• GVHD
• Graft-versus-host disease
• Immunotherapy
• Inflammation
• Macrophages
• Therapeutic targets
• Transplantation

• Graft vs Host Disease/immunology
• Graft vs Host Disease/therapy
• Graft vs Host Disease/pathology
• Humans
• Macrophages/immunology
• Macrophages/drug effects
• Hematopoietic Stem Cell Transplantation/adverse effects
• Animals

• Allogeneic tumor rejection
• Cord blood CD34
• Endothelial disfunction
• Fetal liver CD34
• Graft-versus-tumor
• HSPC
• Humanized mice
• LSEC
• Sinusoidal dilatation

• Animals
• Humans
• Antigens, CD34/metabolism
• Mice
• Liver/metabolism
• Liver/pathology
• Mice, Inbred NOD
• Hematopoietic Stem Cell Transplantation
• Mice, SCID
• Endothelial Progenitor Cells/metabolism
• Endothelial Progenitor Cells/cytology
• Endothelial Progenitor Cells/transplantation
• Hematopoietic Stem Cells/metabolism
• Hematopoietic Stem Cells/cytology
• Fetal Blood/cytology
• Melanoma/pathology
• Melanoma/immunology

• #H16/01/a0/002 Industry Alignment Fund - Pre-positioning Programme (IAF-PP)
• MOH-001221-01, MOH-000932-00 Singapore's Ministry of Health
• CIRG/1484/2018, NMRC CSA (SI)/008/2016, NMRC STaR22jul-0004 National Medical Research Council (SG)
• NRF2017_SISFP09 National Research Foundation (NRF), Immunomonitoring Service Platform (ISP) grant
• Industry Alignment Fund – Pre-positioning Programme (IAF-PP)
• Singapore’s Ministry of Health
• National Research Foundation (NRF), Immunomonitoring Service Platform (ISP) grant

• HER2
• breast cancer
• cell therapy
• immunotherapy
• trastuzumab, universal chimeric antigen receptor

• Humans
• Mice
• Animals
• Trastuzumab/pharmacology
• Trastuzumab/therapeutic use
• Trastuzumab/metabolism
• Biotin/metabolism
• Heterografts
• Receptor, ErbB-2
• Cell Line, Tumor
• T-Lymphocytes
• Antibody-Dependent Cell Cytotoxicity

Burn healing should be regarded as a dynamic process consisting of two main, interrelated phases: (a) the inflammatory phase when neutrophils and monocytes infiltrate the injury site, through localized vasodilation and fluid extravasation, and (b) the proliferative-remodeling phase, which represents a key event in wound healing. In the skin, both canonical autophagy (induced by starvation, oxidative stress, and environmental aggressions) and non-canonical or selective autophagy have evolved to play a discrete, but, essential, “housekeeping” role, for homeostasis, immune tolerance, and survival. Experimental data supporting the pro-survival roles of autophagy, highlighting its Yang, luminous and positive feature of this complex but insufficient explored molecular pathway, have been reported. Autophagic cell death describes an “excessive” degradation of important cellular components that are necessary for normal cell function. This deadly molecular mechanism brings to light the darker, concealed, Yin feature of autophagy. Autophagy seems to perform dual, conflicting roles in the angiogenesis context, revealing once again, its Yin–Yang features. Autophagy with its Yin–Yang features remains the shadow player, able to decide quietly whether the cell survives or dies.

• autophagy
• burn wound healing
• burns
• oxidative stress

• GvHD
• T cells
• fatty acid oxidation
• glycolysis
• metabolism
• therapeutic targets

Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4⁺ and CD8⁺ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34⁺ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

• CAR-T cells
• HCMV
• PD-1
• humanized mice
• transplantation

Humanized mouse models generated with human hematopoietic stem cells (HSCs) and reconstituting the human immune system (HIS-mice) are invigorating preclinical testing of vaccines and immunotherapies. We have recently shown that human engineered dendritic cells boosted bonafide human T and B cell maturation and antigen-specific responses in HIS-mice. Here, we evaluated a cell-free system based on in vivo co-delivery of lentiviral vectors (LVs) for expression of a human leukocyte antigen (HLA-DRA*01/ HLA-DRB1*0401 functional complex, “DR4”), and a LV vaccine expressing human cytokines (GM-CSF and IFN-α) and a human cytomegalovirus gB antigen (HCMV-gB). Humanized NOD/Rag1^null/IL2Rγ^null (NRG) mice injected by i.v. with LV-DR4/fLuc showed long-lasting (up to 20 weeks) vector distribution and expression in the spleen and liver. In vivo administration of the LV vaccine after LV-DR4/fLuc delivery boosted the cellularity of lymph nodes, promoted maturation of terminal effector CD4⁺ T cells, and promoted significantly higher development of IgG⁺ and IgA⁺ B cells. This modular lentigenic system opens several perspectives for basic human immunology research and preclinical utilization of LVs to deliver HLAs into HIS-mice.

