Persistent HIV-1 infection in Persistent HIV-1 infection in duodenal mucosa and memory duodenal mucosa and memory

CD8+ T cell differentiationCD8+ T cell differentiation

Liliana BelmonteLiliana Belmonte11 PhD PhD; Alberto Zalar; Alberto Zalar22 MD; Patricia Baré MD; Patricia Baré11 PhD; Noel PhD; Noel

BadanoBadano1 1 PhD st; Valentina ArayaPhD st; Valentina Araya22 MD; Eduardo Piskorz MD; Eduardo Piskorz22 MD; Maria MD; Maria

Inés FigueroaInés Figueroa33 MD; Cecilia Parodi MD; Cecilia Parodi11 PhD, Beatriz Ruibal-Ares PhD, Beatriz Ruibal-Ares11 MD, MD,

Pedro CahnPedro Cahn33 MD, PhD; Maria Marta de E de Bracco MD, PhD; Maria Marta de E de Bracco11 PhD. PhD.

Laboratory of Immunology and Virology 1, Academia Nacional de Medicina,

Buenos Aires, Argentina.

Gastroenterology2 and Infectious Diseases3 Units, Hospital Juan A Fernandez,

Buenos Aires, Argentina.

IntroductionIntroduction

Despite the clinical improvement associated with

HAART, current antiviral drugs are not able to

eradicate HIV due to the persistence of virus in

tissue reservoirs. Viral reservoirs therefore

represent a potentially life-long site of replication-

competent

forms of HIV that cannot be suppressed by current

antiretroviral treatment

The objectives of the study were:The objectives of the study were:

¤ To verify if HIV-1 persists in the To verify if HIV-1 persists in the intestine of HIV+ patients in spite intestine of HIV+ patients in spite of successful antiretroviral of successful antiretroviral treatment (HAART)treatment (HAART)

¤ To identify the cells that harbour To identify the cells that harbour HIV-1 in the duodenal mucosa HIV-1 in the duodenal mucosa

Material and MethodsMaterial and Methods

A.-A.- Distal duodenal biopsies were obtained from 13 patients with chronic HIV-1 Distal duodenal biopsies were obtained from 13 patients with chronic HIV-1 infection under HAART:infection under HAART: 9 males, 4 females, 9 males, 4 females, 7 with pVL < 50 copies/ml, Median CD4 cell count 354 cell/mm7 with pVL < 50 copies/ml, Median CD4 cell count 354 cell/mm33(31-563) (31-563) 6 with pVL>100.000 copies/ml, Median CD4 cell count 58 cell/mm6 with pVL>100.000 copies/ml, Median CD4 cell count 58 cell/mm33(7-198) (7-198)

Distal duodenal biopsies from 10 HIV-1 seronegative individuals were used as Distal duodenal biopsies from 10 HIV-1 seronegative individuals were used as control (C).control (C).

B.-B.- A biopsy was labeled as A biopsy was labeled as “HIV +”“HIV +” if HIV-DNA was detected by standard if HIV-DNA was detected by standard PCR PCR (using primers SK145 and SKCC1B to define a sequence of 155 nucleotides within (using primers SK145 and SKCC1B to define a sequence of 155 nucleotides within the highly conserved region of the HIV-1 the highly conserved region of the HIV-1 gag gag gene)gene)

C.-C.- The phenotype of leukocytes present in the tissue was assessed by flow The phenotype of leukocytes present in the tissue was assessed by flow cytometry using anti CD4, CD64, CD27, CD28, CD45RO. HIV-1 detection was cytometry using anti CD4, CD64, CD27, CD28, CD45RO. HIV-1 detection was performed by intracellular staining with KC57 antibody (anti-p24). performed by intracellular staining with KC57 antibody (anti-p24).

CD4 depletion in duodenal CD4 depletion in duodenal mucosamucosa

0

10

20

30

40

HIV-1+patients

Controls

p=0.0017

Mu

cosa

l C

D4+

Tce

lls

(%)

n:13

n:10

CD8+ T cells in duodenal CD8+ T cells in duodenal mucosamucosa

0

25

50

75

100

HIV-1+patients

Controls

Mu

cosa

l C

D8+

Tce

lls

(%)

n:13 n:10

Characteristics of duodenal Characteristics of duodenal CD8+ T cell populationCD8+ T cell population

HIV-1 + patients

HIV+ biopsies (n=6)

HIV- biopsies (n=7)

Controls

(n=10)

Early

Differentiated cells

(CD28+,CD27+)

18 ± 8 10 ± 4 13 ± 6

Fully

Differentiated cells

(CD28-, CD27-)

32 ± 8

61 ± 8

24 ± 5

Mean±SEM

p=0.001p=0.027

% % %

% % %

p=NS p=NS

Patients

ND pVL

(n=7)

Patients

pVL > 100.000 copies/ml (n=6)

HIV + HIV + Biopsies Biopsies 44 44

HIV-HIV- BiopsiesBiopsies 33 22

Lack of correlation of pVL with HIV Lack of correlation of pVL with HIV persistence in duodenal mucosapersistence in duodenal mucosa

p=0.72, chi-square test

p24 positivity in duodenal p24 positivity in duodenal CD64+ cellsCD64+ cells

Control BiopsyHIV- biopsy(by PCR)

HIV+ biopsy(by PCR)

Control isotype

p24 expression

KC57 FITC KC57 FITC KC57 FITC

Median FI KC57: 193Median FI isotype: 52

Median FI KC57: 56Median FI isotype: 47

Median FI KC57: 45Median FI isotype: 35

p24 positivity in duodenal p24 positivity in duodenal CD68+ MacrophagesCD68+ Macrophages

HIV+ biopsy(by PCR)

Control Biopsy

Control isotype

p24 expression

Median FI KC57: 158Median FI isotype: 54

Median FI KC57: 67Median FI isotype: 57

ConclusionsConclusions

As reported in other segments of the GI tract, we found that

CD4 T cells were depleted in duodenal mucosa of HIV patients

when compared to controls, while no differences were

observed in

the proportion of CD8 T cells in both groups.

However, the persistence of HIV in the duodenal tissue affects

the differentiation pattern of CD8 T cells. Thus, the proportion

of

fully differentiated CD8+ T cells was higher in the absence of

HIV

in the duodenum of HIV patients.

ConclusionsConclusions

HIV-DNA could be detected in the duodenal mucosa both in

patients who failed treatment and in those with undetectable

pVL.

Concerning the nature of cells that harbour HIV infection, we

have shown that macrophages may host persistent HIV

infection

and could be considered as a reservoir of HIV even after

HAART.