Service Delivery Considerations for Introducing New Injectable Contraceptives Lasting 4 and 6 Months in Nigeria and Uganda: A Qualitative Study.

BACKGROUND: New family planning (FP) product introduction requires understanding the target market and support from stakeholders from across the health sector. We aimed to understand the perspectives of FP providers and other stakeholders on the potential introduction of new subcutaneous (SC) depot medroxyprogesterone acetate (DMPA) injectable contraceptives lasting 4 and 6 months in Nigeria and Uganda. METHODS: Between July 2021 and February 2022, we conducted 48 in-depth interviews (IDIs) and 11 focus group discussions (FGDs) with FP providers and other stakeholders involved with service delivery, program management, and policymaking in Lagos and Abuja in Nigeria and Kampala and Luwero in Uganda. IDIs and FGDs explored respondents' reactions to and preferences for the new injectables lasting 4 and 6 months. RESULTS: Most respondents liked the idea of longer-acting DMPA-SC products, noting the potential for reduced facility visits for clients and workloads for providers, cost savings for users and the health system, and potential for improved commodity logistics. Some nonproviders raised concerns about confusion among providers and clients with the availability of multiple injectable products; however, providers did not share this concern. The greatest interest among all groups was for the 6-month injectable, even without the option for self-injection. Several respondents reported that self-injection is not widespread in either context, and some noted that contact with a provider would be important for products with longer durations. Respondents' acceptability of the new injectables assumed that side effects would be no worse than the existing 3-month product. CONCLUSIONS: Family planning stakeholders in Nigeria and Uganda are supportive of expanding the method mix with new injectables, which they see as having the potential to meet the needs of more users. Concerted engagement of health providers, policymakers, and the community will be necessary for successful introduction once these new contraceptive products are available.

Using Human-Centered Design to Explore Potential Users' and Men's Views of New Injectable Contraceptives in Kampala and Lagos.

BACKGROUND: Injectable contraceptives are the most used method in sub-Saharan Africa. We conducted market research to assess potential user attitudes toward 4- and 6-month injectables. We also present user suggestions for marketing these new injectables once they are available. METHODS: We implemented a 2-phase market research study from October through December 2021 in Kampala, Uganda, and Lagos, Nigeria. We conducted 11 focus group discussions (FGDs) with 51 participants in Kampala and 12 FGDs with 67 participants in Lagos. FGDs included current and potential injectable users and men stratified by marital status and age. Next, 23 women in Kampala and 24 in Lagos participated in cocreation workshops using human-centered design methods to explore marketing and communications strategies for each injectable. Data collection teams completed semistructured data extraction tables that were then analyzed thematically. RESULTS: Participants liked both injectable options due to the reduced number of facility visits that would save time and money and increase privacy. Primary concerns included side effects, delayed return to fertility, cost, self-efficacy to self-inject, and stock-outs. Participants in Kampala preferred a shorter reinjection window (or "grace period") because it is easier to remember and they assumed it meant a quicker return to fertility, but participants in Lagos preferred a longer window because it provides extra time for reinjection. Citing norms around women needing to get pregnant quickly after marriage, participants in both sites felt that the 4-month injectable would benefit young people with busy lifestyles or limited access to facilities, whereas the 6-month injectable would benefit women who already had children. CONCLUSIONS: We found that participants in Kampala and Lagos would prefer additional injectable options to meet the wide-ranging needs of users in different stages of their reproductive lives. Family planning program planners can apply the marketing insights we identified when these new injectables become available.

Exploring public perceptions of vaccine-derived poliovirus and a novel oral polio vaccine in the Democratic Republic of the Congo, Kenya, and Nigeria.

