RESUMEN
During chronic HIV and SIV infections, the majority of viral replication occurs within lymphoid follicles. In a pilot study, infusion of SIV-specific CD4-MBL-CAR-T cells expressing the follicular homing receptor, CXCR5, led to follicular localization of the cells and a reduction in SIV viral loads in rhesus macaques. However, the CAR-T cells failed to persist. We hypothesized that temporary disruption of follicles would create space for CAR-T cell engraftment and lead to increased abundance and persistence of CAR-T cells. In this study we treated SIV-infected rhesus macaques with CAR-T cells and preconditioned one set with anti-CD20 antibody to disrupt the follicles. We evaluated CAR-T cell abundance and persistence in four groups of SIVmac239-infected and ART-suppressed animals: untreated, CAR-T cell treated, CD20 depleted, and CD20 depleted/CAR-T cell treated. In the depletion study, anti-CD20 was infused one week prior to CAR-T infusion and cessation of ART. Anti-CD20 antibody treatment led to temporary depletion of CD20+ cells in blood and partial depletion in lymph nodes. In this dose escalation study, there was no impact of CAR-T cell infusion on SIV viral load. However, in both the depleted and non-depleted animals, CAR-T cells accumulated in and around lymphoid follicles and were Ki67+. CAR-T cells increased in number in follicles from 2 to 6 days post-treatment, with a median 15.2-fold increase in follicular CAR-T cell numbers in depleted/CAR-T treated animals compared to an 8.1-fold increase in non-depleted CAR-T treated animals. The increase in CAR T cells in depleted animals was associated with a prolonged elevation of serum IL-6 levels and a rapid loss of detectable CAR-T cells. Taken together, these data suggest that CAR-T cells likely expanded to a greater extent in depleted/CAR-T cell treated animals. Further studies are needed to elucidate mechanisms mediating the rapid loss of CAR-T cells and to evaluate strategies to improve engraftment and persistence of HIV-specific CAR-T cells. The potential for an inflammatory cytokine response appears to be enhanced with anti-CD20 antibody treatment and future studies may require CRS control strategies. These studies provide important insights into cellular immunotherapy and suggest future studies for improved outcomes.
Asunto(s)
Infecciones por VIH , Receptores Quiméricos de Antígenos , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Macaca mulatta , Receptores Quiméricos de Antígenos/uso terapéutico , Proyectos Piloto , Anticuerpos/uso terapéutico , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies.
Asunto(s)
Inmunoterapia , Receptores Quiméricos de Antígenos , Síndrome de Inmunodeficiencia Adquirida del Simio , Animales , Anticuerpos/uso terapéutico , Formación de Anticuerpos , Antígenos CD28 , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Virus de la Inmunodeficiencia de los SimiosRESUMEN
Vaccine strategies aimed at eliciting human immunodeficiency virus (HIV)-specific CD8+ T cells are one major target of interest in HIV functional cure strategies. We hypothesized that CD8+ T cells elicited by therapeutic vaccination during antiretroviral therapy (ART) would be recalled and boosted by treatment with the interleukin 15 (IL-15) superagonist N-803 after ART discontinuation. We intravenously immunized four simian immunodeficiency virus-positive (SIV+) Mauritian cynomolgus macaques receiving ART with vesicular stomatitis virus (VSV), modified vaccinia virus Ankara strain (MVA), and recombinant adenovirus serotype 5 (rAd-5) vectors all expressing SIVmac239 Gag. Immediately after ART cessation, these animals received three doses of N-803. Four control animals received no vaccines or N-803. The vaccine regimen generated a high-magnitude response involving Gag-specific CD8+ T cells that were proliferative and biased toward an effector memory phenotype. We then compared cells elicited by vaccination (Gag specific) to cells elicited by SIV infection and unaffected by vaccination (Nef specific). We found that N-803 treatment enhanced the frequencies of both bulk and proliferating antigen-specific CD8+ T cells elicited by vaccination and the antigen-specific CD8+ T cells elicited by SIV infection. In sum, we demonstrate that a therapeutic heterologous prime-boost-boost (HPBB) vaccine can elicit antigen-specific effector memory CD8+ T cells that are boosted by N-803. IMPORTANCE While antiretroviral therapy (ART) can suppress HIV replication, it is not a cure. It is therefore essential to develop therapeutic strategies to enhance the immune system to better become activated and recognize virus-infected cells. Here, we evaluated a novel therapeutic vaccination strategy delivered to SIV+ Mauritian cynomolgus macaques receiving ART. ART was then discontinued and we delivered an immunotherapeutic agent (N-803) after ART withdrawal with the goal of eliciting and boosting anti-SIV cellular immunity. Immunologic and virologic analysis of peripheral blood and lymph nodes collected from these animals revealed transient boosts in the frequency, activation, proliferation, and memory phenotype of CD4+ and CD8+ T cells following each intervention. Overall, these results are important in educating the field of the transient nature of the immunological responses to this particular therapeutic regimen and the similar effects of N-803 on boosting T cells elicited by vaccination or elicited naturally by infection.