• B cell maturation
• HLA match
• IgG
• class-switch
• cytomegalovirus
• humanized mice
• lentiviral vector
• stem cell transplantation
• vaccine

• T-regulatory cells
• class III obesity
• follow up
• immunity
• laparoscope gastric sleeve surgery
• peripheral CD4+ T-lymphocytes

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent “induced DCs” (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14⁺ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34⁺ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

• GMP
• WT1
• automated
• dendritic cells
• graft-versus-leukemia
• immunetherapy
• lentiviral vectors
• leukemia
• stem cell transplantation

Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV⁺ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81⁺ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34⁺ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8⁺ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67⁺CD30⁺CD20⁺EBER⁺PD-L1⁺ PTLD with central nervous system (CNS) involvement, mirroring EBV⁺ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4⁺ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3⁺ regulatory CD4⁺ and CD8⁺ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8⁺ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV⁺ PTLD.

• Epstein-Barr Virus (EBV)
• PD-1
• humanized mice
• immune checkpoint inhibition (ICI)
• immuno-oncology
• lymphoma
• pembrolizumab
• post-transplant lymphoproliferative disease (PTLD)

• CAR-T cells
• EBV
• PTLD
• adoptive T cell therapy
• gp350
• humanized mice
• lymphoma
• lymphoproliferation
• lytic infection
• transplantation

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8⁺ and CD4⁺ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4⁺, liver B/IgA⁺ and spleen B/IgG⁺ cells as predictive biomarkers of immunization (≈87% accuracy). CD8⁺ and CD4⁺ T cell responses against gB were validated. Splenic gB-binding IgM^-/IgG⁺ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen. As long as the immune system is functional, T and B cells can control HCMV. Yet, for patients who have debilitated immune functions, HCMV infections and reactivations cause major complications. Vaccines or antibodies to prevent or treat HCMV are not yet approved. Novel animal models for testing new immunization approaches are emerging and are important tools to identify biomedical products with a reasonable chance to work in patients. Here, we used a model based on mice transplanted with human immune cells and infected with a traceable HCMV. We tested a cell vaccine (iDCgB) carrying gB, a potent HCMV antigen. The model showed that iDCgB halted the HCMV infection in more than 90% of the mice. We found that antibodies were key players mediating protection. Using state-of-the-art methods, we were able to use the sequences of the human antibodies generated in the mice to construct and produce monoclonal antibodies in the laboratory. Proof-of-concept experiments indicated that administration of these monoclonal antibodies into mice protected them against HCMV infection. In summary, this humanized mouse model was useful to test a vaccine and to generate and test novel antibodies that can be further developed for human use.

• Animals
• Antibodies, Monoclonal/immunology
• Antibodies, Monoclonal/pharmacology
• Antibodies, Neutralizing/immunology
• Antibodies, Neutralizing/pharmacology
• Antibodies, Viral/immunology
• Antigens, Viral/immunology
• Cytomegalovirus/immunology
• Cytomegalovirus Infections/immunology
• Cytomegalovirus Vaccines/immunology
• Dendritic Cells/immunology
• Disease Models, Animal
• Humans
• Immunization, Passive
• Immunoglobulin G/immunology
• Immunoglobulin G/pharmacology
• Mice

Hematopoietic stem cell transplantation has become a curative choice of many hematopoietic malignancy, but graft-vs-host disease (GVHD) has limited the survival quality and overall survival of hematopoietic stem cell transplantation. Understanding of the immune cells’ reaction in pathophysiology of GVHD has improved, but a review on the role of macrophages in GVHD is still absent. Studies have observed that macrophage infiltration is associated with GVHD occurrence and development. In this review, we summarize and analyze the role of macrophages in GVHD based on pathophysiology of acute and chronic GVHD, focusing on the macrophage recruitment and infiltration, macrophage polarization, macrophage secretion, and especially interaction of macrophages with other immune cells. We could conclude that macrophage recruitment and infiltration contribute to both acute and chronic GVHD. Based on distinguishing pathology of acute and chronic GVHD, macrophages tend to show a higher M1/M2 ratio in acute GVHD and a lower M1/M2 ratio in chronic GVHD. However, the influence of dominant cytokines in GVHD is controversial and inconsistent with macrophage polarization. In addition, interaction of macrophages with alloreactive T cells plays an important role in acute GVHD. Meanwhile, the interaction among macrophages, B cells, fibroblasts, and CD4+ T cells participates in chronic GVHD development.

