Ap₄A Regulates Directional Mobility and Antigen Presentation in Dendritic Cells

Summary

The significance of intracellular Ap₄A levels over immune activity of dendritic cells (DCs) has been studied in Nudt2^fl/fl/CD11c-cre mice. The transgenic mice have been generated by crossing floxed NUDT2 gene mice with DC marker CD11c recombinase (cre) mice. The DCs derived from these mice have higher levels of Ap₄A (≈30-fold) compared with those derived from Nudt2^+/+ mice. Interestingly, the elevated Ap₄A in DCs has led them to possess higher motility and lower directional variability. In addition, the DCs are able to enhance immune protection indicated by the higher cross-presentation of antigen and priming of CD8⁺ OT-I T cells. Overall, the study denotes prominent impact of Ap₄A over the functionality of DCs. The Nudt2^fl/fl/CD11c-cre mice could serve as a useful tool to study the influence of Ap₄A in the critical immune mechanisms of DCs.

Graphical Abstract

Highlights

• •DCs of Nudt2^fl/fl/CD11c-cre mice exhibit low directional variability and high motility
• •DCs elevate proliferation of OVA-specific T cell receptor transgenic CD8⁺ T cells
• •The escalation of Ap₄A levels in DCs could enhance their immune protective activity
• •Mice can serve as useful functional tool to study the role of Ap₄A in various cells

Molecular Mechanism of Behavior; Immunology; Immune Response; Model Organism

Introduction

Transfer ribonucleic acid (tRNA) synthetases play an important role in the central dogma of molecular biology. The specific function of tRNA synthetases is to conjugate tRNAs with the cognate amino acid for correct translation of polypeptides from mRNA. Progressively, highly conserved and non-canonical activities of tRNA synthetases that are unique for each amino acid-charging tRNA synthetase have been discovered. Lysyl-tRNA synthetase (LysRS), a tRNA that charges lysine onto lysine-tRNA for use in ribosome for translation, have also been serving an evolutionarily conserved, non-canonical enzymatic activity to produce diadenosine tetraphosphate (Ap₄A), a small signaling molecule composed of two adenosine moieties joined through a 5′-5′ linkage by a chain of four phosphates. This non-canonical pathway in LysRS is triggered by the phosphorylation of LysRS on serine 207 (P-s207 LysRS) via p38 mitogen-activated protein kinase activity. Phosphorylation leads to the dissociation of P-s207 LysRS from multi-synthetase complex (MSC) and promotes the production of Ap₄A (Ofir-Birin et al., 2013). In turn, the synthesis of Ap₄A is regulated by the housekeeping protein Ap₄A hydrolase (Ap₄AHy) that converts Ap₄A back into its original building blocks (one molecule of ATP and one molecule of AMP) thereby creating a regulatory feedback to maintain intracellular Ap₄A levels (Vollmayer et al., 2003).

Ap₄A synthesis activity by LysRS can directly control specific response programming in immune-specialized cells (Nechushtan et al., 2009). Our group has previously demonstrated that non-canonical LysRS activity can drive increased intracellular Ap₄A and control USF2 transcriptional activity, which up-regulates transforming growth factor-β2 in FcepsilonRI-activated mast cells (Lee and Razin, 2005). Ap₄A can enhance phorbol myristate acetate (PMA)-stimulated reactive oxygen species production in lymphocytes (Schepers et al., 2010) and has been implicated in key immunological responses (Carracedo et al., 2013, Castany et al., 2011, Chang et al., 2014, Louie et al., 1988). Another pathway that is driven by the increase of intracellular Ap₄A is the activation of microphthalmia-associated transcription factor (MITF), a master regulator in melanocyte development (Levy et al., 2006). Hint1, a co-suppressor of MITF, is also inactivated by high intracellular concentration of Ap₄A and dissociates from Hint1. Released MITF can translocate to the nucleus and initiate transcription of downstream genes (Lee et al., 2004a).