BACKGROUND: The Global Polio Eradication Initiative introduced novel oral polio vaccine Type 2 (nOPV2) to address circulating vaccine-derived poliovirus Type 2 (cVDPV2). Although nOPV2 is a more genetically stable vaccine, it may not have the immediate trust of communities and health workers due to its novelty, potential side effects, and introduction under an Emergency Use Listing (EUL). We explored how nOPV2 introduction might be perceived by stakeholders and identified communications barriers related to nOPV2 hesitancy. METHODS: This work was conducted in the Democratic Republic of the Congo, Kenya, and Nigeria between January and March 2020. We used a rapid qualitative approach to conduct focus group discussions and in-depth interviews with four stakeholder groups: caregivers of children under 5, polio frontline workers, healthcare practitioners, and social/health influencers. Data are presented according to awareness, attitudes/beliefs, and concerns about cVDPV2 and nOPV2. RESULTS: Stakeholders were largely unaware of cVDPV2. The causes of recent polio outbreaks were characterized as poor sanitation, under-immunization/in-migration, or poor vaccine management procedures. Caregivers were aware of and concerned by repeated vaccination campaigns. All stakeholder groups anticipated initial hesitancy, fear, and suspicion from caregivers due to nOPV2 introduction, with primary concerns linked to vaccine testing, safety, effectiveness, side effects, and support from authorities. Stakeholders thought the term "genetic modification" could be controversial but that introduction under an EUL would be acceptable given the emergency nature of cVDPV2 outbreaks. Stakeholders called for adequate and timely information to counter concerns. CONCLUSIONS: Despite initial concerns, stakeholders felt nOPV2 would ultimately be accepted by caregivers. However, public health officials have a small window for "getting things right" when introducing nOPV2. Strategic communication interventions addressing key concerns and targeted communications with stakeholder groups, especially frontline workers, could improve community acceptance of nOPV2.

Working Safely with African Swine Fever Virus.

Risk assessment is the cornerstone of working safely with biological agents. The World Health Organization (WHO) Laboratory Biosafety Manual Fourth Edition Monograph on Risk Assessment provides stepwise guidance for carrying out a risk assessment, from gathering information and identifying hazards to evaluating the risks and developing and implementing controls and review.To support the development of a mature safety culture within laboratories, it is important that all staff who handle biological agents understand the process of risk assessment and receive training in identifying hazards and mitigating risk. All personnel can partake in risk assessments, and the guidance is written in such a way that it is applicable to all-not just to biosafety professionals, laboratory scientists, or facility managers.Here we take the guidance from WHO and apply the principles of risk assessment to working with ASFV, illustrating the process using an example activity-the passage of low titer ASFV in cell culture. We discuss other techniques and protocols that you may need to consider when working with ASFV.

What About Methods for Men? A Qualitative Analysis of Attitudes Toward Male Contraception in Burkina Faso and Uganda.

CONTEXT: Male contraceptive options are limited; however, product development efforts tend to focus on female methods. Research on attitudes toward methods for men-particularly in regions of low contraceptive prevalence, such as Sub-Saharan Africa-could inform the development of new male methods. METHODS: Qualitative data were taken from focus group discussions with 80 men aged 23-67 and 398 women aged 15-50 conducted in Burkina Faso and Uganda in 2016. Transcripts were analyzed thematically to explore support among men and women for male contraceptive methods, and to extract suggestions about ideal method characteristics. RESULTS: Male and female participants in both countries expressed support for new male contraceptive options; more positive attitudes were expressed in Uganda than in Burkina Faso. Participants of both sexes recognized that male methods could reduce the family planning burden on women and offer men greater control over their fertility; however, some had concerns about side effects and thought that men would not use contraceptives. Relationship characteristics, such as polygamous unions, were cited as possible challenges. In both countries, various delivery methods (e.g., creams or jellies, injections and implants) and durations (from short-acting to permanent) were proposed. CONCLUSIONS: The acceptability of new male methods among most participants in the two countries indicates a potential demand for male contraceptives. Options should include a variety of method characteristics to maximize choice, engage men, and support men and women's contraceptive needs.