• Macrophage
• Graft-vs-host disease
• Hematopoietic stem cell transplantation
• Polarization
• Cytokine
• Regulation

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and overall survival remains poor. Chemotherapy is the standard of care for intensive induction therapy. Patients who achieve a complete remission require post-remission therapies to prevent relapse. There is no standard of care for patients with minimal residual disease (MRD), and stem cell transplantation is a salvage therapy. Considering the AML genetic heterogeneity and the leukemia immune-suppressive properties, novel cellular immune therapies to effectively harness immunological responses to prevent relapse are needed. We developed a novel modality of immune therapy consisting of monocytes reprogrammed with lentiviral vectors expressing GM-CSF, IFN-α and antigens. Preclinical studies in humanized mice showed that the reprogrammed monocytes self-differentiated into highly viable induced dendritic cells (iDCs) in vivo which migrated effectively to lymph nodes, producing remarkable effects in the de novo regeneration of T and B cell responses. For the first-in-man clinical trial, the patient’s monocytes will be transduced with an integrase-defective tricistronic lentiviral vector expressing GM-CSF, IFN-α and a truncated WT1 antigen. For transplanted patients, pre-clinical development of iDCs co-expressing cytomegalovirus antigens is ongoing. To simplify the product chain for a de-centralized supply model, we are currently exploring a closed automated system for a short two-day manufacturing of iDCs. A phase I clinical trial study is in preparation for immune therapy of AML patients with MRD. The proposed cell therapy can fill an important gap in the current and foreseeable future immunotherapies of AML.

• Immune therapy
• Lentivirus
• Leukemia
• PIVAC 18
• WT1

Syngeneic in vivo tumor models are valuable for the development and investigation of immune-modulating anti-cancer drugs. In the present study, we established a novel syngeneic in vivo model type named mouse-derived isografts (MDIs). Spontaneous MDIs (sMDIs) were obtained during a long-term observation period (more than one to two years) of naïve and untreated animals of various mouse strains (C3H/HeJ, CBA/J, DBA/2N, BALB/c, and C57BL/6N). Primary tumors or suspicious tissues were assessed macroscopically and re-transplanted in a PDX-like manner as small tumor pieces into sex-matched syngeneic animals. Nine outgrowing primary tumors were histologically characterized either as adenocarcinomas, histiocytic carcinomas, or lymphomas. Growth of the tumor pieces after re-transplantation displayed model heterogeneity. The adenocarcinoma sMDI model JA-0009 was further characterized by flow cytometry, RNA-sequencing, and efficacy studies. M2 macrophages were found to be the main tumor infiltrating leukocyte population, whereas only a few T cells were observed. JA-0009 showed limited sensitivity when treated with antibodies against inhibitory checkpoint molecules (anti-mPD-1 and anti-mCTLA-4), but high sensitivity to gemcitabine treatment. The generated sMDI are spontaneously occurring tumors of low passage number, propagated as tissue pieces in mice without any tissue culturing, and thus conserving the original tumor characteristics and intratumoral immune cell populations.

• carcinogen-induced tumors
• experimental cancer
• immune checkpoint inhibitors
• immunocompetent animals
• mouse tumor models
• mouse-derived isografts (MDIs)
• spontaneous tumors
• syngeneic
• therapy

• cell-based immunotherapy
• humanized mice
• mouse/human chimera
• preclinical model
• rheumatoid arthritis
• transgenic mice

Humanized mice developing functional human T cells endogenously and capable of recognizing cognate human leukocyte antigen–matched tumors are emerging as relevant models for studying human immuno-oncology in vivo. Herein, mice transplanted with human CD34⁺ stem cells and bearing endogenously developed human T cells for >15 weeks were infected with an oncogenic recombinant Epstein-Barr virus (EBV), encoding enhanced firefly luciferase and green fluorescent protein. EBV–firefly luciferase was detectable 1 week after infection by noninvasive optical imaging in the spleen, from where it spread rapidly and systemically. EBV infection resulted into a pronounced immunologic skewing regarding the expansion of CD8⁺ T cells in the blood outnumbering the CD4⁺ T and CD19⁺ B cells. Furthermore, within 10 weeks of infections, mice developing EBV-induced tumors had significantly higher absolute numbers of CD8⁺ T cells in lymphatic tissues than mice controlling tumor development. Tumor outgrowth was paralleled by an up-regulation of the programmed cell death receptor 1 on CD8⁺ and CD4⁺ T cells, indicative for T-cell dysfunction. Histopathological examinations and in situ hybridizations for EBV in tumors, spleen, liver, and kidney revealed foci of EBV-infected cells in perivascular regions in close association with programmed cell death receptor 1–positive infiltrating lymphocytes. The strong spatiotemporal correlation between tumor development and the T-cell dysfunctional status seen in this viral oncogenesis humanized model replicates observations obtained in the clinical setting.