Ap₄A is also implicated in the control of antigen presentation. Low levels of Ap₄A are observed in patients with Chediak-Higashi syndrome (Kim et al., 1985), where delayed major histocompatibility complex (MHC) class II-restricted antigen presentation increases pathogen load (Martin-Fernández et al., 2005). The expression of MHC class II is a defining feature of antigen-presenting cells (APCs). Among APCs, dendritic cells (DCs) possess vastly efficient antigen cross-presentation response over all other known immune cell types. DCs possess high motility to bring sampled antigens to naive T cells located in lymph nodes to stimulate adaptive immune response. Hence DCs act as connecting linkers between innate and acquired immunity. Immature DCs, although present in blood, are more prevalent in pathogen-prone peripheral tissues. Mature DCs travel toward lymph nodes for the antigen presentation to T and B cells, thus activating acquired immune response (Clark et al., 2000). Harnessing the ability of DC as a professional APC to enhance antigen-specific T cell immune response with high precision is particularly useful for the development of more effective cancer immunotherapy vaccines (Zamarin and Postow, 2015).

DCs adopt a dynamic behavior by migrating to lymph nodes for naive T or B cell priming and maintaining acquired immune activity (Germain et al., 2012). Hence migratory ability of DC is directly correlated with its ability to stimulate immune response through antigen presentation to naive T and B cells in remotely accessible regions of lymph nodes awaiting stimulation by DCs.

We reasoned that if a highly conserved function of APCs such as antigen presentation can be precisely impaired by low levels of intracellular Ap₄A, APCs may functionally benefit from the increase of intracellular Ap₄A concentration. We hypothesized that Ap₄A is able to enhance functional capacities of APCs by improving either mobility or antigen presentation, or both.

The NUDT2 gene encodes Ap₄A hydrolase, a member of the nudix-type family of enzymes that hydrolyze a wide range of pyrophosphates. CD11c⁺ is preferentially expressed in murine DCs. Ap₄A hydrolase allele was floxed and crossed with a CD11c⁺ promoter-specific Cre mice to generate the deletion of NUDT2 gene in CD11c⁺ cells (Nudt2^fl/fl/CD11c-cre mice). The present study is aimed at investigating the immune modulatory effect of Ap₄A in DCs from Nudt2^fl/fl/CD11c-cre mice. We report the influence of Ap₄A levels over DCs' viability, motility, and expression of immune activation markers.

Results

Selective Knockout of Ap₄A Hydrolase in Dendritic Cells of Nudt2^fl/fl/CD11c⁺ Mice

The intracellular level of Ap₄A is regulated by two enzymes: lysyl tRNA synthetase (LysRS) and Ap₄A hydrolase (Ap₄AHy; Figure 1A). To investigate the function of Ap₄A in DCs, we have utilized a floxed Ap₄A hydrolase allele mouse (Nudt2^fl/fl; Figure 1B), which we crossed with CD11c⁺ transgenic mice thereby targeting deletion of Ap₄A hydrolase to cells that expresses the transmembrane surface protein CD11c, i.e., DCs (Figure 1C). The western blot of splenic DC extracts of the mice showed neither any detectable expression of Ap₄AHy nor any disruption in the expression of MITF and Hint1 in Nudt2^fl/fl/CD11c⁺ cells when compared with Nudt2^+/+ CD11c⁺ cells, confirming successful knockout of Ap₄A hydrolase in DCs of Nudt2^fl/fl/CD11c⁺ mice (Figure 1D).

Figure 1.

Generation of Ap₄A Hydrolase Knockout Mice and Viability of Dendritic Cells

(A) The phosphorylated form of Lysyl-tRNA transferase (phosphoSerine 207 LysRS) synthesizes Ap₄A from ATP and AMP, and is in turn broken down into same by Ap₄A hydrolase (Ap₄AHy).

(B) The gene locus and mRNA structure of NUDT2. The full open reading frame for Ap₄AHy (15,412 bp) consists of three exons interspersed with two introns. The Nudix box is nothing but the enzymatically functional site for Ap₄AHy and is located at exon 3.

(C) The deletion of Ap4_AHy by gene floxing and deletion strategy. The two loxP sites were generated on either side of the gene encompassing the full sequence of exon 3 and a portion of immediate intron upstream to it.

