IL-15-dependent upregulation of GITR on CD8 memory phenotype T cells in the bone marrow relative to spleen and lymph node suggests the bone marrow as a site of superior bioavailability of IL-15

CD8 memory T cells are enriched in the bone marrow, a site where these cells are thought to receive homeostatic signals. However, the primary site where CD8 memory T cells receive their cytokine-induced homeostatic signals has recently come under debate. In this study, we demonstrate that the bone marrow contains a fraction of CD8 memory phenotype T cells with elevated expression of glucocorticoid-induced TNFR-related protein (GITR). In contrast, splenic and lymph node memory phenotype T cells have GITR levels similar to those on naive T cells. The bone marrow GITR(hi) memory T cells have a phenotype indicative of cytokine activation, with higher CD122 and lower CD127 than do the GITR(basal) memory T cells. Remarkably, these bone marrow-specific GITR(hi) cells are almost completely ablated in the absence of IL-15, whereas TNFR2 and 4-1BB expression on the CD8 memory T cells are IL-15 independent. Furthermore, adoptively transferred splenic CD8 memory phenotype T cells show IL-15-dependent GITR upregulation upon entry into the bone marrow. This result implies that the selective appearance of GITR(hi) memory phenotype T cells in the bone marrow reflects the local microenvironment rather than a different subset of memory T cells. GITR(-/-) mice have a lower frequency of CD8 memory phenotype cells in the bone marrow, yet the GITR(-/-) cells hyperproliferate compared with those in wild-type mice. Taken together, these data suggest that GITR plays a role in the survival of CD8 memory phenotype T cells and that GITR upregulation represents a precise marker of cells that have responded to IL-15.     

Selection of a highly aggressive myeloma cell line by an altered bone microenvironment in the C57BL/KaLwRij mouse

In multiple myeloma (MM), bone marrow microenvironment has an important role for the survival and growth of plasma cells. We previously showed that a high bone turnover, induced by ovariectomy, increased MM cells growth in the 5T2MM model. The present study characterized a new plasma cell line (5THL) which was isolated from 5T2MM mice previously ovariectomized. Cells were propagated unchanged in normal C57BL/KaLwRij mice during six generations. 5THL was compared to the original 5T2MM phenotype. Paraproteinemia was detected 6 weeks post injection in 5THL mice and after 8 weeks in 5T2MM mice. All 5THL mice developed a hind-limb paralysis after 10 weeks. 5T2MM mice were euthanized at 16 weeks, due to a more progressive development of the disease. In 5THL mice, osteolytic lesions were observed after 8 weeks and severe bone destruction was evidenced at 10 weeks. In 5T2MM mice, minimal lesions were observed only after 10 weeks. Like in 5T2MM mice, no extra osseous lesions were observed in 5THL mice. The 5THL MM model closely mimics human myeloma with higher and faster bone aggressiveness. This new aggressive cell line, with a preserved phenotype, was selected by an altered microenvironment due to an increased bone turnover.     

The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation

To maintain bone mass turnover and bone mineral density (BMD), bone marrow (BM) mesenchymal stem cells (MSCs) are constantly recruited and subsequently differentiated into osteoblasts. HIV‐infected patients present lower BMD than non‐HIV infected individuals and a higher prevalence of osteopenia/osteoporosis. In antiretroviral treatment (ART)‐naive patients, encoded HIV proteins represent pathogenic candidates. They are released by infected cells within BM and can impact on neighbouring cells. In this study, we tested whether HIV proteins Tat and/or Nef could induce senescence of human BM‐MSCs and reduce their capacity to differentiate into osteoblasts. When compared to nontreated cells, MSCs chronically treated with Tat and/or Nef up to 30 days reduced their proliferative activity and underwent early senescence, associated with increased oxidative stress and mitochondrial dysfunction. The antioxidant molecule N‐acetyl‐ cysteine had no or minimal effects on Tat‐ or Nef‐induced senescence. Tat but not Nef induced an early increase in NF‐κB activity and cytokine/chemokine secretion. Tat‐induced effects were prevented by the NF‐κB inhibitor parthenolide, indicating that Tat triggered senescence via NF‐κB activation leading to oxidative stress. Otherwise, Nef‐ but not Tat‐treated cells displayed early inhibition of autophagy. Rapamycin, an autophagy inducer, reversed Nef‐induced senescence and oxidative stress. Moreover, Tat+Nef had cumulative effects. Finally, Tat and/or Nef decreased the MSC potential of osteoblastic differentiation. In conclusion, our in vitro data show that Tat and Nef could reduce the number of available precursors by inducing MSC senescence, through either enhanced inflammation or reduced autophagy. These results offer new insights into the pathophysiological mechanisms of decreased BMD in HIV‐infected patients.     

