HIV and SIV are capable of infecting thymocytes, resulting in thymocyte depletion and thymic atrophy (2
). In the present study, we used measurement of TREC levels in the peripheral blood as a means to evaluate thymic function. TREC analysis enabled the SIV infection to be divided into two distinct phases. Phase 1 involved stable or increasing TREC levels within both the CD4 and CD8 T-cell populations. Within the SIVmac239-infected macaques, phase 1 lasted throughout the first 16 to 30 weeks. The cross-sectional analysis of SIV-positive macaques (Fig. ) assessed samples at 20 to 34 weeks postinfection. The TREC levels were similar to or higher than the levels in the uninfected macaques, possibly because these macaques were likely at the end of phase 1 or the very beginning of phase 2.
The observation that TREC levels were sometimes elevated within the peripheral T cells, particularly in the CD4+
cells, was not expected based on the studies of HIV-infected patients (9
). Previous studies have assessed thymic tissue from SIV-infected macaques through 50 days postinfection. These studies have identified alterations in the thymocyte subsets, increased thymocyte proliferation, altered thymic architecture, and an increased level of thymocyte apoptosis at the earliest time points (days 7, 14, and 21 postinfection) (40
). However, at 50 days postinfection, many of these changes had resolved, indicating that the thymus could function during an SIV infection (40
). Alternatively, the increase in TREC levels may not be due to any changes within the thymus but rather to an increase in the percentage of cells that contain TREC in the peripheral blood (naïve phenotype). The increased percentage of cells with a naïve phenotype (Fig. ) may result from an increase in the number of cells in the naïve cell pool or a decrease in the number of cells in the memory cell pool. A specific decrease in CD4+
memory/effector T cells has been documented following SIV infection (25
In conclusion, there are two hypotheses that might explain the stable or increasing TREC levels during phase 1: thymic output is maintained or increased to compensate for the T-cell destruction, or thymic output is reduced, but this decrease is not detectable in the periphery until after 16 to 30 weeks of infection.
A decline in TREC levels during HIV infection has been attributed to both decreased thymic output and increased proliferation of peripheral T cells (9
). Within the SIV-infected macaques, TREC levels did not decline until the second phase of infection, after 16 to 30 weeks. This decrease was observed within the four SIVE660-infected macaques (Fig. ) as well as two of the SIVmac239-infected macaques, RM2 and RM3 (RM1 succumbed to AIDS prior to the start of phase 2). The decline in TREC levels did not appear to be dependent on high viral loads, as it declined within the slow progressor RM3 with undetectable viral loads for most of the disease course.
We have now followed macaque RM3 for 120 weeks, and TREC levels in this animal are still low, ranging from undetectable (<20 TREC/100,000 cells) to 200 TREC/100,000 cells within the CD4 and CD8 T cells. However, the decline in TREC levels during phase 2 of the SIV infection does not in itself indicate decreased thymic output. The use of the proliferation marker Ki67 offers a means by which the proliferation status of CD4 and CD8 T cells can be assessed. Our data did not demonstrate a direct association between low TREC levels and increased Ki67 expression. Therefore, dilution of TREC molecules through proliferation is not sufficient to explain all observed phases of TREC decline. This finding is similar to data obtained from HIV-infected patients (9
) and suggests that both T-cell proliferation and thymic output are contributing to the decreased peripheral blood TREC levels observed in SIV-infected macaques.
An earlier cross-sectional study assessed PBMC α1 circles (TREC) in macaques infected at 3 months to 3 years of age and did find a detectable but limited (approximately 0.3 log units) impact of SIV infection in macaques (5
). In addition, the previous study also assessed α1 circles (TREC) at longitudinal time points in six SIVmac251-infected macaques. Three macaques exhibited the phase 1-phase 2 pattern observed here, and three macaques contained stable α1 circle (TREC) levels for 400 to 800 days (5
). Therefore, an infection by SIV does not always result in low peripheral blood TREC levels in macaques. Both viral (strain, dose, and infection route) and host factors are likely influencing the levels of recent thymic emigrants within the peripheral blood of SIV-infected macaques.
Quantification of TREC levels within the peripheral blood represents an indirect assessment of thymic output during SIV infection. Thymic tissue was assessed directly from a macaque that progressed to disease over 65 weeks (RM2). Previous studies have observed increased levels of infiltrating lymphocytes within the perivascular space around the thymic epithelium (18
). We used flow cytometry to observe an increase in the level of memory/effector CD8+
T cells in the SIV-infected thymus. These memory/effector CD8+
T cells may have been located within the perivascular space or within the epithelial space, which was the predominant tissue observed following in situ analysis.
Here, increased levels of apoptosis within the epithelial space were observed. These apoptotic cells were particularly evident within the thymic cortex, where the immature thymocytes reside. Many of the apoptotic cells were CD68+
macrophages, which may be undergoing apoptosis due to an SIV infection (60
) or through an indirect mechanism. However, it is likely that the macrophages in the thymus may be becoming TUNEL-positive due to their role as scavengers responsible for removal of the apoptotic thymocytes. In mice, thymic macrophages have been demonstrated to acquire low-molecular-weight DNA from the apoptotic thymocytes and become TUNEL positive (35
). The increased level of apoptotic T cells within the thymic cortex is unlikely to be directly due to viral infection.
Similar to findings in other studies, levels of SIV DNA were quite low (1 to 10 proviral copies/4,000 thymocytes) (60
). Therefore, an indirect mechanism may be affecting the thymocytes, resulting in an increased level of apoptosis. Rosenzweig and colleagues assessed the thymic tissue from macaques through 50 days of an SIV infection and observed an increase in Fas and Fas ligand, two proteins involved in apoptotic induction (40
). In addition, there was a decrease in Bcl-2, a protein that protects cells from apoptosis-inducing signaling (40
). It is possible that shifts in the cytokines that affect thymic function (52
) may be impacting the expression of these apoptosis-related proteins.
T cells generated from thymopoiesis would be predicted to have a broad T-cell repertoire capable of mounting a T-cell response to novel antigens. In contrast, the peripheral expansion of T cells leads to the expression of limited repertoires with limited abilities to identify novel antigens (31
). The findings presented here demonstrate declining TREC levels (indirect measure of thymic dysfunction) and increased levels of memory/effector CD8+
T cells and apoptosis (direct assessment of thymic dysfunction) in the SIV-positive macaque thymus. Similar findings have been observed for HIV (17
) as well as at early time points post-SIV infection (40
). It is likely that a reduced thymic output is contributing to the skewed T-cell receptor repertoire observed during HIV (7
) and SIV (6
) infection. A limited T-cell receptor repertoire would be predicted to impair the ability of the immune system to respond to a wide range of antigens, possibly resulting in the opportunistic infections and cancers characteristic of AIDS.
There are a number of mechanisms by which HIV and SIV may be inducing thymic dysfunction, including the direct killing of thymocytes (4
), killing of the dendritic cells required for normal thymocyte development (53
), damaging of the thymic epithelial cells required for normal thymopoiesis (46
), and inhibition of thymocyte signaling. SIV infection of macaques will be a useful animal model for distinguishing the influence of each of these mechanisms in lentivirus-induced thymic dysfunction.