(D–F) (D) Western blot showing expression levels of Ap4_AHy, MITF, and Hint1 in splenic dendritic cells of Nudt2^+/+ and Nudt2^fl/fl/CD11c⁺ (Ap₄A hydrolase knockout) mice (represents four independent experiments). Immature bone marrow-derived cells (BM). (E) and (F) Viability percentage of isolated BMDCs and splenic DCs. Results (mean ± SEM) represent two independent experiments. The significant difference of test in comparision to control. ^nsp> 0.05 (Student's t test).

Based on the increased cellular permeability and binding ability with free amines retained in dead cells than live cells, saturation of cells with amines conjugated to florescent APC-Cy7 probe were measured by using flow cytometry to determine the live and dead cell population percentages. The study has revealed a slight decrease in viability of bone marrow DCs (BMDCs) as well as splenic DCs in case of Nudt2^fl/fl/CD11c⁺ mice when compared with Nudt2^+/+ mice and is found to be statistically insignificant (Figures 1E and 1F).

Characterization of Ap₄AHy Nudt2^fl/fl/CD11c⁺ DCs

Higher Accumulation of Ap₄A Is Observed in Nudt2^fl/fl/CD11c⁺DCs

The intracellular Ap₄A level of BMDCs generated from Nudt2^fl/fl/CD11c⁺ and Nudt2^+/+ mice was examined using a diadenosine nucleotide assay. Compared with Nudt2^+/+ CD11c⁺ mice, the BMDCs of Nudt2^fl/fl/CD11c⁺ mice contained prominently higher levels of Ap₄A on their third and sixth days of culture (2.3- and 3.2-folds, respectively), whereas the levels reached peak state on eighth day of culture (approximately 32-fold; Figure 2A).

Figure 2.

Detailed Functional Characterization of Nudt2^fl/fl/CD11c-cre DC

(A) Intracellular concentrations of Ap₄A in Nudt2^+/+ and Nudt2^fl/fl/CD11c-cre BMDC were generated from bone marrow cultured with granulocyte-macrophage colony-stimulating factor and examined on days 3, 6, and 8, using a luciferase assay.

(B and C) (B) Western blot of LysRS, Ap₄AHy, MITF, and Hint1 in Nudt2^+/+ and Nudt2^fl/fl/CD11c-cre CD11c⁺ splenic cells. Immature bone marrow-derived cells (BM) (C). Real-time PCR result of MITF-specific gene (TRACP5) expression in BMDCs. The results (mean ± SEM) are representative of four independent experiments and the significant difference of test in comparison to control. **p < 0.01; ****p < 0.0001 (Mann-Whitney test for multiple comparison).

Another correlative activation from the increase in intracellular Ap₄A concentration is the initiation of LysRS-Ap₄A-Hint1-MITF pathway. The MITF nuclear localization was examined in splenic DCs and was detected prominently (without concomitant increase in expression) in splenic Nudt2^fl/fl/CD11c⁺ cells, but not in Nudt2^+/+ CD11c⁺ cells (Figure 2B). Furthermore, Hint1, a suppressor of MITF, is not present in the nucleus of matured DCs and monocytes and is only present in unmatured BM cells. Hint1 translocation also posits that MITF suppression is released from increased Ap₄A concentration within Nudt2^fl/fl/CD11c⁺ splenic cells.

To validate the translocation of MITF in to nucleus, which leads to gene transcription, MITF-specific gene tartrate-resistant acid phosphatase 5 (TRACP5) was examined for expression (Luchin et al., 2010). TRACP5 is strongly expressed in both Nudt2^+/+ and Nudt2^fl/fl CD11c⁺ cells and is upregulated by 2.97-fold in Nudt2^fl/fl/CD11c⁺ cells compared with Nudt2⁺/⁺ CD11c⁺ cells (Figure 2C).

To review the association of MITF with LysRS, which occurs during activation of MITF, a pull-down of MITF using LysRS antibody via co-immunoprecipitation experiment was performed on BMDCs. MITF is co-immunoprecipitated using LysRS antibody pull-down in both Nudt2^+/+ and Nudt2^fl/fl DCs, indicating the that binding of LysRS to MITF occurs at baseline level in DCs and the association is increased in Nudt2^fl/fl DCs (Figure S1).

