Polymorphic estrogen receptor binding site causes Cd2-dependent sex bias in the susceptibility to autoimmune diseases.

Complex autoimmune diseases are sexually dimorphic. An interplay between predisposing genetics and sex-related factors probably controls the sex discrepancy in the immune response, but the underlying mechanisms are unclear. Here we positionally identify a polymorphic estrogen receptor binding site that regulates Cd2 expression, leading to female-specific differences in T cell-dependent mouse models of autoimmunity. Female mice with reduced Cd2 expression have impaired autoreactive T cell responses. T cells lacking Cd2 costimulation upregulate inhibitory Lag-3. These findings help explain sexual dimorphism in human autoimmunity, as we find that CD2 polymorphisms are associated with rheumatoid arthritis and 17-ß-estradiol-regulation of CD2 is conserved in human T cells. Hormonal regulation of CD2 might have implications for CD2-targeted therapy, as anti-Cd2 treatment more potently affects T cells in female mice. These results demonstrate the relevance of sex-genotype interactions, providing strong evidence for CD2 as a sex-sensitive predisposing factor in autoimmunity.

CD58 Immunobiology at a Glance.

The glycoprotein CD58, also known as lymphocyte-function antigen 3 (LFA-3), is a costimulatory receptor distributed on a broad range of human tissue cells. Its natural ligand CD2 is primarily expressed on the surface of T/NK cells. The CD2-CD58 interaction is an important component of the immunological synapse (IS) that induces activation and proliferation of T/NK cells and triggers a series of intracellular signaling in T/NK cells and target cells, respectively, in addition to promoting cell adhesion and recognition. Furthermore, a soluble form of CD58 (sCD58) is also present in cellular supernatant in vitro and in local tissues in vivo. The sCD58 is involved in T/NK cell-mediated immune responses as an immunosuppressive factor by affecting CD2-CD58 interaction. Altered accumulation of sCD58 may lead to immunosuppression of T/NK cells in the tumor microenvironment, allowing sCD58 as a novel immunotherapeutic target. Recently, the crucial roles of costimulatory molecule CD58 in immunomodulation seem to be reattracting the interests of investigators. In particular, the CD2-CD58 interaction is involved in the regulation of antiviral responses, inflammatory responses in autoimmune diseases, immune rejection of transplantation, and immune evasion of tumor cells. In this review, we provide a comprehensive summary of CD58 immunobiology.

CD2 Is a Novel Immune-Related Prognostic Biomarker of Invasive Breast Carcinoma That Modulates the Tumor Microenvironment.

Female breast cancer (BCa) is the most commonly occurring cancer worldwide. The tumor microenvironment (TME) plays an essential role in tumor invasion, angiogenesis, unlimited proliferation, and even immune escape, but we know little about the TME of BCa. In this study, we aimed to find a TME-related biomarker for BCa, especially for invasive breast carcinoma (BRCA), that could predict prognosis and immunotherapy efficacy. Based on RNA-seq transcriptome data and the clinical characteristics of 1222 samples (113 normal and 1109 tumor samples) from The Cancer Genome Atlas (TCGA) database, we used the ESTIMATE algorithm to calculate the ImmuneScore and StromalScore and then identified differentially expressed genes (DEGs) between the high and low ImmuneScore groups and the high and low StromalScore groups. Thereafter, a protein-protein interaction (PPI) network analysis and univariate Cox regression analyses of overall survival were used to identify potential key genes. Five candidate genes were identified, comprising CD2, CCL19, CD52, CD3E, and ITK. Thereafter, we focused on CD2, analyzing CD2 expression and its association with survival. CD2 expression was associated with tumor size (T stage) to some extent, but not with overall TNM stage, lymph node status (N stage), or distant metastasis (M stage). High CD2 expression was associated with longer survival. METABRIC data were used to validate the survival result (n = 276). Gene set enrichment analysis (GSEA) showed that the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were significantly associated with high CD2 expression were mainly immune-related pathways. Furthermore, CD2 expression was correlated with 16 types of tumor-infiltrating immune cells (TICs). Hence, CD2 might be a novel biomarker in terms of molecular typing, and it may serve as a complementary approach to TNM staging to improve clinical outcome prediction for BCa patients.

Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity.

The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.

Human Oral Epithelial Cells Suppress T Cell Function via Prostaglandin E2 Secretion.

