Association of the killer immunoglobulin-like receptor genes KIR2DS1 and KIR2DS4 with pediatric autoimmune hepatitis type I in Egypt

Background Killer cell immunoglobulin-like receptors (KIR) are considered to be the key receptors that control the development and function of human natural killer cells which play complex mechanisms in autoimmune diseases. We aimed in this study to assess possible associations between killer cell immunoglobulin-like receptors (KIR2DS1 and 2DS4) genes and susceptibility to autoimmune hepatitis type I in Egyptian children. Results In the case-control study conducted on eighty children diagnosed as autoimmune hepatitis (AIH) type I and eighty apparently healthy age and sex-matched control, we found that KIR2DS1, -2DS4, KIR2DS4-full length allele, and homozygous KIR2DS4-full/full variant were significantly associated with AIH-I, while the KIR1D allele and homozygous KIR2DS4-del/del variant were significantly observed in controls (P < 0.05 each). Absence of KIR2DS4 gene was significant among ANA positive AIH-I patients, patients on steroid therapy alone, and patients showing complete disease remission (P < 0.05 each). Higher activity and fibrosis indices were found significantly in patients lacking one or both studied genes. Conclusions Children carrying KIR2DS1, -2DS4 genes, KIR2DS4-full length allele, and homozygous KIR2DS4-full/full variant could be more susceptible to develop autoimmune hepatitis type I.

Proliferation of Highly Cytotoxic Human Natural Killer Cells by OX40L Armed NK-92 With Secretory Neoleukin-2/15 for Cancer Immunotherapy

Adoptive natural killer (NK) cell transfer has been demonstrated to be a promising immunotherapy approach against malignancies, but requires the administration of sufficient activated cells for treatment effectiveness. However, the paucity of clinical-grade to support the for large-scale cell expansion limits its feasibility. Here we developed a feeder-based NK cell expansion approach that utilizes OX40L armed NK-92 cell with secreting neoleukin-2/15 (Neo-2/15), a hyper-stable mimetic with a high affinity to IL-2Rβγ. The novel feeder cells (NK92-Neo2/15-OX40L) induced the expansion of NK cells with a 2180-fold expansion (median; 5 donors; range, 1767 to 2719) after 21 days of co-culture without added cytokines. These cells were highly cytotoxic against Raji cells and against several solid tumors in vivo. Mechanistically, NK92-Neo2/15-OX40L induced OX40 and OX40L expression on expanded NK cells and promoted the OX40-OX40L positive feedback loop, thus boosting NK cell function. Our data provided a novel NK cell expansion mechanism and insights into OX40-OX40L axis regulation of NK cell expansion.

CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.

Prevalence of KIR2DL2/DS2 and KIR2DL3 and Presence of B19V in Patients with Thyroid Disorders

The functions of human natural killer cells are controlled by diverse families of antigen receptors. Prominent among these are the killer cell immunoglobulin-like receptors (KIR), controlled by a family of genes clustered in one of the most variable regions of the human genome — on chromosome 19q13.4. This study aimed to investigate the possible interplay between KIR allotype, B19 infection, and thyroid disorders. Thyroid gland tissue of 30 patients with autoimmune thyroid gland diseases (AITD), 30 patients with non-autoimmune thyroid gland diseases (non-AITD) and 30 deceased subjects whose histories did not show any of autoimmune or thyroid diseases (control group) were enrolled in the study. The presence of B19V, KIR2DL2/DS2, and KIR2DL3 was detected using PCRs (nPCR, PCR). The results showed that 28% of samples of thyroid tissue from patients with AITD and 67% with non-AITD were positive for the presence of B19V, in contrast only 5% control tissue samples harbored B19V DNA. B19V-positive AITD patients had higher frequency of KIR2DL2/DS2 homozygosity and lower frequency of homozygous KIR2DL3 genotype compared to B19V negative cases (33% vs 21% and 17% vs 46%, respectively). Although our data showed that B19V positive patients with AITD had a higher frequency of homozygosity for KIR2DL2/DS2, further studies with larger groups of patients are necessary to confirm the relationship between KIR, B19V and susceptibility to thyroid disease.