Mechanism of human immunodeficiency virus type 1 localization in CD4-negative thymocytes: differentiation from a CD4-positive precursor allows productive infection

Human immunodeficiency virus (HIV) infection of the thymus could have profound effects on development of the immune response, particularly in children. We and others have established that in addition to infecting and depleting CD4-bearing thymocytes, functional HIV proviruses are found in thymocytes lacking surface CD4 expression. Using in vitro thymocyte cultures, we show that neither HIV-mediated down regulation of CD4 nor CD4-independent infection contributes to the localization of HIV in cells lacking the primary virus receptor. Rather, infection of a CD4-positive precursor cell (CD4 positive/CD8 positive) with subsequent differentiation into a mature CD4-negative phenotype results in productively infected CD4-negative cells. This novel mechanism may contribute to pathogenesis by distributing viral sequences into functional subsets of T cells typically refractory to HIV infection and could account for the presence of viral DNA in CD8-positive lymphocytes recently observed in patients.     

Thymic microenvironment and lymphoid responses to sublethal irradiation

Sublethal irradiation of the murine thymus has been a useful tool for depleting the thymus of dividing immature thymocyte subsets, to sequence thymocyte differentiation events occurring from radiation-resistant precursors. This massive reduction in thymocytes also represents a model in which the bidirectional interplay between the thymic stromal cells and lymphocytes can be investigated. The purpose of this study was thus twofold: to precisely map the initiation of thymopoiesis as a prelude to assessing the effects of injected mAb to novel thymic antigens; and to use a panel of mAbs to determine the alterations in the thymic stroma during the T-cell depletion and reconstitution phases. The striking finding from this study was that following T-cell depletion, there was a marked upregulation of specific stromal antigens, which retracted with the reappearance of T cells. Thus, following sublethal irradiation, there are modifications in the thymic microenvironment that may by necessary to support renewed thymopoiesis and the complete restoration of the thymus involved the synchronous development of both the stromal and lymphocytic components.     

The ethics of placebo-controlled trials for perinatal transmission of HIV in developing countries

Structural and phenotypic modifications of rat thymocytes from birth up to one year of age, i.e. during maturation and at the beginning of the involutive process of the thymus are described. Since the biological significance and the mechanisms of thymic involution are still a matter of debate, this study aims at clarifying the complexity of the compensatory events occurring during this relatively neglected period of time. Thymuses from Sprague-Dawley rats were analyzed morphologically and morphometrically by light and electron microscopy. At the same time, thymocyte subsets, isolated from the same animals, were characterized by flow cytometry according to physical parameters and phenotypic markers. Results indicate that major changes occur during the first month from birth and from six months onward. In particular, already during the first weeks after birth, thymocytes undergo a slight reduction of mitoses associated with an increased number of apoptoses. Moreover, during the same period of time, flow cytometry revealed an expansion of small thymocytes and changes in thymocyte subsets such as increase of CD4+CD8+ and CD5+alpha(beta)TCR- and a decrease of CD4-CD8-, CD4-CD8+ cells. The thymus of adult rats was characterized by time-dependent decrease of both mitoses and apoptoses, progressive physical disconnection among cells, increase of necrotic areas and fibrosis. Around one year of age tissue changes were associated with a dramatic reduction of the population of large thymocytes and the rise of numerous small thymocytes that were unexpectedly negative for all tested markers. By contrast, medium-size thymocytes exhibited a marked decrease of CD4+CD8+ and CD5+alpha(beta)TCR- subsets. In conclusion, our data indicate that thymus undergoes, with time, a complex remodeling and suggest that thymic involution is not only a simple shrinkage of the organ but rather the result of a series of compensatory mechanisms among different cell populations in a setting of progressive involution.     

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.     

