Evaluation of the impact of viscosity, injection volume, and injection flow rate on subcutaneous injection tolerance.

AIM: The primary objective of this study was to evaluate the impact of fluid injection viscosity in combination with different injection volumes and flow rates on subcutaneous (SC) injection pain tolerance. METHODS: The study was a single-center, comparative, randomized, crossover, Phase I study in 24 healthy adults. Each participant received six injections in the abdomen area of either a 2 or 3 mL placebo solution, with three different fluid viscosities (1, 8-10, and 15-20 cP) combined with two different injection flow rates (0.02 and 0.3 mL/s). All injections were performed with 50 mL syringes and 27G, 6 mm needles. Perceived injection pain was assessed using a 100 mm visual analog scale (VAS) (0 mm/no pain, 100 mm/extreme pain). The location and depth of the injected fluid was assessed through 2D ultrasound echography images. RESULTS: Viscosity levels had significant impact on perceived injection pain (P=0.0003). Specifically, less pain was associated with high viscosity (VAS =12.6 mm) than medium (VAS =16.6 mm) or low (VAS =22.1 mm) viscosities, with a significant difference between high and low viscosities (P=0.0002). Target injection volume of 2 or 3 mL was demonstrated to have no significant impact on perceived injection pain (P=0.89). Slow (0.02 mL/s) or fast (0.30 mL/s) injection rates also showed no significant impact on perceived pain during SC injection (P=0.79). In 92% of injections, the injected fluid was located exclusively in SC tissue whereas the remaining injected fluids were found located in SC and/or intradermal layers. CONCLUSION: The results of this study suggest that solutions of up to 3 mL and up to 15-20 cP injected into the abdomen within 10 seconds are well tolerated without pain. High viscosity injections were shown to be the most tolerated, whereas injection volume and flow rates did not impact perceived pain.

Inherited IL-12p40 deficiency: genetic, immunologic, and clinical features of 49 patients from 30 kindreds.

Autosomal recessive interleukin (IL)-12 p40 (IL-12p40) deficiency is a rare genetic etiology of mendelian susceptibility to mycobacterial disease (MSMD). We report the genetic, immunologic, and clinical features of 49 patients from 30 kindreds originating from 5 countries (India, Iran, Pakistan, Saudi Arabia, and Tunisia). There are only 9 different mutant alleles of the IL12B gene: 2 small insertions, 3 small deletions, 2 splice site mutations, and 1 large deletion, each causing a frameshift and leading to a premature stop codon, and 1 nonsense mutation. Four of these 9 variants are recurrent, affecting 25 of the 30 reported kindreds, due to founder effects in specific countries. All patients are homozygous and display complete IL-12p40 deficiency. As a result, the patients lack detectable IL-12p70 and IL-12p40 and have low levels of interferon gamma (IFN-γ). The clinical features are characterized by childhood onset of bacille Calmette-Guérin (attenuated Mycobacterium bovis strain) (BCG) and Salmonella infections, with recurrences of salmonellosis (36.4%) more common than recurrences of mycobacterial disease (25%). BCG vaccination led to BCG disease in 40 of the 41 patients vaccinated (97.5%). Multiple mycobacterial infections were rare, observed in only 3 patients, whereas the association of salmonellosis and mycobacteriosis was observed in 9 patients. A few other infections were diagnosed, including chronic mucocutaneous candidiasis (n = 3), nocardiosis (n = 2), and klebsiellosis (n = 1). IL-12p40 deficiency has a high but incomplete clinical penetrance, with 33.3% of genetically affected relatives of index cases showing no symptoms. However, the prognosis is poor, with mortality rates of up to 28.6%. Overall, the clinical phenotype of IL-12p40 deficiency closely resembles that of interleukin 12 receptor ß1 (IL-12Rß1) deficiency. In conclusion, IL-12p40 deficiency is more common than initially thought and should be considered worldwide in patients with MSMD and other intramacrophagic infectious diseases, salmonellosis in particular.

Decreased T-cell repertoire diversity in sepsis: a preliminary study.