The oral mucosa is constantly exposed to a plethora of stimuli including food antigens, commensal microbiota and pathogens, requiring distinct immune responses. We previously reported that human oral epithelial cells (OECs) suppress immune responses to bacteria, using H413 and TR146 OEC lines and primary OECs in co-culture with dendritic cells (DCs) and T cells (OEC-conditioned cells). OECs reduced DCs expression of CD80/CD86 and IL-12/TNFα release and impaired T cell activation. Here, we further evaluated the immunosuppression by these OECs and investigated the underlying mechanisms. OEC-conditioned DCs did not induce CD4 T cell polarization towards Treg, judging by the absence of FoxP3 expression. OECs also repressed T-bet/IFNγ expression in CD4 and CD8 T cells activated by DCs or anti-CD3/CD28 antibodies. This inhibition depended on OEC:T cell ratio and IFNγ repression occurred at the transcriptional level. Time-lapse experiments showed that OECs inhibited early steps of T cell activation, consistent with OECs inability to suppress T cells stimulated with PMA/ionomycin. Blocking CD40/CD40L, CD58/CD2 and PD-L1/PD-1 interactions with specific antibodies did not disrupt T cell suppression by OECs. However, preventing prostaglandin E2 (PGE2) synthesis or blocking PGE2 binding to the cognate EP2/EP4 receptors, restored IFNγ and TNFα production in OEC-conditioned T cells. Finally, treating OECs with poly(I:C), which simulates viral infections, limited T cell suppression. Overall, these results point to an inherent ability of OECs to suppress immune responses, which can nonetheless be eluded when OECs are under direct assault.

CD2 Immunobiology.

The glycoprotein CD2 is a costimulatory receptor expressed mainly on T and NK cells that binds to LFA3, a cell surface protein expressed on e.g., antigen-presenting cells. CD2 has an important role in the formation and organization of the immunological synapse that is formed between T cells and antigen-presenting cells upon cell-cell conjugation and associated intracellular signaling. CD2 expression is upregulated on memory T cells as well as activated T cells and plays an important role in activation of memory T cells despite the coexistence of several other costimulatory pathways. Anti-CD2 monoclonal antibodies have been shown to induce immune modulatory effects in vitro and clinical studies have proven the safety and efficacy of CD2-targeting biologics. Investigators have highlighted that the lack of attention to the CD2/LFA3 costimulatory pathway is a missed opportunity. Overall, CD2 is an attractive target for monoclonal antibodies intended for treatment of pathologies characterized by undesired T cell activation and offers an avenue to more selectively target memory T cells while favoring immune regulation.

Phosphoproteomics of CD2 signaling reveals AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells.

Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity.

BACKGROUND AIMS: Key obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells. METHODS: We delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3+Vα24+Vß11+ iNKT cells. RESULTS: Gene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4+ and CD8+ responder T cells. DISCUSSION: These data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.

Leukemia-associated immunophenotypes subdivided in "categories of specificity" improve the sensitivity of minimal residual disease in predicting relapse in acute myeloid leukemia.

BACKGROUND: The assessment of minimal residual disease (MRD) by flow cytometry (FC) has a prognostic impact in acute myeloid leukemia (AML), despite the low sensitivity in predicting relapse. Nonetheless, the role of leukemic-associated immunophenotypes (LAIPs)-related specificity on the sensitivity of MRD has not been clarified yet. In this respect, we accomplished this study. METHODS: LAIP-frequencies of bone marrow samples from healthy donors and patients after treatment were quantified and subdivided in "categories of specificity" named as: "strong," "good," and "weak." At the following, the diagnostic performance of MRD was investigated in terms of sensitivity, specificity, predictive values, likelihood ratio (LR). RESULTS: "Strong" LAIPs were identified by CD7, CD2, CD4, and CD56 markers while "weak" LAIPs, independently of coexpressed markers, were mainly observed in CD33+ cells. MRD identified patients with significantly low DFS and OS but showed a low sensitivity in predicting relapse. Interestingly, majority of recurrences was noticed in patients with two LAIPs and lacking of "strong" LAIPs or only with one "good" LAIP. Thus, only patients showing one "strong" or two "good" LAIPs were considered suitable for MRD monitoring and selected to be further investigated. In this subset, positive MRD predicted a poor prognosis. Moreover, a higher sensitivity, negative predictive value (NPV) and LR- were observed after comparison with the previous series. CONCLUSIONS: These data highlight the relevant role of LAIP classification in "categories of specificity" in improving the sensitivity of MRD as assessed by FC.

Immunophenotype of pediatric-onset mastocytosis does not correlate with clinical course.

