Single-Cell Transcriptomics Reveals Discrete Steps in Regulatory T Cell Development in the Human Thymus.

CD4+CD25+FOXP3+ regulatory T (Treg) cells control immunological tolerance. Treg cells are generated in the thymus (tTreg) or in the periphery. Their superior lineage fidelity makes tTregs the preferred cell type for adoptive cell therapy (ACT). How human tTreg cells develop is incompletely understood. By combining single-cell transcriptomics and flow cytometry, we in this study delineated three major Treg developmental stages in the human thymus. At the first stage, which we propose to name pre-Treg I, cells still express lineage-inappropriate genes and exhibit signs of TCR signaling, presumably reflecting recognition of self-antigen. The subsequent pre-Treg II stage is marked by the sharp appearance of transcription factor FOXO1 and features induction of KLF2 and CCR7, in apparent preparation for thymic exit. The pre-Treg II stage can further be refined based on the sequential acquisition of surface markers CD31 and GPA33. The expression of CD45RA, finally, completes the phenotype also found on mature recent thymic emigrant Treg cells. Remarkably, the thymus contains a substantial fraction of recirculating mature effector Treg cells, distinguishable by expression of inflammatory chemokine receptors and absence of CCR7. The developmental origin of these cells is unclear and warrants caution when using thymic tissue as a source of stable cells for ACT. We show that cells in the major developmental stages can be distinguished using the surface markers CD1a, CD27, CCR7, and CD39, allowing for their viable isolation. These insights help identify fully mature tTreg cells for ACT and can serve as a basis for further mechanistic studies into tTreg development.

How to Reliably Define Human CD8+ T-Cell Subsets: Markers Playing Tricks.

In recent years, our understanding about the functional complexity of CD8+ T-cell populations has increased tremendously. The immunology field is now facing challenges to translate these insights into phenotypic definitions that correlate reliably with distinct functional traits. This is key to adequately monitor and understand compound immune responses including vaccination and immunotherapy regimens. Here we will summarize our understanding of the current state in the human CD8+ T-cell subset characterization field. We will address how reliably the currently used cell surface markers are connected to differentiation status and function of particular subsets. By restricting ourselves to CD8+ αß T cells, we will focus mostly on major histocompatibility complex (MHC) class I-restricted virus- and tumor-specific T cells. This comes with a major advantage as fluorescently labeled peptide-loaded MHC class I multimers have been widely used to identify and characterize these cells.

CD8 and CD4 T Cell Populations in Human Kidneys.

BACKGROUND: At border sites, and in internal organs, tissue resident memory T cells (TRM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However, information on the presence and function of these cells in the human kidney is scant. In order to better understand the T cell-mediated immunological defense in this organ, we aimed to determine phenotypic and functional aspects of CD8 and CD4 T cells present in healthy and allograft kidney tissue. METHODS: Using multichannel flow cytometry, we assessed the phenotype and function of T cells in healthy renal tissue samples (n = 5) and kidney allograft tissue (n = 7) and compared these aspects to T cells in peripheral blood from healthy controls (n = 13). RESULTS: Kidney tissue samples contained substantial amounts of CD8 and CD4 T cells. In contrast to the circulating cells, kidney T cells frequently expressed CD69 and CD103, and were more often actively cycling. Furthermore, nearly all kidney T cells expressed CXCR3, and often expressed CXCR6 compared to T cells in the circulation. Markedly, kidney T cells produced greater quantities of IFNγ than circulating cells and were frequently polyfunctional. CONCLUSION: Functional T cells with the characteristic traits of TRM reside in human kidney tissues. These cells are more often actively cycling and frequently express CXCR3 and CXCR6.

GITR shapes humoral immunity by controlling the balance between follicular T helper cells and regulatory T follicular cells.