OBJECTIVE: Septic syndromes are the leading causes of mortality in intensive care units. In patients, the occurrence of sepsis-induced immune suppression is associated with delayed mortality, although the exact role of lymphocyte dysfunctions is not well established. The objective of this study was to investigate T-cell receptor diversity, an important feature of T-cell response, in patients with septic shock. DESIGN: Preliminary prospective observational study. SETTING: Adult intensive care units in a university hospital. SUBJECTS: Patients with septic shock (n = 41) sampled twice after the onset of shock (early after inclusion [day 1] and at the end of the first week [day 7]). MEASUREMENTS AND MAIN RESULTS: Using a novel molecular biology technique, the combinatorial diversity of human T-cell receptor ß-chain (TRB locus) was measured in peripheral blood. Patients with septic shock presented with a marked decreased T-cell receptor diversity after the onset of shock in comparison with normal values. Importantly, in paired samples, a very steep recovery slope of T-cell receptor diversity, never described in other clinical situations, was observed between day 1 and day 7 (p < 0.0001, Wilcoxon's paired test). Decreased T-cell receptor diversity was associated with mortality (log-rank test, p = 0.0058; hazard ratio = 4.48; 95% confidence interval 1.96-53.32), and the development of nosocomial infections (p < 0.05, Mann-Whitney U test). CONCLUSION: Our results show for the first time that septic patients present with a marked decreased T-cell receptor diversity that returned rapidly toward normal values over time. This opens novel cognitive research perspectives that deserve to be investigated in experimental models of sepsis. After confirmation in larger cohorts of these preliminary results, T-cell receptor diversity measurements may become a crucial tool to monitor immune functions in ICU patients.

CD4+ T cells rely on a cytokine gradient to control intracellular pathogens beyond sites of antigen presentation.

Effector T cells are critical for clearance of pathogens from sites of infection. Like cytotoxic CD8(+) T cells, CD4(+) helper T cells have been shown to deliver effector molecules directionally toward the immunological synapse, suggesting that infected cells need to be engaged individually to receive effector signals. In contrast, we show here that CD4(+) T cells stably contacted a minority of infected cells, yet these interactions triggered intracellular defense mechanisms in bystander cells in vivo. By using a functional read-out, we provide evidence that this effector bystander activity extends via a gradient of IFN-γ more than 80 µm beyond the site of antigen presentation, promoting pathogen clearance in the absence of immunological synapse formation. Our results thus demonstrate that CD4(+) T cells can exert their protective activity by engaging a minority of infected cells.

Accounting for genetic heterogeneity in homozygosity mapping: application to Mendelian susceptibility to mycobacterial disease.

INTRODUCTION: Genome-wide homozygosity mapping is a powerful method for locating rare recessive Mendelian mutations. However, statistical power decreases dramatically in the presence of genetic heterogeneity. METHODS: The authors applied an empirical approach to test for linkage accounting for genetic heterogeneity by calculating the sum of positive per-family multipoint LOD scores (S) across all positions, and obtaining corresponding empirical p values (EmpP) through permutations. RESULTS: The statistical power of the approach was found to be consistently higher than the classical heterogeneity LOD by simulations. Among 21 first-cousin matings with a single affected child, for five families linked to a locus of interest and 16 families to other loci, S/EmpP achieved a power of 40% versus 28% for heterogeneity LOD at an α level of 0.001. The mean size of peak linkage regions was markedly higher for true loci than false positive regions. The S/EmpP approach was applied to a sample of 17 consanguineous families with Mendelian susceptibility to mycobacterial disease, leading to the identification of two mutations in IL12RB1 and TYK2 from the largest of six linkage regions at p<10(-3). CONCLUSIONS: The S/EmpP approach is a flexible and powerful approach that can be applied to linkage analysis of families with suspected Mendelian disorders.

Revisiting human IL-12Rß1 deficiency: a survey of 141 patients from 30 countries.

Interleukin-12 receptor ß1 (IL-12Rß1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guérin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years ± 9.8 years (range, 0.5-46.4 yr). IL-12Rß1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought.

A dynamic map of antigen recognition by CD4 T cells at the site of Leishmania major infection.

