Phenotype of BTK-lacking myeloid cells during prolonged COVID-19 and upon convalescent plasma.

XLA patient with 7-month course of COVID-19 with persistent plasma SARS-CoV-2 load revealed a sustained non-inflammatory profile of myeloid cells in association with contained severity of disease, arguing in favor of the use of BTK inhibitors in SARS-COV-2 infection.

SARS-CoV2 pneumonia recovery is linked to expansion of innate lymphoid cells type 2 expressing CCR10.

Accelerate lung repair in SARS-CoV-2 pneumonia is essential for pandemic handling. Innate lymphoid cells (ILCs) are likely players, given their role in mucosal protection and tissue homeostasis. We studied ILC subpopulations at two time points in a cohort of patients admitted in the hospital due to SARS-CoV-2 infection. COVID-19 patients with moderate/severe respiratory failure featured profound depletion of circulating ILCs at hospital admission, in agreement with overall lymphocyte depletion. However, ILCs recovered in direct correlation with lung function improvement as measured by oxygenation index and in negative association with inflammatory and lung/endothelial damage markers like RAGE. While both ILC1 and ILC2 expanded, ILC2 showed the most striking phenotype changes, with CCR10 upregulation in strong correlation with these parameters. Overall, CCR10+ ILC2 emerge as relevant contributors to SARS-CoV-2 pneumonia recovery.

miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication.

Cell activation is a vital step for T-cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next-generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up-regulation of miR-34c-5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV-1 and HIV-2 infections. Overexpression of miR-34c-5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T-cell activation. We additionally show that miR-34c-5p promotes HIV-1 replication, suggesting that its down-regulation during HIV infection may be part of an anti-viral host response.

Expanding the Nude SCID/CID Phenotype Associated with FOXN1 Homozygous, Compound Heterozygous, or Heterozygous Mutations.

Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.

Follicular Helper T Cells Are Major Human Immunodeficiency Virus-2 Reservoirs and Support Productive Infection.

Follicular helper T cells (Tfh), CD4 lymphocytes critical for efficient antibody responses, have been shown to be key human immunodeficiency virus (HIV)-1 reservoirs. Human immunodeficiency virus-2 infection represents a unique naturally occurring model for investigating Tfh role in HIV/acquired immune deficiency syndrome, given its slow rate of CD4 decline, low to undetectable viremia, and high neutralizing antibody titers throughout the disease course. In this study, we investigated, for the first time, Tfh susceptibility to HIV-2 infection by combining in vitro infection of tonsillar Tfh with the ex vivo study of circulating Tfh from HIV-2-infected patients. We reveal that Tfh support productive HIV-2 infection and are preferential viral targets in HIV-2-infected individuals.

Defects in memory B-cell and plasma cell subsets expressing different immunoglobulin-subclasses in patients with CVID and immunoglobulin subclass deficiencies.

BACKGROUND: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. OBJECTIVE: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses. METHODS: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age-matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs. RESULTS: Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA+ PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA+ PCs with mild versus severe smIgA+ MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA+ and smIgG+ MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27+ MBCs with almost normal IgG3+ MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG2+ MBCs; and (6) with IgA1+ MBCs. CONCLUSION: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles.

MDSCs in infectious diseases: regulation, roles, and readjustment.

Many pathogens, ranging from viruses to multicellular parasites, promote expansion of MDSCs, which are myeloid cells that exhibit immunosuppressive features. The roles of MDSCs in infection depend on the class and virulence mechanisms of the pathogen, the stage of the disease, and the pathology associated with the infection. This work compiles evidence supported by functional assays on the roles of different subsets of MDSCs in acute and chronic infections, including pathogen-associated malignancies, and discusses strategies to modulate MDSC dynamics to benefit the host.

Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.