Follicular helper CD4+ T-cells (Tfh) control humoral immunity by driving affinity maturation and isotype-switching of activated B-cells. Tfh localize within B-cell follicles and, upon encounter with cognate antigen, drive B-cell selection in germinal centers (GCs) as GC-Tfh. Tfh functionality is controlled by Foxp3-expressing Tfh, which are known as regulatory T follicular cells (Tfr). Thus far, it remains unclear which factors determine the balance between these functionally opposing follicular T-cell subsets. Here, we demonstrate in human and mouse that Tfh and GC-Tfh, as well as their regulatory counterparts, express glucocorticoid-induced TNF receptor related protein (GITR) on their surface. This costimulatory molecule not only helps to identify follicular T-cell subsets, but also increases the ratio of Tfh vs. Tfr, both within and outside the GC. Correspondingly, GITR triggering increases the number of IL-21 producing CD4+ T-cells, which also produce more IFN-γ and IL-10. The latter are known switch factors for IgG2c and IgG1, respectively, which corresponds to a concomitant increase in IgG2c and IgG1 production upon GITR-mediated costimulation. These results demonstrate that GITR can skew the functional balance between Tfh and Tfr, which offers new therapeutic possibilities in steering humoral immunity.

Functional Heterogeneity of CD4+ Tumor-Infiltrating Lymphocytes With a Resident Memory Phenotype in NSCLC.

Resident memory T cells (TRM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103+ TRM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103+CD8+ tumor infiltrating lymphocytes (TILs), with TRM properties, are a positive prognostic marker. To better understand the role of TRM in tumors, we performed a detailed characterization of CD8+ and CD4+ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8+ and CD4+ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in TRM ratios compared to surrounding lung tissue. The majority of both CD4+ and CD8+ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of TRM. While CD103+CD8+ T cells were enriched in tumors, CD103+CD4+ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103+CD4+ and CD103+CD8+ TILs showed multiple characteristics of TRM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8+ and CD4+ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103+ TILs. Strikingly, CD103+CD4+ TILs were the most potent producers of TNF-α and IFN-γ, while other TIL subsets lacked such cytokine production. Whereas, CD103+CD4+PD-1low TILs produced the most effector cytokines, CD103+CD4+PD-1++ and CD69+CD4+PD-1++ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung TRM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103+CD4+ and CD69+CD8+ TILs. Our findings thus provide a rationale to target CD103+CD4+ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.

Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis.

Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions.

Tissue-resident memory T cells at the center of immunity to solid tumors.

Immune responses in tissues are constrained by the physiological properties of the tissue involved. Tissue-resident memory T cells (TRM cells) are a recently discovered lineage of T cells specialized for life and function within tissues. Emerging evidence has shown that TRM cells have a special role in the control of solid tumors. A high frequency of TRM cells in tumors correlates with favorable disease progression in patients with cancer, and studies of mice have shown that TRM cells are necessary for optimal immunological control of solid tumors. Here we describe what defines TRM cells as a separate lineage and how these cells are generated. Furthermore, we discuss the properties that allow TRM cells to operate in normal and transformed tissues, as well as implications for the treatment of patients with cancer.

Clinically Relevant Reactivation of Polyomavirus BK (BKPyV) in HLA-A02-Positive Renal Transplant Recipients Is Associated with Impaired Effector-Memory Differentiation of BKPyV-Specific CD8+ T Cells.

Polyomavirus BK (BKPyV) frequently reactivates in immunosuppressed renal transplant recipients (RTRs) and may lead to graft loss due to BKPyV-induced interstitial nephritis (BKVN). Little is known on the differentiation of CD8+ T cells targeting BKPyV in RTRs. Here we investigated whether BKPyV-specific CD8+ T cell differentiation differs in RTRs with varying degrees of BKPyV reactivation and/or BKVN. Using combinatorial encoding with tetramers carrying BKPyV major capsid protein (VP1) and large T antigen protein (LTAG) epitopes, we investigated CD8+ T cell responses to BKPyV in longitudinally obtained PBMC samples from 46 HLA-A02-positive RTRs and 20 healthy adults. We were also able to isolate BKPyV-specific CD8+ T cells from five renal allografts, two of which were affected by BKVN. Before transplantation, BKPyV-specific CD8+ T cells targeting VP1 and LTAG epitopes appeared predominantly as central-memory and CD27+/CD28+ effector-memory (TEM), and naïve-like PD-1-expressing cells, respectively. After viral reactivation, BKPyV-specific CD8+ T cells assumed CD28- TEM and TEMRA states in patients who were able to control BKPyV, whereas differentiation lagged behind in patients with severe viral reactivation or BKVN. Furthermore, VP1-specific CD69+/CD103+ tissue-resident memory (TRM) cells accumulated in BKVN-affected allografts but lacked signs of effector differentiation. In contrast, granzyme B-expressing effector cells were detected in allografts not affected by BKVN. In conclusion, effector-memory differentiation of BKPyV-specific CD8+ T cells in patients with high viral load or BKVN is impaired. Further characterization of the specific mechanisms behind this altered cellular differentiation is necessary to develop therapies that can prevent the emergence of BKVN.