Interleukin-7 (IL-7): immune function, involvement in the pathogenesis of HIV infection and therapeutic potential

Interleukin 7 (IL-7), which is constitutively produced particularly by stromal cells from the bone marrow and thymus, plays a crucial role in T cell homeostasis. This cytokine is implicated in thymopoiesis since it sustains thymocyte proliferation and survival. It regulates peripheral naive T cell survival by modulating the expression of the anti-apoptotic molecule Bcl-2, and sustains peripheral T cell expansion in response to antigenic stimulation. Infection by the human immunodeficiency virus (HIV) leads to severe T lymphopenia and general immune dysfunction. Increased IL-7 plasma levels are generally observed in HIV-infected patients. The existence of an inverse correlation between IL-7 plasma levels and the CD4+ T cell count suggests that a direct feedback mechanism is working to restore peripheral T cell counts in lymphopenic patients. Here, IL-7 plasma levels are a good predictive marker of CD4+ T cell restoration during therapy. Combinations of antiretroviral treatments considerably slow disease progression. They drastically decrease the viral load and, in most patients, significantly increase peripheral CD4+ T cell counts. However, despite their usual ability to reduce viral replication, such treatments often fail to reverse lymphopenia and do not restore specific antiviral immune responses. IL-7, based therapy, combined with efficient antiretroviral treatment, may be beneficial to HIV-infected patients by promoting thymic output, sustaining naive T cell counts and increasing memory T cell activation.     

Functional differentiation of T cell precursors. I. Parameters of carrier-specific tolerance in murine helper T cell precursors.

Irradiated mice reconstituted with bone marrow from sheep gamma-globulin- (SGG) tolerant syngeneic donors display reduced IgG responsiveness to challenge with trinitrophenylated (TNP)-SGG compared with recipients of normal marrow. This effect is SGG-specific and is due neither to suppressor T cells nor to antigen carryover. "Helper T cell precursor tolerance" can be induced with as little as 40 micrograms tolerogen (SGG). Unlike mature helper T cells, these precursors show both a rapid induction and rapid waning patterns, suggesting a high rate of turnover. Our results imply that marrow helper T cell precursors bear antigen-specific receptors and that the T cell repertoire must be at least partially generated before residence in the thymus.     

Basophils support the survival of plasma cells in mice

We have previously shown that basophils support humoral memory immune responses by increasing B cell proliferation and Ig production as well as inducing a Th2 and B helper phenotype in T cells. Based on the high frequency of basophils in spleen and bone marrow, in this study we investigated whether basophils also support plasma cell survival and Ig production. In the absence of basophils, plasma cells of naive or immunized mice rapidly undergo apoptosis in vitro and produce only low amounts of Igs. In contrast, in the presence of basophils and even more in the presence of activated basophils, the survival of plasma cells is markedly increased and continuous production of Igs enabled. This effect is partially dependent on IL-4 and IL-6 released from basophils. Similar results were obtained when total bone marrow cells or bone marrow cells depleted of basophils were cultured in the presence or absence of substances activating basophils. When basophils were depleted in vivo 6 mo after immunization with an Ag, specific Ig production in subsequent bone marrow cultures was significantly reduced. In addition, depletion of basophils for 18 d in naive mice significantly reduced the number of plasma cells in the spleen. These data indicate that basophils are important for survival of plasma cells in vitro and in vivo.     

Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection

CD4+ Th cells deliver the cognate and cytokine signals that promote the production of protective virus-neutralizing IgG by specific B cells and are also able to mediate direct antiviral effector functions. To quantitatively and qualitatively analyze the antiviral functions of CD4+ Th cells, we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of vesicular stomatitis virus (VSV). The elevated precursor frequency of naive VSV-specific Th cells in tg7 mice led to a markedly accelerated and enhanced class switching to virus-neutralizing IgG after immunization with inactivated VSV. Furthermore, in contrast to nontransgenic controls, tg7 mice rapidly cleared a recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G) from peripheral organs. By adoptive transfer of naive tg7 CD4+ T cells into T cell-deficient recipients, we found that 105 transferred CD4+ T cells were sufficient to induce isotype switching after challenge with a suboptimal dose of inactivated VSV. In contrast, naive transgenic CD4+ T cells were unable to adoptively confer protection against peripheral infection with Vacc-IND-G. However, tg7 CD4+ T cells that had been primed in vitro with VSV-G peptide were able to adoptively transfer protection against Vacc-IND-G. These results demonstrate that the antiviral properties of CD4+ T cells are governed by the differentiation status of the CD4+ T cell and by the type of effector response required for virus elimination.     