BACKGROUND: Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated. OBJECTIVE: We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments. METHODS: B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n = 19) and peripheral blood (n = 215) from healthy donors aged 0 to 90 years by using flow cytometry and nephelometry, respectively. RESULTS: IgH-switched MBCs expressing IgG1, IgG2, IgG3, IgA1, and IgA2 were already detected in cord blood and newborns at very low counts, whereas CD27+IgM++IgD+ MBCs only became detectable at 1 to 5 months and remained stable until 2 to 4 years, and IgD MBCs peaked at 2 to 4 years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG1, IgG3, and IgA1) peaked later during childhood (2-4 years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG2, IgG4, and IgA2) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11 months for IgM, IgG1, IgG2, IgG3, IgA1, and IgA2; until 2 to 4 years for IgD; and until 5 to 9 years for IgG4 and decreasing thereafter. For most IgH isotypes (except IgD and IgG4), maximum plasma levels were reached after PC and MBC counts peaked. CONCLUSIONS: PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of IgH isotype switch recombination through life.

Thymic HIV-2 infection uncovers posttranscriptional control of viral replication in human thymocytes.

UNLABELLED: A unique HIV-host equilibrium exists in untreated HIV-2-infected individuals. This equilibrium is characterized by low to undetectable levels of viremia throughout the disease course, despite the establishment of disseminated HIV-2 reservoirs at levels comparable to those observed in untreated HIV-1 infection. Although the clinical spectrum is similar in the two infections, HIV-2 infection is associated with a much lower rate of CD4 T-cell decline and has a limited impact on the mortality of infected adults. Here we investigated HIV-2 infection of the human thymus, the primary organ for T-cell production. Human thymic tissue and suspensions of total or purified CD4 single-positive thymocytes were infected with HIV-2 or HIV-1 primary isolates using either CCR5 or CXCR4 coreceptors. We found that HIV-2 infected both thymic organ cultures and thymocyte suspensions, as attested to by the total HIV DNA and cell-associated viral mRNA levels. Nevertheless, thymocytes featured reduced levels of intracellular Gag viral protein, irrespective of HIV-2 coreceptor tropism and cell differentiation stage, in agreement with the low viral load in culture supernatants. Our data show that HIV-2 is able to infect the human thymus, but the HIV-2 replication cycle in thymocytes is impaired, providing a new model to identify therapeutic targets for viral replication control. IMPORTANCE: HIV-1 infects the thymus, leading to a decrease in CD4 T-cell production that contributes to the characteristic CD4 T-cell loss. HIV-2 infection is associated with a very low rate of progression to AIDS and is therefore considered a unique naturally occurring model of attenuated HIV disease. HIV-2-infected individuals feature low to undetectable plasma viral loads, in spite of the numbers of circulating infected T cells being similar to those found in patients infected with HIV-1. We assessed, for the first time, the direct impact of HIV-2 infection on the human thymus. We show that HIV-2 is able to infect the thymus but that the HIV-2 replication cycle in thymocytes is impaired. We propose that this system will be important to devise immunotherapies that target viral production, aiding the design of future therapeutic strategies for HIV control.

Human γδ thymocytes are functionally immature and differentiate into cytotoxic type 1 effector T cells upon IL-2/IL-15 signaling.

Cytotoxicity and IFN-γ production by human γδ T cells underlie their potent antitumor functions. However, it remains unclear where and how human γδ T cells acquire these key effector properties. Given the recent disclosure of a major contribution of the thymus to murine γδ T cell functional differentiation, in this study we have analyzed a series of human pediatric thymuses. We found that ex vivo-isolated γδ thymocytes produced negligible IFN-γ and lacked cytolytic activity against leukemia cells. However, these properties were selectively acquired upon stimulation with IL-2 or IL-15, but not IL-4 or IL-7. Unexpectedly, TCR activation was dispensable for these stages of functional differentiation. The effects of IL-2/IL-15 depended on MAPK/ERK signaling and induced de novo expression of the transcription factors T-bet and eomesodermin, as well as the cytolytic enzyme perforin, required for the cytotoxic type 1 program. These findings have implications for the manipulation of γδ T cells in cancer immunotherapy.

Delta-like 1-mediated Notch signaling enhances the in vitro conversion of human memory CD4 T cells into FOXP3-expressing regulatory T cells.