Nudt2^fl/fl/CD11c⁺ BMDCs Possess Greater Motility with Lower Directional Variation

The Nudt2^fl/fl/CD11c⁺ BMDCs have shown vigorous motility as indicated by higher values of distance, speed and displacement compared with Nudt2^+/+ BMDCs (Figures 3A–3D). Less directional variability indicates enhanced organization of cytoskeletal dynamics, and such organization generally requires the activation of small GTPases, such as Rho GTPases. The Rho GTPases play a crucial role in cellular migration, of which Rho, Rac, and Cdc42 are widely studied proteins and are highly conserved among eukaryotes (Ridley, 2015). To examine a possible relationship between intracellular Ap₄A increase in DCs and small GTPase activation, active Rac1, cdc42, and RhoA levels were measured (GTP-bound form). The results show a significant decrease in active Rac1 and cdc42, and an increase in RhoA activation levels in case of BMDCs of Nudt2^fl/fl/CD11c⁺ mice compared with that of Nudt2^+/+ CD11c⁺ mice (Figure 3E).

Figure 3.

Comparative Motility of DCs from Nudt2^fl/fl/CD11c⁺ and Nudt2^+/+ Mice

(A) Live imaging microscopy of granulocyte-macrophage colony-stimulating factor-treated DCs (8 days). The post-treated DCs on observational Petri dish were chosen randomly (n = 20) and traced minute wise (time lap ≈30 min) to find the change in cell movement and the distance moved.

(B) The speed of cellular movement and the ratio between the distance moved and difference of two time frames.

(C) Cellular displacement.

(D) A still frame taken from the video created for live cell imaging of Nudt2^+/+ and Nudt2^fl/fl/CD11c⁺ DCs.

(E) Small GTPase activation state in Nudt2^+/+ and Nudt2^fl/fl/CD11c⁺ DCs (n = 2). Results (mean ± SEM) represent two independent experiments. The significant difference of test in comparision to control. *p < 0.05, ***p < 0.001, and ****p < 0.0001 (Student's t test or Mann-Whitney test for multiple comparison).

Nudt2^fl/fl/CD11c⁺ DCs Possess Higher Antigen-Presenting and CD8⁺ T Cell-Priming Potential

The possibility of NUDT2 gene directly controlling fundamental immune activity of DCs was also investigated. It is equally important to know whether Nudt2 is important for DC maturation because its status directly impacts the immune function as APCs. Although DCs are highly specialized immune cells that specialize as APCs, they are initially phagocytic before maturation. A change in phagocytic potential would indicate aberrant DC maturation signaling compared with Nudt2^+/+ DCs. To measure the phagocytic potential, uptake of fluorescein isothiocyanate (FITC) dextran by DCs was measured. The phagocytic potential of DCs of Nudt2^fl/fl/CD11c⁺ mice was found to be unchanged when compared with that of Nudt2^+/+ DCs as the uptake of FITC dextran DCs from Nudt2^fl/fl/CD11c + mice was identical to Nudt2^+/+ DCs both at 4°C and 37°C (Figure S2). Therefore, these DCs do not differ in phagocytic capacity, a defining attribute in DC maturation status, indicating that DC maturation status is identical. To determine if antigen presentation functionality is affected, the capacity of DCs to trigger proliferation in antigen-specific T cells that requires DCs to present highly specific antigens to these T cells to trigger activation and proliferation was also investigated. The cultures of BMDCs from either the Nudt2^fl/fl/CD11c⁺ or the Nudt2^+/+/CD11c⁺ mice incubated with OT-1 T cell were co-incubated with positive control SIINFEKL peptides, and have given a similar extent of stimulation of proliferation of CD8⁺ T cells, indicating an overall lack of involvement in the activation of T cells by DCs, which does not require antigen cross-presentation (Figures 4A and 4B). Remarkably, when cultured with DCs co-incubated either with whole ovalbumin (OVA) or OVA and cytosine-phosphodiester linked guanine ligodeoxynucleotides (CpG ODN), there was an average of 20% and 15% higher stimulus in the proliferation of CD8⁺ T cell population, respectively, by the BMDCs from Nudt2^fl/fl/CD11c⁺ mice compared with the Nudt2^+/+ BMDCs. Nudt2 gene is therefore involved in the control of antigen cross-presentation machinery in DCs.

