The importance of lymphocyte trafficking in regulating blood lymphocyte levels during HIV and SIV infections.

In humans, blood is commonly monitored to provide surrogates of disease progression and assess immune status. However, the varied, rapid and atypical alterations in lymphocyte subsets which may occur in blood in response to pathogens, are not predictive of changes in the bulk of the immune system. A hallmark of human and simian immunodeficiency virus (SIV) infections is the profound loss of blood CD4(+) lymphocytes, a feature widely accepted as being a consequence of direct or indirect viral killing of CD4(+) cells throughout the body. However, in recording declining CD4 counts and CD4/8 ratios in the blood, little attention has been paid to migratory behaviour or the composition and tissue distribution of various lymphocyte subsets. This article compares the lymphocyte subsets in blood and various tissues in normal and virus-infected individuals prior to and following drug treatment and indicates an absence of selective CD4(+) cell decreases or increases, highlighting the importance of lymphocyte trafficking and compartmentalization in regulating blood T cell levels and suggesting a reevaluation of the currently favoured paradigm.

Enhanced follicular dendritic cell-B cell interaction in HIV and SIV infections and its potential role in polyclonal B cell activation.

Human immunodeficiency virus (HIV) infections have been characterized by both polyclonal B-cell activation and enhanced responsiveness to B-cell growth factors on one hand and the loss of specific antibody (Ab) responses and refractoriness to the normal signals for B-cell activation on the other. Histopathological studies of lymph node from HIV- and simian immunodeficiency virus (SIV)-infected individuals have indicated initial follicular hyperplasia and the appearance of large irregular germinal centers that undergo progressive involution concomitant with follicular dendritic-cell (FDC) disruption. During this process, follicular dendritic-cell-enriched lymph-node-cell cultures exhibit increased ability to induce cluster formation ("in vitro germinal centers"), lymphocyte proliferation and antibody production compared to uninfected controls. This paper discusses how enhanced FDC-B-cell interaction within SIV-infected germinal centers may result in a reduced ability to select high-affinity B cells and alter the dynamics of antibody-producing-cell and memory-cell generation resulting in the observed hyperactivity.

Interpreting data on lymphocyte subsets in the blood of HIV patients - organ distribution, proliferation and migration kinetics are critical factors.

Lymphocytes stay in the blood only a short time before migrating to lymphoid and nonlymphoid organs. They represent only about 2% of all lymphocytes in the body. The ratio of CD4+/CD8+ lymphocytes in the blood depends on age and genetic influences. In HIV infection not only relative but also absolute numbers of lymphocyte subsets should be determined. The different effects of proliferation and apoptosis on lymphocytes in HIV infection have to be considered. Lymphocyte levels in the blood of HIV patients do not mirror alterations in the lamina propria of the gut or lymph nodes. The dynamic aspects of lymphocyte life span and migration during HIV infection and the progression to AIDS as well as the effects of treatment have to be taken into consideration to enable a meaningful interpretation of experimental data. More data from animal models of HIV infection are needed to study these kinetic aspects.

Characteristics of the CD8+ lymphocytosis during primary simian immunodeficiency virus infections.

OBJECTIVE: To investigate the source of the expanded blood CD8+ subsets during an acute primary simian immunodeficiency virus (SIV) infection of macaques and the potential role of these cells in disease progression. DESIGN AND METHODS: The primary CD8+ lymphocytosis, which occurs at 1-2 weeks following infection with SIVsmm/PBj-14, was examined in rhesus and cynomolgus macaques. Extensive subset analysis of the expanded blood CD8+ cell pool in a rhesus macaque was compared phenotypically with those in thymus, lymph nodes, spleen, ileum and lung washouts obtained at necropsy during blood lymphocytosis. The influence of the primary CD8+ cells expansion on disease progression was assessed at days 175-679 post-infection in long-term PBj-14 survivors staged according to immunological, virological and histopathological changes in their lymphoid organs. RESULT: The very rapid and transient blood lymphocytosis following infection consisted of two distinct CD45RA(low), CD8+ and CD28-, lymphocyte function-associated antigen (LFA)-1(high), CD45RA(high), CD8+ populations. These populations were present in low levels in thymus, lymph and spleen but were highly represented in mucosal tissues, such as long washout, in which CD28- LFA-1(high) CD45RA(high) CD8+ cells comprised 86% of CD8+ cells, and gut, which was predominantly CD45RA(low) CD28- CD8+ cells. A comparison of progressor and non-progressor PBj-14-infected rhesus and cynomolgus macaques also indicated that the existence or magnitude of a blood CD8+ lymphocytosis during the acute phase of infection did not by itself appear to influence or be predictive of disease progression. CONCLUSION: The marked blood CD8+ lymphocytosis observed during acute SIV infection did not result from expansion of virus-specific precursors in peripheral lymph node and did not appear to influence the rate of disease progression. The findings provide a novel explanation for the primary CD8+ cell lymphocytosis and invoke a mechanism whereby virus-induced cytokine/chemokine production in mucosal sites initiate the transient migration of a pre-existing CD8+ population into the blood from compartments such as lung and gut. Such results suggest that the magnitude of lymphocytosis may depend on the level of viral replication in mucosal tissues and the presence of other infections, for example, cytomegalovirus.

