Collagen deposition in HIV-1 infected lymphatic tissues and T cell homeostasis (original) (raw)
A total of 11 individuals were enrolled in the study, with seven initiating HAART after the first biopsy and four choosing to defer HAART. The duration of infection and clinical stage of disease are summarized in Table 1. The protocol called for a total of four biopsies over 6 months among the patients choosing HAART, and three biopsies over 1 month for patients choosing to defer therapy. Four of the seven treated subjects (57%) completed all four biopsies, six of seven completed three biopsies, and one subject completed only the first two biopsies. All four untreated subjects completed their three biopsies over a period of 1 month. At baseline, the median CD4+ T cell count for all patients was 400 (range 112–905) cells/mm3, and median plasma HIV-1 RNA was 20,014 (range 10,684–484,694) copies/ml. All HAART regimens among the seven treated subjects met the definition of HAART, with six receiving two nu-cleoside analogues plus at least one protease inhibitor, and the seventh receiving two nucleoside analogues plus one non-nucleoside analogue. Plasma HIV-1 RNA was reduced to less than 500 copies/ml in all treated subjects by approximately 2 weeks after initiation of HAART and remained at that level for the 6-month follow-up period. The seven HAART-treated subjects had a median increase of 198 CD4+ T cells/ mm3 in peripheral blood over the 6-month follow-up period (range, 50–937 CD4+ T cells/mm3), but in three individuals the increase was only ≤ 62 cells/mm3 between baseline and the 6-month follow-up.
Demographic characteristics and change in plasma HIV-1 RNA and peripheral CD4+ T cell count as a response to HAART
Histologic analysis of tissues at baseline and during follow-up. The size and character of B cell follicles and the TZ were assessed at each timepoint for all patients (Figure 1). At baseline in all LTs there was well-described and characteristic follicular hyperplasia except in tissue obtained from 1327, in whom follicles were hypoplastic and decreased in number, consistent with her diagnosis of AIDS (13–15). The TZ was normal in only two patients, 1329 and 1391, both of whom were in the very early stages of HIV-1 infection. In all other biopsies, the TZ was diminished, with a decreased population of cells and prominent vessels, consistent with loss of tissue volume.
Histologic changes in B cell follicles and TZ characteristic of HIV-1 infection. (a) Section of lymph node from an HIV-negative patient. There is one B cell follicle that is normal in size and shape (black arrow). The TZ is normal in size with a normal population of cells (white arrow). (b) Section of a lymph node from an HIV-1–infected patient with presymptomatic HIV-1 infection. The follicles are hyperplastic and disorganized (black arrow), and the TZ is small and somewhat depleted of cells (white arrow). (c) Lymph node section from a patient with AIDS. The follicular space is disorganized and without structure (black arrow), and the TZ is unrecognizable with few cells (white arrow). All images are at a magnification of ×4.
As expected, there was no significant change in the histologic appearance of tissues at the 1-month follow-up for untreated subjects. Among the seven treated subjects, we observed only minimal improvement in B cell follicle hyperplasia or in the size and organization of the TZ. Of interest, among two of four subjects (1329 and 1391) with acute or early HIV infection and one patient with presymptomatic infection (1293), there was a decrease in the size and number of B cell follicles with a trend toward normalcy in the size and organization of the TZ. At the 6-month biopsy we noted continued and significant improvement in these same patients. However, there was no improvement in the tissues obtained 1 month after initiation of HAART from 1324, 1086, or 1327, or in the 6-month sample obtained from 1327 (1324 and 1086 did not have a biopsy at 6 months).
LT CD4+ T cell population. We used immunohistochemistry and quantitative image analysis to quantify the CD4+ T cell population before initiation of and during HAART. Five-micrometer sections were stained with anti-CD4 antibody and counted using the MetaMorph software program as previously described (4, 10–12, 20–23). Based upon previous studies we expected (and found) the number of CD4+ cells to be approximately 13.9 × 10–3 cells/μm2. This is commensurate with the total body estimates of 2.2 × 1011 CD4+ T cells (4) in tissue from normal, immunocompetent adults that was obtained from the Division of Surgical Pathology of Fairview-University Medical Center (Table 2). The median baseline CD4+ T cell population for these HIV-1–infected patients in our study (both treated and untreated) was 6.81 × 10–4 cells/μm2 (range, 2.41 × 10–4 to 9.78 × 10–4, which is approximately 50% of normal (Table 2). While there was general correlation between stage of disease and the size of the LT CD4+ T cell population (the earlier the stage of infection the higher the LT CD4+ T cell count), it was not significant. We found no correlation between the baseline LT CD4+ T cell count and the baseline peripheral CD4+ T cell count or plasma HIV-1 RNA (P = 0.5, Wilcoxin signed rank test, Figure 2). This suggests that among HIV-1–infected persons who are not taking ART, common measures of disease severity (duration or stage of infection, peripheral CD4+ T cell count, and plasma HIV-1 RNA) do not correlate with the size of the baseline LT CD4+ T cell population.
