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Viral Latency and T-cell Responses After HAART Experimental Protocol: Ho et al. Nature 1995 20 HIV-infected individuals with CD4+ T cell counts ranging from 36 to 490 cells (mm-3) Protease Inhibitor:ABT-538 Measure viral RNA in the plasma Measure CD4+ T-cells in the blood Plasma viral load before and after drug treatment RNA copies per ml (x 103) 1000 Slope: -0.21 t 1/2: 3.3 days 100 10 1 -10 -5 Ho et al. Nature 1995 0 5 10 15 Days 20 25 30 35 Viral Half-Life •Extrapolating from the curves: •Between 108 and 109 virions are cleared each day suggesting that viral production is 108 to 109 virions per day •The T ½ is ~2 days (current estimates place it as even shorter) •Unfortunately, not all the virus is cleared Viral Latency •The latent viral pool persists in everyone following Highly Active Anti-Retroviral Therapy (HAART) •Is established soon after infection •T1/2 of replication competent virus is ~44 months therefore eradication could take up to 60 years. Evidence of Viral Reservoirs Plasma Viral RNA Primary Infection Viral Rebound Viral Setpoint Cessation Of HAART HAART 50 copies Infection Forms of HIV-1 “Latency” •Compartmentalization •CD4 + cells •Pre-integration complexes – labile vs. stable •Post-integration proviral DNA •Drug resistance: While drug resistance does occur, there is evidence of wild-type HIV-1 replication. Model for establishment and maintenance of HIV-1 reservoirs Death Activation: antigen Activated T-cell Quiescent T-cell Activated T-cell and renewed viral replication What Researchers Know So Far: Infection of naïve cells occurs infrequently and tends to result in more labile forms of virus. Viral DNA is most abundant in CD45RA- T-cells: Effector or memory? ~ 1 per 106 cells. Viral RNA is more abundant in activated, HLA-DR+, T-cells. Macrophages are also a cellular reservoir for HIV-1, but are short lived, therefore; their contribution to the long-lived reservoir is unclear. But, what about the T-cell compartment? Do individuals regain lost t-cells? Increase in CD4+ cell counts after drug treatment 1000 CD4 cell count (per mm3) Slope: 2.4 100 10 1 -15 -10 Ho et al. Nature 1995 -5 0 5 10 Days 15 20 25 30 35 Autran et al.:Experimental Protocol 8 HIV-infected, previously untreated individuals with advanced HIV-infection. Protease inhibitor; Ritonavir AZT Dideoxycytosine Measured viral RNA in the plasma Determined lymphocyte counts in the blood Phenotyped the cells Determined responses to recall antigens Phenotype of CD4+ T-cells following drug therapy 300 250 Cells (per ml) 200 150 100 50 0 0 5 10 15 20 Weeks Pakker et al. Nature Medicine 1998 25 30 35 40 Naïve CD4+ cells Memory CD4+ cells Total CD4+ cells Phenotype of CD8+ T-cells following drug therapy 900 800 700 Cells per ml 600 500 400 300 200 100 0 0 5 10 15 20 Weeks Pakker et al. Nature Medicine 1998 25 30 35 40 Naïve CD8+ cells Memory CD8+ cells Total CD8+ cells Roederer Nature Medicine 1998 Changes in T-cell subsets with drug therapy CD8+ T-cells Memory Naive CD4+ T-cells Memory