1. Ziv Sevilya1
• Ehud Chorin1
• Orit Gal-Garber2
• Einat Zelinger2
• Yael Yagel1
• Dan Turner1
• Boaz Avidor1
• Gideon Berke3
• David Hassin1
1
Tel-Aviv Sourasky Medical Center, Tel-Aviv University; 2
Interdepartmental Equipment Facility, Robert H. Smith Faculty of Agriculture, Food and Environment, the Hebrew University, Rehovot,
and 3
Department of immunology, Weizmann Institute of Science, Rehovot, Israel
Apoptosis of HIV-infected CD4 T cells caused by autologous
CD8 T cells throughout the course of HIV infection leads to AIDS
The PBMC of HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. We found that CD8 T cells express
perforin, form conjugates and kill by apoptosis autologous CD4 T cells during both the acute and chronic phases of HIV infection. In situ PCR detected HIV DNA in 10-
15% of CD4T cells from chronic, treated and AIDS patients. In chronic and treated patients, 25% of the HIV-infected CD4T cells were conjugated by CD8T cells, exhibiting
low grade apoptosis compared with 75% in AIDS patients in which apoptosis was robust. We propose that CD4 T cell annihilation in HIV-infected patients results from
the interaction of CD8 T cells with latently HIV-infected CD4 T cells. It is likely that the virus manipulates the immune system to maintain a low-grade infection, thus
achieving prolonged survival combined with efficient virus spread.
Fig. 1. Conjugate formation: Table 1: In situ PCR of HIV LTR in a mixture of CD4 and CD8 T cells
from the PBMC of HIV infected patients
Fig. 4. Perforin expression in CD8 T cells
The CD4 T cells in the conjugates are HIV infected.
Fig. 5.
Fig. 6.
Fig. 2. CD8-mediated killing of autologous conjugated CD4 T cells during acute
and chronic HIV infection
Fig. 3. Conjugate formation and lytic activity
Fig. 7. Chronic HIV infected patient on ART
CD4 and CD8 T cells were sorted
from PBMC of HIV-1-infected
patients. Calcein-labeled CD4 T
cells (green florescence) and non-
labeled autologous CD8 T cells
were allowed to form conjugates
(A) Conjugates were counted in a
hemacytometer - magnification
x 20. (B) CD4 (green) and CD8 T
cells in conjugates, magnification
x 100.
(A) FACS analysis of perforin expression in CD8 T cells in acute (blue), chronic (red) HIV infected patients, and
non-infected controls (black). (B) Statistical analysis of perforin expression in sorted CD8 T cells of acute and
chronic HIV-infected patients and healthy controls.
(C) Immunofluorescence of CD8 (blue) and perforin (green) in conjugated CD8-CD4 T cells of acute and (D)
chronic HIV-infected patients. Each scale bar is 10 μm.
We demonstrated that the CD4 T cells in the conjugates are HIV infected using in situ PCR: HIV DNA in the cell
nucleus (green). (A) A mixture of CD8 and CD4 T cells in which 10% of the CD4 T cells have HIV DNA in their
nucleus. (B) conjugates demonstrating an HIV-infected cell within them.
(A-D) We demonstrated apoptosis
of the CD4 T cells in the conjugates
using continuous live cell imaging in
tissue culture and further observed
and measured it using the Annexin
V Fitc Apoptosis Detection Kit. Three
representative films and snapshots
fromthefilmsareshown.(A)acuteHIV-
infected patient. (B and C) chronic
HIV-infectedppatients.
(D 1-6) CD8 T cell (blue) and the
apoptosis marker annexin V (green)
staining of conjugates. during both the
acute (D 1-3) and chronic stage (D 4-6)
ofHIVinfection.Eachscalebaris10μm.
Killing activity was measured by an
annexin V-FITC apoptosis detection kit
and analyzed by FACS where annexin
and PI-positive cells represent cells in
different stages of apoptosis. (A) Two
different experiments are presented:
(B) Statistical analysis of the CD8-
CD4 T cell conjugate formation and
cytotoxic activity between 11 acute and
11 chronic HIV-infected patients and
healthycontrols.Average±SE;barswith
different letters differ at p < 0.05 (Tukey-
Kramer test).
A B
A
C E
B
D F
In Situ PCR of CD4 and CD8 T cells conjugates
procured from: Acute HIV infected patient (A),
ChronicHIVinfectedpatient(B),Chronicpatient
on ART (C) and AIDS patient. (D-F): conjugation
mixture of CD4 and CD8 T cells (D,E) and CD4 T
cells without CD8 T cells (F). HIV DNA in green,
Perforin in red.
C O N C L U S I O N S
1. WeproposethatCD4TcellannihilationinHIV-infectedpatientsresults
from the interaction of CD8 T cells with HIV-infected CD4 T cells.
2. WesuggestthepresenceofequilibriumbetweentheCTLandthevirus,
mediated by the HIV Nef protein.
Acknowledgment
This work was supported by a joint grant Weizmann Institute of Science -Tel Aviv Sourasky Medical Center.
Correspondence: Dr. David Hassin, davidh@tasmc.health.gov.il‫‫‬
AIDS
(n=3)
On ART
(n=6)
Chronic HIV
(n=3)
9.5±2.0%12±1.5%10±2.0%CD4 T cells with HIV DNA
7.0±0.9%3±0.3%3±0.6%Fraction of CD4 T cells conjugated with CD8 T cells
75±8.0%25±3.0%30±5%Fraction of HIV positive CD4 T cells conjugated with CD8 T cells
Very highLowLowCD4 T cells in apoptosis
A B