Virus-induced cytokines regulate circulating lymphocyte levels during primary SIV infections.

Decline in blood CD4+ lymphocytes during primary symptomatic infections with HIV is usually attributed to viral killing, and has not been considered in terms of altered lymphocyte migration and sequestration. We therefore sought to examine whether CD4+ cell loss from blood of macaques undergoing an acute primary SIV infection might be due to increased synthesis of cytokines, known to profoundly affect lymphocyte trafficking, rather than to direct lymphocyte destruction by virus. The findings indicate that rapid lymphocyte depletion following acute infection is not selective for CD4+ cells, correlates precisely with increased plasma IFN-gamma and tumor necrosis factor-alpha levels, and is reversible. CD4/CD8 ratios in lymph nodes with high viral burdens remain relatively unchanged despite lymphocyte loss from blood. Levels of cytokine mRNA measured in lymphoid organs reflect neither cytokine plasma levels nor their potential to induce sequestration. These results support a model of cytokine-induced lymphocyte extravasation to account for the acute HIV/SIV-induced CD4+ cell lymphopenia and raise questions regarding the extent to which altered lymphocyte migration plays a role in the gradual CD4+ cell depletion throughout infection.

Enhanced follicular dendritic cell function in lymph nodes of simian immunodeficiency virus-infected macaques: consequences for pathogenesis.

HIV and simian immunodeficiency virus (SIV) infections are characterized by several abnormalities in B cell function. Pathogenesis is also associated with marked changes within germinal centers (GC) including hypertrophy and degeneration of follicular dendritic cells (FDC) and accumulation of both viral antigen and activated CD45RO+ CD8+ cells. Since FDC are critical to the generation of antibody-forming cells and specific B cell memory, the simplest assumption is that such B cell defects directly result from virus-induced changes in the GC environment. The present study examined FDC-enriched mesenteric lymph node lymphocyte preparations from early and late stage SIV-infected and uninfected macaques for their ability to support GC reactions in vitro. The results indicate that FDC function as measured by cluster formation, B cell proliferation and SIV-specific antibody production is enhanced in SIV-infected macaques suggesting that, despite FDC atrophy, virus accumulation induces increased FDC-B cell interactions resulting in B cell hyperactivity. The activation and proliferation of CD8+ cells in FDC-enriched cultures further suggest that the infiltrating CD8+ population observed in situ in GC of late-stage SIV/HIV-infected individuals may also benefit from FDC-derived growth signals. Thus, in addition to enhanced B cell proliferation and antibody production, hyperactivity of FDC may potentially promote their own self destruction via the infiltrating CD8+ cells. The increased B cell responsiveness may further exacerbate the disease process due to an overall decrease in the affinity of anti-HIV/SIV antibody, a loss of crucial protective antibodies to other infectious agents and the creation of an environment in which increased trapping of virions facilitates more extensive infection of CD4+ T cells.

Viral DNA burden and decline in percentage of CD4-positive cells in the lymphoid compartment of SIV-infected macaques.

The decline in CD4+ cells and increased viral DNA and RNA burden in the blood of human immunodeficiency virus (HIV)-infected individuals have been used as closely related correlates of disease progression. However, little is known about levels of total or unintegrated viral DNA in lymphoid tissue of HIV-infected patients and how they relate to CD4+ cell decline or disease progression. Exploiting the similarities between HIV- and simian immunodeficiency virus (SIV)-induced disease, we examined lymphoid organs and peripheral blood from SIV-infected macaques for total (pol) and unintegrated 2-LTR circular viral DNA by polymerase chain reaction (PCR). Two SIV isolates (SIVmac/251 and SIVmne/E11S) that differ markedly in their biological and clinical properties were studied. The results indicate that total viral DNA burdens vary considerably between isolates. There was no strong association between total viral DNA levels and CD4% in lymphoid tissues when isolates were compared and death was not associated with any particular level of viral pol DNA. In contrast, accumulation of unintegrated viral DNA was closely associated with decline in CD4/CD8 ratios in lymphoid organs and AIDS. The appearance of both pol and unintegrated viral DNA in thymus of infected macaques also emerged as one of the single best correlates or possible predictors of advanced disease yet studied. Their roles in pathogenesis are discussed.

