Lymphocytes In Rhesus Monkeys Research Articles

Long-Term Persistence of Increased Number of γH2AX+ Peripheral Blood Lymphocytes in Monkeys Exposed to Negative Factors of Space Flights: Ionizing Radiation and Simulated Hypogravity.

We studied the influence of ionizing radiation and hypogravity as negative factors of space flights on DNA damage in peripheral blood lymphocytes of rhesus monkeys at different times after exposure (from 1 to 446 days). The proportion of cells with high numbers of DNA double-strand breaks (DSB), positive for the surrogate DSB marker-protein γH2AX, was monitored using flow cytometry. Some animals were exposed to 7-day antiorthostatic hypokinesia simulating hypogravity, the others to a combined effect of antiorthostatic hypokinesia, whole-body γ-irradiation (2.34 cGy/h, dose 1 Gy), and irradiation of the head with 12C ions (450 MeV, dose 1 Gy). Exposure to antiorthostatic hypokinesia led to a significant increase in the proportion of γH2AX+ lymphocytes only on the first day after exposure, whereas after combined exposure, increased numbers of damaged lymphocytes were recorded up to 42 days after exposure.

Effects of transportation, relocation, and acclimation on phenotypes and functional characteristics of peripheral blood lymphocytes in rhesus monkeys (Macaca mulatta).

Nonhuman primates from domestic sources constitute a small, but critical, proportion of animals studied in research laboratories. Many of these nonhuman primates are raised at one facility and subsequently transported/relocated to another facility for research purposes. We examined the effects of transport, relocation, and acclimation on the phenotype and function of peripheral blood mononuclear cells (PBMCs) in a group of rhesus monkeys that were transported by road for approximately 21 hours from one facility to another. Using a panel of human antibodies and a set of standardized human immune assays, we evaluated the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of circulating cytokines and cortisol in plasma at various time points including immediately before transport, immediately upon arrival, and after approximately 30 days of acclimation. Analyses of blood samples revealed that CD3+ T-cell and CD20+ B-cell populations had decreased significantly immediately after relocation but had recovered within 30 days after arrival at the new facility. Similarly, circulating cortisol and cytokine levels in plasma were significantly higher immediately after relocation; and by the 30-day time point, these differences were no longer significant. However, immune assays of PBMCs indicated that mitogen-specific responses for proliferation, interferon γ (IFN-γ), and perforin were significantly higher after relocation and 30 days of acclimation. These findings have implications on the research participation of transported and relocated nonhuman primates in immunologic research studies, suggesting that 30 days is not sufficient to ensure return to baseline immune homeostasis. These data should be considered when planning research studies in order to minimize potential confounding factors associated with relocation and to maximize study validity.

EV71-infected CD14+ cells modulate the immune activity of T lymphocytes in rhesus monkeys

Preliminary studies of the major pathogen enterovirus 71 (EV71), a member of the Picornaviridae family, have suggested that EV71 may be a major cause of fatal hand, foot and mouth disease cases. Currently, the role of the pathological changes induced by EV71 infection in the immunopathogenic response remains unclear. Our study focused on the interaction between this virus and immunocytes and indicated that this virus has the ability to replicate in CD14+ cells. Furthermore, these EV71-infected CD14+ cells have the capacity to stimulate the proliferation of T cells and to enhance the release of certain functional cytokines. An adaptive immune response induced by the back-transfusion of EV71-infected CD14+ cells was observed in donor neonatal rhesus monkeys. Based on these observations, the proposed hypothesis is that CD14+ cells infected by the EV71 virus might modulate the anti-EV71 adaptive immune response by inducing simultaneous T-cell activation.Emerging Microbes & Infections (2013) 2, e44; doi:10.1038/emi.2013.44

Trace amine associated receptor 1 signaling in activated lymphocytes.

Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function.

Vaccine-Induced CD8+ T Lymphocytes of Rhesus Monkeys Recognize Variant Forms of an HIV Epitope but Do Not Mediate Optimal Functional Activity

The sequence diversity of HIV-1 presents a challenge for the development of an effective HIV-1 vaccine, because such a vaccine must confer protection against diverse forms of the virus. The present studies were initiated to explore how vaccine-induced clonal populations of CD8(+) T lymphocytes of rhesus monkeys recognize variants of an HIV-1 envelope epitope sequence. Evaluating a subset of variants of a selected epitope peptide that retain their binding to the MHC class I molecule of rhesus monkeys that presents this epitope peptide, we show that vaccine-elicited CD8(+) T lymphocytes comparably recognize the wild-type and a number of variant epitope peptides as determined by tetramer binding assays. In fact, the same clonal populations of CD8(+) T lymphocytes recognize the wild-type and variant epitope peptides. However, functional assays show that many of these variant epitope peptides stimulate suboptimal cytokine production by the vaccine-elicited CD8(+) T lymphocytes. These findings suggest that vaccine-induced CD8(+) T lymphocyte populations may recognize diverse forms of a viral epitope, but may not function optimally to confer protection against viruses expressing many of those variant sequences.