CD4 T helper cells play a central role in the control of infection by intracellular parasites. How efficiently pathogen-specific CD4 T cells detect infected cells in vivo is unclear. Here, we employed intravital two-photon imaging to examine the behavior of pathogen-specific CD4 T cells at the site of Leishmania major infection. While activated CD4 T cells enter the inflamed tissue irrespective of their antigen specificity, pathogen-specific T cells preferentially decelerated and accumulated in infected regions of the dermis. Antigen recognition by CD4 T cells was heterogeneous, involving both stable and dynamic contacts with infected phagocytes. However, not all infected cells induced arrest or deceleration of pathogen-specific T cells, and dense clusters of infected cells were poorly accessible to migrating T cells. Thus, disparities in the dynamics of T cell contacts with infected cells and local variation in T cell access to infected cells are important elements of the host-pathogen interplay.

Mutations in STAT3 and IL12RB1 impair the development of human IL-17-producing T cells.

The cytokines controlling the development of human interleukin (IL) 17--producing T helper cells in vitro have been difficult to identify. We addressed the question of the development of human IL-17--producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic traits affecting transforming growth factor (TGF) beta, IL-1, IL-6, or IL-23 responses. Activating mutations in TGFB1, TGFBR1, and TGFBR2 (Camurati-Engelmann disease and Marfan-like syndromes) and loss-of-function mutations in IRAK4 and MYD88 (Mendelian predisposition to pyogenic bacterial infections) had no detectable impact. In contrast, dominant-negative mutations in STAT3 (autosomal-dominant hyperimmunoglobulin E syndrome) and, to a lesser extent, null mutations in IL12B and IL12RB1 (Mendelian susceptibility to mycobacterial diseases) impaired the development of IL-17--producing T cells. These data suggest that IL-12Rbeta1- and STAT-3--dependent signals play a key role in the differentiation and/or expansion of human IL-17-producing T cell populations in vivo.

A role for interleukin-12/23 in the maturation of human natural killer and CD56+ T cells in vivo.

Natural killer (NK) cells have been originally defined by their "naturally occurring" effector function. However, only a fraction of human NK cells is reactive toward a panel of prototypical tumor cell targets in vitro, both for the production of interferon-gamma (IFN-gamma) and for their cytotoxic response. In patients with IL12RB1 mutations that lead to a complete IL-12Rbeta1 deficiency, the size of this naturally reactive NK cell subset is diminished, in particular for the IFN-gamma production. Similar data were obtained from a patient with a complete deficit in IL-12p40. In addition, the size of the subset of effector memory T cells expressing CD56 was severely decreased in IL-12Rbeta1- and IL-12p40-deficient patients. Human NK cells thus require in vivo priming with IL-12/23 to acquire their full spectrum of functional reactivity, while T cells are dependent upon IL-12/23 signals for the differentiation and/or the maintenance of CD56(+) effector memory T cells. The susceptibility of IL-12/23 axis-deficient patients to Mycobacterium and Salmonella infections in combination with the absence of mycobacteriosis or salmonellosis in the rare cases of human NK cell deficiencies point to a role for CD56(+) T cells in the control of these infections in humans.

BCG-osis and tuberculosis in a child with chronic granulomatous disease.

A few known primary immunodeficiencies confer predisposition to clinical disease caused by weakly virulent mycobacteria, such as BCG vaccines (regional disease, known as BCG-itis, or disseminated disease, known as BCG-osis), or more virulent mycobacteria, such as Mycobacterium tuberculosis (pulmonary and disseminated tuberculosis). We investigated the clinical and genetic features of a 12-year-old boy with both recurrent BCG-osis and disseminated tuberculosis. The patient's phagocytic cells produced no O(2)(-). A hemizygous splice mutation was found in intron 5 of CYBB, leading to a diagnosis of X-linked chronic granulomatous disease. Chronic granulomatous disease should be suspected in all children with BCG-osis, even in the absence of nonmycobacterial infectious diseases, and in selected children with recurrent BCG-itis or severe tuberculosis.

A novel X-linked recessive form of Mendelian susceptibility to mycobaterial disease.

