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Introduction The expansion of insulin-producing beta cells during pregnancy is critical to maintain glucose homeostasis in the face of increasing insulin resistance. Prolactin receptor (PRLR) signaling is one of the primary mediators of beta cell expansion during pregnancy, and loss of PRLR signaling results in reduced beta cell mass and gestational diabetes. Harnessing the proliferative potential of prolactin signaling to expand beta cell mass outside of the context of pregnancy requires quantitative understanding of the signaling at the molecular level. Methods A mechanistic computational model was constructed to describe prolactin-mediated JAK-STAT signaling in pancreatic beta cells. The effect of different regulatory modules was explored through ensemble modeling. A Bayesian approach for likelihood estimation was used to fit the model to experimental data from the literature. Results Including receptor upregulation, with either inhibition by SOCS proteins, receptor internalization, or both, allowed the model to match experimental results for INS-1 cells treated with prolactin. The model predicts that faster dimerization and nuclear import rates of STAT5B compared to STAT5A can explain the higher STAT5B nuclear translocation. The model was used to predict the dose response of STAT5B translocation in rat primary beta cells treated with prolactin and reveal possible strategies to modulate STAT5 signaling. Conclusions JAK-STAT signaling must be tightly controlled to obtain the biphasic response in STAT5 activation seen experimentally. Receptor up-regulation, combined with SOCS inhibition, receptor internalization, or both is required to match experimental data. Modulating reactions upstream in the signaling can enhance STAT5 activation to increase beta cell survival.

The in vivo tissue distribution and trafficking patterns of natural killer (NK) cells remain understudied. Animal models can help bridge the gap, and rhesus macaque (RM) primates faithfully recapitulate key elements of human NK cell biology. Here, we profiled the tissue distribution and localization patterns of three NK cell subsets across various RM tissues. We utilized serial intravascular staining (SIVS) to investigate the tissue trafficking kinetics at steady state and during recovery from CD16 depletion. We found that at steady state, CD16+ NK cells were selectively retained in the vasculature while CD56+ NK cells had a shorter residence time in peripheral blood. We also found that different subsets of NK cells had distinct trafficking kinetics to and from the lymph node as well as other lymphoid and non-lymphoid tissues. Lastly, we found that following administration of CD16-depleting antibody, CD16+ NK cells and their putative precursors retained a high proportion of continuously circulating cells, suggesting that regeneration of the CD16 NK compartment may take place in peripheral blood or the perivascular compartments of tissues.

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Recent phenotypic, functional and transcriptomic analyses of natural killer (NK) cells in human and animals have established the presence of tissue resident NK(trNK) cells with specific characteristics and a central role in NK memory. The lack of endogenous clonal markers on NK cells impedes understanding the clonal genesis of trNKs. Transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPCs) has allowed tracking of NK cells at a clonal level in rhesus macaques (RM). We reported large KIR-restricted clonal expansions of mature CD56 -CD16 +NK in the peripheral blood (PB) of RM, clonally distinct from myeloid, T, B and CD56 +16 -NK, persisting for months to years, suggesting self-renewal independent of ongoing production from HSPCs (Wu et al, Cell Stem Cell, 2014 and Science Imm, 2018). We have now used this model to investigate the clonal distribution of NK cell populations in bone marrow (BM), liver, spleen, lymph nodes (LN), jejunum, colon and bronchoalveolar lavage (BAL). Serial of tissue biopsies were obtained from 3 barcoded monkeys by endoscopy and laparotomy overtime at steady state post transplantation , as well as tissues from necropsy. We compared clonal patterns between various trNKs and PB NKs collected from barcoded RM. Tissue or PB NK were defined as CD3-CD14-CD20-NKG2+ (NKG2A+ and NKG2C+), and NK subsets were further sorted for CD16, CD56, CD49a (putative liver tissue memory-like NK marker), and CXCR3 (critical for NK cell migration into tumor or normal tissues). The same expanded CD56 -CD16 +NK clones found in the PB were also detected at high abundance within BM, LN, liver and/or spleen CD56 -CD16 +NK, but not found in tissue CD56 +16 -NK and CD56 -16 -NK subsets. The liver and spleen bulk NK clonal patterns were highly correlated, and distinct from other tissues. We also observed tissue specific barcoded NK clones in BAL, jejunum and colon samples with no or very low abundance in other tissues and PB. Strikingly, a group of markedly expanded trNK clones, distinct from the expanded CD56 -CD16 +NK clones present concurrently or previously in PB, were present and shared across all tissues examined. These clones were enriched in CD56 -16 + trNK and absent in CD56 +16 - trNK. Notably, in both tissue and PB these clones were expanded in NKG2+ CD56-CD16- NK. These common expanded trNK cells were specifically enriched in both tissue and PB CD56 -16 -CXCR3 +NK, suggesting a role for this chemokine receptor and the ability of these clones to move between tissues. In contrast, CD49a expression did not enrich for these expanded clones. Clonally-expanded and persistent mature trNK cells, shared across multiple tissues but not present within PB mature CD56 -CD16 +NK subsets, combined with prior functional data suggesting NK memory is restricted to liver or other trNK cells, suggests these clonally-expanded trNK cells may be of interest. The pattern of shared clones across tissues, together with identification of a rare PB CD56-CD16-NK subpopulation harboring the clones, suggests preferential hematogenous homing of these clones to multiple tissues. Further analyses of gene expression and clonal dynamics are ongoing and should shed light on the ontology of trNK cells, with implications for the development of NK based immunotherapies and NK memory. Disclosures No relevant conflicts of interest to declare.