Figure 4.

Nudt2^fl/fl/CD11c⁺ DCs' Potential in Influencing OT-I CD8⁺ T Cells Antigen Cross-Priming

(A) The percentage of OT-I CD8⁺ T cells that have proliferated following co-culture for 3 days either with Nudt2^fl/fl/CD11c⁺ BMDCs or Nudt2^+/+ CD11c⁺ BMDCs (1:10) incubated with SIINFEKL peptide, OVA and CpG ODN. The harvested cells were analyzed by flow cytometry to quantify CD8⁺ CellTrace Violet⁺ population for proliferation.

(B) Graph represents the percentage of OT-I CD8⁺ T cells proliferated when cultured with Nudt2^+/+ or Nudt2^fl/fl/CD11c⁺ DCs in response to different antigenic preparations.

(C) IL-12 p40 production by DCs during the above co-culture conditions.

(D) mRNA levels for TAP1, TAP2, and TAPASIN in Nudt2^+/+ and Nudt2^fl/fl/CD11c-cre in splenic DCs measured by RT-PCR. Results (mean ± SEM) represent three independent experiments and the significant difference of test in comparision to control. *p < 0.05 (Student's t test or Mann-Whitney test for multiple comparison).

To determine the involvement of interleukin (IL)-12 p40, which is a DC-specialized T cell activation cytokine that controls enhancement of antigen cross-presentation by Nudt2^fl/fl/CD11c⁺ BMDCs, the cytokine levels were measured by ELISA technique. The results revealed a lack of involvement of IL-12 p40 in enhancing the priming of CD8⁺ T cells by Nudt2^fl/fl/CD11c⁺ BMDCs as there was no observable difference in the levels of IL-12 p40 between Nudt2^fl/fl/CD11c⁺ BMDCs and Nudt2^+/+ BMDCs (Figure 4C).

Antigen cross-presentation can also be skewed by the change in expression of TAP proteins required for assembly of short peptides onto MHC-I molecules. The mRNA levels of TAP1, TAP2, and TAPASIN were measured in Nudt2^fl/fl/CD11c⁺ DCs as well as in Nudt2^+/+ DCs. The results have shown a lack of difference in TAP1, TAP2, or TAPASIN between Nudt2^fl/fl/CD11c-cre or Nudt2^+/+ DCs indicating lack of their involvement (Figure 4D). Taken together, Nudt2 gene is involved in controlling antigen cross-presentation machinery that directly signals T cell proliferation.

Discussion

This study shows that intracellular Ap₄A can be upregulated in immune cells of a specific lineage (DCs) within an in vivo model. The DCs from these mice are lacking Ap₄A hydrolase, and a consequent rise in intracellular Ap₄A amounts is observed, revealing an obvious substantial regulation of Ap₄A concentrations in DCs by Ap₄A hydrolase through the NUDT2 gene. The Nudt2^fl/fl/CD11c⁺ mice did not exhibit any observable physiological differences to wild-type, leading to the inference that they harbor similar small population of CD11c⁺ cells as are normally present. In addition, their viability remained almost similar.

DCs play a pivotal role in adaptive immunity and tolerance. Efficient migration for accurate positioning to capture antigens from invading pathogens and capacity to process the foreign peptides into recognizable MHC-I-bound complexes through antigen-cross presentation are two immutable purposes of DCs. These functions are therefore critical for immune protection. Most of the immature and highly phagocytic DCs begin their voyage from the bone marrow where they are generated, followed by entry into the blood and sequential movement into peripheral lymphoid tissues (PLTs) and non-lymphoid tissues (NLTs). The DCs possess advanced migratory skills, and their main function in NLTs is the carriage and presentation of antigenic components into and within secondary lymphoid organs (Alvarez et al., 2008). Once primed by the phagocytosis of foreign antigens the enrouted Ag-bearing DCs in lymph nodes begin to build up an immune-stimulatory phenotype, exhibiting increased expression of MHC complexes as well as upregulation of the co-stimulatory molecules and cytokines required for effective T cell priming. The non-retained DCs enter into the blood stream (blood-borne DCs) and deliver the antigenic components to the spleen (Mullins et al., 2003) and also to PLTs (Cavanagh et al., 2005).