Immunological and virological changes associated with decline in CD4/CD8 ratios in lymphoid organs of SIV-infected macaques.

The decline in CD4/CD8 ratios in lymph nodes (LNs) of SIV macaques and HIV-infected individuals occurs later than that in blood. In a previous study, long-term SIV-infected macaques were delineated into two groups: (1) those whose LNs had normal CD4/CD8 ratios and (2) those whose LNs had low CD4/CD8 ratios. In the present investigation, LNs, spleens, and blood from these groups have been further analyzed to ascertain the cellular and virological events, particularly those involving CD8+ cells, that occur concomitantly with LN CD4% decline. An increase in the percent of CD69-, IL-2R(p75)-, CD45RA1o CD8+ cells was the most constant event observed in lymphoid tissue from mid- to late-stage SIV-infected monkeys. Such cells were sometimes observed in LNs prior to any other immunological or morphological changes. However, decline in LN CD4/CD8 ratios and the associated degeneration of follicular dendritic cells (FDCs) in the germinal centers (GCs) of these nodes were observed only when both CD8+ cell infiltration of GCs and accumulation of viral antigens within the FDC network could be demonstrated. These dramatic changes were also associated with significantly reduced responsiveness to mitogens throughout the lymphoid compartment. In terms of viral burden, immunological and structural collapse of LNs was not always associated with increased viral DNA levels. Despite the CD4+ cell decline in blood during HIV and SIV infections, the immunological and architectural collapse of the lymphoid compartment, which comprises the bulk of the lymphocytes in the body, appears to be a critical event leading to the onset of AIDS. The present findings suggest that increased CD8+ cell activity as well as decrease in CD4+ cell function both contribute to this process.

Decline in the CD4+ lymphocyte population in the blood of SIV-infected macaques is not reflected in lymph nodes.

Although loss of CD4+ lymphocytes in peripheral blood is a standard criterion for evaluating the course of HIV disease, little is known about changes within lymphoid organs, which contain the bulk (> 50%) of the body's lymphocytes. Because such studies are feasible only by using non-human primates, we have examined lymph nodes (LNs), spleen, and blood from monkeys infected with two isolates of simian immunodeficiency virus (SIV). During both the acute and chronic phases of these infections, characteristic reductions in the blood CD4+ cell levels are not reflected in LN, where the CD4+ pool remains within normal levels. However, when circulating CD4/CD8 ratios have consistently fallen to approximately 0.5, striking decreases in the percentage of CD4 cells (CD4%) and CD4/CD8 ratios in LN occur concomitantly with dramatic increases in viral antigen expression on follicular dendritic cells within LN germinal centers (GCs). The data suggest that loss from the total T cell pool in minimal until the final stages of SIV and HIV disease and that the immunological deterioration of LN is the event that precipitates the increased susceptibility to infections and progression to AIDS.

Variation in the CD4+ and CD8+ populations in lymph nodes does not reflect that in the blood during SIVMNE/E11S infection of macaques.

The decline in the CD4% and CD4/CD8 ratios have been compared in lymph nodes and blood from SIVMNE/E11S infected rhesus macaques. The results indicate that loss from the LN CD4+ cell pool does not occur until CD4/CD8 ratios of less than 0.5 is reached in blood. These changes also correlate with the ability to isolate virus from the blood and the transition of CD45RAhi to highly activated CD45RAlo CD8+ cells both of which may play a role in eliminating CD4+ cells. In end-stage disease, CD8+ cells also decline in LN and mitogen responsiveness no longer exists in any nodes. Interestingly at this stage, the circulating CD8% increases significantly and represents the only source of functional T cells remaining in the body.

Variation in T-lymphocyte activation and susceptibility to SIVPBj-14-induced acute death in macaques.

