Deficiency of innate and acquired immunity caused by an IKBKB mutation.

BACKGROUND: Severe combined immunodeficiency (SCID) comprises a heterogeneous group of heritable deficiencies of humoral and cell-mediated immunity. Many patients with SCID have lymphocyte-activation defects that remain uncharacterized. METHODS: We performed genetic studies in four patients, from four families of Northern Cree ancestry, who had clinical characteristics of SCID, including early onset of severe viral, bacterial, and fungal infections despite normal B-cell and T-cell counts. Genomewide homozygosity mapping was used to identify a candidate region, which was found on chromosome 8; all genes within this interval were sequenced. Immune-cell populations, signal transduction on activation, and effector functions were studied. RESULTS: The patients had hypogammaglobulinemia or agammaglobulinemia, and their peripheral-blood B cells and T cells were almost exclusively of naive phenotype. Regulatory T cells and γδ T cells were absent. All patients carried a homozygous duplication--c.1292dupG in exon 13 of IKBKB, which encodes IκB kinase 2 (IKK2, also known as IKKß)--leading to loss of expression of IKK2, a component of the IKK-nuclear factor κB (NF-κB) pathway. Immune cells from the patients had impaired responses to stimulation through T-cell receptors, B-cell receptors, toll-like receptors, inflammatory cytokine receptors, and mitogens. CONCLUSIONS: A form of human SCID is characterized by normal lymphocyte development despite a loss of IKK2 function. IKK2 deficiency results in an impaired response to activation stimuli in a variety of immune cells, leading to clinically relevant impairment of adaptive and innate immunity. Although Ikk2 deficiency is lethal in mouse embryos, our observations suggest a more restricted, unique role of IKK2-NF-κB signaling in humans. (Funded by the German Federal Ministry of Education and Research and others.).

Soluble BAFF levels inversely correlate with peripheral B cell numbers and the expression of BAFF receptors.

The TNF family member protein BAFF/BLyS is essential for B cell survival and plays an important role in regulating class switch recombination as well as in the selection of autoreactive B cells. In humans, increased concentrations of soluble BAFF are found in different pathological conditions, which may be as diverse as autoimmune diseases, B cell malignancies, and primary Ab deficiencies (PAD). Because the mechanisms that regulate BAFF levels are not well understood, we newly developed a set of mAbs against human BAFF to study the parameters that determine the concentrations of soluble BAFF in circulation. Patients with PAD, including severe functional B cell defects such as BTK, BAFF-R, or TACI deficiency, were found to have higher BAFF levels than asplenic individuals, patients after anti-CD20 B cell depletion, chronic lymphocytic leukemia patients, or healthy donors. In a comparable manner, mice constitutively expressing human BAFF were found to have higher concentrations of BAFF in the absence than in the presence of B cells. Therefore, our data strongly suggest that BAFF steady-state concentrations mainly depend on the number of B cells as well as on the expression of BAFF-binding receptors. Because most patients with PAD have high levels of circulating BAFF, the increase in BAFF concentrations cannot compensate defects in B cell development and function.

Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects.

BACKGROUND: Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history. OBJECTIVE: We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency. METHODS: Molecular, immunologic, and functional assays were performed. RESULTS: The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells. CONCLUSION: Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response in human subjects.

T and B lymphocyte abnormalities in bone marrow biopsies of common variable immunodeficiency.

In common variable immunodeficiency (CVID) defects in early stages of B-cell development, bone marrow (BM) plasma cells and T lymphocytes have not been studied systematically. Here we report the first morphologic and flow cytometric study of B- and T-cell populations in CVID BM biopsies and aspirates. Whereas the hematopoietic compartment showed no major lineage abnormalities, analysis of the lymphoid compartment exhibited major pathologic alterations. In 94% of the patients, BM plasma cells were either absent or significantly reduced and correlated with serum immunoglobulin G levels. Biopsies from CVID patients had significantly more diffuse and nodular CD3(+) T lymphocyte infiltrates than biopsies from controls. These infiltrates correlated with autoimmune cytopenia but not with other clinical symptoms or with disease duration and peripheral B-cell counts. Nodular T-cell infiltrates correlated significantly with circulating CD4(+)CD45R0(+) memory T cells, elevated soluble IL2-receptor and neopterin serum levels indicating an activated T-cell compartment in most patients. Nine of 25 patients had a partial block in B-cell development at the pre-B-I to pre-B-II stage. Because the developmental block correlates with lower transitional and mature B-cell counts in the periphery, we propose that these patients might form a new subgroup of CVID patients.

