James P. Di Santo

Biological predictors of variable vaccine responses are lacking. We hypothesized that variability in prevaccine innate immune responses, specifically for type I interferons (IFN-I), is predictive of postvaccine antigen-specific responses. To test this, we assessed prevaccine immune responses at protein and transcriptomic levels following whole blood stimulation with Toll-like receptor (TLR) viral agonists in healthy adolescents and adults. Four weeks after the second vaccine dose, with either the BNT162b2 mRNA or CoronaVac inactivated virus vaccine, we assessed antigen-specific T cell cytokine responses and plasma antibody levels. BNT162b2 vaccinees had increased production of the antigen-specific T cell cytokines interleukin-2 (IL-2), interferon-γ, and IL-21 after severe acute respiratory syndrome coronavirus 2 spike stimulation, as well as increased antibody levels and serum pseudo-neutralization compared with CoronaVac recipients. In direct support of our hypothesis, we find that prevaccine poly(I:C) (polyinosine-polycytidylic acid; TLR3 viral agonist) IFN-I responses were significantly associated with the postvaccine T cell cytokine responses. In an independent cohort of 990 healthy donors, we confirmed the significant association between poly(I:C)-induced IFN-α and spike-induced cytokines in mRNA vaccine recipients. We further confirmed this specific association in a cohort of healthy Europeans and identified a common genetic polymorphism in TLR3 that affects IFN-I induction and subsequent vaccine-specific T cell responses. This study shows that preexisting innate immune variability can predict the effectiveness of vaccine responses and identifies pathways relevant to mRNA vaccination. Targeting the specific innate immune pathway relevant for a vaccine may provide a new approach for tailoring vaccines to different populations.

Tumor infiltration by XCR1⁺ conventional dendritic cells (cDC1) correlates strongly with favorable prognosis and improved responses to immunotherapy. Yet, tumor-driven immunosuppressive programs restrict efficient cDC1 recruitment, highlighting the need for strategies to increase cDC1 access to the tumor microenvironment. Here, we establish a proof-of-concept cell-based immunotherapy that enhances the infiltration of circulating cDC1 progenitors and supports their local expansion. Intratumoral engraftment of autologous mesenchymal stromal cells engineered to express membrane bound FLT3L promotes cDC1 recruitment when combined with poly(I:C). We identify poly(I:C)-induced CXCL9 and CCL5 as essential chemokines controlling intratumoral cDC1 infiltration. Stromal cell–mediated local delivery of FLT3L together with CXCL9 and CCL5 is sufficient to enhance cDC1 infiltration in mice or humanized mice settings. Finally, this approach activates antitumor immunity and partially overcomes resistance to immune checkpoint blockade. Collectively, our data support the therapeutic potential of expanding intratumoral cDC1s through local and sustained delivery of FLT3L, CXCL9, and CCL5.

Tumor infiltration by dendritic cells (DCs) has been identified as a biomarker associated with favorable outcomes and improved responses to immunotherapies by T cell immune checkpoint blockade. However, tumor induced immunosuppressive pathways contrive efficient DC infiltration. This calls for the development of DC-targeted interventions purposed to stimulate DC infiltration and local expansion. Here we deliver a proof-of-concept for a cell-based immunotherapeutic approach promoting circulating DC progenitors infiltration in the tumor together with their local expansion. We report that engraftment of autologous mesenchymal stromal cells engineered to express the membrane bound form of FLT3L (eMSC-FLT3L) uniquely induce DC infiltration, T cells activation and anti-tumor immunity, when combined with an adjuvant like the poly(I:C) double stranded RNA mimic. Mechanistic experiments demonstrate that poly(I:C)-induced CXCR3 and CCR5 ligands (CXCL9, CCL5) are required for intra-tumoral DC and T cell recruitment and for the therapeutic effect of eMSC-FLT3L + poly(I:C) immunotherapy. Stromal cell-based delivery of CXCL9 and CCL5, together with FLT3L, by-passes the requirement for poly(I:C) agonist and is sufficient to induce T cell and DC recruitment within tumors. These findings are transferrable to human settings as we show that eMSCs-FLT3L-CXCL9-CCL5 stimulate tissue infiltration by human XCR1⁺ DC1s in mice harboring a ‘Human Immune System’ (HIS mice). Lastly, we report that engraftment of eMSC-FLT3L-CXCL9-CCL5 in the tumor induces anti-tumor immunity and partially overcomes resistance to immune checkpoint blockade. Altogether, these data support the therapeutic potential of expanding intra-tumoral DC1s using a continuous and local source of FLT3L, CXCL9 and CCL5. One sentence summary Intra-tumoral delivery of FLT3L synergizes with CXCL9 and CCL5 to activate the expansion of intra-tumoral DCs.

Entry of viral capsids into the nucleus induces the formation of biomolecular condensates called HIV-1 membraneless organelles (HIV-1-MLOs). Several questions remain about their persistence, in vivo formation, composition, and function. Our study reveals that HIV-1-MLOs persisted for several weeks in infected cells, and their abundance correlated with viral infectivity. Using an appropriate animal model, we show that HIV-1-MLOs were formed in vivo during acute infection. To explore the viral structures present within these biomolecular condensates, we used a combination of double immunogold labeling, electron microscopy and tomography, and unveiled a diverse array of viral core structures. Our functional analyses showed that HIV-1-MLOs remained stable during treatment with a reverse transcriptase inhibitor, maintaining the virus in a dormant state. Drug withdrawal restored reverse transcription, promoting efficient virus replication akin to that observed in latently infected patients on antiretroviral therapy. However, when HIV-1 MLOs were deliberately disassembled by pharmacological treatment, we observed a complete loss of viral infectivity. Our findings show that HIV-1 MLOs shield the final reverse transcription product from host immune detection.