RESUMEN Contexto: Las opciones de anticonceptivos masculinos son limitadas; sin embargo, los esfuerzos de desarrollo de productos tienden a enfocarse en los métodos femeninos. La investigación sobre las actitudes hacia los métodos para hombres, ­ particularmente en las regiones de baja prevalencia de anticoncepción, como el África subsahariana­ podría dar sustento al desarrollo de nuevos métodos masculinos. Métodos: Se tomaron datos cualitativos a partir de discusiones de grupos focales que se llevaron a cabo en Burkina Faso y Uganda en 2016, con la participación de 80 hombres de 23 a 67 años y de 398 mujeres de 15 a 50 años. Las transcripciones se analizaron temáticamente para explorar el apoyo de hombres y mujeres a los métodos anticonceptivos masculinos, así como para extraer sugerencias sobre las características ideales del método. Resultados: Los participantes masculinos y femeninos en ambos países expresaron su apoyo a las nuevas opciones de anticonceptivos masculinos; se expresaron más actitudes positivas en Uganda que en Burkina Faso. Los participantes de ambos sexos reconocieron que los métodos masculinos podrían reducir la carga de planificación familiar para las mujeres y ofrecer a los hombres un mayor control sobre su fecundidad; sin embargo, algunos participantes plantearon sus preocupaciones sobre los efectos secundarios y consideraron que los hombres no usarían anticonceptivos. Las características de la relación, como las uniones polígamas, se mencionaron como posibles desafíos. En ambos países, se propusieron varios tipos de métodos anticonceptivos (por ejemplo, cremas o jaleas, la inyección y el implante) y de distintas duraciones (de acción corta a permanente). Conclusiones: La aceptabilidad de los nuevos métodos masculinos en la mayoría de los participantes en los dos países indica una demanda potencial de anticoncepción masculina. Las opciones deberían incluir una variedad de características del método para maximizar la elección, involucrar a los hombres y apoyar las necesidades de anticonceptivos de hombres y mujeres.

RÉSUMÉ Contexte: Les options contraceptives masculines ne sont guère nombreuses. Le fait est, cependant, que les efforts de développement de produits se concentrent généralement sur les méthodes féminines. L'étude des attitudes à l'égard des méthodes masculines ­ en particulier dans les régions à faible prévalence contraceptive telles que l'Afrique subsaharienne ­ permettrait d'éclairer le développement de nouvelles méthodes pour les hommes. Méthodes: Les données qualitatives requises ont été extraites de discussions de groupe menées en 2016 avec 80 hommes âgés de 23 à 67 ans et 398 femmes âgées de 15 à 50 ans au Burkina Faso et en Ouganda. Elles ont été transcrites et analy-sées thématiquement pour examiner le soutien des hommes et des femmes à l'égard des méthodes contraceptives masculines et en dégager les suggestions possibles sur les caractéristiques des méthodes idéales. Résultats: Dans les deux pays, les participants et participantes ont exprimé leur appui de nouvelles options de contraception masculine; plus d'attitudes positives ont été exprimées en Ouganda qu'au Burkina Faso. Les participants des deux sexes ont reconnu que les méthodes masculines pourraient alléger la charge de la planification familiale portée par les femmes et offrir aux hommes un meilleur contrôle de leur fécondité. Certains s'inquiétaient cependant des effets secondaires et pensaient que les hommes n'utiliseraient pas les contraceptifs. Les caractéristiques de relation, telles que les unions polygames, ont été citées comme difficultés possibles. Dans les deux pays, différentes méthodes (par exemple, crèmes ou gels, injection ou implant) et durées (de courte à permanente) ont été proposées. Conclusions: L'acceptabilité de nouvelles méthodes masculines aux yeux de la plupart des participants dans les deux pays révèle une demande potentielle de contraception masculine. Les options proposées doivent inclure diverses caractéristiques de méthode pour maximiser le choix, engager les hommes et soutenir les hommes et les femmes dans leurs besoins contraceptifs.

Rapid identification of novel functional promoters for gene therapy.