The relationship between Nudt2 and DC is unknown, but its association with immune function has been speculated. A recent RNA sequencing analysis of Nudt2 knockout myelogenous leukemia cells revealed that the majority of the target genes are linked to immune-specific processes such as interferon-associated inflammatory responses, MHC-II antigen presentation, allograft rejection, and B cell development (Marriott et al., 2016). The current study focused on the influence of Ap₄A over immune activity of DCs from Nudt2^fl/fl/CD11c⁺ mice and has confirmed that Nudt2 gene has influence over MHC-II antigen presentation.

Ap₄A regulates MITF activation through the release of Hint1 suppression and consequent nuclear localization of MITF. The nuclear localization of MITF, as a result of increase of Ap₄A, is a hallmark of the LysRS-Ap₄A-Hint1-MITF pathway (Carmi-Levy et al., 2008, Carmi-Levy et al., 2011, Huete et al., 2011, Lee and Razin, 2005, Lee et al., 2004b, Tshori et al., 2013). The LysRS-Ap₄A-Hint1-MITF pathway has been described in mast cells (Lee et al., 2004a), but it was not clear whether the same pathway is active in DCs.

The LysRS-Ap₄A-Hint1-MITF pathway was examined in DCs in this study, and it was revealed that the increase in MITF nuclear localization, but not HINT-1, in response to the elevated Ap₄A levels is due to the knockout of NUDT2 gene. The binding of MITF to LysRS using co-immunoprecipitation pull down of MITF using LysRS antibody establishes the existence of LysRS-Ap₄A-Hint1-MITF pathway in DCs and further confirms that Hint1 suppression is lifted so that LysRS can bind active MITF.

The BMDCs from Nudt2^fl/fl/CD11c-cre mice cultured from undifferentiated monocytes did not show any morphological or phenotypic differences to Nudt2^+/+ DCs. Interestingly, the DCs of Nudt2^fl/fl/CD11c⁺ mice exhibited a unique pattern of cellular motility with higher velocity and lower directional variability. This suggests that the majority population of DCs of Nudt2^fl/fl/CD11c⁺ mice is having uniform directional imprint that may indicate effective migration toward the immune reaction zones. Furthermore, we found such behavior was correlated to the variation in their expression of small GTPases, i.e., cdc42, Rac1, and RhoA. Our findings provide evidence that reduced activation of Rac1 in DCs leads to increased directional motility, consistent with the earlier studies over fibroblasts that had shown their enhancement of directional motility by the inactivation of the same GTPases (Hanna and El-Sibai, 2013). Among small GTPases that are important in cell motility, Rho GTP is critical for inducing actomyosin contraction and inhibiting actin filament disassembly for cell polarity and directional migration (Kimura et al., 1998, Maekawa et al., 1999). Upregulation of active Rho is observed in CD11c⁺ DC derived from Nudt2^fl/fl mice. Pharmacologic Rho effector blockades (e.g., ROCK inhibitors) can profoundly decrease DC migration capability. Hence increased bioavailability of RhoGTP can correlate with the increased mobility observed in CD11c⁺ DC derived from Nudt2^fl/fl mice. Increase in Rho activity also causes decreased availability of Rac (Ohta et al., 2006), and this is likewise also observed in CD11c⁺ DC derived from Nudt2^fl/fl mice.