FOXP3-expressing regulatory T cells (Treg) are essential for the prevention of autoimmunity and were shown to be reduced and/or dysfunctional in several autoimmune diseases. Although Treg-based adoptive transfer represents a promising therapy, the large cell number required to achieve clinical efficacy constitutes an important limitation. Therefore, novel strategies to generate bona fide in vitro-induced Treg (iTreg) are critical. In this study, we report that human memory CD4 T cells can be efficiently converted into iTreg, and that Delta-like 1 (DL1)-mediated Notch signaling significantly enhances this process. The iTreg generated in the presence of DL1 featured higher levels of Treg function-associated molecules and were efficient suppressors. Importantly, these iTreg displayed a stable phenotype in long-term cultures, even in the presence of proinflammatory cytokines. Additionally, DL1 potentiated FOXP3 acquisition by memory CD4 cells through the modulation of the TGF-ß signaling pathway and of Foxp3 transcription. Our data demonstrate that iTreg can be efficiently induced from memory CD4 cells, a subset enriched in relevant specificities for targeting in autoimmune diseases, and that DL1 enhances this process. DL1 also enhanced the proliferation and Treg function-associated marker expression of ex vivo-stimulated human circulating FOXP3(+) cells. Manipulation of the Notch signaling pathway constitutes a promising approach to boost the in vitro generation of iTreg and ex vivo Treg expansion, thus facilitating the establishment of effective Treg-based adoptive therapy in autoimmune diseases.

Recent thymic emigrants are the preferential precursors of regulatory T cells differentiated in the periphery.

Most Forkhead box P3(+) (Foxp3(+)) CD4 regulatory T cell (Treg) precursors are newly formed thymocytes that acquire Foxp3 expression on antigen encounter in the thymus. Differentiation of Treg, however, can also occur in the periphery. What limits this second layer of self- and nonself-reactive Treg production in physiological conditions remains to be understood. In this work, we tested the hypothesis that, similarly to thymic Treg, the precursors of peripheral Treg are immature T cells. We show that CD4(+)CD8(-)Foxp3(-) thymocytes and recent thymic emigrants (RTEs), contrarily to peripheral naïve mature cells, efficiently differentiate into Treg on transfer into lymphopenic mice. By varying donor and recipient mice and conducting ex vivo assays, we document that the preferential conversion of newly formed T cells does not require intrathymic preactivation, is cell-intrinsic, and correlates with low and high sensitivity to natural inhibitors and inducers of Foxp3 expression, such as IL-6, T-cell receptor triggering, and TGF-ß. Finally, ex vivo analysis of human thymocytes and peripheral blood T cells revealed that human RTE and newly developed T cells share an increased potential to acquire a FOXP3(bright)CD25(high) Treg phenotype. Our findings indicating that RTEs are the precursors of Tregs differentiated in the periphery should guide the design of Treg-based therapies.

Human regulatory T-cell development is dictated by Interleukin-2 and -15 expressed in a non-overlapping pattern in the thymus.

Thymus-derived FOXP3-expressing regulatory T-cells (tTregs) are master orchestrators of physiological and pathological immune responses, thus constituting ideal targets for the treatment of autoimmunity. Despite their clinical importance, the developmental program governing their differentiation in the human thymus remains poorly understood. Here, we investigated the role of common gamma-chain cytokines in human tTreg differentiation, by performing gain- and loss-of-function experiments in 3D and 2D postnatal thymic cultures. We identified IL-2 and IL-15 as key molecular determinants in this process and excluded a major function for IL-4, IL-7 and IL-21. Mechanistically, IL-2 and IL-15 were equally able to drive tTreg precursor differentiation into FOXP3(+) cells, and promote tTreg proliferation and survival. Both cytokines also increased the expression levels of molecules associated with effector function within FOXP3(+) subsets, supporting their involvement in tTreg functional maturation. Furthermore, we revealed that IL-2 and IL-15 are expressed in a non-overlapping pattern in the human thymus, with the former produced mainly by mature αß and γδ thymocytes and the latter by monocyte/macrophages and B lymphocytes. Our results identify core mechanisms dictating human tTreg development, with IL-2 and IL-15 defining specific niches required for tTreg lineage stabilization and differentiation, with implications for their therapeutic targeting in autoimmune conditions.

Enteric mucosa integrity in the presence of a preserved innate interleukin 22 compartment in HIV type 1-treated individuals.