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Linfocitos T CD8-positivos , Vacunas contra el SIDAS , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Proliferación Celular , Macaca mulatta/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Vacunación , Virus VacciniaRESUMEN
Despite mounting a robust antiviral CD8 T cell response to HIV infection, most infected individuals are unable to control HIV viral load without antiretroviral therapy (ART). Chimeric Ag receptor (CAR) T cell treatment is under intensive investigation as an alternative therapy for ART-free remission of chronic HIV infection. However, achieving durable remission of HIV will require a successful balance between CAR T cell effector function and persistence. CAR T cells with CD28 costimulatory domains have robust effector function but limited persistence in vivo, whereas CAR T cells with 4-1BB costimulatory domains present a more undifferentiated phenotype and greater in vivo persistence. We compared the in vitro phenotype and function of rhesus macaque and human CAR T cells that contained either the CD28 or 4-1BB costimulatory domain; both constructs also included CARs that are bispecific for gp120 of HIV or SIV and the CXCR5 moiety to promote in vivo homing of CAR/CXCR5 T cells to B cell follicles. Cells were transduced using a gammaretroviral vector and evaluated using flow cytometry. 4-1BB-CAR/CXCR5 T cells were phenotypically distinct from CD28-CAR/CXCR5 T cells and showed increased expression of CAR and CD95. Importantly, both CD28- and 4-1BB-CAR/CXCR5 T cells retained equal capacity to recognize and suppress SIV in vitro. These studies provide new insights into rhesus macaque and human 4-1BB- and CD28-bearing CAR T cells.
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Infecciones por VIH , Receptores Quiméricos de Antígenos , Virus de la Inmunodeficiencia de los Simios , Animales , Antivirales , Antígenos CD28 , Infecciones por VIH/terapia , Humanos , Macaca mulattaRESUMEN
During chronic human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection prior to AIDS progression, the vast majority of viral replication is concentrated within B cell follicles of secondary lymphoid tissues. We investigated whether infusion of T cells expressing an SIV-specific chimeric antigen receptor (CAR) and the follicular homing receptor, CXCR5, could successfully kill viral-RNA+ cells in targeted lymphoid follicles in SIV-infected rhesus macaques. In this study, CD4 and CD8 T cells from rhesus macaques were genetically modified to express antiviral CAR and CXCR5 moieties (generating CAR/CXCR5-T cells) and autologously infused into a chronically infected animal. At 2 days post-treatment, the CAR/CXCR5-T cells were located primarily in spleen and lymph nodes both inside and outside of lymphoid follicles. Few CAR/CXCR5-T cells were detected in the ileum, rectum, and lung, and no cells were detected in the bone marrow, liver, or brain. Within follicles, CAR/CXCR5-T cells were found in direct contact with SIV-viral RNA+ cells. We next infused CAR/CXCR5-T cells into ART-suppressed SIV-infected rhesus macaques, in which the animals were released from ART at the time of infusion. These CAR/CXCR5-T cells replicated in vivo within both the extrafollicular and follicular regions of lymph nodes and accumulated within lymphoid follicles. CAR/CXR5-T cell concentrations in follicles peaked during the first week post-infusion but declined to undetectable levels after 2 to 4 weeks. Overall, CAR/CXCR5-T cell-treated animals maintained lower viral loads and follicular viral RNA levels than untreated control animals, and no outstanding adverse reactions were noted. These findings indicate that CAR/CXCR5-T cell treatment is safe and holds promise as a future treatment for the durable remission of HIV.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores CXCR5/inmunología , Receptores Quiméricos de Antígenos/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Linfocitos B/inmunología , Centro Germinal/inmunología , Humanos , Inmunoterapia , Ganglios Linfáticos/inmunología , Macaca mulatta , ARN Viral , Receptores CXCR5/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Carga ViralRESUMEN
