Why do CD4 cell counts fail to rise in some patients with undetectable viral loads?

Low-level regeneration of CD4 cell counts in patients with prolonged undetectable viral loads may occur due to a combination of increased cell death and reduced production of new cells in the thymus, according to a prospective case-control study published in the on-line edition of The Journal of Infectious Diseases. Despite this, the study suggested that responses to some disease-causing organisms are not impaired in these patients.

Between 5 and 27% of patients taking highly active antiretroviral therapy (HAART) have persistently low CD4 cell counts despite having undetectable viral loads for long periods. Since increases in CD4 cell count are believed to be important in preventing illness, there is concern that these patients may be at risk of disease progression even while taking a treatment regimen that is virologically successful.

Failure of CD4 cell counts to rise could occur due to low-level production of new cells, or excessive destruction of CD4 T-cells. Production of new CD4 T-cells occurs through division of long-lived ‘memory’ T-cells that have already been activated by a foreign substance, as well as production of new ‘naïve’ cells in the thymus, a gland found under the breastbone.

In contrast, destruction of CD4 T-cells can occur as a direct result of HIV infection, by being killed by ‘cytotoxic’ T-cells or through cell suicide or ‘apoptosis’.

To understand possible mechanisms for discordant CD4 cell count responses, investigators from Paris identified 19 adult ‘case’ patients with viral loads below 200 copies/ml for over a year, but with CD4 cell counts below 250 cells/mm³. They compared the biological properties of these patients’ white blood cells to a group of ‘control’ patients who had CD4 cell counts over 500 cells/mm³.

“The present study suggests that HIV-1-infected patients with low CD4⁺ cell counts despite long-term undetectable plasma viral loads have several immunological abnormalities,” write the investigators. “Compared with the control patients, the case patients had fewer naïve CD4⁺ cells in relation to a lower thymic output, and their increased T cell death by apoptosis was probably related to a higher level of activation of these cells.”

To limit differences between the patients in the two groups, each patient in the low CD4 cell count group was matched to a control patient based on sex, age and lowest-ever CD4 cell count. There were no significant differences between the groups in terms of risk factors for HIV infection, length of HIV infection, disease stage, hepatitis B or C co-infection or anti-HIV drug exposure.

The investigators used a cell separation technique called flow cytometry to measure the number of each type of cell in the patients’ blood. As expected, the patients with low CD4 cell counts had fewer total lymphocytes (mean: 1484 vs. 2734 cells/mm³; p 3; p

The case patients showed a decrease in the proportion of naïve CD4 T-cells (mean: 23 vs. 47%; p

To confirm this finding, the researchers measured the number of naïve T-cells being produced by the thymus, by counting the number of ‘T-cell rearrangement excision circles’ (TRECs) in the blood using a polymerase chain reaction (PCR)-based technique. TRECs are fragments of DNA that are released when the thymus produces new T-cells.

They found that the patients with low CD4 cell counts had evidence of lower output of naïve T-cells from the thymus (1.28 vs. 3.95 TRECs/mm³; p = 0.002).

“The population of naïve CD4⁺ cells is predominantly affected in case patients, which suggests that thymus function is impaired,” write the investigators. “We found a good correlation between the decreased numbers of TRECs per CD4⁺ cells [sic] and the decreased numbers of CD4⁺ naïve cells in naïve patients.

“Our results do not, however, exclude that an additional impairment of extrathymic expansion of naïve cells could also contribute to a decrease in the number of naïve CD4⁺ T cells.”

The investigators also found more CD4 T-cells undergoing apoptosis in the patients with low CD4 cell counts. This included cells undergoing apoptosis spontaneously (23 vs. 12%; p = 0.02) and those induced by the presence of a cellular factor called Fas (39 vs. 16%; p = 0.004). CD8 T-cells showed no differences in rates of apoptosis between the groups.

The authors suggest that this increased rate of apoptosis may be due to persistent HIV replication within the body. “Despite the apparent control of plasma viral loads in these patients, T cell activation is probably still related to the persistence of in vivo HIV-1 replication,” the explain. “That replication of HIV-1 in plasma is undetectable by the most sensitive method available does not exclude the possibility that replication does persist somewhere, perhaps in a compartment inaccessible for measurement.”

The investigators went on to examine the role of cytokines known to affect rates of apoptosis in CD4 cell proliferation. They failed to find any differences in the levels of a range of cytokines including tumour necrosis factor α (TNFα), tumour growth factor β (TGFβ) and interleukins 2, 3, 7, 10 and 12.

However, they did observe higher levels of plasma soluble tumour necrosis factor receptor II in the case patients (mean: 9.6 vs. 5.3 ng/ml; p = 0.006). As this ‘death receptor’ can trigger cell suicide when it is stimulated by cytokines in the blood, its increased levels could offer an explanation for the increased rates of CD4 T-cell apoptosis they observed.

Finally, the investigators compared the responses of the patients’ white blood cells to exposure to antigens from disease-causing organisms. They found that similar proportions of patients in both groups responded to cytomegalovirus (82% vs. 79%) and the fungus Candida albicans, which causes thrush (76 vs. 84%). These values were similar to the proportions of HIV-negative patients’ cells that showed a response (70 and 100%, respectively).

Responses to tuberculosis were lower in the patients with low CD4 cell counts (76% vs. 89%; p = 0.042), with both groups’ responses being lower than HIV-negative volunteers (100%, p = 0.001). However, responses to tetanus were similar in the two HIV-positive groups (41 and 47% respectively), but both were lower than the HIV-negative comparator group (100%; p

The investigators conclude: “Despite their low CD4⁺ cell counts, [the case group’s] T cell responses to some antigens appear to be as efficient as those in patients with high CD4⁺ cell counts, and this response may contribute to lowering the risk of opportunistic infections in both groups.”

Although this study does not extend to future treatment options for patients with discordant viral load and CD4 cell count responses to HAART, it may pave the way for future investigations into strategies to boost CD4 T-cell numbers, including the use of interleukin-2.