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.     

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.     

Neutrophil expression of fas ligand and perforin directs effector CD8 T cell infiltration into antigen-challenged skin

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin(-/-) mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin(-/-) recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin(-/-) mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.     

Correction or transfer of immunodeficiency due to TNF-LT alpha deletion by bone marrow transplantation.

BACKGROUND: Mice with inactivated tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha) genes have profound abnormalities of the immune system including lymphocytosis, lack of lymph nodes, undifferentiated spleen, hypoimmunoglobulinaemia, and defective Ig class switch. Here, we asked whether this phenotype is due to incompetent lymphohemopoietic progenitors or to a defective environment. MATERIALS AND METHODS: Lethally irradiated TNF-LT alpha-deficient and wild-type mice received bone marrow cells from either TNF-LT alpha-deficient or wild-type mice. The reconstitution and transfer of the phenotype was followed by morphological and functional analyses. RESULTS: Bone marrow cells from wild-type mice restored the synthesis of TNF and LT alpha, corrected the splenic microarchitecture, normalized the lymphocyte counts in the circulation, and repopulated the lamina propria with IgA-producing plasma cells of TNF-LT alpha-deficient mice. Furthermore, the formation of germinal centers in the spleen and the defective Ig class switch in response to a T-cell dependent antigen was corrected, while no lymph nodes were formed. Conversely, the TNF-LT alpha phenotype could be transferred to wild-type mice by bone marrow transplantation after lethal irradiation. CONCLUSIONS: These data demonstrate that most TNF- and LT alpha-producing cells are bone marrow derived and radiosensitive, and that the immunodeficiency due to TNF-LT alpha deletion can be corrected to a large extent by normal bone marrow cell transplantation. The genotype of the donor bone marrow cells determines the functional and structural phenotype of the TNF-LT alpha-deficient adult murine host, with the exception of lymph node formation. These findings may have therapeutic implications for the restoration of genetically defined immunodeficiencies in humans.     

Transgenic galectin-1 induces maturation of dendritic cells that elicit contrasting responses in naive and activated T cells

Dendritic cells (DC) are professional APC that control the balance between T cell immunity and tolerance. Genetic engineering of DC to regulate the outcome of the immune response is an area of intense research. Galectin (gal)-1 is an endogenous lectin that binds to glycoproteins and exerts potent regulatory effects on T cells. Consequently, gal-1 participates in central deletion of thymocytes and exerts therapeutic effects on experimental models of T cell-mediated autoimmune disorders and graft-vs-host disease. Together, these observations strongly indicate that engineering DC to express transgenic (tg) gal-1 may be beneficial to treat T cell-mediated disorders. In this study, we have investigated the impact of the expression of high levels of tg gal-1 on maturation/activation of DC and on their T cell stimulatory function. Murine DC were transduced with a recombinant adenovirus encoding hu gal-1 (gal-1-DC). Tg gal-1 was exported by a nonclassical pathway through exosomes and was retained on the DC surface inducing segregation of its ligand CD43. Expression of tg gal-1 triggered activation of DC determined by induction of a more mature phenotype, increased levels of mRNA for proinflammatory cytokines, and enhanced ability to stimulate naive T cells. Conversely, gal-1-DC induced rapid apoptosis of activated T cells. In vivo, gal-1-DC increased significantly the sensitization phase of contact hypersensitivity assays while inducing a drastic inhibition of the elicitation phase by triggering apoptosis of activated T cells in the dermis. Gal-1-DC represent a novel tool to control differentially the afferent and efferent arms of the T cell response.     