BACKGROUND: The increasing prevalence and expanding geographical range of the chronic wasting disease (CWD) panzootic in cervids is threatening human, animal, environmental and economic health. The pathogenesis of CWD in cervids is, however, not well understood. We used RNA sequencing (RNA-seq) to compare the brain transcriptome from white-tailed deer (WTD; Odocoileus virginianus) clinically affected with CWD (n = 3) to WTD that tested negative (n = 8) for CWD. In addition, one preclinical CWD+ brain sample was analyzed by RNA-seq. RESULTS: We found 255 genes that were significantly deregulated by CWD, 197 of which were upregulated. There was a high degree of overlap in differentially expressed genes (DEGs) identified when using either/both the reference genome assembly of WTD for mapping sequenced reads to or the better characterized genome assembly of a closely related model species, Bos taurus. Quantitative PCR of a subset of the DEGs confirmed the RNA-seq data. Gene ontology term enrichment analysis found a majority of genes involved in immune activation, consistent with the neuroinflammatory pathogenesis of prion diseases. A metagenomic analysis of the RNA-seq data was conducted to look for the presence of spiroplasma and other bacteria in CWD infected deer brain tissue. CONCLUSIONS: The gene expression changes identified highlight the role of innate immunity in prion infection, potential disease associated biomarkers and potential targets for therapeutic agents. An association between CWD and spiroplasma infection was not found.
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Ciervos , Priones , Enfermedad Debilitante Crónica , Animales , Bovinos , Ciervos/genética , Humanos , Transcriptoma , Enfermedad Debilitante Crónica/genéticaRESUMEN
HIV-specific chimeric antigen receptor (CAR) T cells that target lymphoid follicles have the potential to functionally cure HIV infection. CD8+ T cells, NK cells, or peripheral blood mononuclear cells (PBMC) may be modified to express HIV-specific CARs as well as follicular homing molecules such as CXCR5 to target the virally infected T follicular helper cells that concentrate within B cell follicles during HIV infection. This chapter outlines methods utilizing a simian immunodeficiency virus (SIV) rhesus macaque model of HIV to produce transduced T cells from primary PBMCs. Methods are presented for production of an SIV-specific CAR/CXCR5-encoding retrovirus used to transduce primary rhesus macaque PBMCs. Procedures to evaluate the functionality of the expanded CAR/CXCR5 T cells in vitro and ex vivo are also presented. An in vitro migration assay determines the ability of the T cells expressing CAR/CXCR5 to migrate to the CXCR5 ligand CXCL13, while an ex vivo migration assay allows measurement of the transduced T cell migration into the B cell follicle. Antiviral activity of the CAR/CXCR5 transduced T cells is determined using a viral suppression assay. These methods can be used to produce T cells for immunotherapy in SIV-infected rhesus macaques and to evaluate the functionality of the cells prior to infusion. Similar procedures can be used to produce HIV-specific CAR/CXCR5 T cells.