The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

Restoration of T cell function in chronic hepatitis B patients upon treatment with interferon based combination therapy.

BACKGROUND & AIMS: Chronic hepatitis B virus (HBV) infection is characterized by functional impairment of HBV-specific T cells. Understanding the mechanisms behind T cell dysfunction and restoration is important for the development of optimal treatment strategies. METHODS: In this study we have first analysed the phenotype and function of HBV-specific T cells in patients with low viral load (HBV DNA <20,000IU/ml) and spontaneous control over the virus. Subsequently, we assessed HBV-specific T cells in patients with high viral load (HBV DNA >17,182IU/ml) treated with peginterferon/adefovir combination therapy who had various treatment outcomes. RESULTS: HBV-specific T cells could be detected directly ex vivo in 7/22 patients with low viral load. These showed an early differentiated memory phenotype with reduced ability to produce IL-2 and cytotoxic molecules such as granzyme B and perforin, but with strong proliferative potential. In a cohort of 28 chronic hepatitis B patients with high viral load treated with peginterferon and adefovir, HBV-specific T cells could not be detected directly ex vivo. However, HBV-specific T cells could be selectively expanded in vitro in patients with therapy-induced HBsAg clearance (HBsAg loss n=7), but not in patients without HBsAg clearance (n=21). Further analysis of HBV-specific T cell function with peptide pools showed broad and efficient antiviral responses after therapy. CONCLUSIONS: Our results show that peginterferon based combination therapy can induce HBV-specific T cell restoration. These findings may help to develop novel therapeutic strategies to reconstitute antiviral functions and enhance viral clearance.

Molecular characterization of HCMV-specific immune responses: Parallels between CD8(+) T cells, CD4(+) T cells, and NK cells.

CD8(+) T cells are important for immunity against human cytomegalovirus (HCMV). The HCMV-specific CD8(+) T-cell response is characterized by the accumulation of terminally differentiated effector cells that have downregulated the costimulatory molecules CD27 and CD28. These HCMV-specific CD8(+) T cells maintain high levels of cytotoxic molecules such as granzyme B and rapidly produce the inflammatory cytokine IFN-γ upon activation. Remarkably, HCMV-specific CD8(+) T cells are able to persist long term as fully functional effector cells, suggesting a unique differentiation pathway that is distinct from the formation of memory CD8(+) T cells after infection with acute viruses. In this review, we aim to highlight the most recent developments in HCMV-specific CD8(+) T-cell differentiation, maintenance, tissue distribution, metabolism and function. HCMV also induces the differentiation of effector CD4(+) T cells and NK cells, which share characteristics with HCMV-specific CD8(+) T cells. We propose that the overlap in differentiation of NK cells, CD4(+) and CD8(+) T cells after HCMV infection may be regulated by a shared transcriptional machinery. A better understanding of the molecular framework of HCMV-specific CD8(+) T-cell responses may benefit vaccine design, as these cells uniquely combine the capacity to rapidly respond to infection with long-term survival.

Enhanced CD8 T cell responses through GITR-mediated costimulation resolve chronic viral infection.