Naive Years after infection HAART Re-expression of CD25 on CD4+ T-cells After Drug CD4+ CD25+ Therapy CD45RO+ CD25+ CD45RA+ CD25+ Total CD4+ T cells 30 % Positive Cells 25 20 15 10 5 0 0 2 4 6 8 Months Autran et a. Science 1997 10 12 14 Decrease in HLA-DR expression in T-cells after CD3+8+DR+ drug therapy Infectious Cells CD45RO+DR+ CD4+DR+ Total CD4+ cells 30 % Positive Cells 25 20 15 10 5 0 0 2 4 6 8 Months Autran et al. Science 1997 10 12 14 Restoration of CD4+ T-cell responses to tuberculin Stimulation Index 100 10 1 0.1 -1 0 1 2 3 Months Autran et al. Science 1997 4 5 6 7 Response of CD8+CD38+ T-cells To HAART CD38 molecules on CD8T cells Median Number of Molecules/cell 10000 8000 6000 4000 2000 0 25 26 27 28 29 Visit 30 31 32 33 Summary •With the addition of a protease inhibitor, plasma viral load drops but is not completely eliminated = viral latency •As viral load drops CD4+ T-cells increase •CD8+ T-cells increase then decline slightly •Activation antigens decrease on both subsets •CD4+ T-cells re-express CD25 and regain proliferative responses to recall antigens •The greatest T-cell increases are in the memory phenotype cells •After about six weeks slow, but steady, increases are observed in the naïve T-cell compartment Where Do The T-cells Come From? •Homeostasis mechanisms trigger proliferation of surviving cells. •Redistribution of cells from inflammed tissues, back into blood causes an observed increase in cells. •Naïve cells: Thymus vs. gut vs. proliferation? Can The Thymus of Adults Contribute To T-cell Reconstitution? Phenotypic markers of T-cell development in the Thymus BM CD34+ CD1CD3CD4CD5+/CD8- Thymus CD34+/CD1+ CD3CD4+ CD5+ CD8- CD34CD1++ CD3CD4+ CD5+ CD8+ CD34CD1++ CD3+ CD4+ CD5+ CD8+ CD69+ PB CD1+ CD3+ CD4+ CD8CD69+ CD1CD3+ CD4+ CD8CD69+ CD1CD3+ CD4+ CD8CD69- CD1+ CD3+ CD4CD8+ CD69+ CD1CD3+ CD4CD8+ CD69+ CD1CD3+ CD4CD8+ CD69- Summary of Adult Thymus Specimens Thymus# Fetal Fetal 1* 2* 3 4 5 6 7 8 9 10 11 12* Age 0 0 23 23 23 33 33 36 36 37 48 52 54 56 CD4+CD8+ 85 82 86 70 65 35 88 76 15 2 48 79 22 69 *Corrective Cardiac Surgery Human Thymocyte Differentiation CD4+ TCR+ CD4+/- CD4+ CD4+ CD8+ TCR+ CD8+ TCR+ CD34 CD5 CD1a CD69 CD3 CD62L Adult And Fetal Thymocytes Demonstrate Similar Subset Distribution Age 11% 85% 4% 88% Fetal 10% 86% 6% CD1a-PE 23 yrs. CD4-TC 4% 2% 3% 13% 79% 89% 8% 2% 83% 52 yrs. 6% CD8-FITC 14% CD5-FITC Adult And Fetal Thymocytes Demonstrate Similar Subset Distribution Age 8% 15% 80% Fetal 11% 8% 16% 6% CD3-PE 23 yrs. CD69-PE 72% 14% 6% 72% 12% 22% 77% 14% 69% 52 yrs. 62% CD8-FITC 14% CD8-FITC Co-stimulation of Adult Thymocytes Unstimulated Medium Stimulated Medium Anti-CD28 Anti-CD3 3 Days 3 Days Harvest Stain for activation markers Adult Thymocytes Respond To Costimulatory Signals Thymus # Fetal SCID-hu 1 3 4 5 8 11 Age 0 0 23 23 33 33 37 54 CD25 Unstim Stim 8 74 1 47 2 80 1 49 9 96 2 46 <1 62 ND ND CD69 Unstim Stim 10 86 18 93 22 67 20 57 49 96 17 80 7 75 29 67 10% CD8+ 8% 6% 4% 2% Adult 0% Thymocytes 8% 6% Display A Diverse Vb Repertoire 4% 2% 0% 8% 6% 4% 2% 0% Adult