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14⁺, CD169⁺, and CD34⁺ cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4⁺ T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG⁺ plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4⁺ T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.

• B cell class switch
• HCMV
• T cell maturation
• humanized mice
• linear discriminant analyses
• optical imaging analyses
• principal component analyses
• reactivation

• T‐cell targeting
• cytokine release syndrome
• gene delivery
• humanized mouse

• Animals
• Antigens, CD19/metabolism
• B-Lymphocytes/immunology
• Cytokines/metabolism
• Graft vs Host Disease/immunology
• HEK293 Cells
• Humans
• Leukocytes, Mononuclear/transplantation
• Lymphocyte Depletion
• Mice
• Receptors, Chimeric Antigen/immunology
• Syndrome
• T-Lymphocytes/immunology

The skin is a highly organized first line of defense that stretches up to 1.8 m² and is home to more than a million commensal bacteria. The microenvironment of skin is driven by factors such as pH, temperature, moisture, sebum level, oxidative stress, diet, resident immune cells, and infectious exposure. The skin has a high turnover of cells as it continually bares itself to environmental stresses. Notwithstanding these limitations, it has devised strategies to adapt as a nutrient-scarce site. To perform its protective function efficiently, it relies on mechanisms to continuously remove dead cells without alarming the immune system, actively purging the dying/senescent cells by immunotolerant efferocytosis. Both canonical (starvation-induced, reactive oxygen species, stress, and environmental insults) and non-canonical (selective) autophagy in the skin have evolved to perform astute due-diligence and housekeeping in a quiescent fashion for survival, cellular functioning, homeostasis, and immune tolerance. The autophagic “homeostatic rheostat” works tirelessly to uphold the delicate balance in immunoregulation and tolerance. If this equilibrium is upset, the immune system can wreak havoc and initiate pathogenesis. Out of all the organs, the skin remains under-studied in the context of autophagy. Here, we touch upon some of the salient features of autophagy active in the skin.

• autophagy
• selective autophagy
• skin autoimmunity
• skin cancers
• skin diseases

• Adult
• Aged
• Bariatric Surgery
• Female
• Humans
• Immunologic Memory
• Male
• Middle Aged
• Obesity, Abdominal/blood
• Obesity, Abdominal/immunology
• Obesity, Abdominal/surgery
• Severity of Illness Index
• T-Lymphocytes, Regulatory/immunology
• T-Lymphocytes, Regulatory/metabolism
• Th17 Cells/immunology
• Th17 Cells/metabolism

Mice transplanted with human cord blood-derived hematopoietic stem cells (HSCs) became a powerful experimental tool for studying the heterogeneity of human immune reconstitution and immune responses in vivo. Yet, analyses of human T cell maturation in humanized models have been hampered by an overall low immune reactivity and lack of methods to define predictive markers of responsiveness. Long-lived human lentiviral induced dendritic cells expressing the cytomegalovirus pp65 protein (iDCpp65) promoted the development of pp65-specific human CD8⁺ T cell responses in NOD.Cg-Rag1^tm1Mom-Il2rγ^tm1Wj humanized mice through the presentation of immune-dominant antigenic epitopes (signal 1), expression of co-stimulatory molecules (signal 2), and inflammatory cytokines (signal 3). We exploited this validated system to evaluate the effects of mouse sex in the dynamics of T cell homing and maturation status in thymus, blood, bone marrow, spleen, and lymph nodes. Statistical analyses of cell relative frequencies and absolute numbers demonstrated higher CD8⁺ memory T cell reactivity in spleen and lymph nodes of immunized female mice. In order to understand to which extent the multidimensional relation between organ-specific markers predicted the immunization status, the immunophenotypic profiles of individual mice were used to train an artificial neural network designed to discriminate immunized and non-immunized mice. The highest accuracy of immune reactivity prediction could be obtained from lymph node markers of female mice (77.3%). Principal component analyses further identified clusters of markers best suited to describe the heterogeneity of immunization responses in vivo. A correlation analysis of these markers reflected a tissue-specific impact of immunization. This allowed for an organ-resolved characterization of the immunization status of individual mice based on the identified set of markers. This new modality of multidimensional analyses can be used as a framework for defining minimal but predictive signatures of human immune responses in mice and suggests critical markers to characterize responses to immunization after HSC transplantation.

• T cell maturation
• artificial neural network
• cord blood
• dendritic cell
• gender
• hematopoietic stem cell transplantation
• humanized mice
• lymphatic