Genetic CD21 deficiency is associated with hypogammaglobulinemia.

BACKGROUND: Complement receptor 2 (CR2/CD21) is part of the B-cell coreceptor and expressed by mature B cells and follicular dendritic cells. CD21 is a receptor for C3d-opsonized immune complexes and enhances antigen-specific B-cell responses. OBJECTIVE: Genetic inactivation of the murine CR2 locus results in impaired humoral immune responses. Here we report the first case of a genetic CD21 deficiency in human subjects. METHODS: CD21 protein expression was analyzed by means of flow cytometry and Western blotting. CD21 transcripts were quantified by using real-time PCR. The CD21 gene was sequenced. Wild-type and mutant CD21 cDNA expression was studied after transfection of 293T cells. Binding of EBV-gp350 or C3d-containing immune complexes and induction of calcium flux in CD21-deficient B cells were analyzed by means of flow cytometry. Antibody responses to protein and polysaccharide vaccines were measured. RESULTS: A 28-year-old man presented with recurrent infections, reduced class-switched memory B cells, and hypogammaglobulinemia. CD21 receptor expression was undetectable. Binding of C3d-containing immune complexes and EBV-gp350 to B cells was severely reduced. Sequence analysis revealed a compound heterozygous deleterious mutation in the CD21 gene. Functional studies with anti-immunoglobulin- and C3d-containing immune complexes showed a complete loss of costimulatory activity of C3d in enhancing suboptimal B-cell receptor stimulation. Vaccination responses to protein antigens were normal, but the response to pneumococcal polysaccharide vaccination was moderately impaired. CONCLUSIONS: Genetic CD21 deficiency adds to the molecular defects observed in human subjects with hypogammaglobulinemia.

B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans.

B-cell survival depends on signals induced by B-cell activating factor (BAFF) binding to its receptor (BAFF-R). In mice, mutations in BAFF or BAFF-R cause B-cell lymphopenia and antibody deficiency. Analyzing BAFF-R expression and BAFF-binding to B cells in common variable immunodeficiency (CVID) patients, we identified two siblings carrying a homozygous deletion in the BAFF-R gene. Removing most of the BAFF-R transmembrane part, the deletion precludes BAFF-R expression. Without BAFF-R, B-cell development is arrested at the stage of transitional B cells and the numbers of all subsequent B-cell stages are severely reduced. Both siblings have lower IgG and IgM serum levels but, unlike most CVID patients, normal IgA concentrations. They also did not mount a T-independent immune response against pneumococcal cell wall polysaccharides but only one BAFF-R-deficient sibling developed recurrent infections. Therefore, deletion of the BAFF-R gene in humans causes a characteristic immunological phenotype but it does not necessarily lead to a clinically manifest immunodeficiency.

Clinical and immunologic consequences of a somatic reversion in a patient with X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency is a life-threatening disorder caused by mutations in the gene encoding the interleukin-2 receptor gamma chain (IL2RG). Hypomorphic mutations and reversion of mutations in subpopulations of cells can result in variant clinical phenotypes, making diagnosis and treatment difficult. We describe a 5-year-old boy with mild susceptibility to infection who was investigated for a mutation in IL2RG due to persistent natural killer (NK)- and T-cell lymphopenia. A functionally relevant novel T466C point mutation was found in B, NK, and epithelial cells, whereas alpha/beta and gamma/delta T cells showed the normal gene sequence, suggesting reversion of the mutation in a common T-cell precursor. This genetic correction in T cells resulted in a diverse T-cell repertoire and significant immunity despite failure to produce specific antibodies linked to an intrinsic defect of mutant B cells. These observations confirm the potential of revertant T-cell precursors to reconstitute immune function, but questions remain on the longevity of revertant cells implicating the need for careful follow up and early consideration of hematopoietic stem cell transplantation (HSCT).