BACKGROUND: Interleukin 22 (IL-22) is emerging as a key cytokine for gut epithelial homeostasis and mucosal repair. Gut disruption is a hallmark of human immunodeficiency virus (HIV) infection. Here, we investigated IL-22 production and gut mucosal integrity in HIV type 1 (HIV-1)-infected individuals receiving long-term antiretroviral therapy (ART). METHODS: Biopsy specimens from 37 individuals who underwent colonoscopy primarily for cancer screening and from 17 HIV-1-infected and 20 healthy age-matched controls were assessed. RESULTS: We found significant depletion of sigmoid IL-22-producing CD4(+) T cells (T-helper type 22 [Th22] cells) even after prolonged ART, contrasting with the apparently normal compartments of regulatory and interleukin 17 (IL-17)-producing CD4(+) T cells, as well as total mucosal CD4(+) T cells. Despite the preferential Th22 cell depletion, IL-22 production by innate lymphoid cells (ILCs) was similar to that observed in HIV-1-seronegative subjects, and transcription of genes encoding molecules relevant for IL-22 production (ie, AHR, IL23, IL23R, IL1B, IL6, and TGFB1) was preserved. Remarkably, levels of transcripts of IL-22-target genes (ie, REG3G, DEFB4A, S100A9, MUC1, and MUC13) were unaltered, suggesting an adequate production of antimicrobial peptides and mucins. In agreement, enteric epithelial architecture was fully preserved. CONCLUSIONS: Despite the reduced Th22 cell subset, innate IL-22-mediated mechanisms, essential for sigmoid mucosa integrity, were fully operational in long-term-treated HIV-1-infected individuals. Our data highlight IL-22 production by ILCs as an important target for therapies aimed at facilitating human mucosal reconstitution.

Reduced BAFF-R and increased TACI expression in common variable immunodeficiency.

PURPOSE: B-cell survival and differentiation critically depend on the interaction of BAFF-R and TACI with their ligands, BAFF and APRIL. Mature B-cell defects lead to Common Variable Immunodeficiency (CVID), which is associated with elevated serum levels of BAFF and APRIL. Nevertheless, BAFF-R and TACI expression in CVID and their relationship with ligand availability remain poorly understood. METHODS AND RESULTS: We found that BAFF-R expression was dramatically reduced on B cells of CVID patients, relative to controls. BAFF-R levels inversely correlated with serum BAFF concentration both in CVID and healthy subjects. We also found that recombinant BAFF stimulation reduced BAFF-R expression on B cells without decreasing transcript levels. On the other hand, CVID subjects had increased TACI expression on B cells in direct association with serum BAFF but not APRIL levels. Moreover, splenomegaly was associated with higher TACI expression, suggesting that perturbations of TACI function may underlie lymphoproliferation in CVID. CONCLUSIONS: Our results indicate that availability of BAFF determines BAFF-R and TACI expression on B cells, and that BAFF-R expression is controlled by BAFF binding. Identification of the factors governing BAFF-R and TACI is crucial to understanding CVID pathogenesis, and B-cell biology in general, as well as to explore their potential as therapeutic targets.

Monocyte and myeloid dendritic cell activation occurs throughout HIV type 2 infection, an attenuated form of HIV disease.

Monocytes and myeloid dendritic cells (mDCs) are important orchestrators of innate and human immunodeficiency virus (HIV)-specific immune responses and of the generalized inflammation that characterizes AIDS progression. To our knowledge, we are the first to investigate monocyte and mDC imbalances in HIV type 2 (HIV-2)-positive patients, who typically feature reduced viremia and slow disease progression despite the recognized ability of HIV-2 to establish viral reservoirs and overcome host restriction factors in myeloid cells. We found a heightened state of monocyte and mDC activation throughout HIV-2 infection (characterized by CD14(bright)CD16(+) expansion, as well as increased levels of soluble CD14, HLA-DR, and CD86), together with progressive mDC depletion. Importantly, HIV-2-positive patients also featured overexpression of the inhibitory molecule PD-L1 on monocytes and mDCs, which may act by limiting the production of proinflammatory molecules. These data, from patients with a naturally occurring form of attenuated HIV disease, challenge current paradigms regarding the role of monocytes in HIV/AIDS and open new perspectives regarding potential strategies to modulate inflammatory states.

Decoding mutational hotspots in human disease through the gene modules governing thymic regulatory T cells.