Dominant CD8+T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus

Emerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8(+) T-lymphocyte responses in Mamu-A*01(+) rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinated Mamu-A*01(+) rhesus monkeys with a SHIV Gag Deltap11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8(+) T-lymphocyte response in the absence of secondary Gag p11C-specific CD8(+) T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8(+) T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8(+) T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.

Blocking T cell co-stimulation using a CD80 blocking small molecule reduces delayed type hypersensitivity responses in rhesus monkeys

Blockade of co-stimulation signals between T cells and antigen-presenting cells could be an important approach for treatment of autoimmune diseases and transplant rejection. Recently a series of small compound inhibitors which bind human CD80 (B7-1) and inhibit T cell co-stimulation has been described. To investigate their potency for clinical use, one of these compounds, RhuDex, was evaluated for reactivity with rhesus monkey CD80. The in vitro biological effect on rhesus monkey lymphocytes, the potency for suppression of an inflammatory recall response and the protein-induced delayed type hypersensitivity (DTH) response in the skin were studied. In a rhesus monkey T cell co-stimulation assay RhuDex inhibited proinflammatory cytokine release and cellular proliferation with micromolar potency. Systemic administration of RhuDex to rhesus monkeys inhibited the DTH response significantly, indicating that this compound may inhibit autoimmune mediated inflammatory processes where the target, CD80, is up-regulated.

The Anti-Non-Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells Is Encoded by a Restricted Number of Germline Progenitors

Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into nonhuman primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgV(H) genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3-21 germline progenitor encode xenoantibodies directed at non-gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine alpha1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of 'anti-non-gal' xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti-non-gal xenoantibodies cross react with carbohydrate xenoantigens. The galactosyltransferase two gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti-non-gal xenoantibodies in primates are encoded by IgV(H) genes that are restricted to IGHV3-21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.

Use of an anti‐CD16 antibody for in vivo depletion of natural killer cells in rhesus macaques

Non-human primates serve as key animal models for a variety of viral infections. To evaluate the contribution of natural killer (NK) cells to the immune-mediated control of these viruses in macaque monkeys, we have described a method for depleting NK cells in vivo by administration of anti-human CD16 mouse monoclonal antibody. Using a fluorometric NK-cell cytotoxicity assay, we show that most NK-cell cytotoxicity in rhesus monkey peripheral blood mononuclear cells resides in the CD16(+) and/or CD159A(+) subset of lymphocytes. The anti-human CD16 antibody, 3G8, binds to subsets of rhesus monkey lymphocytes and monocytes but not to neutrophils. Intravenous administration of 10-50 mg/kg of 3G8 to normal rhesus monkeys resulted in anti-CD16 antibody persistence in the plasma for 1-3 weeks. This treatment also depleted 80-90% of CD3(-) CD159A(+) lymphocytes, putative NK cells, from blood for at least 1 week and was associated with the loss of NK-cell cytotoxicity when evaluated by in vitro assays. Using this method, transient depletion of NK cells from two rhesus monkeys chronically infected with simian immunodeficiency virus failed to cause changes in virus replication. These studies describe a non-human primate model for in vivo NK-cell depletion and suggest a limited role for cytotoxic CD16(+) NK cells in controlling AIDS virus replication during chronic infection.

Clonal Focusing of Epitope-Specific CD8+T Lymphocytes in Rhesus Monkeys following Vaccination and Simian-Human Immunodeficiency Virus Challenge

To afford the greatest possible immune protection, candidate human immunodeficiency virus (HIV) vaccines must generate diverse and long-lasting CD8(+) T lymphocyte responses. In the present study, we evaluate T-cell receptor Vbeta (variable region beta) gene usage and a CDR3 (complementarity-determining region 3) sequence to assess the clonality of epitope-specific CD8(+) T lymphocytes generated in rhesus monkeys following vaccination and simian-human immunodeficiency virus (SHIV) challenge. We found that vaccine-elicited epitope-specific CD8(+) T lymphocytes have a clonal diversity comparable to those cells generated in response to SHIV infection. Moreover, we show that the clonal diversity of vaccine-elicited CD8(+) T-lymphocyte responses is dictated by the epitope sequence and is not affected by the mode of antigen delivery to the immune system. Clonal CD8(+) T-lymphocyte populations persisted following boosting with different vectors, and these clonal cell populations could be detected for as long as 4 years after SHIV challenge. Finally, we show that the breadth of these epitope-specific T lymphocytes transiently focuses in response to intense SHIV replication. These observations demonstrate the importance of the initial immune response to SHIV, induced by vaccination or generated during primary infection, in determining the clonal diversity of cell-mediated immune responses and highlight the focusing of this clonal diversity in the setting of high viral loads. Circumventing this restricted CD8(+) T-lymphocyte clonal diversity may present a significant challenge in the development of an effective HIV vaccine strategy.

