Testing Cancer Immunotherapy in a Human Immune System Mouse Model: Correlating Treatment Responses to Human Chimerism, Therapeutic Variables and Immune Cell Phenotypes.

Over the past decade, immunotherapies have revolutionized the treatment of cancer. Although the success of immunotherapy is remarkable, it is still limited to a subset of patients. More than 1500 clinical trials are currently ongoing with a goal of improving the efficacy of immunotherapy through co-administration of other agents. Preclinical, small-animal models are strongly desired to increase the pace of scientific discovery, while reducing the cost of combination drug testing in humans. Human immune system (HIS) mice are highly immune-deficient mouse recipients rtpeconstituted with human hematopoietic stem cells. These HIS-mice are capable of growing human tumor cell lines and patient-derived tumor xenografts. This model allows rapid testing of multiple, immune-related therapeutics for tumors originating from unique clinical samples. Using a cord blood-derived HIS-BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) mouse model, we summarize our experiments testing immune checkpoint blockade combinations in these mice bearing a variety of human tumors, including breast, colorectal, pancreatic, lung, adrenocortical, melanoma and hematological malignancies. We present in-depth characterization of the kinetics and subsets of the HIS in lymph and non-lymph organs and relate these to protocol development and immune-related treatment responses. Furthermore, we compare the phenotype of the HIS in lymph tissues and tumors. We show that the immunotype and amount of tumor infiltrating leukocytes are widely-variable and that this phenotype is tumor-dependent in the HIS-BRGS model. We further present flow cytometric analyses of immune cell subsets, activation state, cytokine production and inhibitory receptor expression in peripheral lymph organs and tumors. We show that responding tumors bear human infiltrating T cells with a more inflammatory signature compared to non-responding tumors, similar to reports of "responding" patients in human immunotherapy clinical trials. Collectively these data support the use of HIS mice as a preclinical model to test combination immunotherapies for human cancers, if careful attention is taken to both protocol details and data analysis.

Human innate lymphoid cell precursors express CD48 that modulates ILC differentiation through 2B4 signaling.

Innate lymphoid cells (ILCs) develop from common lymphoid progenitors (CLPs), which further differentiate into the common ILC progenitor (CILP) that can give rise to both ILCs and natural killer (NK) cells. Murine ILC intermediates have recently been characterized, but the human counterparts and their developmental trajectories have not yet been identified, largely due to the lack of homologous surface receptors in both organisms. Here, we show that human CILPs (CD34+CD117+α4ß7+Lin-) acquire CD48 and CD52, which define NK progenitors (NKPs) and ILC precursors (ILCPs). Two distinct NK cell subsets were generated in vitro from CD34+CD117+α4ß7+Lin-CD48-CD52+ and CD34+CD117+α4ß7+Lin-CD48+CD52+ NKPs, respectively. Independent of NKPs, ILCPs exist in the CD34+CD117+α4ß7+Lin-CD48+CD52+ subset and give rise to ILC1s, ILC2s, and NCR+ ILC3s, whereas CD34+CD117+α4ß7+Lin-CD48+CD52- ILCPs give rise to a distinct subset of ILC3s that have lymphoid tissue inducer (LTi)-like properties. In addition, CD48-expressing CD34+CD117+α4ß7+Lin- precursors give rise to tissue-associated ILCs in vivo. We also observed that the interaction of 2B4 with CD48 induced differentiation of ILC2s, and together, these findings show that expression of CD48 by human ILCPs modulates ILC differentiation.

Prolactin Acts on Myeloid Progenitors to Modulate SMAD7 Expression and Enhance Hematopoietic Stem Cell Differentiation into the NK Cell Lineage.

Numerous cell types modulate hematopoiesis through soluble and membrane bound molecules. Whether developing hematopoietic progenitors of a particular lineage modulate the differentiation of other hematopoietic lineages is largely unknown. Here we aimed to investigate the influence of myeloid progenitors on CD34+ cell differentiation into CD56+ innate lymphocytes. Sorted CD34+ cells cultured in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L) give rise to numerous cell types, including progenitors that expressed the prolactin receptor (PRLR). These CD34+PRLR+ myeloid-lineage progenitors were derived from granulocyte monocyte precursors (GMPs) and could develop into granulocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Moreover, CD34+PRLR+ myeloid progenitors lacked lymphoid developmental potential, but when stimulated with prolactin (PRL) they increased the differentiation of other CD34+ cell populations into the NK lineage in a non-contact dependent manner. Both mRNA and protein analyses show that PRL increased mothers against decapentaplegic homolog 7 (SMAD7) in CD34+PRLR+ myeloid cells, which reduced the production of transforming growth factor beta 1 (TGF-ß1), a cytokine known to inhibit CD56+ cell development. Thus, we uncover an axis whereby CD34+PRLR+ GMPs inhibit CD56+ lineage development through TGF-ß1 production and PRL stimulation leads to SMAD7 activation, repression of TGF-ß1, resulting in CD56+ cell development.

Transient Expression of GATA3 in Hematopoietic Stem Cells Facilitates Helper Innate Lymphoid Cell Differentiation.

