AIDSWEEKLY Plus, 26 August 1996
Daniel J. DeNoon, Senior Editor
A breakthrough study shows that proper stimulation of CD4(+) T cells cultured from HIV infected individuals not only causes them to proliferate but also reduces viral load.
Moreover, the same stimulation of CD4(+) T cells from uninfected donors makes them resistant to HIV infection.
"We made the surprising observation that you didn't have to add antiretroviral drugs to the media to see this antiviral effect," said Carl H. June of the Naval Medical Research Institute, Bethesda, Maryland.
June announced the findings at the XI International Conference on AIDS, held July 7-12, 1996 in Vancouver, British Columbia, Canada, and in the journal Science ("Antiviral Effect and Ex Vivo CD4(+) T-Cell Proliferation in HIV Positive Patients as a Result of CD28 Costimulation," Levine et al., Science, 1996;272(5270):1939-1943).
The findings suggest several approaches to the treatment of HIV disease:
A clinical trial is already underway, June said at the AIDS conference.
"We are just beginning to test growing CD4 cells this way in a clinical trial where cells will be grown ... and administered at increasing doses up to 3x10(10) per patient to test the safety and feasibility of this approach," he said.
In their studies, June and colleagues set out to explore a previously reported phenomenon: the ability of CD28 signal transduction to prevent apoptosis in cultures of HIV infected cells.
In order for a T cell to become active after an antigenic molecule interacts with its major histocompatibility complex (MHC) receptors, it must receive other signals as well.
"The most important of the costimulatory signals identified to date is provided by the interaction of CD28 on T cells with its ligands CD80 and CD86 on antigen-presenting cells," noted Bruce L. Levine, June, et al. in their Science article.
To present a proper costimulatory signal to CD4(+) T cells, June and colleagues used monoclonal antibodies against CD28 and CD3. So that the antibodies would provide simultaneous signals, the researchers immobilized them on tiny beads.
June and colleagues took purified CD4 cells from the peripheral blood of a patient with intermediate-stage HIV disease and divided them into two cultures. One culture was activated with the anti-CD28/anti-CD3 beads and the other was activated in the traditional manner using phytohemagglutinin (PHA) and interleukin 2 (IL-2).
For the first 10 days, both cultures exhibited exponential growth. As is usual with CD4 cultures from people with HIV infection, growth in the PHA/IL-2-stimulated culture dropped off and the cells soon succumbed to the deadly effects of HIV infection.
But the cells stimulated with the immobilized CD28/CD3 antibodies continued their exponential growth for 50 days.
"We showed that this could occur routinely in patients with intermediate stage HIV infection," June said, noting that similar results were obtained with CD4 cells from 10 HIV(+) individuals with CD4 counts ranging from 350 to 550 cells/(micro)L.
Cells in these experiments continued expanding for up to 70 days.
"The cultures were primarily CD4 cells," June said. "The average expansion in these 10 different cultures is 8,000 fold. The cultures weren't taken to termination but there was a wide variation between donors."
Viral DNA was measured at regular intervals in eight of the cultures. In all of these CD4 cells cultured from HIV infected donors, the RNA levels continually decreased. In most, the viral levels dropped to undetectable levels: that is, to less than five copies of HIV DNA per 100,000 cells.
To test the validity of this astonishing result, the researchers performed large-scale cultures of CD4(+) cells using three-liter bags and small-volume flasks. The cells in these studies came from an HIV(+) patient with a CD4 count of 393 cells/(micro)L.
At baseline, the cultures contained 800 copies of HIV DNA per 100,000 cells. Regardless of the culture conditions, viral DNA dropped to undetectable levels in all cases.
June and colleagues then wanted to see what would happen if they exposed CD4(+) T cells from normal, uninfected donors to the anti-CD28/anti-CD3-coated beads and then exposed them to HIV.
Despite exposure to high infectious titers of HIV-1, the cells resisted infection. This resistance was shown to be specific for CD28 stimulation.
"CD28 costimulation conferred marked resistance to HIV-1 infection," Levine et al. wrote. "The effect did not appear to depend on the strain of virus used for infection, which is consistent with the ability of CD28 costimulation to increase the number of CD4(+) cells from multiple patients infected with HIV-1."
To explore the mechanism of this antiviral effect, the researchers exposed cultures of uninfected CD4(+) cells to various combinations of either soluble or bead-immobilized CD3 or CD28. The cultures were then exposed to infectious HIV-1.
There were three different outcomes:
"We don't yet know the mechanism for this," June said. "We suspect it represents different cell-signalling pathways that are elicited depending on whether cells encounter unbound or immobilized antibody."
Interestingly, supernatants from six-day cultures of the costimulated CD4 cells had anti-HIV activity.
The recently recognized anti-HIV chemokines RANTES, MIP1(alpha), and MIP1(beta) could be detected in the supernatant, but when antibodies were added to neutralize the antibodies the anti-HIV effect remained. Moreover, stimulation conditions that enhanced virus replication also induced chemokine secretion.
"We don't think the chemokines themselves are sufficient to explain the effect," June said.
Further studies showed that the anti-HIV activity affected an early stage of the HIV life cycle.
"It is a pre-integration event but post binding," June said.
There appears to be a window of opportunity for the antiviral effect.
"Preliminary results from a limited number of patients indicate that the antiviral effect may be less potent in late-stage HIV infection, even though CD28 costimulation still enhances CD4(+) T-cell proliferation," Levine et al. noted.
A high level of excitement was evident among researchers at the Vancouver AIDS conference.
"This is the most important paper I've seen in Science for years," enthused one researcher in response to June's presentation.
The scientists themselves are only slightly less restrained.
"Our results indicate that in vivo manipulation of CD28 interaction with B7 counterreceptors has the potential to enhance CD4(+) T-cell proliferation and prevent or limit HIV-1 viral spread in patients," Levine et al. wrote.
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