BACKGROUND: Pediatric mastocytosis differs from adult mastocytosis in its presentation and clinical course. However, the data regarding the immunophenotypic characterization of mast cells in children are limited. Our objective was to evaluate the immunophenotype of mast cells in pediatric mastocytosis and correlate it with the clinical course. METHODS: Biopsy specimens of children with cutaneous mastocytosis were retrieved from the institutions of pathology and were stained for CD25, CD2, and CD30. The percentage of mast cells and the staining intensity were correlated with the clinical data. RESULTS: Twenty-five biopsy specimens were included in the study. Patients' average age was 15.4 at presentation and 37.5 months at biopsy performance. Clinical presentations included maculopapular cutaneous mastocytosis in 79% and mastocytoma in 21% of cases. CD25, CD2, and CD30 were positive in 60%, 44%, and 84% of the biopsy specimens, respectively. The staining score was significantly higher for CD30 as compared to those for CD25 and CD2 (P = 0.02). No correlation was found between the immunophenotype and the clinical form or course of disease. CONCLUSIONS: Our results confirm that CD30 is a sensitive marker for pediatric-onset mastocytosis. Nevertheless, its expression does not correlate with clinical subtype or clinical course. The sensitivity of CD25 is higher than that of CD2 in skin lesions.

In vitro Evidence That Combination Therapy With CD16-Bearing NK-92 Cells and FDA-Approved Alefacept Can Selectively Target the Latent HIV Reservoir in CD4+ CD2hi Memory T Cells.

Elimination of the latent HIV reservoir remains the biggest hurdle to achieve HIV cure. In order to specifically eliminate HIV infected cells they must be distinguishable from uninfected cells. CD2 was recently identified as a potential marker enriched in the HIV-1 reservoir on CD4+ T cells, the largest, longest-lived and best-characterized constituent of the HIV reservoir. We previously proposed to repurpose FDA-approved alefacept, a humanized α-CD2 fusion protein, to reduce the HIV reservoir in CD2hi CD4+ memory T cells. Here, we show the first evidence that alefacept can specifically target and reduce CD2hi HIV infected cells in vitro. We explore a variety of natural killer (NK) cells as mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) including primary NK cells, expanded NK cells as well as the CD16 transduced NK-92 cell line which is currently under study in clinical trials as a treatment for cancer. We demonstrate that CD16.NK-92 has a natural preference to kill CD2hi CD45RA- memory T cells, specifically CD45RA- CD27+ central memory/transitional memory (TCM/TM) subset in both healthy and HIV+ patient samples as well as to reduce HIV DNA from HIV+ samples from donors well controlled on antiretroviral therapy. Lastly, alefacept can combine with CD16.NK-92 to decrease HIV DNA in some patient samples and thus may yield value as part of a strategy toward sustained HIV remission.

Transcription factors early growth response gene (Egr) 2 and 3 control inflammatory responses of tolerant T cells.

INTRODUCTION: Impaired proliferation and production of IL2 are the hallmarks of experimental T cell tolerance. However, in most autoimmune diseases, auto-reactive T cells do not display hyper proliferation, but inflammatory phenotypes. METHODS: We have now demonstrated that the transcription factors Egr2 and 3 are important for the control of inflammatory cytokine production by tolerant T cells, but not for tolerance induction. RESULTS: In the absence of Egr2 and 3, T cell tolerance, as measured by impaired proliferation and production of IL2, can still be induced, but tolerant T cells produced high levels of inflammatory cytokines. Egr2 and 3 regulate expression of differentiation repressors and directly inhibit T-bet function in T cells. Indeed, decreased expression of differentiation repressors, such as Id3 and Tcf1, and increased expression of inflammatory transcription factors, such as RORγt and Bhlhe40 were found in Egr2/3 deficient T cells under tolerogenic conditions. In addition, T-bet was co-expressed with Egr2 in tolerant T cells and Egr2/3 defects leads to production of high levels of IFNγ in tolerant T cells. CONCLUSIONS: Our findings demonstrated that despite impaired proliferation and IL2 production, tolerant T cells can display inflammatory responses in response to antigen stimulation and this is controlled at least partly by Egr2 and 3.

Spontaneous chondroma formation in CD2-Cre-driven Erk-deficient mice.