Mitochondrial toxicity of nucleoside analogues in primary human lymphocytes.

OBJECTIVE: To evaluate if nucleoside analogue reverse transcriptase inhibitors (NRTIs) and polymerase-gamma inhibitors deplete mitochondrial DNA (mtDNA) in cultured primary lymphocytes and if such depletion might be associated with functional defects. METHODS: Primary peripheral blood CD4 and CD8 lymphocytes were purified from six healthy humans (three male and three female), stimulated mitotically (CD3/CD28) and cultured for 10 days in the presence or absence of NRTIs. Lymphocyte proliferation, mtDNA content, the expression of mtDNA-encoded cytochrome c-oxidase II (COXII) and lactate production were assessed. RESULTS: In CD4 lymphocytes, 10-day exposure to zalcitabine (1.77 microM), didanosine (118 microM) and stavudine (361 microM) induced a time-dependent decline of mtDNA. Compared with controls, residual mtDNA levels were 25%, 21% and 40%, respectively. COXII was reduced to 55%, 35% and 70% of control values. Lactic acid production was increased (by 214%, 294% and 175%, respectively). At day 10, lymphocyte counts were reduced (to 60%, 51%, and 41%, respectively). Zidovudine (71 microM) also reduced lymphocyte counts to 34% and increased lactic acid production by 170%, but did not induce mtDNA and COXII depletion. All these changes were highly significant. Lower NRTI concentrations (0.177 microM of zalcitabine, 11.8 microM of didanosine, 3.6 microM of stavudine and 7.1 microM of zidovudine) had effects at the border of significance. Similar observations were made in CD8 lymphocytes. CONCLUSIONS: In human lymphocytes, zalcitabine, didanosine and stavudine induce dose- and time-dependent mtDNA depletion, which is associated with decreased cell proliferation and increased lactate production. Zidovudine impairs lymphocyte division without inducing mtDNA depletion.

Expansion of CD19(hi)CD21(lo/neg) B cells in common variable immunodeficiency (CVID) patients with autoimmune cytopenia.

Common variable immunodeficiency (CVID) is characterized by a severe hypogammaglobulinemia. While the clinical picture is dominated by recurrent respiratory and gastrointestinal infections, a subgroup of up to 30% of the patients develops additional autoimmune phenomena, including thrombocytopenia and autoimmune hemolytic anemia. So far no classification allowed a prediction of the coincidence of immunodeficiency and autoimmunity. Here, we propose the size of the peripheral CD19(hi)CD2(lo/neg) B cell pool as a marker for CVID patients with autoimmune cytopenia and splenomegaly. Interestingly similar B cell populations are also found in patients with SLE and may not only be an epiphenomenon of the disease.

Analysis of specific IgG titers against tick-borne encephalitis in patients with primary antibody deficiency under immunoglobulin substitution therapy: impact of plasma donor origin.

Immunoglobulin (Ig) replacement therapy is effective in reducing infections in patients with primary antibody deficiency (PAD). Diversity of specific antibodies is achieved by pooling plasma from over 1000 donors usually of a given geographic region. However, there is no agreement with regard to an optimal vaccination schedule for plasma donors. Especially for tick-borne encephalitis (TBE), regional vaccination rates differ widely among populations due to the epidemiology of the disease. We analyzed specific antibody titers against TBE in comparison to total IgG levels in 162 serum samples collected from 110 PAD patients substituted with polyvalent intravenous IgG or subcutaneous IgG. Some patients received different IgG products over time leading to a total number of 122 different patient-IgG product combinations. Positive TBE-specific IgG levels were detected in 35 cases when measured by standard ELISA and could be confirmed by demonstration of neutralizing antibodies in 31 cases. The detection of specific antibody levels correlated with the geographic origin of the IgG preparations. No titers were detectable in patients substituted with IgG products from North-American donors, whereas variable degrees of anti-TBE titers were observed in patients receiving products from different European countries. We suggest considering the patients' personal risk for TBE when selecting an appropriate Ig preparation. These data support regional plasma donation in order to address the diverse local infection profile.

