Interferon-Producing Plasmacytoid Dendritic Cells and the Pathogenesis of AIDS
Interferon-alpha (IFN-a) generation by peripheral blood mononuclear cells (PBMC) responding in vitro to HSV was first found to be deficient in patients with severe ulcerative herpes simplex virus (HSV) infections early in the AIDS epidemic. Such deficits were soon found to be associated with all opportunistic infections (OI). Further studies during the natural history of HIV infection indicated that OI did not occur so long as IFN generation remained relatively intact. OI occurred only when both IFN-a generation and CD4 T-cell counts were sufficiently depressed. The IFN-a response to HSV was innate, not adaptive. Evidence that the IFN-a response to HSV was derived from a rare and previously undefined cell type prompted studies eventually revealing that the IFN-producing cells were identical to the "enigmatic plasmacytoid T cells" described by Lennert in lymphoid tissues in 1958. The normal functions of these cells appear to be diverse, but one such function involves initiation of the Th-1 pathway in response to certain microbial antigens. The IFN-producing cells are now known as plasmacytoid dendritic cells (pDCs), because they differentiate following appropriate stimulation, into type-2 dendritic cells. During therapy for HIV infection, pDCs recover somewhat more rapidly than CD4 T cells to levels associated with resistance to OI, and their renewed response appears closely associated with clinically apparent immune reconstitution. Increased pDCs have been associated with nonprogressor status. In HIV infection and in certain other clinical states, PBMC IFN-a generation and pDC numbers correlate closely, suggesting that numerical depletion of circulating pDCs is an important component of the immune deficiency of AIDS. Losses of pDCs during HIV progression and repletion during antiretroviral therapy could be involved in both the progressive loss and reconstitution of the Th-1 pathway.
Frederick Siegal, a physician at the St. Vincent Catholic Medical Center in New York City, began his presentation by discussing his first encounter with AIDS in 1980 when he and colleagues began seeing young homosexual males with severe HSV-induced ulcers; prolonged HSV infection of this type only occurs in severely immunocompromised people. Collaborating with Carlos Lopez and Patricia Fitzgerald-Bocarsly, Siegal began investigating herpes-specific immune responses in patients with AIDS. The group initially studied natural killing of HSV-infected cells, but quickly focused their efforts on identifying a specific type of peripheral blood mononuclear cell (PBMC) that possessed the innate ability to generate IFN-a in response to viral infection. The researchers referred to these elusive cells as "natural interferon producing cells" or "NIPCs." Interferons are proteins rapidly generated in virus-infected cells and they suppress infection by interfering with viral replication in neighboring cells. The researchers believed that identifying and understanding these NIPCs could provide insight into viral-induced immune responses.
Work in Siegal's and Bocarsly's laboratories demonstrated that HSV selectively stimulates NIPCs to generate IFN-a. The HSV response provided an excellent marker for studying these unusual cells. Siegal's group used herpes-infected target cells as triggers for IFN-a production in PBMCs from healthy volunteers and patients with AIDS. Preliminary studies in cells from patients with AIDS revealed a relationship between the inability to make IFN-a and the likelihood of experiencing an OI. When patients who had an OI were prospectively studied, marked deficits in CD4 T-cells counts and IFN-a production were observed. The researchers also analyzed cells from patients who had not yet experienced an OI, and discovered that these patients did not get an OI within 4 months of follow-up unless their CD4 T-cell count dropped to fewer than 250 cells/mm3 and IFN-a production decreased to less than 300 IU/mL, a value subsequently determined to be a critical level for preventing an OI. Patients with deficits in both CD4 T-cell counts and IFN-a production tended to get an OI or die within 24 to 30 months, while patients with a deficit in just one of these factors were unlikely to experience an OI or die during the ensuing 36 months. In patients with AIDS, failure to generate IFN-a appeared to be just as important as failure to produce enough CD4 T cells when predicting clinical outcome.
Questioning whether the relationship between IFN-a production and susceptibility to intracellular pathogens and OIs was specific to diseases involving retroviruses, the researchers examined IFN-a production in patients with other immune-compromised conditions. Defects in IFN-a production occur in hairy cell leukemia (HCL), spindle cell thymoma with immunodeficiency, and in some cases of idiopathic CD4 T cell lymphocytopenia (ICL). Data collected from patients with HCL provided some insight into the origin of these cells. Untreated patients had very low levels of IFN-a production; however, when patients were treated with chlorodeoxyadenosine, normal levels of IFN-a were detected one year later. This time frame coincided with complete remission in the bone marrow, suggesting that the bone marrow could be the origin of NIPCs.
