A new study from Marty Markowitz’s research group at the Aaron Diamond AIDS Research Center, recently published in the advance online section of J. Virology, offers a detailed analysis of events in the gut-associated lymphoid tissue (GALT) during acute HIV infection.
Participants included 32 HIV-infected gay men identified prior to full seroconversion (some participants had been infected for less than three weeks) and 18 uninfected controls. The researchers used biopsies of colonic mucosal tissue and peripheral blood samples to compare virological and immunological events in the different compartments. Viral burden was consistently higher (3-90 fold) in the gut; the mean amount of viral DNA in gut CD4 T cells was 163.5 copies per 500 cells compared to a mean of 11.9 copies per 500 cells in the peripheral blood. In addition to HIV DNA, the amount of messenger RNA was also compared. The mean HIV mRNA copy number was 16,542 copies in gut CD4 T cells versus 1,594 copies in peripheral blood CD4 T cells. Cumulative analysis of all study participants showed a mean 10-fold greater HIV mRNA levels in gut CD4 T cells compared to peripheral blood CD4 T cells (range 3-fold to 102-fold). The subject displaying the greatest (102-fold) difference between compartments had been infected only 18 days. There was a strong correlation between the level of HIV mRNA in gut CD4 T cells and peripheral blood viral load as measured with the standard assay (r2=0.90).
Immunological analyses showed that the percentage of CD4 T cells defined as memory cells (based on the expression of the surface marker CD45RO) was reduced in both the blood and gut tissue compared to the uninfected control subjects. Conversely, the percentage of activated CD4 T cells (defined by CD38 expression) was significantly increased. To assess CD4 T cell proliferation in the different compartments, the researchers measured the percentage of cells expressing the proliferation marker Ki67. In peripheral blood, 2.9% of CD4 T cells were Ki67+ versus 1.1% in uninfected participants, in the gut the difference was 11.1% versus 3.1%. The researchers also used a technique called immunohistochemistry to ascertain the proportion of CD4 T cells that expressed Ki67 in a given area of gut tissue. This technique demonstrated that there were always more proliferating CD4 T cells in the tissue sections from the HIV-infected participants, averaging 17.7% per unit area compared 8.6% per unit area in the uninfected controls.
An important part of the antiviral immune response is the cytotoxic (cell killing) activity of virus-specific T cells (usually mediated mainly by CD8 T cells). To get a sense of the amount of cytotoxic activity in the gut during acute HIV infection, the researchers measured the levels of three substances released by cytotoxic cells: perforin, granzyme B and TIA-1. Levels of all three were elevated in the HIV-infected study participants compared to controls. To further characterize the cytotoxic cells, expression of the CD8 receptor and perforin were analyzed simultaneously. In the infected individuals, 26.6% of gut CD8 T cells co-expressed perforin compared to 11.7% in the controls. The researchers also found that 41% of the perforin-expressing cells were negative for CD8. These cells may represent natural killer cells but could also include CD4 T cells. Notably, Australian researchers John Zaunders and Anthony Kelleher have documented the presence of perforin-expressing CD4 T cells in the blood during acute HIV infection and have suggested that these cells may contribute significantly to the primary immune response to HIV. The Aaron Diamond team is conducting additional analyses to try and define the nature and specificity of the cytotoxic responses seen in this study.
The authors conclude that the loss of CD4 T cells from the gut that occurs in HIV infection is likely multifactorial, with activation-induced cell death, cytotoxic responses and viral cytopathicity all contributing. What is unclear from the study is whether events in the gut are really dramatically different from occurrences in the lympoid tissue generally; it has long been known that many more CD4 T cells are HIV infected in lymph tissue compared to the peripheral blood. A recent SIV study found comparable numbers of SIV-infected CD4 T cells in the gut and lymph nodes (mesenteric and inguinal). The question of whether CD4 T cell depletion is more extensive in the gut is also complicated by the vastness of the GI tract and the difficulty of counting absolute numbers of CD4 T cells as compared to assessing declines in the percentage of CD4 T cells. Most studies report gut CD4 T cell depletion in terms of the percentage of CD4 cells among total T cells, but interpretation of the data is complicated by the expansion of CD8 T cells that occurs in acute HIV infection. These unresolved issues will need to be addressed by future studies.
Study abstract:
JVI Accepts, published online ahead of print on 25 October 2006
J. Virol. doi:10.1128/JVI.01739-06
Mechanisms of Gastrointestinal CD4+ T Cell Depletion During Acute and Early HIV-1 Infection
Saurabh Mehandru, Michael A. Poles, Klara Tenner-Racz, Victoria Manuelli, Patrick Jean-Pierre, Peter Lopez, Anita Shet, Andrea Low, Hiroshi Mohri, Daniel Boden, Paul Racz, and Martin Markowitz
During acute and early HIV-1 infection (AEI) more than 50% of CD4+T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. Thirty-two AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+ T cells derived from the GI tract and PB using real time polymerase chain reaction (PCR). The phenotype of infected cells was characterized using combinations of immunohistochemistry and in-situ hybridization. Markers of immunological memory, activation and proliferation were examined using flow-cytometry and immunohistochemistry and host-derived cytotoxic cellular response was examined using immunohistochemistry. GI CD4+ T cells harbored on average, 13-fold greater HIV-1 viral DNA levels and 10-fold greater HIV-1 RNA levels compared to PB CD4+ T cells during AEI. HIV-1 RNA was detected in both ‘activated’ and ‘non-activated’ mucosal CD4+ T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB and, a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract, as early as 18 days post infection. Mucosal CD4+ T cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+ T cells. Subsequently, ongoing infection of susceptible CD4+ T cells along with activation induced cellular death and host cytotoxic cellular response are responsible for persistence of the lesion.
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