Control of the thymic microenvironment by growth hormone/insulin-like growth factor-I-mediated circuits

The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo maturation, eventually leading to the migration of positively selected thymocytes to the T-dependent areas of peripheral lymphoid organs. This process occurs under the influence of the thymic microenvironment, by means of secretory polypeptides and cell-cell contacts. The thymic microenvironment is a tridimensional cellular network composed of epithelial cells (its major component), macrophages, dendritic cells, fibroblasts and extracellular matrix elements. The epithelial reticulum is a heterogeneous tissue, in which a particular lymphoepithelial structure has been isolated in vitro: the thymic nurse cell complex, which possibly creates particular microenvironmental conditions for thymocyte differentiation. Additionally, thymic nurse cells are useful tools to study mechanisms involved in intrathymic T cell migration, including neuroendocrine influences. Previous data showed that thymic hormonal function can be modulated by hormones and neuropeptides, including growth hormone. Interestingly, GH acts pleiotropically on the thymic epithelium increasing cell growth and expression of extracellular matrix ligands and receptors, the latter resulting in an enhancement of thymocyte adhesion to the epithelial cells and thymocyte release from thymic nurse cells. The role of GH on thymus development is further stressed by the findings obtained with GH-deficient dwarf mice. Besides the precocious decline in serum thymulin found in these animals, a progressive thymic hypoplasia occurs, with decreased numbers of CD4+CD8+thymocytes, both defects being largely restored by long-term GH treatment. The effects of GH in the thymus are apparently mediated by IGF-1. Enhancement of thymulin secretion induced by GH, as well as the stimulation of thymocyte adhesion to thymic epithelial cells can be prevented in vitro by treatment with antibodies for IGF-I or IGF-I receptor. Moreover, in both systems IGF-I alone can yield similar effects. Also, the enhanced concanavalin-A mitogenic response and IL-6 production by thymocytes observed in GH-treated mice can be detected in animals treated with IGF-I. Lastly, mouse substrains selected for high or low IGF-I circulating levels exhibited differential thymus developmental patterns correlating with IGF-I levels. A further conceptual aspect concerning the GH-IGF-I-mediated control of thymus physiology is the recent demonstration of an intrathymic production of these molecules, leading to the hypothesis that, in addition to the classical endocrine pathway, GH-IGF-I-mediated paracrine and autocrine pathways may also be implicated in the control of thymus physiology. In any case, such control is exerted pleiotropically, with modulation in the expression of several genes in different cell types of the organ. In this respect, it is exciting to imagine a role of GH-IGF-I loops in shaping the intrathymically generated T cell repertoire.     

CCR5- and CXCR4-utilizing strains of human immunodeficiency virus type 1 exhibit differential tropism and pathogenesis in vivo

CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.     

Ecologic studies of rodent reservoirs: their relevance for human health

To gain insight into the immunomodulatory effects of vitamin E (VE), immune cell population analyses were conducted using thymus and spleen from male broilers fed diets with various levels of VE supplementation (0, 17, 46, and 87 mg dl-alpha-tocopherol acetate/kg of feed). At 2 and 7 wk of age, the percentages of B cells, macrophages, and T cell subsets, delineated by the expression of CD4, CD8, and T cell receptor (TCR) isotype, in thymus and spleen were determined by flow cytometry. The percentages of thymic and splenic B cells and macrophages from 2- and 7-wk-old chickens, as well as the percentage of thymic T cells in 2-wk-old chickens, were unaffected by VE treatment. However, 7-wk-old broilers maintained on 87 mg VE/kg feed had a higher percentage of CD4+CD8- thymocytes, a higher CD4+CD8- to CD4-CD8+ thymocyte ratio, and a lower percentage of CD4+CD8+ thymocytes than chickens receiving no dietary VE supplementation. The VE-induced increase in the percentage of CD4+CD8- thymocytes was due to an increase in the TCR2+CD4+CD8- thymocyte subset, whereas the decrease in the percentage of CD4+CD8+ thymocytes involved all TCR defined T cell subsets. In the spleen, the percentage of CD4+CD8- T cells was lower in 2-wk-old chickens and higher in 7-wk-old chickens maintained on 87 mg/kg feed than in chickens receiving no dietary VE supplementation. The decrease in CD4+CD8- splenocytes at 2 wk of age was due to a decline in the percentage of TCR2+CD4+CD8- splenocytes, whereas the increase in CD4+CD8- splenocytes in 7-wk-old chicks was due to an increase in the percentages of all TCR defined CD4+CD8- T cell subsets. These data support an immunomodulatory effect of VE on CD4+CD8- T cells.     

