In most cases, progression of HIV to AIDS is preceded by approximately 2 years of substantial decline in circulating counts of CD3+
T cells. The onset of this decline, termed TCH failure, appears to be an important milestone in the progression of HIV-1 disease to AIDS; however, its cause is not known. Therefore, we performed the most comprehensive longitudinal assessment of naive and memory T cell numbers, sjTREC levels, and T cell proliferation to date. The data obtained, coupled with recent studies validating sjTREC levels as a measure of thymic output, provide new insight into the mechanisms involved in TCH failure. In addition, segmented regression modeling allowed the first adjustment of sjTREC data for the possible effect of changes in directly measured T cell proliferation fractions. Like previous studies,27–29
we found lower levels of circulating sjTREC-bearing T cells with disease progression. We extended these studies, which were almost exclusively cross-sectional, by analyzing the timing of sjTREC loss. The most striking finding was the close temporal relationship between a very large decline (median 1109-fold) in circulating sjTRECs and the failure of T cell homeostasis.
Declines in sjTREC levels may be precipitated by multiple factors, acting alone or in combination. Chief among these are (1) increased cellular proliferation, (2) increased sequestration of sjTREC+ cells, (3) declines in overall T cell counts, (4) decreased production of new T cells by the thymus, and (5) decreased survival of sjTREC+ cells. Our data, and previous reports, argue against some of these possibilities as major mechanisms for the decline in sjTREC observed with TCH failure.
The possibility that proliferation of TREC-bearing cells contributed to the reduction in circulating TRECs19,30
should be considered, but several considerations argue that this mechanism is not a major contributor to our findings. In the present study, only a small fraction (4%) of the decline in sjTREC observed at TCH failure was accounted for by naive cell proliferation. This finding is consistent with most studies that have looked specifically at this question. Many studies found only weak correlations between expression of proliferation markers (such as Ki-67) and levels of circulating sjTRECs.19,29,31,32
Moreover, Dion, et al.31
recently described trends in two species of TREC (sjTRECs, which are produced late in thymic development, and β
TRECs, which are formed earlier in thymic development) that were not consistent with a dilution effect caused by enhanced division of naive cells. In that study, people who became infected with HIV exhibited a significant decrease in the ratio of sjTRECs to β
TRECs, indicating decreased proliferation of thymocytes, only a few months after infection.
It is true that some patients in our study exhibited substantially increased cellular proliferation after TCH failure. However, these large increases are unlikely to have influenced our findings, for at least four reasons. First, as shows, most of the loss of sjTRECs occurred within the first year after TCH failure. During this time, changes in the proportion of proliferating cells were generally small. Changes in proliferating cells after this time, though large in a few cases, were therefore too late to account for the loss of sjTRECs. Second, the large increases in proliferation were confined to memory cell subsets, which contain very low levels of TRECs. Thus, changes in memory cell proliferation are unlikely to contribute substantially to the massive change in total TREC levels that we observed. To confirm this, we compared the TREC decline in total PBMC to that in naive cells, and found that they were the same. This finding supports the interpretation that the overall TREC decline is not attributable to high levels of memory cell proliferation. Third, when all cellular proliferation data were included in a statistical model, only a small effect of proliferation on TREC levels was found. Finally, some of the increase in the percentage of Ki-67+ cells (i.e., the fraction of proliferating cells) was due to the declines in absolute numbers of CD4+ or CD8+ T cells (the denominators from which the proliferation fractions were calculated). In absolute terms, the number of proliferating cells did not increase over the study period. Taken together, the evidence from this and previous studies suggests that it is unlikely that increases in cellular proliferation are sufficient to account for the large decline in sjTREC at the time of TCH failure.