The defining feature of DCs is their ability to take up antigen in the periphery, through their dendrites; to withdraw those dendrites; and then to migrate to lymph nodes. Upon regaining their former shapes in lymph node, DCs will present antigenic peptides to CD4⁺ and CD8⁺ T cells. Thus DCs may be regarded as the “shape-shifters” of the immune system. Cross-presentation is the intracellular degradation of extrinsic antigen and its presentation by MHC-I. This important DC function activates CD8⁺ T cells during an immunological response to intracellular pathogens (Murphy and McLennan, 2004, Murphy et al., 2000) and is a highly energy-dependent process and requires movement of the cells into finding suitable T cells. Though identical levels of phagocytic potential, Class-II MHC display and expression of co-stimulatory molecules were seen in DCs of Nudt2^fl/fl/CD11c⁺ mice, there was an enhancement in their immune functionality. These DCs have increased immune activity in terms of antigen processing and cross-presentation in priming the proliferation of CD8⁺ T cells. In vitro antigen presentation assay using OT-1 CD8⁺ T cells is well suited to validate this result as an antigen-specific response. The activation of CD8⁺ T cells of OT-1 mice is exclusive for antigen presentation of OVA through DC cross-presentation machinery, and the subsequent trigger of proliferation in OT-1 CD8⁺ T cells is a highly specific response against cross-presented MHC-I antigen on APCs. CD11c⁺ DC derived from Nudt2^fl/fl mice are capable of driving the proliferation of antigen-specific CD8⁺ T cells without the need for CpG ODN, a potent immune agonist (Ramírez-Pineda et al., 2004). However, this observation was found not associated with the expected alteration in the elements of MHC-I machinery, reflecting the possible positive influence of directional motility making effective contact to the T cells and also the rise in the duration of functioning in antigen presentation process. There is direct evidence in support of one of our assumption. Other groups have also reported that overexpression of Rho can directly increase the ability of DC to present OVA peptide to specific T cells (Shurin et al., 2005). Our studies indicate that the increased CD8⁺ T cell cross-priming potential of DC from Nudt2^fl/fl/CD11c⁺ mice compared with Nudt2^+/+ is more likely the result of RhoA activation and RAC1 repression. This is in agreement with the earlier studies (Hanna and El-Sibai, 2013, Shurin et al., 2005, Wu et al., 2009).

Another important feature of DCs is their capacity to steer the immune response into clinically beneficial Th1-type immune response by the production of IL-12 (Muller-Berghaus et al., 2005). Without stimulation, IL-12 production is not observed to be different than Nudt2^+/+ for CD11c⁺ DC derived from Nudt2^fl/fl mice, indicating that the resultant DCs are not activated immune response without stimuli. Presence of CpG ODN increased IL-12 production but by an identical amount to Nudt2^+/+ mice, suggesting that the CD11c⁺ DC derived from Nudt2^fl/fl mice are neither functionally impaired or overactivated compared with Nudt2^+/+ DC counterparts.

In conclusion, increased Ap₄A concentration in DC of Nudt2^fl/fl/CD11c⁺ mice led to the localization of MITF into the nucleus during restive conditions compared with Nudt2^+/+ DC. These Ap₄A enriched DCs have prominently exhibited alteration in small GTPases rising their motility and antigen presenting potentiality. This study documents an in vivo model that can modulate the intracellular Ap₄A concentration by knocking out Ap₄A hydrolase and enhancing antigen cross-presentation by DC. Fundamentally, enhanced antigen cross-presentation in a controlled manner will benefit a wide variety of APC-associated functions such as earlier pathogen recognition by the immune system and better loading and presentation of tumor antigens to cytotoxic CD8⁺ T cells in response to cancer. The highly specific control of intracellular Ap₄A concentration through manipulation of NUDT2 gene makes this mechanism an attractive pharmacological target to enhance antigen presentation while keeping the complex repertoire of transcriptional activation of APCs as intact as possible.

Limitations of the Study

The currently studied Nudt2^fl/fl/CD11c⁺ mice model is an excellent in vivo system to understand the consequences of increased intracellular Ap₄A. It is important and yet to be determined whether the circulatory DCs in Nudt2^fl/fl/CD11c⁺ mice exhibit elevation in antigen presentation to T cells. Also, the identification of key cytokines responsible for DC-specialized T cell activation would become significant. In addition, this study necessitates the elucidation of Ap₄A-binding proteins that are associated with cellular migration in future.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Published: June 28, 2019