection 67% reduction in CD8 T- cells expressing IL-7Rα in HIV+ patients. Partial recovery with sustained HIV suppression on cart. CD127 Expression on CD8 T Cells Magnitude of recovery proportional to time on suppressive treatment. Healthy Controls HIV + (Untreated) HIV+ HAART MacPherson et al., JAIDS 2001

14 Summary Active HIV replication is associated with a marked decrease in the level of expression of CD127 on CD8 T-cells. Suppression of HIV replication with effective antiretroviral therapy is associated with an apparent recovery of CD127 on CD8 T-cells. Given that CD127 (IL-7R ) is necessary for optimal CD8 T- cell proliferation and cytolytic activity, this may explain in part the reduced CTL activity seen in HIV infection and its recovery on antiretroviral therapy MacPherson et al JAIDS 2001

15 How does HIV infection lead to a down regulation of the IL-7 Receptor -chain on CD8 T-cells?

16 Viral factor: HIV-1 Tat 14 kdal Protein Secreted by HIV-infected cells and can be detected in serum of HIV+ patients. Taken up by uninfected T-cells through heparin sulfate receptors and clathrin-dependent endocytosis. Soluble Tat suppresses T-cell activity in vitro Anti-Tat antibodies are associated with low viral loads and slower disease progression in HIV+ patients

17 Hypothesis The decrease in IL-7 Receptor -chain expression on CD8 T-cells is mediated by soluble HIV Tat protein

18 CD8 T-cells isolated from healthy donors PBMC were isolated over Ficoll CD8+ cells were purified using the AutoMACS magnetic bead cell isolation system Purity by flow cytometry 95-99% pure CD8+

19 Tat down regulates CD127 on CD8 T-cells

20 CD127 is not down regulated by other HIV proteins

21 HIV Tat induces a time and dose dependant decrease in CD127 expression

22 Tat does not effect IL-7R γ-chain

23 Tat specifically targets CD127

24 %CD127 surface expression Effect of Tat on CD127 expression is reversible media Tat W 24 Tat W 48 Tat W 72 Tat (10 µg/ml) Incubation Time (hours)

25 Tat induces a down-regulation of IL7Rα expression on CD8 T-cells: Dose and Time Dependant Specific for CD127 Effect is blocked with anti-tat antibodies Effects both naïve and memory cells Reversible: requires continuous presence of Tat Down regulation is not due to cell stimulation or apoptosis

26 Tat down regulates surface expression of CD127 on CD8 T-Cells in vitro Is this reflected in vivo?

27 Healthy controls Healthy HIV+ patients recover CD127 on the surface of CD8 T-cells 24h HIV+ media CD127-PE

28 Tat protein inhibits recovery of CD127 on CD8 T-Cells isolated from HIV+ patients 24 hours 48 hours 72 hours Media Media Media Media Tat 2 μg/ml Tat 2 μg/ml Tat 2 μg/ml Tat 2mg/ml CD127-PE

29 Tat down regulates surface expression of CD127 on CD8 T-Cells Is there any functional relevance to this decreased expression of IL-7R on CD8 T-cells?

30 Tat inhibits IL-7 induced proliferation

31 Tat inhibits IL-7 induced Perforin production

32 By down regulating the IL-7R at the cell surface, Tat is able to induce anergy in CD8 T-cells.

33 How does HIV Tat down regulate the IL-7 Receptor on CD8 T-cells?

34 CD127 is stable in resting CD8 T-cells with a half-life of 55 hours

35 CD127 decays from the surface of resting CD8 T-cells at a slow rate Incubation time (hours)

36 Tat decreases the half-life of CD127 protein

37 Tat increases the rate of decay of CD127 from the surface of resting CD8 T-cells

38 CD127 decays from the surface of resting CD8 T-cells with a half life of 55 hours Tat increases the rate of decay of CD127 from the surface of CD8 T-cells How does Tat exert its effect?

39 Tat enters Lymphocytes through receptor mediated endocytosis, followed by exit from late endosomes to exert effect in the cytoplasm (Vendeville et al., Mol. Biol. Cell, 2004) endosomal acidification is required for early to late endosomal development and release of Tat into the cytoplasm.

40 Tat is internalized by CD8 T-cells 1.5 hours 3 hours 6 hours 12 hours

41 Tat trapped in endocytic vesicles cannot down regulate CD127 at the cell surface

42 Tat entry into the cell and subsequent exit from late endosomes are required for Tat to exert its effect on CD127. What happens after Tat enters the Cytosol?

43 Tat co-localizes with CD127 DAPI CD127 Tat Overlay media Tat

44 Tat trapped in endocytic vesicles cannot down regulate CD127 at the cell surface

45 Tat associates with CD127 and induces receptor aggregation. Do Tat and CD127 interact?

46 Anti-CD127 antibodies co-immunoprecipitate the HIV Tat Protein kda Tat (15 kda) CD8 CD8 + Tat

47 Tat binds to the cytoplasmic tail of CD127 and induces receptor endocytosis.

48 Once inside the cell, what is the destination of CD127?

49 Proteasome inhibitors block Tat s effect Lysosome inhibitors do not block Tat s effect

50 1 Summary 1. Tat enters the cell by receptor (HS) mediated endocytosis Tat exits late endosomes to enter the cytosol. 3. Tat binds the cytoplasmic tail of CD127 inducing receptor endocytosis CD127 is internalized and degraded by the proteasome. 4 Faller et al J Immunol 2010

51 Mutational Analysis of the Tat Protein wttat Acidic C-Rich Core Basic Q-Rich C-Term HIV-2N10 Acidic Acidic C-Rich Core Basic Q-Rich C-Term D17-21 Acidic C-Rich Core Basic Q-Rich C-Term D22-26 Acidic C-Rich Core Basic Q-Rich C-Term DCore D37-50 Acidic C-Rich Basic Q-Rich C-Term DBasic D48-57 Acidic C-Rich Core Q-Rich C-Term DGln-Rich D63-78 Acidic C-Rich Core Basic C-Term DC-term D73-86 Acidic C-Rich Core Basic Q-Rich Basic N-Term C-Rich Core Q-Rich C-Term

52 CD127 Surface Expression Normalized to Media Deletions of amino acids 1-10, or the basic domain abolish Tat s ability to down regulate CD127 on the cell surface B Mock wttat D DBasic HIV-2N Hours

53 Tat binds via its amino terminal domain to the cytoplasmic tail of CD127 and directs it to the proteasome for degradation.

54 Current Model E3 Ligase By binding to the cytoplasmic tail of CD127, Tat recruits an E3-ubiquitin ligase to the receptor thus inducing receptor internalization and degradation.

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56 Elliott Faller Scott Sugden Feras Al-Ghazawi Hafsa Cherid Juzer Kakal Mark McVey Denny McLaughlin Acknowledgements A special thanks to all of our wonderful and willing blood donors!