Homeostatic proliferation of lymphocytes results in augmented memory-like function and accelerated allograft rejection

Homeostatic proliferation is a normal physiological process triggered by lymphopenia to maintain a constant level of T cells. It becomes the predominant source of new T cells in adulthood after thymus regression. T cells that have undergone homeostatic proliferation acquire the memory phenotype, cause autoimmune disease, and are resistant to tolerance induction protocols. Transplantation is a rare example in which lymphopenia is deliberately induced for its immunosuppressive effect. However, it is not known whether the homeostatic proliferation that follows will have the opposite effect and accelerate rejection. We show that T cells that have undergone homeostatic proliferation acquire a memory phenotype, spontaneously skews toward the Th1 phenotype, even in the absence of antigenic stimulus. Interestingly, in contrast, the percentage of Foxp3(+) regulatory T cells increased by 28-fold following homeostatic proliferation. Using a mouse life-sustaining kidney transplant model, we showed that T cells that have gone through homeostatic proliferation in lymphopenic hosts transformed chronic rejection to acute rejection of a single MHC class II-mismatched kidney allograft. T cells that have undergone homeostatic proliferation consistently cause reliable rejection even when bona fide memory T cells cannot. These functional changes are long-lasting and not restricted to the acute phase of homeostatic proliferation. Our findings have important implications for tolerance induction or graft-prolonging protocols involving leukocyte depletion such as irradiation bone marrow chimera, T cell-depleting Abs, and lymphopenia induced by infections such as CMV and HIV.     

IL-7/STAT5 cytokine signaling pathway is essential but insufficient for maintenance of naive CD4 T cell survival in peripheral lymphoid organs

Constitutive expression of suppressors of cytokine signaling (SOCS)1 in T lineage in vivo attenuated cytokine signaling and resulted in a dramatic reduction in the number of naive CD44(low)CD62L(high) CD4 T cells in the spleen. After adoptive transfer of thymocytes from SOCS1 transgenic mice into normal recipients, naive CD4 T cells rapidly disappeared from the spleen within 1 wk. Likewise, T cell-specific deletion of STAT5a/b in vivo resulted in a similar phenotype characterized by loss of naive CD4 T cells. Thus, STAT5-mediated signaling is crucial for promoting naive T cell survival. However, forced expression of constitutively active STAT5 failed to rescue CD4 T cells in SOCS1 transgenic mice, implying that STAT5 activation is necessary but not sufficient for naive CD4 T cell survival. Although blockade of the IL-7R, a SOCS1 target, resulted in clear inhibition of naive T cell survival, the effect occurred 3 wk after anti-IL-7R Ab treatment, but not at earlier time points. These results suggest that IL-7-mediated STAT5 activation is essential for long-term survival of naive CD4 cells after export from thymus, and that another SOCS1-sensitive cytokine is critical for short-term naive T cell survival.     

Kinetics of in vivo proliferation and death of memory and naive CD8 T cells: parameter estimation based on 5-bromo-2'-deoxyuridine incorporation in spleen, lymph nodes, and bone marrow

To study naive and memory CD8 T cell turnover, we performed BrdU incorporation experiments in adult thymectomized C57BL/6 mice and analyzed data in a mathematical framework. The following aspects were novel: 1) we examined the bone marrow, in addition to spleen and lymph nodes, and took into account the sum of cells contained in the three organs; 2) to describe both BrdU-labeling and -delabeling phase, we designed a general mathematical model, in which cell populations were distinguished based on the number of divisions; 3) to find parameters, we used the experimentally determined numbers of total and BrdU(+) cells and the BrdU-labeling coefficient. We treated mice with BrdU continuously via drinking water for up to 42 days, measured by flow cytometry BrdU incorporation at different times, and calculated the numbers of BrdU(+) naive (CD44(int/low)) and memory (CD44(high)) CD8 T cells. By fitting the model to data, we determined proliferation and death rates of both subsets. Rates were confirmed using independent sets of data, including the numbers of BrdU(+) cells at different times after BrdU withdrawal. We found that both doubling time and half-life of the memory population were approximately 9 wk, whereas for the naive subset the doubling time was almost 1 year and the half-life was roughly 7 wk. Our findings suggest that the higher turnover of memory CD8 T cells as compared with naive CD8 T cells is mostly attributable to a higher proliferation rate. Our results have implications for interpreting physiological and abnormal T cell kinetics in humans.     

NZB mice exhibit a primary T cell defect in fetal thymic organ culture

Defects in T cell development have been suggested to be a factor in the development of systemic autoimmunity in NZB mice. However, the suggestion of a primary T cell defect has often been by extrapolation, and few direct observations of T cell precursors in NZB mice have been performed. Moreover, the capacity of NZB bone marrow T cell precursors to colonize the thymus and the ability of the NZB thymic microenvironment to support T lymphopoiesis have not been analyzed. To address this important issue, we employed the fetal thymic organ culture system to examine NZB T cell development. Our data demonstrated that NZB bone marrow cells were less efficient at colonizing fetal thymic lobes than those of control BALB/c or C57BL/6 mice. In addition, NZB bone marrow cells did not differentiate into mature T cells as efficiently as bone marrow cells from BALB/c or C57BL/6 mice. Further analysis revealed that this defect resulted from an intrinsic deficiency in the NZB Lin-Sca-1+c-kit+ bone marrow stem cell pool to differentiate into T cells in fetal thymic organ culture. Taken together, the data document heretofore unappreciated deficiencies in T cell development that may contribute to the development of the autoimmune phenotype in NZB mice.     