Convinced that the NIPCs were a crucial factor in the clinical outcome of patients with AIDS or other immunodeficiencies, Siegal and colleagues pursued identifying the specific cell type responsible for IFN-a production. Preliminary experiments showed that NIPCs were not natural killer or T cells. Study of these cells was complicated because NIPCs were present in only a small fraction of PBMC and were difficult to keep alive. Several laboratories were trying to enrich and isolate these cells. These efforts were only partially successful; data were difficult to interpret because of the large numbers of contaminating cells. At the same time, a future collaborator of Siegal's, Yong-Jun Liu, had developed a technique to separate a cell type he believed to be precursors of dendritic cells and recognized that these cells were similar to a cell described in the 1950s by Lennert, a German pathologist. Lennert had observed that there were plasmacytoid cells in deep cortical tissue of secondary lymphoid tissues that tended to cluster in areas with extensive apoptosis. Liu and his group also witnessed these cells (once purified to nearly 99%) undergo rapid apoptotic death that was prevented if cells were cultured with interleukin-3 (IL-3), a critical survival factor. When cultured in the presence of CD40 ligand and IL-3, these cells made an astonishing morphological transformation from smooth plasma-like cells to extremely complex dendritic cells, which the researchers referred to as "type 2 dendritic cells" or "DC2s." Moreover, they discovered that DC2s tended to promote Th-2 immune responses in vitro when co-cultured with T cells. Liu and his laboratory struggled to determine what factor these secretory cells were producing. During this time, Siegal attended a presentation given by Liu discussing this work and was struck by the similarities between Liu's precursor DC2s and his NIPCs. A series of experiments showed that these cells were, in fact, the same and they referred to these cells as "pDC2s." They now had identified the origin of the elusive cell responsible for IFN-a production and published these findings in Science in 1999.
Siegal discussed a simplified model of how pDCs may play a role at the interface of innate and adaptive immunity (see Figure 1). These cells circulate in the blood until they encounter a microbial stimulus, such as a virus. This microbe acts as a trigger for the subsequent immune response. Using toll-like receptors (TLR) and other receptors, pDCs may pick up these antigens through pattern recognition. Following migration to T-cell areas of lymphoid tissues, these cells produce IFN-a, which causes immature T cells to express IL-12 receptors. These T cells then receive an IL-12 signal from DC1s; however others believe that pDCs are also capable of generating this signal. This entire process fosters the Th-1 pathway. When cultured with IL-3, CD40-ligand, and virus, these cells can differentiate into mature DC2s, which foster the Th-2 pathway.
Figure 1. A model of how interferon-producing cells (IPCs or pDCs) may play a role at the interface of innate and adaptive immunity.
Questions still remained about how pDC2s and IFN-a production affect HIV pathogenesis. As previously discussed, IFN-a production is substantially decreased in patients with AIDS, which the researchers discovered was caused by both decreased production per cell as well as a decreased number of pDCs. When Siegal's group examined IFN-a production in patients with AIDS over the last decade, they noticed an increase in IFN-a production that coincided with the introduction of AZT therapy; however, that increase subsequently waned. Antiretroviral therapy appeared capable of causing immune reconstitution of CD4 T-cell counts and IFN-a production, which is reflected in the clinical outcomes of these patients. When patients are treated with antiretroviral therapy and made aviremic, CD4 T-cell counts slowly recover in approximately 10 months. However, IFN-a production returns much faster, typically in about 4 months. Siegal's data demonstrated that when there is immune reconstitution of either IFN-a or CD4 T cells (usually with viral suppression), AIDS patients no longer experience OIs. However, when immune reconstitution does not occur, approximately half the patients experience an OI and die.
Siegal proposed a plausible model to illustrate the hypothetical role of pDCs in the loss of Th-1 immunity in HIV infection and how these events lead to HIV pathogenesis (see Figure 2). HIV is capable of infecting pDCs, as well as triggering pDCs to generate IFN-a. Once infected, pDCs traffic HIV into the T-cell areas of lymphoid tissues, where they generate IFN-a, upregulate IL-12 receptors, and produce HIV. Neighboring CD4 T cells are subsequently infected. As a result, the Th-1 immune response is completely abrogated and the specific Th-1 response to HIV is selectively eliminated. Moreover, as HIV infection progresses, pDCs are depleted, and IFN-a production becomes inefficient as viremia increases. This may explain why immature, naive T cells do not become part of the Th-1 pathway. In this situation, the HIV-specific immune response is eliminated, as well as other specific Th-1 immune responses.
Figure 2. A model of the hypothetical role of interferon-producing cells (IPCs or pDCs) in the loss of Th-1 immunity in HIV infection and how these events lead to HIV pathogenesis.
Recent work by Soumelis, Levy, Liu, and others independently confirms that IFN-a production is an important predictor of clinical outcome in AIDS. For example, data collected from HIV-positive nonprogressors show that some of these patients have an abundance of circulating IFN-a-producing cells, which may explain why their disease has not progressed. Siegal and colleagues recently published data showing that there is a selective decrease in the number of pDCs as a person ages, though each cell continues to generate the same amount of IFN-a. People with an innate ability to make higher than normal levels of IFN-a, such as the young, may be more intrinsically resistant to primary HIV infection. Many questions remain about the specific mechanisms of pDCs in vivo. Whether pDCs differentiate into DCs in vivo, or if these cells produce as much IFN-a in vivo as they do in cell culture, remains unclear.
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Frederick Siegal, M.D. is from the St. Vincent Catholic Medical Center.