Helper Innate lymphoid cells (ILCs) are tissue resident lymphocytes that play a critical role in a number of biological processes. Several transcription factors are required for the differentiation of hematopoietic stem cells (HSCs) into ILCs. Recent studies demonstrate GATA3 as a transcriptional regulator that plays an essential role in ILC development. We aimed to modulate the differentiation of human cord blood-derived CD34+ cells into ILCs by transient and ectopic expression of mRNA encoding transcription factors known to be important for ILC lineage differentiation, including GATA3, TOX, NFIL3, ID2, and RORγt. Using this experimental protocol, only GATA3 significantly modulated HSCs to differentiate into helper ILCs. Transient overexpression of GATA3 drove the emergence of CD34+α4ß7+ early ILC progenitors during the first few days of culture. These ILC progenitors further acquired IL-7Rα and CD117 to give rise to immediate ILC precursors. In support of these findings, analysis of the genes induced by GATA3 in HSCs showed an upregulation of those associated with ILC development. Moreover, we show GATA3 also acts on more committed progenitors and significantly shifts the differentiation of progenitors away from the ILC1/NK lineage to the ILC2 and ILC3 lineage. In summary, transient overexpression of GATA3 mRNA in CD34+ HSCs enhances the differentiation of HSCs into the helper ILC lineages, at the expense of NK cell development.

Aryl hydrocarbon receptor inhibition promotes hematolymphoid development from human pluripotent stem cells.

The aryl hydrocarbon receptor (AHR) plays an important physiological role in hematopoiesis. AHR is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and inhibition of AHR results in a marked expansion of human umbilical cord blood-derived HSPCs following cytokine stimulation. It is unknown whether AHR also contributes earlier in human hematopoietic development. To model hematopoiesis, human embryonic stem cells (hESCs) were allowed to differentiate in defined conditions in the presence of the AHR antagonist StemReginin-1 (SR-1) or the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We demonstrate a significant increase in CD34+CD31+ hematoendothelial cells in SR-1-treated hESCs, as well as a twofold expansion of CD34+CD45+ hematopoietic progenitor cells. Hematopoietic progenitor cells were also significantly increased by SR-1 as quantified by standard hematopoietic colony-forming assays. Using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-engineered hESC-RUNX1c-tdTomato reporter cell line with AHR deletion, we further demonstrate a marked enhancement of hematopoietic differentiation relative to wild-type hESCs. We also evaluated whether AHR antagonism could promote innate lymphoid cell differentiation from hESCs. SR-1 increased conventional natural killer (cNK) cell differentiation, whereas TCDD treatment blocked cNK development and supported group 3 innate lymphoid cell (ILC3) differentiation. Collectively, these results demonstrate that AHR regulates early human hematolymphoid cell development and may be targeted to enhance production of specific cell populations derived from human pluripotent stem cells.

Brief Report: HIV-1 Infection Results in Increased Frequency of Active and Inflammatory SlanDCs that Produce High Level of IL-1ß.

HIV infection is marked by phenotypic and functional alterations of immune cells. Different studies have shown both numerical and functional deterioration of dendritic cells in HIV-1-infected patients. In this study, we report an increase of inflammatory 6-sulfo LacNAc dendritic cells (slanDCs) that are more activated and produce higher amounts of interleukin (IL)-1ß during HIV-1 infection as compared with healthy controls. IL-1ß plays a regulatory role in chronic inflammatory disorders. Therefore, our findings might reveal a compensatory regulatory function of slanDCs during HIV-1 infection.

The TLR-2/TLR-6 agonist macrophage-activating lipopeptide-2 augments human NK cell cytotoxicity when PGE2 production by monocytes is inhibited by a COX-2 blocker.

Macrophage-activating lipopeptide-2 (MALP-2) is a potent inducer of proinflammatory cytokine secretion by macrophages, monocytes, and dendritic cells. MALP-2 was reported to be involved in natural killer (NK) cell activation and ensuing tumor rejection. However, the mechanism of MALP-2-mediated NK cell activation remained unclear. Therefore, we studied the effects of MALP-2 on cultured human NK cells. We found that MALP-2 had no direct effect on NK cells. Instead, MALP-2 acted on monocytes and triggered the release of different molecules such as interleukin (IL)-1ß, IL-6, IL-10, IL-12, IL-15, interferon gamma-induced protein (IP-10), and prostaglandin (PG)-E2. Our data show that monocyte-derived IP-10 could significantly induce NK cell cytotoxicity as long as the immunosuppression by PGE2 is specifically inhibited by cyclooxygenase (COX)-2 blockade. In summary, our results show that MALP-2-mediated stimulation of monocytes results in the production of several mediators which, depending on the prevailing conditions, affect the activity of NK cells in various ways. Hence, MALP-2 administration with concurrent blocking of COX-2 can be considered as a promising approach in MALP-2-based adjuvant tumor therapies.

TNFR2 and IL-12 coactivation enables slanDCs to support NK-cell function via membrane-bound TNF-α.

Human blood NK cells exert strong cytotoxicity against transformed cells and produce different cytokines and chemokines with an important role in modulating immune responses. However, the nature of NK-cell function depends on NK-cell interaction with other immune cells. One type of immune cells that communicate with NK cells are 6-sulfo LacNAc DCs (slanDCs), which comprise a major subpopulation of proinflammatory human blood DCs. In this study, we investigated the molecular mechanisms by which slanDCs interact with NK cells. Our in vitro studies demonstrate that LPS-stimulated slanDCs enhance activation and function of NK cells essentially via membrane-bound TNF-α (mTNF-α). LPS stimulation upregulates expression of mTNF-α in slanDCs, and surface TNF receptor 2 (TNFR2) is upregulated on NK cells after coincubation with slanDCs. IL-12 secreted by slanDCs increases surface expression of TNFR2 in NK cells. TNFR2 signaling in NK cells leads to activation of NF-kB, a transcription factor for cytokines such as GM-CSF. GM-CSF provided by NK cells is responsible for enhancing IL-12 secretion in slanDCs. In conclusion, TNFR2 and IL-12 signaling, which support one another, enables slanDCs to enhance NK-cell function through mTNF-α, thereby regulating immune responses.