IFN-gamma promotes Fas ligand- and perforin-mediated liver cell destruction by cytotoxic CD8 T cells

To study liver cell damage by CTL, CD8 T cells from P14 TCR transgenic (tg) mice specific for the gp33 epitope of lymphocytic choriomeningitis virus with either deficiency in IFN-gamma (P14.IFN-gamma(null)), functional Fas ligand (P14.gld), or perforin (P14.PKO) were transferred into H8 tg mice ubiquitously expressing gp33 Ag. Treatment of H8 recipient mice with agonistic anti-CD40 Abs induced vigorous expansion of the transferred P14 T cells and led to liver cell destruction determined by increase of glutamate dehydrogenase serum levels and induction of caspase-3 in hepatocytes. Liver injury was mediated by the Fas/Fas ligand (FasL) pathway and by perforin, because P14.gld and P14.PKO T cells failed to induce increased glutamate dehydrogenase levels despite strong in vivo proliferation. In addition, H8 tg mice lacking Fas were resistant to the pathogenic effect of P14 T cells. Besides FasL and perforin, IFN-gamma was also required for liver cell damage, because P14.IFN-gamma(null) T cells adoptively transferred into H8 mice failed to induce disease. Moreover, Fas expression on hepatocytes from H8 recipient mice was increased after transfer of wild-type compared with P14.IFN-gamma(null) T cells, and wild-type P14 T cells expressed higher levels of FasL than P14 T cells lacking IFN-gamma. Thus, our data suggest that IFN-gamma released by activated CD8 T cells upon Ag contact facilitates liver cell destruction.     

Primary human immunodeficiency virus type 1 nef alleles show major differences in pathogenicity in transgenic mice

We previously reported that the human immunodeficiency virus type 1 NL4-3 Nef is necessary and sufficient to induce a severe AIDS-like disease in transgenic (Tg) mice when the protein is expressed under the regulatory sequences of the human CD4 gene. We have now assayed additional Nef alleles (SF2, JR-CSF, YU10x, and NL4-3 [T71R] Nef alleles), including some from long-term nonprogressors (AD-93, 032an, and 039nm alleles) in the same Tg system and compared their pathogenicities. All these Nef alleles downregulated cell surface CD4 in human cells in vitro and also, with the exception of Nef(YU10x), in Tg CD4(+) T cells. Depletion of double-positive and single-positive thymocytes occurred with all alleles but was less pronounced in Nef(YU10x) Tg mice. A loss of peripheral CD4(+) T cells was observed with all alleles but was minimal in Nef(YU10x) Tg mice. In Nef(032an) and Nef(SF2) Tg mice, T-cell loss was severe despite lower levels of Tg expression, suggesting a higher virulence of these alleles. All Nef alleles except the Nef(YU10x) and Nef(NL4-3(T71R)) alleles induced an enhanced activated memory (CD25(+) CD69(+) CD44(high) CD45RB(low) CD62L(low)) and apoptotic phenotype. Also, all could interact with and/or activate PAK2 except the Nef(JR-CSF) allele. Organ (lung and kidney) diseases were present in Nef(NL4-3(T71R)), Nef(032an), Nef(039nm), and Nef(SF2) Tg mice, despite very low levels of Tg expression for the last strain. However, no organ disease or minimal organ disease developed in Nef(YU10x) and Nef(AD-93) Tg mice and Nef(JR-CSF) Tg mice, respectively, despite high levels of Tg expression. Our data show that important differences in the pathogenicities of various Nef alleles can be scored in Tg mice. Interestingly, our results also revealed that some phenotypes can segregate independently, such as CD4(+) T-cell depletion and activation, as well as severe depletion of thymic CD4(+) T cells and peripheral CD4(+) T cells. Therefore, expression of Nef alleles in Tg mice under the CD4C regulatory elements represents a novel assay for measuring their pathogenicity. Because of the very high similarity of this murine AIDS-like disease to human AIDS, this assay may have a predictive value regarding the behavior of Nef in infected humans.