Computational strategies to extract meaningful biological information from multiomics data are in great demand for effective clinical use, particularly in complex immune-mediated disorders. Regulatory T cells (Tregs) are essential for immune homeostasis and self-tolerance, controlling inflammatory and autoimmune processes in many diseases with a multigenic basis. Here, we quantify the Transcription Factor (TF) differential occupancy landscape to uncover the Gene Regulatory Modules governing lineage-committed Tregs in the human thymus, and show that it can be used as a tool to prioritise variants in complex diseases. We combined RNA-seq and ATAC-seq and generated a matrix of differential TF binding to genes differentially expressed in Tregs, in contrast to their counterpart conventional CD4 single-positive thymocytes. The gene loci of both established and novel genetic interactions uncovered by the Gene Regulatory Modules were significantly enriched in rare variants carried by patients with common variable immunodeficiency, here used as a model of polygenic-based disease with severe inflammatory and autoimmune manifestations. The Gene Regulatory Modules controlling the Treg signature can, therefore, be a valuable resource for variant classification, and to uncover new therapeutic targets. Overall, our strategy can also be applied in other biological processes of interest to decipher mutational hotspots in individual genomes.

Long-term immune reconstitution of naive and memory t cell pools after haploidentical hematopoietic stem cell transplantation.

Haploidentical hematopoietic stem cell transplantation (HSCT) constitutes an important alternative for patients lacking a human leukocyte antigen (HLA)-matched donor. Although the use of haploidentical donors is increasingly common, the long-term impact of generating a donor-derived immune system in the context of an HLA-mismatched thymic environment remains poorly characterized. We performed an in-depth assessment of immune reconstitution in a group of haploidentical HSCT recipients 4 to 6 years posttransplantation, in parallel with the respective parental donors and age-matched healthy control subjects. Our data show that the proportion of naive and memory subsets in the recipients, both within CD8(+) and CD4(+) T cells, more closely resembled that observed in age-matched control subjects than in the donors. HSCT recipients displayed relatively high signal-joint T cell-receptor excision circle levels and a high frequency of the recent thymic emigrant-enriched CD31(+) subset within naive CD4(+) and naive regulatory T cells. Moreover, CD8(+), CD4(+), and regulatory T cells from HSCT recipients displayed a diverse T cell repertoire. These results support a key role for thymic output in T cell reconstitution. Nevertheless, HSCT recipients had significantly shorter telomeres within a naive-enriched CD4(+) T cell population than age-matched control subjects, despite the similar telomere length observed within the most differentiated CD8(+) and CD4(+) T cell subsets. Overall, our data suggest that long-term immune reconstitution was successfully achieved after haploidentical HSCT, a process that appears to have largely relied on de novo T cell production.

First use of thymus transplantation therapy for FOXN1 deficiency (nude/SCID): a report of 2 cases.

FOXN1 deficiency is a primary immunodeficiency characterized by athymia, alopecia totalis, and nail dystrophy. Two infants with FOXN1 deficiency were transplanted with cultured postnatal thymus tissue. Subject 1 presented with disseminated Bacillus Calmette-Guérin infection and oligoclonal T cells with no naive markers. Subject 2 had respiratory failure, human herpes virus 6 infection, cytopenias, and no circulating T cells. The subjects were given thymus transplants at 14 and 9 months of life, respectively. Subject 1 received immunosuppression before and for 10 months after transplantation. With follow up of 4.9 and 2.9 years, subjects 1 and 2 are well without infectious complications. The pretransplantation mycobacterial disease in subject 1 and cytopenias in subject 2 resolved. Subject 2 developed autoimmune thyroid disease 1.6 years after transplantation. Both subjects developed functional immunity. Subjects 1 and 2 have 1053/mm(3) and 1232/mm(3) CD3(+) cells, 647/mm(3) and 868/mm(3) CD4(+) T cells, 213/mm(3) and 425/mm(3) naive CD4(+) T cells, and 10 200 and 5700 T-cell receptor rearrangement excision circles per 100 000 CD3(+) cells, respectively. They have normal CD4 T-cell receptor ß variable repertoires. Both subjects developed antigen-specific proliferative responses and have discontinued immunoglobulin replacement. In summary, thymus transplantation led to T-cell reconstitution and function in these FOXN1 deficient infants.