Asunto(s)
Virus de la Inmunodeficiencia de los Simios , Linfocitos T , Animales , Linfocitos T CD8-positivos , Infecciones por VIH , Leucocitos Mononucleares , Macaca mulatta , Receptores CXCR5/genéticaRESUMEN
A vaccine to ameliorate cytomegalovirus (CMV)-related pathogenicity in transplantation patients is considered a top priority. A therapeutic vaccine must include components that elicit both neutralizing antibodies, and highly effective CD8 T-cell responses. The most important translational model of vaccine development is the captive-bred rhesus macaque (Macaca mulatta) of Indian origin. There is a dearth of information on rhesus cytomegalovirus (rhCMV)-specific CD8 T cells due to the absence of well-defined CD8 T-cell epitopes presented by classical MHC-I molecules. In the current study, we defined two CD8 T-cell epitopes restricted by high-frequency Mamu alleles: the Mamu-A1*002:01 restricted VY9 (VTTLGMALY aa291-299) epitope of protein IE-1, and the Mamu-A1*008:01 restricted NP8 (NPTDRPIP aa96-103) epitope of protein phosphoprotein 65-2. We developed tetramers and determined the level, phenotype, and functional capability of the two epitope-specific T-cell populations in circulation and various tissues. We demonstrated the value of these tetramers for in situ tetramer staining. Here, we first provided critical reagents and established a flow cytometric staining strategy to study rhCMV-specific T-cell responses in up to 40% of captive-bred rhesus macaques. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
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Infecciones por Citomegalovirus , Virus de la Inmunodeficiencia de los Simios , Animales , Linfocitos T CD8-positivos , Citomegalovirus , Epítopos de Linfocito T , Antígenos de Histocompatibilidad Clase I , Humanos , Inmunofenotipificación , Macaca mulattaRESUMEN
Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.
Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Técnicas de Cultivo de Célula , Supervivencia Celular , Inmunoterapia Adoptiva , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Fenotipo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/metabolismo , Transducción GenéticaRESUMEN
Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.
Asunto(s)
Antirretrovirales/administración & dosificación , Linfocitos B/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Interleucina-15/antagonistas & inhibidores , Células Asesinas Naturales/efectos de los fármacos , Proteínas/administración & dosificación , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/fisiopatología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Interleucina-15/genética , Interleucina-15/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Macaca mulatta , Proteínas Recombinantes de Fusión , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus/efectos de los fármacosRESUMEN
Chimeric antigen receptor (CAR)-T cells show great promise in treating cancers and viral infections. However, most protocols developed to expand T cells require relatively long periods of time in culture, potentially leading to progression toward populations of terminally differentiated effector memory cells. Here, we describe in detail a 9-day protocol for CAR gene transduction and expansion of primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Cells produced and expanded with this method show high levels of viability, high levels of co-expression of two transduced genes, retention of the central memory phenotype, and sufficient quantity for immunotherapeutic infusion of 1-2 × 108 cells/kg in a 10 kg rhesus macaque. This 9-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches to decrease culture time and increase maintenance of central memory populations.