Transcriptional control of transgene expression is crucial to successful gene therapy, yet few promoter/enhancer combinations have been tested in clinical trials. We created a simple, desktop computer database and populated it with promoter sequences from publicly available sources. From this database, we rapidly identified novel CpG-free promoter sequences suitable for use in non-inflammatory, non-viral in vivo gene transfer. In a simple model of lung gene transfer, five of the six promoter elements selected, chosen without prior knowledge of their transcriptional activities, directed significant transgene expression. Each of the five novel promoters directed transgene expression for at least 14 days post-delivery, greatly exceeding the duration achieved with the commonly used CpG-rich viral enhancer/promoters. Novel promoter activity was also evaluated in a more clinically relevant model of aerosol-mediated lung gene transfer and in the liver following delivery via high-pressure tail vein injection. In each case, the novel CpG-free promoters exhibited higher and/or more sustained transgene expression than commonly used CpG-rich enhancer/promoter sequences. This study demonstrates that novel CpG-free promoters can be readily identified and that they can direct significant levels of transgene expression. Furthermore, the database search criteria can be quickly adjusted to identify other novel promoter elements for a variety of transgene expression applications.

CpG-free plasmid expression cassettes for cystic fibrosis gene therapy.

Clinical studies are underway for the aerosol delivery of plasmid DNA complexed with Genzyme Lipid GL67A to the lungs of patients with cystic fibrosis (CF). Plasmid vectors contain several functional elements all of which play a role in determining the efficacy of the final clinical product. To optimise the final plasmid, variations of CpG-free 5' enhancer elements and 3'UTR regions were inserted into a common CpG-free, plasmid backbone containing Luciferase or CFTR transgenes. Plasmids were compared in immortalised cell culture, human airway liquid interface primary cell cultures, and mouse lung models to determine which design directed optimal transgene expression. Following aerosol delivery to mouse lung, plasmids containing the murine CMV enhancer showed higher peak Luciferase activity than the human CMV enhancer, but the human version resulted in persistent expression. In cell culture, the SV40 3'UTR and a novel BGH2 3'UTR exhibited up to 20-fold higher Luciferase activity than the commonly used BGH 3'UTR, but in mouse lung aerosol studies the activity and duration was greater for BGH 3'UTR. Systematic evaluation of each functional component of the plasmid has resulted in an improved design, exhibiting superior levels and duration of lung gene expression.

The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes.

Aerosol gene therapy offers great potential for treating acquired and inherited lung diseases. For treatment of chronic lung diseases such as cystic fibrosis, asthma and emphysema, non-viral gene therapy will likely require repeated administration to maintain transgene expression in slowly dividing, or terminally differentiated, lung epithelial cells. When complexed with plasmid DNA (pDNA), the synthetic polymer, 25 kDa branched Polyethylenimine (PEI), can be formulated for aerosol delivery to the lungs. We show that pDNA/PEI aerosol formulations can be repeatedly administered to airways of mice on at least 10 occasions with no detectable toxicity. Interestingly, peak reporter gene activity upon repeated delivery was significantly reduced by up to 75% compared with a single administration, despite similar pDNA lung deposition at each subsequent aerosol exposure. Although the precise mechanism of inhibition is unknown, it is independent of mouse strain, does not involve an immune response, and is mediated by PEI. Importantly, using a dosing interval of 56 days, delivery of a fourth-generation, CpG-free plasmid generated high-level, sustained transgene expression, which was further boosted at subsequent administrations. Together these data indicate that pDNA/PEI aerosol formulations offer a versatile platform for gene delivery to the lung resulting in sustained transgene expression suitable for treatment of chronic lung diseases.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer.

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.

Limitations of the murine nose in the development of nonviral airway gene transfer.

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression.

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (>or=56 d) in vivo transgene expression in the absence of lung inflammation.

Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.