B cell homeostasis is disturbed by immunosuppressive therapies in patients with ANCA-associated vasculitides.

B-lymphocytes play a pivotal role in ANCA-associated vasculitides (AAV). The homeostasis of peripheral human B-lymphocyte subpopulations is tightly regulated, but may be disturbed in autoimmune disease or following immunosuppressive therapies. To elucidate the effect of immunosuppression and the relevance of B-lymphocyte disturbances, the B-lymphocyte compartment was analysed in 61 AAV patients. After immunosuppressive treatment a general B-lymphocytopenia developed in AAV patients. Within the B-lymphocyte subpopulations transitional B cells are the first maturation stage found in the peripheral blood. Transitional B-lymphocytes were significantly lower in AAV patients after immunosuppressive therapy compared to healthy controls. Furthermore, marginal zone B cells--a B-lymphocyte population protecting against encapsulated bacteria--were markedly lowered after immunosuppressive therapy in AAV patients. AAV patients treated with immunosuppressants had lower numbers of naïve and memory B-lymphocytes. Numbers of marginal zone B cells, memory B cells and plasmablasts correlated with concentrations of immunoglobulins. We evaluated plasmablasts for a potential correlation with disease activity. Different from what has been reported for e.g. large vessel vasculitis, absolute numbers of plasmablasts were not increased in patients with AAV and showed no correlation to disease activity. As low transitional B cells after treatment with immunosuppressants indicated an impaired early B-lymphocyte development, seven patients treated with the B cell depleting agent rituximab (RTX) because of relapsing disease activity were analysed for their B cell repopulation kinetics. In the majority of these patients repopulation of the peripheral B cell compartment by newly formed transitional B cells after RTX treatment was constricted and delayed.

Impact of rituximab on immunoglobulin concentrations and B cell numbers after cyclophosphamide treatment in patients with ANCA-associated vasculitides.

OBJECTIVE: To assess the impact of immunosuppressive therapy with cyclophosphamide (CYC) and rituximab (RTX) on serum immunoglobulin (Ig) concentrations and B lymphocyte counts in patients with ANCA-associated vasculitides (AAVs). METHODS: Retrospective analysis of Ig concentrations and peripheral B cell counts in 55 AAV patients. RESULTS: CYC treatment resulted in a decrease in Ig levels (median; interquartile range IQR) from IgG 12.8 g/L (8.15-15.45) to 9.17 g/L (8.04-9.90) (p = 0.002), IgM 1.05 g/L (0.70-1.41) to 0.83 g/L (0.60-1.17) (p = 0.046) and IgA 2.58 g/L (1.71-3.48) to 1.58 g/L (1-31-2.39) (p = 0.056) at a median follow-up time of 4 months. IgG remained significantly below the initial value at 14.5 months and 30 months analyses. Subsequent RTX treatment in patients that had previously received CYC resulted in a further decline in Ig levels from pre RTX IgG 9.84 g/L (8.71-11.60) to 7.11 g/L (5.75-8.77; p = 0.007), from pre RTX IgM 0.84 g/L (0.63-1.18) to 0.35 g/L (0.23-0.48; p<0.001) and from pre RTX IgA 2.03 g/L (1.37-2.50) to IgA 1.62 g/L (IQR 0.84-2.43; p = 0.365) 14 months after RTX. Treatment with RTX induced a complete depletion of B cells in all patients. After a median observation time of 20 months median B lymphocyte counts remained severely suppressed (4 B-cells/µl, 1.25-9.5, p<0.001). Seven patients (21%) that had been treated with CYC followed by RTX were started on Ig replacement because of severe bronchopulmonary infections and serum IgG concentrations below 5 g/L. CONCLUSIONS: In patients with AAVs, treatment with CYC leads to a decline in immunoglobulin concentrations. A subsequent RTX therapy aggravates the decline in serum immunoglobulin concentrations and results in a profoundly delayed B cell repopulation. Surveying patients with AAVs post CYC and RTX treatment for serum immunoglobulin concentrations and persisting hypogammaglobulinemia is warranted.