Resting naïve CD4 + T cells are massively infected and eliminated by X4-tropic simian–human immunodeficiency viruses in macaques

Unlike HIV-1 and simian immunodeficiency virus (SIV), which induce a slow, unrelenting loss of immune function spanning several years, highly pathogenic simian-human immunodeficiency viruses (SHIVs) induce a rapid, complete, and irreversible depletion of CD4(+) T lymphocytes in rhesus monkeys within weeks of infection, leading to death from immunodeficiency. We recently reported that, because these SHIVs exclusively use the CXCR4 coreceptor for cell entry, they target naive CD4(+) T cells for depletion in infected monkeys, whereas SIVs, which use CCR5, not CXCR4, cause the selective loss of memory CD4(+) T lymphocytes in vivo. Here we show both by DNA PCR analyses and infectivity assays, using live sorted CD4(+) T lymphocyte subsets, that 30-90% of circulating naive cells were productively infected by day 10 after inoculation. This result implies that direct cell killing, not bystander apoptosis, is responsible for the massive loss of CD4(+) T cells in the X4-tropic SHIV model. Furthermore, we directly demonstrate that >96% of virus producing cells did not express the Ki-67 proliferation marker on day 10 after inoculation using confocal microscopic analysis of lymph nodes samples. This finding is consistent with the prodigious levels of plasma viremia measured during acute X4-tropic SHIV infections of macaques being generated almost entirely by resting naive CD4(+) T cells.

Dysfunction of Simian Immunodeficiency Virus/Simian Human Immunodeficiency Virus-Induced IL-2 Expression by Central Memory CD4+ T Lymphocytes

Production of IL-2 and IFN-gamma by CD4+ T lymphocytes is important for the maintenance of a functional immune system in infected individuals. In the present study, we assessed the cytokine production profiles of functionally distinct subsets of CD4+ T lymphocytes in rhesus monkeys infected with pathogenic or attenuated SIV/simian human immunodeficiency virus (SHIV) isolates, and these responses were compared with those in vaccinated monkeys that were protected from immunodeficiency following pathogenic SHIV challenge. We observed that preserved central memory CD4+ T lymphocyte production of SIV/SHIV-induced IL-2 was associated with disease protection following primate lentivirus infection. Persisting clinical protection in vaccinated and challenged monkeys is thus correlated with a preserved capacity of the peripheral blood central memory CD4+ T cells to express this important immunomodulatory cytokine.

Variable NKG2 expression in the peripheral blood lymphocytes of rhesus monkeys

To provide a basis for beginning to explore the CD94/NKG2 family of molecules in rhesus monkeys, we sought to characterize the expression of these inhibitory and activating cell signalling molecules in peripheral blood mononuclear cells (PBMCs) from healthy rhesus monkeys. We developed and employed a semiquantitative polymerase chain reaction (PCR)-based assay to evaluate mRNA expression levels of nine NKG2 molecules in PBMCs from the monkeys. In addition to quantitating NKG2A, NKG2B, NKG2C2, NKG2C and NKG2D expression, mRNA expression of transmembrane-deleted forms of these molecules was also evaluated. Significant variability in NKG2 mRNA expression in the PBMCs was detected, with 15 unique NKG2 expression level profiles detected in a study of 15 monkeys. We also found that the ratio of the expressed levels of mRNA of the four NKG2 splice variants, NKG2A, NKG2B, NKG2ADeltatm, and NKG2BDeltatm, was variable between the monkeys as well as in an individual monkey over a period of 1.5 years. These findings indicate the dynamic nature of NKG2 mRNA expression in the rhesus monkey.

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys.

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.

A commonly recognized simian immunodeficiency virus Nef epitope presented to cytotoxic T lymphocytes of Indian-origin rhesus monkeys by the prevalent major histocompatibility complex class I allele Mamu-A*02.