RESUMEN
Paliperidone palmitate is a second generation antipsychotic, approved for the treatment of schizophrenia in the form of the long-acting injectable (LAI) products INVEGA SUSTENNA® (once monthly injection) and INVEGA TRINZA® (once every 3 months injection). Paliperidone palmitate dissolves slowly after deep intramuscular injection before being hydrolyzed to paliperidone and absorbed into the systemic circulation. The pharmacokinetic (PK) profile of the INVEGA SUSTENNA® formulation is biphasic, comprised of an initial relatively fast zero-order input, which allows rapid attainment of therapeutic concentrations without oral supplementation; and a subsequent maintained second-stage, first-order input, allowing for once monthly administration. Changes to the manufacturing processes can substantially alter the release characteristics of paliperidone palmitate LAI and consequently its PK profile. As an example, larger or smaller particle sizes of paliperidone palmitate can result in a delayed or accelerated release of paliperidone into the systemic circulation, respectively. Such changes are clinically relevant, as transient excursions above therapeutic plasma concentrations can be associated with an increased risk of adverse effects, including tachycardia, hypotension, QT prolongation, and extrapyramidal symptoms. Conversely, a delay in attaining therapeutic plasma concentrations of paliperidone on initiation of treatment, or a return to low plasma concentrations before the end of a dosing interval during repeated dosing, increases the risk of relapse. Given the integral relationship of the PK profile to the product's clinical effects, it is important to have bioequivalence standards that reflect the complexity of the paliperidone palmitate LAI PK profile if one is to consider therapeutic equivalence based on simple bioequivalence testing. Although both the EMA and U.S. FDA have product-specific guidelines to determine bioequivalence, their requirements differ substantially. In Canada, no LAI product-specific bioequivalence guidance exists for multiphasic medication delivery systems, and the recently revised Comparative Bioavailability Standards: Formulations Used for Systemic Effects guidance applies only to oral and non-injectable formulations. We recommend that new Canadian standards be developed for multiphasic and biphasic intramuscular / subcutaneous (IM/SC) products, including paliperidone palmitate LAI products, because, similar to modified-release oral dosage forms, a different PK profile in modified-release IM/SC products can result in clinically meaningful differences in safety, efficacy, and tolerability. To ensure bioequivalence for both newly initiated and switch patients, this paper proposes bioequivalence standards that could be adopted in Canada that include two studies, a multiple-dose cross-over study, and a single-dose study with partial AUC metrics.
Asunto(s)
Antipsicóticos/farmacocinética , Palmitato de Paliperidona/farmacocinética , Antipsicóticos/administración & dosificación , Antipsicóticos/uso terapéutico , Canadá , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Humanos , Inyecciones Intramusculares , Palmitato de Paliperidona/administración & dosificación , Palmitato de Paliperidona/uso terapéutico , Esquizofrenia/tratamiento farmacológico , Suspensiones/administración & dosificación , Suspensiones/farmacocinética , Suspensiones/uso terapéutico , Equivalencia TerapéuticaRESUMEN
The development of in situ major histocompatibility complex (MHC) tetramer (IST) staining to detect antigen (Ag)-specific T cells in tissues has radically revolutionized our knowledge of the local cellular immune response to viral and bacterial infections, cancers, and autoimmunity. IST combined with immunohistochemistry (IHC) enables determination of the location, abundance, and phenotype of T cells, as well as the characterization of Ag-specific T cells in a 3-dimensional space with respect to neighboring cells and specific tissue locations. In this review, we discuss the history of the development of IST combined with IHC. We describe various methods used for IST staining, including direct and indirect IST and IST performed on fresh, lightly fixed, frozen, and fresh then frozen tissue. We also describe current applications for IST in viral and bacterial infections, cancer, and autoimmunity. IST combined with IHC provides a valuable tool for studying and tracking the Ag-specific T cell immune response in tissues.