Lineage-specific Cre Tg mice are widely used to delineate the functions of genes in a tissue-specific manner. Several T-cell-specific promoter cassettes have been developed; however, the activities of those promoters in non-T cells have not been investigated extensively. Here, we report that CD2-Cre-mediated deletion of Erk proteins by generating CD2-Cre × Erk1-/-Erk2flox/flox (Erk∆CD2-Cre) mice results in abnormal cartilage hyperplasia. Histological analysis revealed that this abnormality is caused by aberrant hyperplasia of chondrocytes. The presence of Erk-deficient T cells is not required for this chondroma formation, as it was similarly observed in the absence of T cells in a CD3ε-deficient background. In addition, adoptive transfer of bone marrow cells from Erk∆CD2-Cre mice to wild-type recipients did not cause chondroma formation, suggesting that Erk-deficient non-immune cells are responsible for this abnormality. By tracing Cre-expressed tissues using a ROSA26-STOP-RFP allele, we found that the chondroma emitted RFP fluorescence, indicating that functional Cre is expressed in hyperplastic chondrocytes in Erk∆CD2-Cre mice. Furthermore, RFP+ chondrocytes were also found in an Erk-sufficient background, albeit without aberrant growth. These results suggest that unexpected expression of CD2-driven Cre in chondrocytes generates Erk-deficient chondrocytes, resulting in hyperplastic cartilage formation. Recently, two independent reports showed that CD4-Cre-mediated Ras-Erk signaling ablation led to similar abnormal cartilage formation (Guittard, G., Gallardo, D. L., Li, W. et al. 2017. Unexpected cartilage phenotype in CD4-Cre-conditional SOS-deficient mice. Front. Immunol. 8:343; Wehenkel, M., Corr, M., Guy, C. S. et al. 2017. Extracellular signal-regulated kinase signaling in CD4-expressing cells inhibits osteochondromas. Front. Immunol. 8:482). Together with these reports, our study suggests that an unexpected link exists between T-like cell and chondrocyte lineages during ontogeny.

[The influence of methylprednisolone on the ability of CD4+CD95+HLA-DR+ T-cells to produce proinflammatory medators in cultures of TCR-activated CD3+CD45RO+ T-lymphocytes from patients with rheumatoid arthritis]. / Vliianie metilprednizolona na sposobnost' CD4+CD95+HLA-DR+ T-kletok v kul'turakh TCR-aktivirovannykh CD3+CD45RO+ T-limfotsitov bol'nykh revmatoidnym artritom produtsirovat' provospalitel'nye mediatory.

The effect of different concentrations of the glucocorticoid (GC) methylprednisolone (MP) on CD4+CD95+HLA-DR+ T-cells and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes in the in vitro system was investigated. T cells were obtained from healthy donors and patients with rheumatoid arthritis (RA).Under conditions of TCR-activation, MP increased the number of CD4+HLA-DR+CD95+ cells in CD3+CD45RO+ cultures obtained from RA patients and did not change their content in the control group. In general, MP decreased production of proinflammatory factors (IFN-, IL-2, IL-17, IL-21 and TNF-) by TCR-activated CD3+CD45RO+ cells from healthy donors and RA, consistent with the overall immunosuppressive mechanism of GC action. The correlation between CD4+CD45RO+HLA-DR+CD95+ T-cell contents and parameters reflecting production of proinflammatory mediators (IL-17, IL-21 and TNF-) in RA patients indicates maintenance of the pro-inflammatory potential of this T-cell population exposed to GC action. We suggest that relative resistance of CD4+CD45RO+CD95+HLA-DR+ T-cells of RA patients to the suppressor effect of GC leads to maintenance and even enhancement in the functional capacities of autoreactive cells in the pathogenesis of RA.

Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9.

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.

Morphometric abnormalities in the spleen of the progeny of mice fed epigallocatechin during gestation and nursing.

It is very difficult to cure pregnant females suffering from infections, because of the risk which might occur during treatment by several, even herbal, medications. Many of these substances, among them extracts from plants, have antimicrobial, anti-inflammatory and immunostimulatory properties owing to their polyphenols content, but also may reveal unwanted effects on the fetal development because of their anti-angiogenic properties. The aim of the present study was to elucidate whether daily feeding pregnant and nursing mice 0.2 mg/kg epigallocatechin (EGC), previously recognized as angiogenesis inhibitor, may lead to abnormalities in morphology of spleen and in some parameters of immune function of their adult, 6-week old progeny. Morphometry of EGC offspring spleens revealed lower number of lymphatic nodules and their larger diameter than those found in the control offspring. Cellularity of spleens was lower in EGC offspring than in the controls. Cytometric analysis showed that this decline concerns lymphocytes with CD335 (p<0.001), CD19 (p<0.01) and CD4 (p<0.05) markers. No differences were observed in the humoral response to the immunization with SRBC, and in the proliferative response of splenocytes to mitogens PHA, ConA and LPS.