In contrast to pig-tailed and cynomolgus macaques, which die in 6-10 days following infection with the SIV-PBj-14 isolate, only about 50% of rhesus succumbed to rapid disease. Using a CD45RA MAb that delineates memory (CD45RAlo), naive (CD45RAmed) and "activated" (CD45RAhi) T-cell subsets, it was seen that PBMC from pig-tailed and cynomolgus monkeys, unlike rhesus, have reduced CD4/CD8 ratios and a skewing of T cells towards CD45RAhi expression. Such preactivation of CD4+ cells could lead to enhanced viral replication and early death.

IL-2 receptor expression in autoimmune MRL-lpr/lpr mice. The expanded L3T4-, Lyt-2- population does not express p75 and cannot generate functional high-affinity IL-2 receptors.

Autoimmune MRL-lpr/lpr mice develop an SLE-like disease characterized by a profound lymphadenopathy within an L3T4-, Lyt-2- (DN), B220+ T-cell population. Despite its immature phenotype this subset expresses mature alpha beta TCR belonging predominantly to the V beta 8 gene family and appears to be identical to an activated form of a minor T cell population present in both the thymus and periphery of normal mice. However, the mechanisms underlying the greatly increased cellularity in lpr/lpr-bearing mice are not understood. In this study, the IL-2R expression of lpr/lpr T cells was examined to assess the contribution of IL-2-mediated division to their expansion. The lpr/lpr DN T cells lacked high-affinity IL-2R, even after stimulation, suggesting that IL-2-dependent proliferation plays no role in the expansion of these cells and demonstrating the existence of this unusual T cell phenotype in vivo.

Growth regulation of transformed T cells by nonactivated macrophages: the role of Ia expression.

We studied the influence of unactivated mouse peritoneal macrophages on the proliferative capacity of a spontaneously transformed MRL-lpr/lpr T cell clone. Macrophages, 25%, induced a reduction in proliferative rate from 20% to 95% measured by [3H]thymidine incorporation and microscopic cytometry. MHC-compatible (H-2k) macrophages caused growth inhibition reciprocal to the amount of Ia expression on the macrophage. Thus, with increasing preculture of the macrophages there was both decreasing Ia and increasing suppression. H-2-incompatible macrophages had maximal inhibitory capacity without preincubation. Macrophages derived from the peritoneum of MRL-lpr/lpr mice were less suppressive than macrophages from other H-2k mice. In contrast to the case of activated macrophages in other studies, in the present system there was no killing of T cells, only reduction in proliferation. The inhibitory effect of the macrophages correlated with the spontaneous formation of rosettes between the macrophages and the T cell clone. The number of rosettes forming a single layer of T cells around the macrophages, but not the number of rosettes with multiple layers of cells, was reciprocally related to the amount of Ia expression. The results suggest that macrophages bear a surface structure that influences and modulates the growth of T cells.

Aberrant lymphocyte trafficking in murine systemic lupus erythematosus.

The patterns of migration of lymphoid cells from autoimmune-prone MRL-lpr/lpr, C57BL/6-lpr/lpr, MRL-+/+, and NZB mice were compared to those from sex and age-matched, normal CBA, C57BL/6, and BALB/C mice. Chromium-51-labelled spleen and lymph node cells from all autoimmune mice tested homed preferentially to the spleen relative to lymph node of the recipient strain. The data indicate that defects in lymphocyte trafficking are widespread in murine lupus and suggest a role for abnormal lymphocyte migration in the pathogenesis of this disease.

Unusual expression of IL 2 receptors and both the c-myb and c-raf oncogenes in T cell lines and clones derived from autoimmune MRL-lpr/lpr mice.

Concomitant with their disease, autoimmune MRL-lpr/lpr mice develop a profound lymphadenopathy composed of an unusual dull Lyt-1+ population of T cells. To examine the unusual growth properties and origin of these T cells, as well as their potential role in disease, very rapidly growing T cell lines and clones have been developed from cultures of MRL-lpr/lpr spleen and LN cells. These were studied for growth receptors, oncogene expression, and surface markers. The results further demonstrate the unique nature of lpr-derived T cells and show that i) all lines and clones exhibit greatly elevated expression of both the c-myb and the c-raf oncogenes, ii) despite the reported defect in IL 2 receptor expression of mitogen-activated fresh MRL-lpr/lpr T cells, all long-term lines or clones bear large numbers of IL 2 receptors continuously and without stimulation, although iii) unlike slower growing IL 2-dependent lines from MRL-lpr/lpr mice, these rapidly growing lines and clones are poorly inhibited by anti-IL 2 receptor antibody. Such IL 2 receptor-bearing, nontransformed T cells that are easily maintained have been useful in growth factor studies.