Influence of detergents on the function of cloned potassium channels

The human thymus is a lymphoepithelial organ in which T cells develop during fetal life. After maturation and selection in the fetal thymic microenvironment, T cells emigrate to peripheral lymphoid tissues such as the spleen, gut, and lymph nodes, and establish the peripheral T cell repertoire. Although the thymus has enormous regenerative capacity during fetal development, the regenerative capacity of the human postnatal thymus decreases over time. With the advent of intensive chemotherapy regimens for a variety of cancer syndromes, and the discovery that infection with the Human Immunodeficiency Virus (HIV) leads to severe loss of CD4+ T cells, has come the need to understand the role of the human thymus in reconstitution of the immune system in adults. During a recent study of the thymus in HIV infection, we observed many CD8+ T cells in AIDS thymuses that had markers consistent with those of mature effector cytotoxic T cells usually found in peripheral immune tissues, and noted these CD8+ effector T cells were predominately located in a thymic zone termed the thymic perivascular space. This article reviews our own work on the thymus in HIV-1 infection, and discusses the work of others that, taken together, suggest that the thymus contains peripheral immune cell components not only in the setting of HIV infection, but also in myasthenia gravis, as well as throughout normal life during the process of thymus involution. Thus, the human thymus can be thought of as a chimeric organ comprised of both central and peripheral lymphoid tissues. These observations have led us to postulate that the thymic epithelial atrophy and decrease in thymopoiesis that occurs in myasthenia gravis, HIV-1 infection, and thymic involution may in part derive from cytokines or other factors produced by peripheral immune cells within the thymic perivascular space.     

Development of a method of thymocyte differentiation of bone marrow-enriched CD34+CD38- cells in postnatal allogeneic cultured thymic epithelia to evaluate immunodeficiency disorders

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.     

Human immunodeficiency virus inhibits multilineage hematopoiesis in vivo

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4(+) lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.     

Early intrathymic precursor cells acquire a CD4(low) phenotype

C4Dlow cells are a population of lymphoid lineage-restricted progenitor cells representing the earliest precursors present in the adult thymus. Paradoxically, thymic progenitors with a similar phenotype in fetal mice and adult RAG-2-deficient (RAG-2-/-) mice lack this characteristic low-level expression of CD4. We now show that radiation-induced differentiation of CD4+ CD8+ double positive thymocytes in RAG-2-/- mice results in the appearance of low levels of CD4 on thymocytes that are phenotypically identical to C4Dlow progenitor cells present in the normal adult thymus. This suggests that CD4 surface expression can be passively transferred from double positive cells to early progenitor thymocytes. Analysis of mixed bone marrow chimeras, reconstituted with hematopoietic stem cells from both CD4-/- (CD45.2) and CD4wt (CD45.1) congenic mice, revealed a CD4low phenotype on cells derived from CD4-/- bone marrow cells. Furthermore, these CD4-/- -derived "C4Dlow" progenitors were capable of reconstituting lymphocyte-depleted fetal thymi, with all thymocytes displaying a CD4-/- phenotype. This directly demonstrates that genetically CD4-deficient thymic progenitor cells can passively acquire a C4Dlow phenotype. Moreover, CD4 expression on C4Dlow progenitor thymocytes is sensitive to mild acid treatment, indicating that CD4 may not exist as an integral cell surface molecule on this thymocyte population. Our findings demonstrate that low-level CD4 surface expression can be passively acquired by intrathymic progenitor cells from the surrounding thymic microenvironment, suggesting that other cell surface molecules expressed at low levels may also result from an acquired phenotype.     

