AIDS TREATMENT NEWS #222, May 5, 1995

Two studies by researchers at Stanford University have questioned "conventional wisdom" on AIDS by finding a relatively early, selective loss of "naive" CD4 and CD8 cells in both HIV infected adults and children, respectively. In view of these results, important beliefs about the pathogenesis of HIV disease will need to be re-examined. AIDS may be primarily a disease not of loss of CD4 cells, as in generally believed, but of loss of naive cells of all types. "This study forces us to re-evaluate all of the experiments that have been done in the past 10 to 12 years on T-cell function with cells from HIV patients," according to Mario Roederer, the lead author of the adult study.

Both CD4 and CD8 cells can be subdivided into "naive" and "memory" subsets. The ratio of naive to memory cells has now been found to change greatly with HIV infection. For example, about 50% of CD8 cells in uninfected adults are naive cells -- while less then 15% are naive in most HIV-infected adults.

Naive cells are cells which have never encountered the antigen (foreign substance such as a protein produced by a bacterium or virus) to which they are genetically able to respond. These cells, therefore, are important because they allow the body to develop immune responses to new challenges. Loss of naive cells is expected to result in greater susceptibility to certain opportunistic infections. In addition, this loss may also erode the body's ability to control HIV, since many different variants of HIV evolve in each patient, and without enough naive cells the body cannot respond effectively to the new ones.

The new findings are important for clinical trials, because trials might be more accurate if volunteers were grouped according to how many naive cells they have, as well as by how many CD4 (T-helper) cells. And these findings may be especially important for vaccine trials, since the lack of naive cells will probably prevent the vaccines from working, due to lack of immune response to the new antigen which a vaccine provides.

Naive and memory cells also produce different cytokines. For example, naive cells tend to produce IL-2, while memory cells produce other cytokines such as IL-4, IL-10, and gamma interferon. Therefore, measurements of the proportions of different cytokines (the basis of theories about the switch from a "Th1" to a "Th2" immune response) may be reflecting differences in the number of naive and memory cells.

The study in adults tested blood samples from 266 HIV-infected study volunteers, and from 44 uninfected healthy adults who served as controls. The HIV-positive subjects were being screened for a study of NAC (n-acetylcysteine), which was seeking patients with a CD4 count under 500, and who were not taking large amounts of antioxidants, vitamins, or minerals.

The parallel naive cell/memory cell study in children tested 19 HIV-infected children (who were recruited for a separate study of glutathione levels) and 17 HIV-negative controls.

How the Measurements Were Made Many different kinds of blood cells are best distinguished by "markers" on their surface. These markers are large protein molecules which are at the surface of the cell, and can therefore interact with substances outside the cell. For example, the CD4 (T-helper) and CD8 cells are distinguished by the presence of CD4 or CD8 markers on their surfaces, respectively. There are a number of other kinds of markers in addition. Some kinds of cells have several different kinds of markers on them. Usually each cell will have 10,000 to 100,000 copies of a marker; for example, CD4 or CD8 cells usually have 50,000 to 100,000 copies each.

These markers are detected by specially prepared antibodies which are chemically attached to a substance which fluoresces (glows) when exposed to a certain wavelength of light. Each antibody is selected to attach only to a specific kind of marker. Therefore, when one of the antibodies is mixed with cells, many antibody molecules will attach to each cell which expresses the target marker. These cells will then glow under the special light, and they can be counted under a microscope. This is how T-cell counts used to be done.

But several years ago a much better way of measuring T-cells (and others cells distinguished by their markers) became available. In this technology, called flow cytometry, a stream of cells, one by one, rapidly moves in front of an ultraviolet laser. When the laser hits the cell, the attached antibodies glow, and this glow is measured and recorded in a computer; the intensity of the glow indicates how many markers are on that individual cell. Different antibodies can be engineered to glow in different colors, allowing two or three different markers to be measured simultaneously. Thousands of cells are typically measured in one run; and the results can be plotted on graph paper as clusters of dots, where each dot represents one cell. By looking at these charts, it is possible to see if certain populations of immune cells are missing or deficient, resulting in specific immune defects. Two patients can be very different in reality, even if their usual blood-count numbers are the same.

Some previous studies have failed to find a selective loss of naive T-cells. The Stanford researchers -- many of whom work at the laboratory at Stanford where flow cytometry was developed -- believe that earlier researchers failed to measure naive cells correctly. Naive cells have the CD45RA marker. But measuring that one alone may not be enough, since two research groups previously determined that it is necessary to measure three markers simultaneously (CD45RA, CD62L, and either CD4 or CD8) to get an accurate count of naive cells. This requires a "three color" flow cytometry machine, which many though not all labs have.

Questions Remaining Many important questions remain. For example, nobody knows why the naive cells are selectively depleted. One theory is that they are killed by HIV before they get out of the thymus gland, where they develop. Damage to or deficiency of the thymus might also contribute. But a completely different possibility is that the immune system is overstimulated, and abnormally causing naive cells to mature into memory cells.

Also, nobody knows what maintains the level of T-cells in anyone's blood, whether or not they have HIV. More basic research will be needed to answer these questions.

The new Stanford studies show the value of conducting basic research in conjunction with clinical trials. Here, a basic research study was piggybacked onto an ongoing trial of the effect of NAC in persons with HIV; additional expense was minimized, since their blood was being drawn and tested anyway. But usually it is hard to include basic research in a drug trial, since the pharmaceutical company sponsoring the trial only want to know how well its drug works, and is unlikely to pay for gathering knowledge which will help design better treatments in the future.

References Rabin RL, Roederer M, Maldonado Y, Petru A, Herzenberg LA, and Herzenberg LA. Altered representation of naive and memory CD8 T cell subsets in HIV-infected children. JOURNAL OF CLINICAL INVESTIGATION. May 1995; volume 95, pages 2054-2060 Roederer M, Dubs JG, Anderson MT, Raju PA, Herzenberg LA, and Herzenberg LA. CD8 naive T cell counts decrease progressively in HIV-infected adults. JOURNAL OF CLINICAL INVESTIGATION. May 1995; volume 95, pages 2061-2066.