Chronic infections are characterized by the inability to eliminate the persisting pathogen and often associated with functional impairment of virus-specific T-cell responses. Costimulation through Glucocorticoid-induced TNFR-related protein (GITR) can increase survival and function of effector T cells. Here, we report that constitutive expression of GITR-ligand (GITRL) confers protection against chronic lymphocytic choriomeningitis virus (LCMV) infection, accelerating recovery without increasing pathology. Rapid viral clearance in GITRL transgenic mice coincided with increased numbers of poly-functional, virus-specific effector CD8+ T cells that expressed more T-bet and reduced levels of the rheostat marker PD-1. GITR triggering also boosted the helper function of virus-specific CD4 T cells already early in the infection, as was evidenced by increased IL-2 and IFNγ production, and more expression of CD40L and T-bet. Importantly, CD4-depletion experiments revealed that the expanded pool of virus-specific effector CD8 T cells and the ensuing viral clearance in LCMV-infected GITRL tg mice was entirely dependent on CD4 T cells. We found no major differences for NK cell and regulatory T cell responses, whereas the humoral response to the virus was increased in GITRL tg mice, but only in the late phase of the infection when the virus was almost eradicated. Based on these findings, we conclude that enhanced GITR-triggering mediates its protective, anti-viral effect on the CD8 T cell compartment by boosting CD4 T cell help. As such, increasing costimulation through GITR may be an attractive strategy to increase anti-viral CTL responses without exacerbating pathology, in particular to persistent viruses such as HIV and HCV.

Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization, and function.

UNLABELLED: Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8(+) T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα(+) hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα(+) and IL-7Rα(-) subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8(+) T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8(+) T cell pool during latency or upon antigen recall. IL-7Rα(+) PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8(+) effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8(+) T cell pool. IMPORTANCE: Insight into the self-renewal properties of long-lived memory CD8(+) T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8(+) T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8(+) T cell pool.

Better safe than sorry: TOB1 employs multiple parallel regulatory pathways to keep Th17 cells quiet.

Th17 cells are key players in antibacterial and antifungal immunity, but have also been implicated in autoimmunity. Interestingly, Th17 cells poorly proliferate upon stimulation, a phenotype that was attributed to a decreased sensitivity to T-cell receptor (TCR) stimulation, and to low IL-2 production by Th17 cells. In this issue of the European Journal of Immunology, Santarlasci et al. [Eur. J. Immunol. 2014. 44: 654-661] shed further light on the molecular mechanism that keeps Th17 cells at bay. They identify the transcriptional regulator TOB1, which not only impairs IL-2 production in Th17 cells, but also blocks the expression of cell cycle genes. Strikingly, TOB1 suppresses Th17-cell proliferation through several pathways, including impaired signal transduction, transcription, and possibly also post-transcriptional regulation.

Blood and beyond: properties of circulating and tissue-resident human virus-specific αß CD8(+) T cells.

CD8(+) αß T-cell responses form an essential line of defence against viral infections. An important part of the mechanisms that control the generation and maintenance of these responses have been elucidated in experimental mouse models. In recent years it has become clear that CD8(+) T-cell responses in humans not only show similarities, but also display differences to those occurring in mice. Furthermore, while several viral infections occur primarily in specialised organ systems, for obvious reasons, most human CD8(+) T-cell investigations were performed on cells deriving from the circulation. Indeed, several lines of evidence now point to essential functional differences between virus-specific CD8(+) memory T cells found in the circulation and those providing protection in organ systems, such as the lungs. In this review, we will focus on summarising recent insights into human CD8(+) T-cell differentiation in response to several viruses and emphasise that for a complete understanding of anti-viral immunity, it is pivotal to scrutinize such responses in both blood and tissue.

CMV-specific CD8+ T-cell function is not impaired in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), CD8(+) T cells exhibit features of exhaustion and impaired functionality. Yet, reactivations of latent viruses such as cytomegalovirus (CMV) are uncommon in untreated CLL, suggesting that antiviral responses are uncompromised. We analyzed phenotypical and functional characteristics of CMV-specific CD8(+) T cells in CLL patients in comparison with age-matched healthy controls (HCs). Despite increased expression of the inhibitory receptors PD1, CD160, and CD244 on total CD8(+) T cells in CLL, expression levels of these markers were decreased on CMV-tetramer(+)CD8(+) T cells. Second, cytokine production upon stimulation with both phorbol 12-myristate 13-acetate/ionomycin and CMV-peptide-loaded antigen-presenting cells was intact in CMV-tetramer(+)CD8(+) T cells. Third, CMV-tetramer(+)CD8(+) T cells of CLL patients and HCs were equally effective in killing CMV-peptide-loaded target cells. Finally, quantitative imaging flow cytometry revealed that the proportion of CD8(+) T cells forming immunologic synapses with CMV-peptide-loaded B cells was intact. In conclusion, despite evidence for global T-cell dysfunction in CLL, we show here that CLL-derived CMV-specific CD8(+) T cells display lower expression of exhaustion markers and are functionally intact. These data indicate that the changes in the T-cell compartment in CLL may be more heterogeneous than presently assumed.