Distinct roles of the interleukin-7 receptor alpha chain in fetal and adult thymocyte development revealed by analysis of interleukin-7 receptor alpha-deficient mice

Mouse mutants lacking expression of the IL-7 receptor (IL-7R) alpha chain are defective in thymopoiesis. The adult thymus has multiple defects, including reduced cell numbers and proportions of the more mature thymocyte subsets, a complete absence of CD25+ cells and a reduced level of RAG1 and RAG2 expression. We show here that, in contrast to the profound developmental arrest observed in the adult thymus, fetal thymocytes from IL-7Ralpha-/- mice have normal proportions of all of the major thymocyte subpopulations, including CD25+ thymocytes and the most mature single-positive subsets. Moreover, normal levels of RAG1 and RAG2 were observed. Total thymocyte numbers, however, remained reduced. These data suggest that the IL-7Ralpha chain is a key regulator of both survival and proliferation during thymocyte development but that it is not essential for the production of T cells during fetal thymopoiesis.     

Circadian rhythm in the hypothermic response to serotonin1A receptor agonist 8-OH-DPAT in rats

The gene encoding the CD2 mouse cell surface antigen was retrovirally transduced into cord blood CD34+ cells. On infection by culture at the contact of retrovirus-packaging cells, the mCD2 marker was expressed by 30-40% CD34+ cells, that included the most primitive stem cell-enriched Thy-1+ and CD38- subsets. Accordingly, sorted cord blood CD34+Thy-1+ cells could be directly infected in the same conditions. mCD2- transgenic cord blood CD34+ cells were then used to reconstitute human fetal thymus implanted in SCID mice. Five to 8 weeks later, the mCD2 antigen was detected on approximately 10% of the human thymocytes repopulating the thymus grafts and the transgene genome was detected in graft cell DNA by Southern blot. These results demonstrate efficient gene transfer into primitive cord blood hematopoietic cells endowed with lymphoid potential and suggest gene therapy schemes in neonates suffering inherited or acquired-such as HIV infection-disorders of the T-cell lineage.     

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.     

Differential tropism and replication kinetics of human immunodeficiency virus type 1 isolates in thymocytes: coreceptor expression allows viral entry, but productive infection of distinct subsets is determined at the postentry level

Human thymocytes are readily infected with human immunodeficiency virus type 1 (HIV-1) in vivo and in vitro. In this study, we found that the kinetics of replication and cytopathic effects of two molecular isolates, NL4-3 and JR-CSF, in postnatal thymocytes are best explained by the distribution of chemokine receptors used for viral entry. CXCR4 was expressed at high levels on most thymocytes, whereas CCR5 expression was restricted to only 0.1 to 2% of thymocytes. The difference in the amount of proviral DNA detected after infection of fresh thymocytes with NL4-3 or JR-CSF correlated with the levels of CXCR4 and CCR5 surface expression. Anti-CCR5 blocking studies showed that low levels of CCR5 were necessary and sufficient for JR-CSF entry in thymocytes. Interleukin-2 (IL-2), IL-4, and IL-7, cytokines normally present in the thymus, influenced the expression of CXCR4 and CCR5 on thymocytes and thus increased the infectivity and spread of both NL4-3 and JR-CSF in culture. NL4-3 was produced by both immature and mature thymocytes, whereas JR-CSF production was restricted to the mature CD1(-)/CD69(+) population. Although CXCR4 and CCR5 distribution readily explained viral entry in mature CD69(+) and immature CD69(-) cells, and correlated with proviral DNA distribution, we found that viral production was favored in CD69(+) cells. Therefore, while expression of CD4 and appropriate coreceptors are essential determinants of viral entry, factors related to activation and stage-specific maturation contribute to HIV-1 replication in thymocyte subsets. These results have direct implications for HIV-1 pathogenesis in pediatric patients.