Increased sequestration of TREC+
cells in the lymph nodes of HIV+
individuals has been suggested by reports of higher TREC levels in the lymph nodes compared to the peripheral blood.33,34
However, there is no evidence that such sequestration increases with disease progression, and a recent study of SIV-infected animals indicated that T cell dynamics in blood and lymph node mirror each other.35
Moreover, the observation that proliferating lymphocytes labeled with bromodeoxyuridine or deuterium rapidly equilibrate between the blood and lymph nodes36
also argues strongly against the sequestration of TREC-bearing cells as an explanation for our data.
It is also unlikely that declines in sjTREC merely reflect a general decline in total T cell or naive T cell numbers, because the loss of sjTREC at the time of TCH failure was much more rapid than the loss of either CD3+
or naive T cells. One might wonder if the rapid decline in TRECs we observed is plausible, given the long half-life and low turnover of naive T cells.37
In fact, a precedent for the occurrence of large changes in TREC levels over the course of a few weeks to months, as we found, is provided by Dion, et al.31
An additional explanation for the rapid loss of TREC-bearing T cells is that most sjTRECs are contained in the T cell population that has recently emerged from the thymus, and this population turns over much more rapidly than the general naive T cell pool.38–40
Therefore, although the decline of sjTRECs may be partially caused by some combination of the factors discussed above, we favor the interpretation that sjTREC decline at the time of TCH failure is largely attributable to decreased survival of TREC-bearing cells and/or decreased thymic production of T cells. Decreased survival of TREC-bearing cells might involve the emergence of X4 strains of HIV, which use the CXCR4 coreceptor and have long been associated with accelerated progression of HIV disease.41
Of the four study participants for whom data are available, two individuals experienced a switch from CCR5-tropic to CXCR4-tropic viruses (0.25 and 0.75 years before T cell homeostasis failure); the remaining two participants did not42
(Shepherd and Margolick, unpublished observations). However, in larger and more definitive studies, we previously presented evidence that X4 viruses become abundant, on average, about 0.6 years before the failure of TCH42,43
and are present in the vast majority of HIV-infected people who reach this stage of disease progression.42
X4 viruses might contribute to the depletion of TRECs through both thymic and extrathymic mechanisms, since both thymocytes and naive T cells express CXCR4 more than CCR5.44–46
In the thymus, X4 strains could infect (or kill by other means) double-positive thymocytes, thus reducing production of naive T cells expressing TRECs. In the peripheral blood, X4 strains could kill both CD4+
T cells, although depletion of TREC-bearing cells might not be as selective if the mechanism of cytotoxicity depends on CXCR4 expression on mononuclear phagocytes rather than on T cells.47
Decreased thymic output might be attributed to other mechanisms, as well. For example, Dion et al.31
observed higher TREC levels in the peripheral blood of people with early HIV infection, compared to HIV-negative donors, possibly reflecting compensation for the loss of sjTREC-bearing cells. They reasoned that one or more of the following mechanisms could cause increased levels of early thymocytes: (1) release of more stem cells from the bone marrow, (2) enhanced survival of thymocytes, or (3) enhanced survival of peripheral recent thymic emigrants. According to this line of reasoning, failure of one (or more) of these mechanisms, coupled with the rapid turnover of TREC-bearing naive T cells relative to other naive T cells, could account for the rapid loss of TRECs that accompanied progression to AIDS in the present study.
It remains unclear to what extent primary thymic failure accounts for loss of T cells and failure of T cell homeostasis in late-stage HIV disease. The fact that naive T cells, TREC levels in PBMC, and thymic size all rose after institution of highly active antiretroviral therapy (HAART), even in people with CD4+
lymphocyte counts <200/μ
suggests that at least some thymic function remains even in people with very low T cell counts and (presumably) T cell homeostasis failure. On the other hand, the clinical benefit of HAART is reduced when it is initiated at CD4+
lymphocyte counts <200/μ
consistent with at least some irreversible thymic damage late in HIV disease.14–16
Thus, measurements of TRECs may be helpful in defining more precisely the stage of HIV disease by which HAART should, optimally, be initiated.