CXCR5+CD4+ follicular helper T cells accumulate in resting human lymph nodes and have superior B cell helper activity.

Although many relevant immune reactions are initiated in the lymph nodes, this compartment has not been systematically studied in humans. Analyses have been performed on immune cells derived from tonsils, but as this tissue is most often inflamed, generalization of these data is difficult. Here, we analyzed the phenotype and function of the human CD4(+) T-cell subsets and lineages in paired resting lymph node and peripheral blood samples. Naive, central memory cells and effector memory cells as well as Th1, Th2, Th17 and Treg cells were equally represented in both compartments. On the other hand, cytotoxic CD4(+) T cells were strikingly absent in the lymph nodes. CXCR5(+)CD4(+) T cells, representing putative follicular Th (Tfh) cells were over-represented in lymph nodes and expressed higher levels of Tfh markers than their peripheral blood counterparts. Compared with the circulating pool, lymph-node-derived CXCR5(+)CD4(+) T cells were superior in providing help to B cells. Thus, functionally competent Tfh cells accumulate in resting human lymph nodes, providing a swift induction of naive and memory antibody responses upon antigenic challenge.

IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells.

Chronic lymphocytic leukemia (CLL) cells multiply in secondary lymphoid tissue, but the mechanisms leading to their proliferation are still uncertain. In addition to B-cell receptor (BCR)-triggered signals, other microenvironmental factors might well be involved. In proliferation centers, leukemic B cells are in close contact with CD4(+)CD40L(+) T cells. Therefore, we here dissected the signals provided by autologous activated T cells (Tact) to CLL cells. Although the gene expression profile induced by Tact was highly similar to that induced by sole CD40 signaling, an obvious difference was that Tact induced proliferation of CLL cells. We determined that stimulation with only CD40L+IL-21 was sufficient to induce robust proliferation in CLL cells. We then defined an interleukin (IL)-21-induced gene signature in CLL, containing components of Janus kinase/signal transducer and activator of transcription and apoptosis pathways, and this signature could be detected in lymph node (LN) samples from patients. Finally, we could detect IL-21 RNA and protein in LN, and IL-21 production ex vivo by LN CD4(+)CXCR5(+) follicular helper T cells. These results indicate that in addition to BCR signaling, activated T cells might contribute to CLL cell proliferation via CD40 and IL-21. Targeting these signaling pathways might offer new venues for treatment of CLL.

Aberrant humoral immune reactivity in DOCK8 deficiency with follicular hyperplasia and nodal plasmacytosis.

Mutations in the DOCK8 gene define the most common form of autosomal-recessive Hyper-IgE-syndrome (AR-HIES/OMIM#243700). In a patient with extensive molluscum contagiosum lesions, a homozygous DOCK8 gene deletion was demonstrated. In-vivo 18-FDG uptake showed multiple non-enlarged lymph nodes without uptake in the spleen. Lymph node biopsies for subsequent immunohistochemistry showed clear differences with the mouse model of DOCK8 deficiency in which these mice show no GCs. Unexpectedly, the patient's lymph nodes demonstrated lymphocyte polyclonality, follicular hyperplasia and an unusual IgE(+) plasma cell expansion. In contrast, the proliferative capacity of circulating B-cells was almost absent with little in-vitro Ig production or plasmablast formation. Also the T-cell proliferation indicated a partial defect. Hematopoietic stem cell transplantation (HSCT) was performed resulting in the disappearance of the molluscum contagiosum lesions. In sum, DOCK8 deficiency results in defective antibody responses and undirected plasma cell expansion in the lymph nodes, as part of a combined immunodeficiency cured by HSCT.