A candidate gene study identifies a haplotype of CD2 as novel susceptibility factor for systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is a rare autoimmune disease (AID) with a complex genetic etiology. Evidence for a shared pathogenesis across AIDs is given by the well-known pleiotropism of autoimmune genes. Recently, several unbiased approaches have identified an association between polymorphisms of the CD2 gene, and rheumatoid arthritis (RA) susceptibility. The objective of this study was to investigate whether CD2 polymorphisms are associated with SSc. METHODS: Two SNPs of CD2, rs624988 and rs798036, were genotyped in a total of 1,786 SSc patients and 2,360 healthy individuals from two European populations (France and Italy). Meta-analyses were performed to assess whether an association exists between CD2 polymorphisms or haplotypes and SSc or its main subtypes. RESULTS: The combined analyses revealed an association between the rs624988 A allele and SSc susceptibility: padj=0.023, OR=1.14 (95%CI 1.04-1.25). Single marker analysis did not reveal any association between rs798036 and SSc. Haplotype analysis identified that the A-T haplotype, previously described in RA, was associated with higher susceptibility for SSc (padj=0.029, OR=1.14, 95%CI 1.04-1.25) and with the positive anti-centromere antibody sub-group of SSc patients (padj=0.009, OR=1.19 95%CI 1.07-1.32). Genotype-mRNA expression correlations revealed that the CD2 risk haplotype was associated with decreased CD2 mRNA expression in SSc patients. CONCLUSIONS: Our study establishes CD2 as a new susceptibility factor for SSc, in a European Caucasian population, confirming the sharing of autoimmune risk factors by SSc and RA.

Critical Role of CD2 Co-stimulation in Adaptive Natural Killer Cell Responses Revealed in NKG2C-Deficient Humans.

Infection by human cytomegalovirus (HCMV) leads to NKG2C-driven expansion of adaptive natural killer (NK) cells, contributing to host defense. However, approximately 4% of all humans carry a homozygous deletion of the gene that encodes NKG2C (NKG2C(-/-)). Assessment of NK cell repertoires in 60 NKG2C(-/-) donors revealed a broad range of NK cell populations displaying characteristic footprints of adaptive NK cells, including a terminally differentiated phenotype, functional reprogramming, and epigenetic remodeling of the interferon (IFN)-γ promoter. We found that both NKG2C(-) and NKG2C(+) adaptive NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Notably, CD2 co-stimulation was critical for the ability of adaptive NK cells to respond to antibody-coated target cells. These results reveal an unexpected redundancy in the human NK cell response to HCMV and suggest that CD2 provides "signal 2" in antibody-driven adaptive NK cell responses.

T cell-specific inactivation of mouse CD2 by CRISPR/Cas9.

The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4(+) and CD8(+) lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo.

Expression of CD2 and CD25 on mast cell populations can be seen outside the setting of systemic mastocytosis.

BACKGROUND: Systemic mastocytosis (SM) is a diagnosis made using clinical, laboratory, and histologic parameters. Aberrant CD2 and/or CD25 expression on mast cells provides one minor criterion for a diagnosis of SM. To validate a tube (CD45/CD117/CD2/CD25) for mast cell evaluation, flow cytometry (FC) on residual material from marrow aspirates samples submitted to the hematopathology laboratory was performed. METHODS: Samples evaluated (n = 98) had no clinical or morphologic suspicion for SM. Samples were excluded if there was history of a myeloid stem cell neoplasm. Ten documented cases of SM were evaluated for comparison. RESULTS: Among cases without history of SM, 17.3% (n = 17) showed expression of CD2 and/or CD25 on ≥10% of the mast cell population (CD25 alone in 14 cases, CD2 alone in 2 cases, both in one case), while 82.6% (n = 81) showed no expression of these antigens. The percentage of mast cells showing aberrant CD2 and/or CD25 expression respectively ranged from 12.1% to 98.8% and 22.2% to 95.7% Interestingly, all of the cases with evidence of aberrant antigen expression on mast cells were collected post-therapy while 22.1% of the negative samples were collected pre-therapy. A cut-off of 60% CD25 expression on mast cells identified all cases of SM while minimizing false positives. CONCLUSIONS: These findings demonstrate that aberrant expression of CD2 and/or CD25 may be seen on mast cells outside of the setting of SM. The data suggests that this phenomenon may be seen more commonly following chemotherapy and that FC of mast cells should be interpreted with caution in the post-chemotherapy setting. © 2015 International Clinical Cytometry Society.