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By virtue of their direct cytotoxicity to transformed and virus infected cells, Natural Killer (NK) cells play crucial roles in immunity. NK cells modulate and coordinate innate and adaptive responses through the release of chemokines and cytokines. Although NK cells are endowed only with germ-line encoded receptors, evidence has been accumulating, that subsets of NK cells can bestow adoptively transferable, long-lasting and antigen-specific immune responses to certain haptens and viruses. Growing evidence suggests that adaptive immune responses lie on a spectrum. Rechallenge of cells, canonically belonging to the innate immune system, can result in enhanced responsiveness - a process termed 'trained immunity' and thought to be maintained by epigenetic and metabolic reprogramming. In previous work, our lab studied the role of NK cell responses to rhesus cytomegalovirus (rhCMV) in a genetic barcoding model. We found that new clones arose in the CD16 + NK compartment after primary rhCMV infection. There was rapid clearance without the emergence of new clones in subsequent rechallenge with rhCMV. In this study we used 3'-end single cell RNA-seq (3'-scRNA-seq) with CITE-seq to profile NK and T cells from an initially CMV-naïve rhesus macaque (RM) at four time points before and after primary and secondary infections with rhCMV. We immunophenotypically sorted NK and T cells from peripheral Blood (PB) samples at 'baseline', 30 days after initial rhCMV infection ('primary infection'), ca. 500 days after initial rhCMV infection ('steady state') and 10 days after rmCMV reinfection ('secondary infection'). Alongside the PB samples at 'steady state' and after 'secondary infection', we also sorted NK and T cells from lymph nodes (LN). We applied CD16 and CD56 CITE-seq antibodies to NK cells from all samples; NK cells from the 'steady state' and 'secondary infection' samples were also labeled with CX3CL1 CITE-seq antibodies. We multiplexed NK and T cells from each time point in 4:1 ratios before preparing 3'-scRNA-seq libraries. We used scanpy and scvi-tools as well as custom python code to demultiplex NK from T cells, harmonize 3'-scRNA-seq with CITE-seq data and integrate the 6 different samples. We used scvelo and cellrank to compute RNA velocities and infer trajectories, respectively. We obtained a total of 35,523 high-quality cells. We identified 20 clusters of NK and T cells, on the basis of community detection via the Leiden algorithm. All clusters contained cells from both tissue sources. The 4 clusters characterized by expression of CD56 exhibit higher expression of KLRC1 (protein: NKG2A), IL7R and the transcription factors LEF1 and MYC. The 8 clusters of CD16 + cells are distinguished by high expression of the transcription factors ZEB2 and TBX21/T-BET, cytotoxicity markers, GZMB and PRF1, and activating receptors, KLRC2 (protein: NKG2C), KLRC3 (protein: NKG2E) and NCR3 (protein: NKp30). An adaptive population of NK cells is identified on the basis of high KLRC2 and low FCER1G expression. We analyzed changes in the proportions of cells in each cluster of the time course of CMV infection using a binomial generalized linear model. Clusters associated with proliferation and acute inflammation were increased in proportion after primary rhCMV infection; the proportion of the adaptive population did not significantly change during the acute phase of primary infection but increased markedly by the later 'steady state' samples. RNA velocity and inferred developmental trajectories suggest transitions between the adaptive, proliferating CD16 + and mature effector subsets; the predominant path into the adaptive population occurring from the proliferating CD16 + subset after primary infection. There is a notable paucity of inferred transitions between the CD56 + and CD16 +subpopulations under all the experimental conditions we observed. We have characterized the single cell transcriptional states and dynamics of RM NK cells in response to rhCMV infection. We focus on a subset transcriptionally resembling a previously identified subset with adaptive function and find it arises from a proliferating population of effector cells after primary infection. This may be analogous to the dedifferentiation of effector CD8 T cells into memory T cells proposed by Youngblood et al. Confirmatory experiments to analyze the reconstitution of the CD16 + compartment after treatment with a depleting antibody are on-going. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.