1. HIV-1 infection decreases CD127 and PD1 expression on duodenal CD8+T cells Liliana Belmonte, PhD Academia Nacional de Medicina Buenos Aires, Argentina

2. Background The GI tract is a major site of HIV replication and pathogenesis, regardless of the specific tissue site of viral entry. Persistence of HIV in the duodenal mucosa even when effective therapy suppresses HIV-RNA in blood to undetectable levels, has been demonstrated by previous studies from our group. (Belmonte L et al, AIDS 2007;21(15):2106-8).

3. Chronic HIV infection often results in ineffective CD8 T-cell responses. However the mechanisms triggered by persisting virus infection and their impact on CD8 T-cell effector functions and tissue distribution, remain unknown. Specific Aim To analyse the quality of duodenal CD8 T cells in chronically HIV-infected patients.

4. Distal Duodenal biopsies 26 patients with chronic HIV-1 infection under HAART 13 HIV-1 seronegative individuals were included as control (C) biopsy was labeled as “HIV +” if HIV-DNA was detected by standard PCR We analyzed the presence of HIV-DNA in the duodenal tissue. A biopsy was labeled as “HIV +” if HIV-DNA was detected by standard PCR (using the primers SK145 and SKCC1B to define a sequence of 155 nucleotides within the highly conserved region of the HIV-1 gag gene)

5. Distal Duodenal biopsies 26 patients with chronic HIV-1 infection under HAART 13 HIV-1 seronegative individuals were included as control (C) Endoscopic material were disrupted mechanically and a cell suspension was prepared. Expression of CD4, CD8, CD27, CD28, CD45RO, CD45RA, CCR7, CD127, PD-1 (BD) was analysed by flow cytometry (FacScan, BD). biopsy was labeled as “HIV +” if HIV-DNA was detected by standard PCR We analyzed the presence of HIV-DNA in the duodenal tissue. A biopsy was labeled as “HIV +” if HIV-DNA was detected by standard PCR (using the primers SK145 and SKCC1B to define a sequence of 155 nucleotides within the highly conserved region of the HIV-1 gag gene)

6. Percentages of CD4 and CD8 T cells in the duodenal mucosa

7. Duodenal Memory CD8 T cell differentiation (CD45RA, CCR7 expression) CD8+ CD45RA+ CCR7+ CD8+ CD45RA- CCR7+ CD8+ CD45RA- CCR7- CD8+ CD45RA+ CCR7- Precursor memory T cells Terminally differentiated cells Pre-terminally differentiated cells

8. Duodenal Memory CD8 T cell differentiation (CD27, CD28 expression) CD8+ CD28+ CD27+ CD8+ CD28- CD27+ CD8+ CD28- CD27- Late effector cells Intermediate Differentiated cells Early Differentiated cells HIV-1 + patients HIV+ biopsies (n=10) HIV- biopsies (n=6) Controls (n=12) Early Differentiated cells (CD28+,CD27+) (%) 12 ± 5 10 ± 4 12 ± 5 Late Effector cells (CD28-, CD27-) (%) 39 ± 6 61 ± 8 23 ± 4 * p=0.0003 p=NS * p=0.04 p=NS Mean  SEM

9. High Programmed-death 1 (PD-1) expression on duodenal CD8 T cells from HIV+ patients HIV+ biopsies HIV- biopsies Controls p=ns

10. Decreased CD127 high (IL7R) expression on duodenal CD8+ T cells from HIV+ patients HIV+ biopsies HIV- biopsies Controls p=ns

11. Conclusions  Our results demonstrate an accumulation of the pre-terminally differentiated subset of memory cells. This findings could be due to a depletion of terminally differentiated CD8 T cells and/or lack of CD4 helper activity.

12. Conclusions  The pool of late effector CD8 T cells (CD27-, CD28-) was higher in HIV+ patients than in C. However, in HIV+ patients with HIV+ biopsies this value was lower than in those with HIV- biopsies, suggesting that continuous Ag exposure leads to late effector cell loss.  Our results demonstrate an accumulation of the pre-terminally differentiated subset of memory cells. This findings could be due to a depletion of terminally differentiated CD8 T cells and/or lack of CD4 helper activity.

13. Conclusions  PD-1 was significantly up-regulated on duodenal CD8 T cells in HIV+ patients. Functional impairment (exhaustion) could lead to ineffective viral control, since in the biopsies where HIV persisted, the % of exhausted cells tended to be higher.

14. Conclusions  PD-1 was significantly up-regulated on duodenal CD8 T cells in HIV+ patients. Functional impairment (exhaustion) could lead to ineffective viral control, since in the biopsies where HIV persisted, the % of exhausted cells tended to be higher.  HIV infection suppressed CD127 high expression on duodenal CD8 T lymphocytes. The proportion of CD127-expressing cells was slightly lower in the biopsies where HIV persisted and this could be related to T cell exhaustion. These data support further the concept that HIV infection can alter memory CD8 differentiation.

15. Aknowledgements Gastroenterology Unit- City Hospital “Juan A Fernandez”  Alberto Zalar, MD  Norma Correa, MD Infectious Diseases Unit-City Hospital “Juan A Fernandez”  Pedro Cahn, MD, PhD  Maria Inés Figueroa, MD Laboratory of Immunology- Academia Nacional de Medicina  Ana Coraglia, PhD st  Maria Marta de E de Bracco, PhD  ANPCyT-MinCyT and Roemmers Foundation for financial support