Fatal adult-onset antibody deficiency syndrome in a patient with cartilage hair hypoplasia.

Cartilage hair hypoplasia (CHH) is an autosomal recessive disorder caused by mutations in the ribonuclease mitochondrial RNA-processing (RMRP) gene. Although its most constant feature is metaphyseal dysplasia with short stature, CHH is associated with extraskeletal defects such as thin hair, anemia, immunodeficiency, and increased incidence of lymphomas. The spectrum of immunologic phenotypes in CHH translates into clinical severity. Whereas T-cell deficiency may remain subclinical or may result in severe combined immunodeficiency or Omenn syndrome, humoral immunodeficiency has only rarely been noted in these patients. Here we report the diagnosis of CHH in a woman who presented with severe short stature and a full-blown antibody deficiency, clinically resembling common variable immunodeficiency. Sequencing of the RMRP gene revealed compound heterozygosity for two novel mutations (g.68_69delinsTT and g.76C>T). Despite the late onset of immunodeficiency in the patient, its clinical course was severe, and the patient died 3 years after the first diagnosis.

Flowcytometric phenotyping of common variable immunodeficiency.

BACKGROUND: Common variable immunodeficiency (CVID) comprises heterogeneous antibody deficiency disorders. To classify this heterogeneous syndrome, clinical as well as immunologic parameters have been combined. Flowcytometric analysis of circulating T and B lymphocyte subpopulations has become an important tool in this endeavor of disease classification. METHODS: Multicolor flowcytometric analysis of circulating lymphocytes. RESULTS: The flowcytometric analysis of B and T cell subpopulations in the blood of CVID patients has contributed significantly to the identification of separate groups within the CVID population. In addition, the flowcytometric analysis of the inducible costimulator on activated T cells, CD19 and BAFF-R on B cells are valid screening methods for three of the four known genetic defects associated with CVID. Only TACI deficiency can not be sufficiently detected by flowcytometric measures. CONCLUSIONS: Flowcytometric classification of patients with CVID has become a standard procedure during the diagnostic work up. This should be performed according to common guidelines to guarantee world wide comparability between different immunodeficiency centers.

The EUROclass trial: defining subgroups in common variable immunodeficiency.

The heterogeneity of common variable immunodeficiency (CVID) calls for a classification addressing pathogenic mechanisms as well as clinical relevance. This European multicenter trial was initiated to develop a consensus of 2 existing classification schemes based on flowcytometric B-cell phenotyping and the clinical course. The clinical evaluation of 303 patients with the established diagnosis of CVID demonstrated a significant coincidence of granulomatous disease, autoimmune cytopenia, and splenomegaly. Phenotyping of B-cell subpopulations confirmed a severe reduction of switched memory B cells in most of the patients that was associated with a higher risk for splenomegaly and granulomatous disease. An expansion of CD21(low) B cells marked patients with splenomegaly. Lymphadenopathy was significantly linked with transitional B-cell expansion. Based on these findings and pathogenic consideration of B-cell differentiation, we suggest an improved classification for CVID (EUROclass), separating patients with nearly absent B cells (less than 1%), severely reduced switched memory B cells (less than 2%), and expansion of transitional (more than 9%) or CD21(low) B cells (more than 10%). Whereas the first group contains all patients with severe defects of early B-cell differentiation, severely reduced switched memory B cells indicate a defective germinal center development as found in inducible constimulator (ICOS) or CD40L deficiency. The underlying defects of expanded transitional or CD21(low) B cells remain to be elucidated. This trial is re-gistered at http://www.uniklinik-freiburg.de/zks/live/uklregister/Oeffentlich.html as UKF000308.