Comparison of peripheral versus LT CD4+ T cell population at baseline shows no significant relationship.
Quantitative comparison of CD4+ T cells in LT and peripheral blood
We observed a general trend toward expansion of the LT CD4+ T cell population with HAART in all treated patients, and a significant increase in the LT CD4+ T cell population among the four treated individuals who provided LT at 6 months (two-sample paired Student t test, P = 0.0039) (Table 2). At baseline, the mean population size for all patients was 6.81 × 10–4 cells/μm2 and among the treated patience, at 1 month it was 7.51 × 10–4 cells/μm2 (range 2.75 × 10–4 to 10.30 × 10–4) cells/μm2 (n = 6), representing only a 2% increase over baseline to 54% of the normal population size. After 6 months of HAART, the median CD4+ T cell population size was 11.1 × 10–4 cells/μm2 (n = 4), representing a mean 29% increase in the overall size of the LT population, with some patients (1329, 1293, and 1391) having a near-normal CD4+ T cell population and others (1327) remaining signifi-cantly diminished.
Measurement of percent area collagen as a marker of fibrotic changes in LT. We consistently found prominent foci of scar tissue in LTs, suggesting that inflammation-associated damage might be reducing the capacity of the TZniche to maintain cellular homeostasis. We hypothesized that the damage might correlate with the size of the CD4+ T cell population, and tested this hypothesis by measuring the percentage of the TZ area occupied by collagen.
We used a trichrome stain to identify collagen deposition in the TZ and observed significantly greater deposition among all samples from HIV-1–infected subjects than was found in the HIV-1–negative sample (Figure 3). In the tissue obtained from the HIV-1–negative patient (Nl in Table 3), less than 1% of the TZ area consisted of collagen; however, among tissues from all HIV-1–infected persons, the values ranged from 2.2% to 19.9% (median 10.0%). To determine whether there was an association between the amount of fibrosis in the TZ and the size of the CD4+ T cell population in the TZ, we compared the percent area collagen in the TZ with the size of the CD4+ T cell population in the baseline LT biopsy specimen. We found a significant inverse relationship (Figure 4a). The greater the area occupied by collagen, the fewer CD4+ T cells were found in the LT (_R_2 = 0.72, P < 0.0001, Spearman rank correlation). There was no association between percent area colla-gen and duration or stage of HIV-1 infection, baseline peripheral CD4+ T cell count, or plasma HIV-1 RNA, which are the markers of disease severity that are often used in the clinic.
Quantitative analysis of collagen deposition in the TZ in HIV-1–infected persons. Tissues were stained with a modified trichrome stain using the Masson method to identify collagen fibers as blue. (a) HIV-negative patient. Collagen makes up 0.23% of the tissue section. (b) Patient 1407. Section shows collagen deposition (stained blue) in the wall of the high endothelial venule. Percent area collagen was calculated to be 2.2%. (c) Patient 1327, the patient with AIDS. In contrast to b, this section shows significant deposition of collagen in the TZ. Percent area collagen was calculated to be 18.6%. A total of 18 images from each tissue sample, imported through the Photoshop program (Adobe, Version 6.0), are displayed (magnification, ×40). (d) The transformed Photoshop image from c. All shades of blue representing collagen in c were averaged and displayed in black; non-collagen area was removed. This image was imported into the MetaMorph imaging program and the percent area in black was calculated.
Relationship between the percentage of LT occupied by collagen and two parameters: baseline LT CD4+ T cell population and change in peripheral CD4+ T cell count after 6 months. (a) Size of the baseline LT CD4+ T cell population is compared with the percent area of the TZ occupied by collagen. There is a significant correlation between these values (_R_2 = 0.72, P < 0.0001, Spearman rank correlation). (b) Change in the peripheral CD4+ T cell population after 6 months of HAART is compared with the percent area of the TZ occupied by collagen. There is significant correlation between these measurements (_R_2 = 0.91, P < 0.001).
Percent area collagen and change in peripheral CD4+ cell count and plasma HIV-1 RNA over 6 months of follow-up
To determine the impact of TZ fibrosis on the response to HAART and immune reconstitution, we compared the percent area collagen with the change in peripheral CD4+ T cell count over the 6-month follow-up period. We used the peripheral count for two reasons: this is the most common marker of HAART efficacy used in clinical practice, and we did not have a 6-month lymph node biopsy from all of the HAART-treated patients. Using multiple regression, we found a significant relationship between the change in the peripheral CD4+ T cell count over 6 months and the baseline percent area collagen, controlling for baseline peripheral CD4+ T cell count and baseline viral load (_R_2 = 0.91, P < 0.001) (Figure 4b). This suggests that a single baseline measurement of percent area of LT occupied by collagen before initiation of HAART has predictive power to estimate the change in peripheral CD4+ T cell count, independent of baseline peripheral CD4+ T cell count or plasma HIV-1 RNA.