Fetal Can Adult Thymocytes Migrate To The Periphery? Signal joint (sj) and coding joint (cj) TREC production from the a/d locus a locus V a d locus a locus Vd dRec Dd Jd Cd yJa Ja Ca sj PCR cj PCR Adapted from Martie C. M. Verschuren, Thesis TREC: -T-cell receptor Rearrangement Excision Circles - are detected in phenotypically naive T-cells, but not in memory T-cells or in B-cells. - decline in the peripheral blood with age, after thymectomy, and in HIV-infected individuals. - increase in HIV-infected individuals undergoing HAART. Douek et al. Nature 1998 TREC Positive Cells In The Periphery of Adults Peripheral CD4+ T-cells Thymocytes Sj TRECs per mg of DNA 100000 10000 1000 100 0 10 20 30 40 50 Age (years) 60 70 80 TREC changes during HAART 1 4 A -3 6 12 10 8 6 4 2 0 8 7 6 5 4 3 2 1 C -2 5 B -2 8 1 06 - 1 05 + 10 4 103 50 10 0 15 0 20 0 0 25 0 D - 29 50 1 00 150 20 0 25 0 0 50 10 0 2 00 2 250 1 0 6 10 F-3 6 E -2 9 1 50 + 1 05 10 - 4 103 0 50 100 150 2 00 2 50 0 8 G -2 7 7 6 5 4 3 2 1 0 1 00 150 20 0 25 0 H -3 2 0 50 100 15 0 200 I-2 2 25 0 102 1 06 1 05 104 10 3 50 10 0 15 0 20 0 8 J-6 3 7 6 5 4 3 2 1 0 50 250 1 06 1 05 10 4 103 50 10 0 15 0 20 0 250 1 0 0 50 1 00 150 20 0 25 0 0 50 100 15 0 200 25 0 1 0 2 Sj TREC x103 per mg naïve CD4 cell DNA Naïve CD4 T cells x102 per ml blood HIV RNA copies per ml of plasma 2 Douek et al. Nature 98 Conclusions 1. Thymopoiesis is ongoing in adults, resulting in functional T-cells with a diverse TCR-Vb repertoire. 2. CD4+, phenotypically naïve, TREC positive T-cells increase following HAART suggesting that the adult thymus contributes to T-cell reconstitution. 3. The thymic microenvironment remains functional after exposure to HIV. Douek et al. 1998 Nature 396: 690 Withers-Ward et al. 1997 Nature Med. 3: 1102 Jamieson et al. 1999 Immunity, 10: 569 Conclusions cont. Taken together, these results suggest that therapeutic strategies to enhance thymopoiesis in the adult may increase T-cell reconstitution in HIV-infected individuals treated with HAART, or in cancer patients undergoing chemotherapy. In addition, these strategies may improve T-cell responses in the elderly. Strategies to Improve T-cell reconstitution Vishwa Deep Dixit Current Opinion in Immunology Volume 22, Issue 4, August 2010: 521-528 Therapeutic Strategies 1. 2. 3. 4. 5. 6. 7. IL-7: Modulates thymic output, expands and supports survival of naïve and memory T-cells. In HIV-1 infected individuals see increases in functional T-cells(1). IL-15: Supports survival of NK, NKT and CD8+ memory T-cells(2) Growth Hormone: acts as a pro-thymopoietic agent (3) Ghrelin: Reduces pro-inflammatory cytokines and reverses thymic involution (3) Leptin: Increases peripheral IGF1, results in thymic regeneration (3) IGF1: expands progenitors in bone marrow, increases thymic epithelial niches (3) CR mimetics: Maintains TCR diversity and increases thymopoiesis (3) 1. 2. 3. Levy et al. Journal of Clinical Investigation 2009, 119:997 Van den Brink et al. Nature 2004, 4:856 Vishwa Deep Dixit, Current Opinion in Immunology, 2010: 521