Infants with biallelic IL7R loss-of-function variants have severe combined immune deficiency (SCID) characterized by the absence of autologous T lymphocytes, but normal counts of circulating B and NK cells (T ^– B ⁺ NK ⁺ SCID). We report 6 adults (aged 22 to 59 years) from 4 kindreds and 3 ancestries (Colombian, Israeli Arab, Japanese) carrying homozygous IL7 loss-of-function variants resulting in combined immunodeficiency (CID). Deep immunophenotyping revealed relatively normal counts and/or proportions of myeloid, B, NK, and innate lymphoid cells. By contrast, the patients had profound T cell lymphopenia, with low proportions of innate-like adaptive mucosal-associated invariant T and invariant NK T cells. They also had low blood counts of T cell receptor (TCR) excision circles, recent thymic emigrant T cells and naive CD4 ⁺ T cells, and low overall TCR repertoire diversity, collectively indicating impaired thymic output. The proportions of effector memory CD4 ⁺ and CD8 ⁺ T cells were high, indicating IL-7–independent homeostatic T cell proliferation in the periphery. Intriguingly, the proportions of other T cell subsets, including TCRγδ ⁺ T cells and some TCRαβ ⁺ T cell subsets (including Th1, Tfh, and Treg) were little affected. Peripheral CD4 ⁺ T cells displayed poor proliferation, but normal cytokine production upon stimulation with mitogens in vitro. Thus, inherited IL-7 deficiency impairs T cell development less severely and in a more subset-specific manner than IL-7R deficiency. These findings suggest that another IL-7R–binding cytokine, possibly thymic stromal lymphopoietin, governs an IL-7–independent pathway of human T cell development.

Summary: Group 3 innate lymphoid cells (ILC3s) are tissue‐resident immune lymphocytes that critically regulate intestinal homeostasis, organogenesis, and immunity. ILC3s possess the capacity to “sense” the inflammatory environment within tissues, especially in the context of pathogen challenges that imprints durable non‐antigen‐specific changes in ILC3 function. As such, ILC3s become a new actor in the emerging field of trained innate immunity. Here, we summarize recent discoveries regarding ILC3 responses to bacterial challenges and the role these encounters play in triggering trained innate immunity. We further discuss how signaling events throughout ILC3 ontogeny potentially control the development and function of trained ILC3s. Finally, we highlight the open questions surrounding ILC3 “training” the answers to which may reveal new insights into innate immunity. Understanding the fundamental concepts behind trained innate immunity could potentially lead to the development of new strategies for improving immunity‐based modulation therapies for inflammation, infectious diseases, and cancer.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1 -expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4 -deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR ⁺ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4 ⁺ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.

Human immune system (HIS) mice provide a model to study human immune responses in vivo. Currently available HIS mouse models may harbor mouse Fc Receptor (FcR)‐expressing cells that exert potent effector functions following administration of human Ig. Previous studies showed that the ablation of the murine FcR gamma chain (FcR‐γ) results in loss of antibody‐dependent cellular cytotoxicity and antibody‐dependent cellular phagocytosis in vivo. We created a new FcR‐γ‐deficient HIS mouse model to compare host (mouse) versus graft (human) effects underlying antibody‐mediated immune responses in vivo. FcR‐γ‐deficient HIS recipients lack expression and function of mouse activating FcRs and can be stably and robustly reconstituted with human immune cells. By screening blood B‐cell depletion by rituximab Ig variants, we found that human FcγRs‐mediated IgG1 effects, whereas mouse activating FcγRs were dominant in IgG4 effects. Complement played a role as an IgG1 variant (IgG1 K322A) lacking complement binding activity was largely ineffective. Finally, we provide evidence that FcγRIIIA on human NK cells could mediate complement‐independent B‐cell depletion by IgG1 K322A. We anticipate that our FcR‐γ‐deficient HIS model will help clarify mechanisms of action of exogenous administered human antibodies in vivo.

An orchestrated cellular network, including adaptive lymphocytes and group 3 innate lymphoid cells (ILC3s), maintains intestinal barrier integrity and homeostasis. T cells can monitor environmental insults through constitutive circulation, scanning tissues and forming immunological contacts, a process named immunosurveillance. In contrast, the dynamics of intestinal ILC3s are unknown. Using intravital imaging, we observed that villus ILC3s were largely immotile at steady state but acquired migratory ‘patrolling’ attributes and enhanced cytokine expression in response to inflammation. We showed that T cells, the chemokine CCL25 and bacterial ligands regulated intestinal ILC3 behavior and that loss of patrolling behavior by interleukin-22 (IL-22)-producing ILC3s altered the intestinal barrier through increased epithelial cell death. Collectively, we identified notable differences between the behavior of ILC3s and T cells, with a prominent adaptation of intestinal ILC3s toward mucosal immunosurveillance after inflammation.