Gene therapy is being investigated in the treatment of lung-related aspects of the genetic disease, Cystic fibrosis (CF). Clinical studies have demonstrated CF transmembrane conductance regulator (CFTR) expression in the airways of adults with CF using a variety of gene transfer agents. In utero gene therapy is an alternative approach that facilitates vector transduction of rapidly expanding populations of target cells while avoiding immune recognition of the vector. In CF, in utero gene transfer could potentially delay the onset of disease symptoms in childhood and compensate for the role, if any, that CFTR plays in the developing organs. Previously published studies have suggested that transient expression of CFTR in utero was sufficient to rescue the fatal intestinal defect in S489X Cftr(tm1Unc)/Cftr(tm1Unc) knockout mice. We replicated these studies using an identical CFTR-expressing adenoviral vector and CF mouse strain in sufficiently large numbers to provide robust Kaplan-Meier survival data. Although each step of the procedure was carefully controlled and vector-specific CFTR expression was confirmed in the fetal organs after treatment, there was statistically no significant improvement in the survival of mice treated in utero with AdCFTR, compared with contemporaneous control animals.

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung.

BACKGROUND: Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. METHODS: Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. RESULTS: Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. CONCLUSIONS: These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.

Optimizing aerosol gene delivery and expression in the ovine lung.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

The mouse CD1d cytoplasmic tail mediates CD1d trafficking and antigen presentation by adaptor protein 3-dependent and -independent mechanisms.

The short cytoplasmic tail of mouse CD1d (mCD1d) is required for its endosomal localization, for the presentation of some glycolipid Ags, and for the development of Valpha14i NKT cells. This tail has a four-amino acid Tyr-containing motif, Tyr-Gln-Asp-Ile (YQDI), similar to those sequences known to be important for the interaction with adaptor protein complexes (AP) that mediate the endosomal localization of many different proteins. In fact, mCD1d has been shown previously to interact with the AP-3 adaptor complex. In the present study, we mutated each amino acid in the YQDI motif to determine the importance of the entire motif sequence in influencing mCD1d trafficking, its interaction with adaptors, and its intracellular localization. The results indicate that the Y, D, and I amino acids are significant functionally because mutations at each of these positions altered the intracellular distribution of mCD1d and reduced its ability to present glycosphingolipids to NKT cells. However, the three amino acids are not all acting in the same way because they differ with regard to how they influence the intracellular distribution of CD1d, its rate of internalization, and its ability to interact with the mu subunit of AP-3. Our results emphasize that multiple steps, including interactions with the adaptors AP-2 and AP-3, are required for normal trafficking of mCD1d and that these different steps are mediated by only a few cytoplasmic amino acids.

The Third Way: Progress on pathways of antigen processing and presentation by CD1.

CD1 proteins are a third family of antigen presenting molecules that bind bacterial and autologous lipid antigens for presentation to T cells. With the solution of the crystal structures of several complexes of CD1 molecules with lipids, a greater appreciation has been gained of the adaptability of CD1 in binding lipid antigens with diverse structural features. Biochemical studies of the interactions between the TCR and CD1-lipid complexes have revealed striking contrasts with TCR that bind to peptides presented by MHC-encoded class I and class II molecules. The sphingolipid activating proteins (SAP) have recently been found to facilitate the transfer of lipid antigens onto CD1 molecules. This helps to provide an explanation as to how the thermodynamic barrier, caused by loading hydrophobic lipid antigens in a hydrophilic environment, can be overcome. Mechanisms of CD1 endosomal trafficking are being delineated, including the means by which adaptor proteins induce the localization of some types of CD1 molecules to lysosomes, where they bind antigens. Unlike MHC class I and class II proteins, specialized molecules that function solely in chaperoning CD1 molecules, or in facilitating their antigen loading, have not been found. This suggests that the CD1 antigen presenting system, which diverged early in vertebrate evolution from MHC antigen presenting molecules, is a simpler system with a character closer to the primordial antigen presenting function.

The adaptor protein AP-3 is required for CD1d-mediated antigen presentation of glycosphingolipids and development of Valpha14i NKT cells.

Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3-deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3-deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II-reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II pathway, NKT cell development and antigen presentation by CD1d are not reduced by invariant chain deficiency. These data demonstrate that the AP-3 requirement is a particular attribute of the CD1d pathway in mice and that, although MHC class II molecules and CD1d are both found in late endosomes or lysosomes, different pathways mediate their intracellular trafficking.