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Epítopos de Linfocito T/inmunología , Complejo Mayor de Histocompatibilidad , Multimerización de Proteína , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/inmunología , Linfocitos T/metabolismo , Humanos , Inmunohistoquímica , Sensibilidad y Especificidad , Coloración y EtiquetadoRESUMEN
CD8+ T cells play an important role in controlling of HIV and SIV infections. However, these cells are largely excluded from B cell follicles where HIV and SIV producing cells concentrate during chronic infection. It is not known, however, if antigen-specific CD8+ T cells are excluded gradually as pathogenesis progresses from early to chronic phase, or this phenomenon occurs from the beginning infection. In this study we determined that SIV-specific CD8+ T cells were largely excluded from follicles during early infection, we also found that within follicles, they were entirely absent in 60% of the germinal centers (GCs) examined. Furthermore, levels of SIV-specific CD8+ T cells in follicular but not extrafollicular areas significantly correlated inversely with levels of viral RNA+ cells. In addition, subsets of follicular SIV-specific CD8+ T cells were activated and proliferating and expressed the cytolytic protein perforin. These studies suggest that a paucity of SIV-specific CD8+ T cells in follicles and complete absence within GCs during early infection may set the stage for the establishment of persistent chronic infection.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Centro Germinal/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Enfermedad Aguda , Animales , Linfocitos B/fisiología , Linfocitos T CD8-positivos/metabolismo , Centro Germinal/inmunología , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Virus de la Inmunodeficiencia de los Simios/inmunología , Carga Viral/inmunología , Replicación ViralRESUMEN
Data from SIV-infected macaques indicate that virus-specific cytotoxic T lymphocytes (CTL) are mostly present in the extrafollicular (EF) compartment of the lymphoid tissue, with reduced homing to the follicular (F) site. This contributes to the majority of the virus being present in the follicle and represents a barrier to virus control. Using mathematical models, we investigate these dynamics. Two models are analyzed. The first assumes that CTL can only become stimulated and expand in the extrafollicular compartment, with migration accounting for the presence of CTL in the follicle. In the second model, follicular CTL can also undergo antigen-induced expansion. Consistent with experimental data, both models predict increased virus compartmentalization in the presence of stronger CTL responses and lower virus loads, and a more pronounced rise of extrafollicular compared to follicular virus during CD8 cell depletion experiments. The models, however, differ in other aspects. The follicular expansion model results in dynamics that promote the clearance of productive infection in the extrafollicular site, with any productively infected cells found being the result of immigration from the follicle. This is not observed in the model without follicular CTL expansion. The models further predict different consequences of introducing engineered, follicular-homing CTL, which has been proposed as a therapeutic means to improve virus control. Without follicular CTL expansion, this is predicted to result in a reduction of virus load in both compartments. The follicular CTL expansion model, however, makes the counter-intuitive prediction that addition of F-homing CTL not only results in a reduction of follicular virus load, but also in an increase in extrafollicular virus replication. These predictions remain to be experimentally tested, which will be relevant for distinguishing between models and for understanding how therapeutic introduction of F-homing CTL might impact the overall dynamics of the infection.
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Interacciones Huésped-Patógeno/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Linfocitos T Citotóxicos , Animales , Biología Computacional , Macaca , Modelos Inmunológicos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virologíaRESUMEN
There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh) located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV)-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh) cells using antiviral chimeric antigen receptor (CAR) T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure) of HIV and SIV infections.
Asunto(s)
Linfocitos B/inmunología , Receptores Quiméricos de Antígenos , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/inmunología , Transducción Genética , Replicación Viral/inmunología , Animales , Linfocitos B/patología , Quimiocina CXCL13/genética , Quimiocina CXCL13/inmunología , Gammaretrovirus , VIH-1/genética , VIH-1/inmunología , Macaca mulatta , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Linfocitos T/patología , Replicación Viral/genéticaRESUMEN
The majority of HIV replication occurs in secondary lymphoid organs (SLOs) such as the spleen, lymph nodes, and gut-associated lymphoid tissue. Within SLOs, HIV RNA+ cells are concentrated in the B-cell follicle during chronic untreated infection, and emerging data suggest that they are a major source of replication in treated disease as well. The concentration of HIV RNA+ cells in the B-cell follicle is mediated by several factors. Follicular CD4+ T-cell subsets including T-follicular helper cells and T-follicular regulatory cells are significantly more permissive to HIV than extrafollicular subsets. The B cell follicle also contains a large reservoir of extracellular HIV virions, which accumulate on the surface of follicular dendritic cells (FDCs) in germinal centers. FDC-bound HIV virions remain infectious even in the presence of neutralizing antibodies and can persist for months or even years. Moreover, the B-cell follicle is semi-immune privileged from CTL control. Frequencies of HIV- and SIV-specific CTL are lower in B-cell follicles compared to extrafollicular regions as the majority of CTL do not express the follicular homing receptor CXCR5. Additionally, CTL in the B-cell follicle may be less functional than extrafollicular CTL as many exhibit the recently described CD8 T follicular regulatory phenotype. Other factors may also contribute to the follicular concentration of HIV RNA+ cells. Notably, the contribution of NK cells and γδ T cells to control and/or persistence of HIV RNA+ cells in secondary lymphoid tissue remains poorly characterized. As HIV research moves increasingly toward the development of cure strategies, a greater understanding of the barriers to control of HIV infection in B-cell follicles is critical. Although no strategy has as of yet proven to be effective, a range of novel therapies to address these barriers are currently being investigated including genetically engineered CTL or chimeric antigen receptor T cells that express the follicular homing molecule CXCR5, treatment with IL-15 or an IL-15 superagonist, use of bispecific antibodies to harness the killing power of the follicular CD8+ T cell population, and disruption of the follicle through treatments such as rituximab.