BACKGROUND: Mendelian susceptibility to mycobacterial disease (MSMD) is associated with infection caused by weakly virulent mycobacteria in otherwise healthy people. Causal germline mutations in five autosomal genes (IFNGR1, IFNGR2, STAT1, IL12RB1, IL12B) and one X-linked (NEMO) gene have been described. The gene products are physiologically related, as they are involved in interleukin 12/23-dependent, interferon gamma-mediated immunity. However, no genetic aetiology has yet been identified for about half the patients with MSMD. METHODS: A large kindred was studied, including four male maternal relatives with recurrent mycobacterial disease, suggesting X-linked recessive inheritance. Three patients had recurrent disease caused by the bacille Calmette-Guérin vaccine, and the fourth had recurrent tuberculosis. The infections showed tropism for the peripheral lymph nodes. RESULTS: Known autosomal and X-linked genetic aetiologies of MSMD were excluded through genetic and immunological investigations. Genetic linkage analysis of the X-chromosome identified two candidate regions, on Xp11.4-Xp21.2 and Xq25-Xq26.3, with a maximum LOD score of 2. CONCLUSION: A new X-linked recessive form of MSMD is reported, paving the way for the identification of a new MSMD-causing gene.

IRAK4 and NEMO mutations in otherwise healthy children with recurrent invasive pneumococcal disease.

BACKGROUND: About 2% of childhood episodes of invasive pneumococcal disease (IPD) are recurrent, and most remain unexplained. OBJECTIVE: To report two cases of otherwise healthy, unrelated children with recurrent IPD as the only clinical infectious manifestation of an inherited disorder in nuclear factor-kappaB(NF-kappaB)-dependent immunity. RESULTS: One child carried two germline mutations in IRAK4, and had impaired cellular responses to interleukin (IL)1 receptor and toll-like receptor (TLR) stimulation. The other child carried a hemizygous mutation in NEMO, associated with a broader impairment of NF-kappaB activation, with an impaired cellular response to IL-1R, TLR and tumour necrosis factor receptor stimulation. The two patients shared a narrow clinical phenotype, associated with two related but different genotypes. CONCLUSIONS: Otherwise healthy children with recurrent IPD should be explored for underlying primary immunodeficiencies affecting the IRAK4-dependent and NEMO-dependent signalling pathways.

T cell-dependent activation of dendritic cells requires IL-12 and IFN-gamma signaling in T cells.

Patients presenting with genetic deficiencies in IFNGR1, IFNGR2, IL-12B, and IL-12RB1 display increased susceptibility to mycobacterial infections. We analyzed in this group of patients the cross-talk between human CD4+ T lymphocytes and dendritic cells (DCs) that leads to maturation of DC into producers of bioactive IL-12 and to activation of T cells into IFN-gamma producers. We found that this cross-talk is defective in all patients from this group. Unraveling the mechanisms underlying this deficiency, we showed that IL-12 signaling in T cells is required to induce expression of costimulatory molecules and secretion of IL-12 by DCs and that IFNGR expression is required on both DCs and CD4+ T cells to induce IL-12 secretion by DCs. These data suggest that CD4+ T cell-mediated activation of DCs plays a critical role in the defense against mycobacterial infections in humans.

Inborn errors of IL-12/23- and IFN-gamma-mediated immunity: molecular, cellular, and clinical features.

Mendelian susceptibility to mycobacterial diseases confers predisposition to clinical disease caused by weakly virulent mycobacterial species in otherwise healthy individuals. Since 1996, disease-causing mutations have been found in five autosomal genes (IFNGR1, IFNGR2, STAT1, IL12B, IL12BR1) and one X-linked gene (NEMO). These genes display a high degree of allelic heterogeneity, defining at least 13 disorders. Although genetically different, these conditions are immunologically related, as all result in impaired IL-12/23-IFN-gamma-mediated immunity. These disorders were initially thought to be rare, but have now been diagnosed in over 220 patients from over 43 countries worldwide. We review here the molecular, cellular, and clinical features of patients with inborn errors of the IL-12/23-IFN-gamma circuit.

X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production.

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappaB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappaB/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappaB activators, such as tumor necrosis factor-alpha, IL-1beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappaB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.

Human complete Stat-1 deficiency is associated with defective type I and II IFN responses in vitro but immunity to some low virulence viruses in vivo.