The ability to monitor vaccine-elicited CD8(+) cytotoxic T-lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys has been limited by our knowledge of viral epitopes predictably presented to those lymphocytes by common rhesus monkey MHC class I alleles. We now define an SIV and SHIV Nef CTL epitope (YTSGPGIRY) that is presented to CD8(+) T lymphocytes by the common rhesus monkey MHC class I molecule Mamu-A*02. All seven infected Mamu-A*02(+) monkeys evaluated demonstrated this response, and peptide-stimulated interferon gamma Elispot assays indicated that the response represents a large proportion of the entire CD8(+) T-lymphocyte SIV- or SHIV-specific immune response of these animals. Knowledge of this epitope and MHC class I allele substantially increases the number of available rhesus monkeys that can be used for testing prototype HIV vaccines in this important animal model.

Detection of viral RNA in CD4(-)CD8(-) and CD4(-)CD8(+) lymphocytes in vivo in rhesus monkeys infected with simian immunodeficiency virus of macaques.

A definition of the specific cell types that support HIV replication early in the course of infection will be important for understanding AIDS pathogenesis and designing strategies for preventing infection. Observations have indicated that the population of lymphocytes susceptible to productive infection extends beyond activated CD4(+) T cells. To explore this issue, we have employed laser scanning cytometry technology and the techniques of lymphocyte surface immunophenotyping followed by fluorescent in situ hybridization to detect simian immunodeficiency virus of macaques (SIVmac) RNA in phenotypically defined rhesus monkey lymphocytes. The immunophenotype of productively infected cells in either a rhesus monkey T cell line or in PBMCs infected in vitro with SIVmac was remarkably similar to that observed in productively infected PBMCs obtained from monkeys during primary infection. We observed low levels or no detectable expression of CD4 on cells infected in vitro or on PBMCs of infected monkeys. However, a substantial number of SIVmac-infected PBMCs both in cultured lymphocytes and sampled directly from infected monkeys expressed CD8 but not CD4. These observations are consistent with the possibility that the CD4 molecule may be modulated off the surface of CD4(+)CD8(-) or CD4(+)CD8(+) lymphocytes after infection or that infection occurred via a CD4-independent mechanism. Moreover, there was no preferential expression of CD25 on cells positive for SIVmac RNA, which might have been predicted if replication of the virus was occurring selectively in activated lymphocytes. These results broaden the range of lymphocytes that support productive SIVmac infection to include CD4(-)CD8(-) and CD4(-)CD8(+) subsets, and are consistent with virus replication occurring in nonactivated cells.

Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans.

The highly pathogenic simian immunodeficiency virus/HIV type 1 (SHIV) chimeric virus SHIV(DH12R) induces a systemic depletion of CD4(+) T lymphocytes in rhesus monkeys during the initial 3-4 weeks of infection. Nonetheless, high levels of viral RNA production continue unabated for an additional 2-5 months. In situ hybridization and immunohistochemical analyses revealed that tissue macrophage in the lymph nodes, spleen, gastrointestinal tract, liver, and kidney sustain high plasma virus loads in the absence of CD4(+) T cells. Quantitative confocal immunofluorescence analysis indicated that greater than 95% of the virus-producing cells in these tissues are macrophage and less than 2% are T lymphocytes. Interestingly, the administration of a potent reverse transcriptase inhibitor blocked virus production during the early T cell phase but not during the later macrophage phase of the SHIV(DH12R) infection. When interpreted in the context of HIV-1 infections, these results implicate tissue macrophage as an important reservoir of virus in vivo. They become infected during the acute infection, gradually increase in number over time, and can be a major contributor to total body virus burden during the symptomatic phase of the human infection.

Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans

The highly pathogenic simian immunodeficiency virus/HIV type 1 (SHIV) chimeric virus SHIV(DH12R) induces a systemic depletion of CD4(+) T lymphocytes in rhesus monkeys during the initial 3-4 weeks of infection. Nonetheless, high levels of viral RNA production continue unabated for an additional 2-5 months. In situ hybridization and immunohistochemical analyses revealed that tissue macrophage in the lymph nodes, spleen, gastrointestinal tract, liver, and kidney sustain high plasma virus loads in the absence of CD4(+) T cells. Quantitative confocal immunofluorescence analysis indicated that greater than 95% of the virus-producing cells in these tissues are macrophage and less than 2% are T lymphocytes. Interestingly, the administration of a potent reverse transcriptase inhibitor blocked virus production during the early T cell phase but not during the later macrophage phase of the SHIV(DH12R) infection. When interpreted in the context of HIV-1 infections, these results implicate tissue macrophage as an important reservoir of virus in vivo. They become infected during the acute infection, gradually increase in number over time, and can be a major contributor to total body virus burden during the symptomatic phase of the human infection.

Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate and n-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.

Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.

Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.