Asunto(s)
Linfocitos B/virología , Centro Germinal/citología , Infecciones por VIH/inmunología , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos B/inmunología , Células Dendríticas Foliculares/virología , Humanos , Linfocitos Intraepiteliales/inmunología , Células Asesinas Naturales/inmunología , Replicación ViralRESUMEN
Sequestering of latent HIV in follicular helper T cells within B-cell follicles that largely exclude cytotoxic T cells is a major barrier to cellular immune-based approaches to eradicate HIV. Here, we show that the clinical-grade human interleukin-15 (IL-15) superagonist ALT-803 activates and redirects simian immunodeficiency virus (SIV)-specific CD8+ T cells from the peripheral blood into B-cell follicles. In agreement with the increased trafficking of SIV-specific cytotoxic T cells to sites of cryptic viral replication, lymph nodes of elite controlling macaques contained fewer cells expressing SIV RNA or harboring SIV DNA post-ALT-803 treatment. These data establish ALT-803 as an immunotherapeutic for HIV and other chronic viral pathogens that evade host immunity by persisting in B-cell follicles.
Asunto(s)
Linfocitos B/virología , Linfocitos T CD8-positivos/inmunología , Proteínas/uso terapéutico , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , VIH/efectos de los fármacos , Humanos , Evasión Inmune/efectos de los fármacos , Interleucina-15/agonistas , Macaca/virología , Proteínas Recombinantes de Fusión , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Tissue-resident memory (TRM) CD8+ T-cells are non-recirculating, long-lived cells housed in tissues that can confer protection against mucosal pathogens. Human immunodeficiency virus-1 (HIV-1) is a mucosal pathogen and the gastrointestinal tract is an important site of viral pathogenesis and transmission. Thus, CD8+ TRM cells may be an important effector subset for controlling HIV-1 in mucosal tissues. This study sought to determine the abundance, phenotype, and functionality of CD8+ TRM cells in the context of chronic HIV-1 infection. We found that the majority of rectosigmoid CD8+ T-cells were CD69+CD103+S1PR1- and T-betLowEomesoderminNeg, indicative of a tissue-residency phenotype similar to that described in murine models. HIV-1-specific CD8+ TRM responses appeared strongest in individuals naturally controlling HIV-1 infection. Two CD8+ TRM subsets, distinguished by CD103 expression intensity, were identified. CD103Low CD8+ TRM primarily displayed a transitional memory phenotype and contained HIV-1-specific cells and cells expressing high levels of Eomesodermin, whereas CD103High CD8+ TRM primarily displayed an effector memory phenotype and were EomesoderminNeg. These findings suggest a large fraction of CD8+ T-cells housed in the human rectosigmoid mucosa are tissue-resident and that TRM contribute to the anti-HIV-1 immune response. Further exploration of CD8+ TRM will inform development of anti-HIV-1 immune-based therapies and vaccines targeted to the mucosa.