The autosomal recessive form of human complete Stat-1 deficiency is a rare disorder, thus far reported in two unrelated patients, both of whom developed disseminated bacillus Calmette-Guérin (BCG) and subsequently died of viral illnesses before detailed studies of the condition could be performed. It is associated with impaired cellular responses to both IFN-gamma and IFN-alphabeta via Stat-1-containing complexes. We describe a third patient with complete Stat-1 deficiency and disseminated BCG infection, who died 3 mo after bone marrow transplantation. The patient's EBV-transformed B cells did not express Stat-1 protein and did not activate Stat-1-containing transcription factors. We also report the ex vivo responses of a Stat-1-deficient patient's fresh blood cells to IFN-gamma and the in vitro responses of a SV40-transformed fibroblastic cell line to IFN-gamma and IFN-alphabeta. There was no response to IFN-gamma in terms of IL-12 production and HLA class II induction, accounting for vulnerability to BCG. Moreover, IFN-alphabeta did not suppress HSV and vesicular stomatitis virus replication in fibroblasts, although in vivo the patient was able to successfully clear at least some viruses. This study broadens our understanding of complete Stat-1 deficiency, a severe form of innate immunodeficiency. Stat-1 deficiency should be suspected in children with severe infections, notably but not exclusively patients with mycobacterial or viral diseases.

The NEMO mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation.

Amorphic mutations in the NF- kappa B essential modulator (NEMO) cause X-dominant incontinentia pigmenti, which is lethal in males in utero, whereas hypomorphic mutations cause X-recessive anhidrotic ectodermal dysplasia with immunodeficiency, a complex developmental disorder and life-threatening primary immunodeficiency. We characterized the NEMO mutation 110_111insC, which creates the most-upstream premature translation termination codon (at codon position 49) of any known NEMO mutation. Surprisingly, this mutation is associated with a pure immunodeficiency. We solve this paradox by showing that a Kozakian methionine codon located immediately downstream from the insertion allows the reinitiation of translation. The residual production of an NH(2)-truncated NEMO protein was sufficient for normal fetal development and for the subsequent normal development of skin appendages but was insufficient for the development of protective immune responses.

Inherited disorders of the IL-12-IFN-gamma axis in patients with disseminated BCG infection.

Disseminated BCG infection is a rare complication of vaccination that occurs in patients with impaired immunity. In recent years, a series of inherited disorders of the IL-12-IFN-gamma axis have been described that predispose affected individuals to disseminated disease caused by BCG, environmental Mycobacteria, and non-typhoidal Salmonella. The routine immunological work-up of these patients is normal and the diagnosis requires specific investigation of the IL-12-IFN-gamma circuit. We report here the first two such patients originating from and living in Iran. The first child is two years old and suffers from complete IFN-gamma receptor 2 deficiency and disseminated BCG infection. He is currently in clinical remission thanks to prolonged multiple antibiotic therapy. The other, a 28-year-old adult, suffers from IL-12p40 deficiency and presented with disseminated BCG infection followed by recurrent episodes of systemic salmonellosis. He is now doing well. A third patient of Iranian descent, living in North America, was reported elsewhere to suffer from IL-12Rbeta1 deficiency. These three patients thus indicate that various inherited defects of the IL-12-IFN-gamma circuit can be found in Iranian people. In conclusion we recommend to consider the disorders of the IL-12-IFN-gamma circuit in all patients with severe BCG infection, disseminated environmental mycobacterial disease, or systemic non-typhoidal salmonellosis, regardless of their ethnic origin and country of residence.

Gains of glycosylation comprise an unexpectedly large group of pathogenic mutations.

Mutations involving gains of glycosylation have been considered rare, and the pathogenic role of the new carbohydrate chains has never been formally established. We identified three children with mendelian susceptibility to mycobacterial disease who were homozygous with respect to a missense mutation in IFNGR2 creating a new N-glycosylation site in the IFNgammaR2 chain. The resulting additional carbohydrate moiety was both necessary and sufficient to abolish the cellular response to IFNgamma. We then searched the Human Gene Mutation Database for potential gain-of-N-glycosylation missense mutations; of 10,047 mutations in 577 genes encoding proteins trafficked through the secretory pathway, we identified 142 candidate mutations ( approximately 1.4%) in 77 genes ( approximately 13.3%). Six mutant proteins bore new N-linked carbohydrate moieties. Thus, an unexpectedly high proportion of mutations that cause human genetic disease might lead to the creation of new N-glycosylation sites. Their pathogenic effects may be a direct consequence of the addition of N-linked carbohydrate.

Bacillus Calmette Guerin triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.

The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.