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Regulatory CD4+ T cells (Treg) are important modulators of the immune response. Different types of Treg have been identified based on whether they are thymically derived (natural Treg) or induced in the periphery (adaptive Treg). We recently reported on an adaptive Treg phenotype that can be induced by the concomitant stimulation of human CD4+ T cells through CD3 and the membrane complement regulator CD46. These complement (CD46)-induced regulatory T cells (cTreg) potently inhibit bystander T-cell proliferation through high-level secretion of IL-10. In addition, cTreg express granzyme B and exhibit cytotoxic effects toward activated effector T cells. Here, we analyzed the effect of cTreg on B-cell functions in a co-culture system. We found that cTreg enhance B-cell Ab production. This B-cell support is dependent on cell/cell contact as well as cTreg-derived IL-10. In addition, we show that T cells from a CD46-deficient patient are not capable of promoting B-cell responses, whereas CD46-deficient B cells have no intrinsic defect in Ig production. This finding may relate to a subset of CD46-deficient patients, who present with common variable immunodeficiency. Thus, the lack of cTreg function in optimizing B-cell responses could explain why some CD46-deficient patients develop common variable immunodeficiency.
CD46, or membrane co-factor protein, is a cell surface receptor, which is expressed by all human cells except erythrocytes. CD46 is a co-factor for degradation of complement fragments C3b and C4b deposited on cells and thereby protects the cell upon which CD46 is expressed from complement attack 1. In addition to this complement regulatory function, CD46 is a receptor for a number of pathogenic bacteria and viruses 2 and can modulate adaptive immune responses 3, 4. In particular, CD46 functions as a co-stimulator during T-cell stimulation: activation of human CD4+ T cells by simultaneous Ab crosslinking of the T-cell receptor and CD46 in the presence of IL-2 induces T-cell proliferation 5, 6 and a phenotype resembling that of adaptive regulatory T cells 7. Characteristics of these regulatory T cells are their high-level synthesis of IL-10, granzyme B and perforin 7, 8. These regulatory T cells, here termed complement-induced regulatory T cells (cTreg), suppress effector CD4+ T cell (Teff) responses through release of immunosuppressive IL-10, consumption of IL-2 as well as through a contact-dependent mechanism involving granzyme B-mediated cytotoxicity 3, 9.
The cytokine pattern secreted by cTreg, although suppressing T-cell responses, does not impair the maturation and T-cell-stimulating function of dendritic cells 10. This is in contrast to other regulatory T cells, which mostly exert suppressive functions toward APC 11, 12. This is an important finding because it provides a means to suppress/modulate T cells without affecting antigen processing. The effects of cTreg on other immunocompetent cells such as B cells have not been studied. cTreg secrete factors 10 that are known to promote B-cell activation and growth, such as IL-10 and soluble CD40L 13. In addition, cTreg produce cytokines that support Ig isotype switching, such as IFN-γ and IL-10 14, 15. However, cTreg also express high amounts of granzyme B and can kill a range of activated target cells 8, 16, 17. Also, suppression of B-cell responses via a cytotoxic pathway has been demonstrated for natural Treg 18. Thus, we could envision two possible scenarios as to how cTreg could impact Ab production during an immune response: cTreg supporting B-cell activation via their strong IL-10/CD40L production but also cTreg-mediated suppression of B-cell responses through granzyme B-mediated killing.
We began studying the interplay between cTreg and B cells because of the recent finding that a subset of CD46-deficient individuals presents with common variable immunodeficiency (CVID) (19–21 and unpublished data, V. F.-B.). CVID is a genetically heterogenous syndrome characterized by low serum levels of Ig and variable T-cell defects 22. Genetic causes of CVID include mutations in B-cell-related genes, such as those coding for CD19 and the B-cell activating factor (BAFF) receptors TACI and BAFF-R, as well as mutations in T-cell costimulatory molecules, such as ICOS 22. Although a role for CD46 in B-cell responses has not yet been described, the CVID phenotype indicates a possible participation of CD46 in Ab production. We therefore analyzed the effect of crosslinking CD46 on B cells on cell proliferation and Ig production as well as on the effect of CD46-induced cTreg on B-cell activation and responses.
In these studies, CD46 activation of B cells did not induce changes in B-cell proliferation or Ab production. In contrast, CD3/CD46-activated T cells, cTreg, induced superior B-cell activation and increased Ig production compared with Teff. B-cell support by cTreg required cTreg-derived IL-10 as well as B cell/cTreg cell contact. Interestingly, and unexpectedly, cTreg/B cell contact did not lead to B-cell killing. A supportive role for cTreg in B-cell responses was further suggested by the finding that CD3/CD46-activated T cells isolated from a CD46-deficient patient are unable to provide B-cell help.
cTreg-derived cytokines do not induce changes in B-cell proliferation or Ig production
The effect of cTreg on B-cell function is unknown. As cTreg display characteristics that could either promote (IL-10/sCD40L secretion) or suppress (granzyme B/perforin production) B-cell function 7, 8, 10, we analyzed the impact of cTreg on B-cell activation in two systems: we first assessed the effect of cTreg-derived soluble factors/cytokines on B-cell proliferation and Ig production and then studied cTreg function toward B cells in a co-culture system.
To determine if cTreg-derived cytokines provide potentially sufficient (co)-stimulatory signals to drive BCR-dependent Ig production, we activated freshly isolated human B cells with pansorbin, which is a BCR crosslinking mitogen in the presence of supernatants derived from either CD3-, CD3/CD28- or CD3/CD46-activated T cells and measured Ig production at days 5 and 10. Activated B cells cultured in fresh media served as control. None of these conditions led to B-cell proliferation or Ig production (data not shown). We next repeated these experiments with the addition of immobilized mAb to CD40 to provide an essential B-cell co-stimulatory signal 23. As expected, B-cell activation under these conditions induced vigorous B-cell proliferation and Ab production in control media (Supporting Information Fig. 1). However, neither cTreg (CD3/CD46-activated) supernatant, nor Teff (CD3/28-activated) supernatant nor supernatant from CD3 (alone)-activated T cells led to changes in Ig production at the end of the incubation period (Supporting Information Fig. 1). These results indicate that the substantial amounts of IL-10 and sCD40L 10 secreted by cTreg do not provide sufficient additional signals to drive BCR-dependent B-cell activation.
Thus, although we used a number of different activation conditions known to induce B-cell responses, we have not been able to show an effect of cTreg-derived cytokines/soluble factors alone on BCR-induced Ab production in vitro.
cTreg do not impair B-cell survival in a co-culture system
In the previous studies, we have demonstrated that cTreg up-regulate adhesion molecules and express high levels of activation markers and co-stimulatory molecules, which could possibly facilitate B-cell activation (7, 10, 24 and our unpublished data). However, cTreg also express high amounts of intracellular granzyme B and perforin and display potent cytotoxicity toward activated Teff, dendritic cells and monocytes 8, 17, making it feasible that cTreg might also kill B cells upon cell contact.
To test for the effects of direct cell–cell contact between cTreg and B cells, we developed an in vitro co-culture system in which freshly isolated autologous B and CD4+ T cells were cultured under conditions that allowed concomitant activation and interaction of both cell types. Plates were coated with Ab to CD3 and either CD28 or CD46 to induce T-cell differentiation into Teff or cTreg, respectively 7. Anti-IgM, -IgA and -IgG Ab were coated on the same plates to elicit polyclonal B-cell activation by crosslinking the BCR 23. Autologous CD4+ T cells and B cells were mixed at a ratio of 1 T cell:2 B cells (conditions that have previously been shown to induce effective killing of other target cells by cTreg 8) and cultured on the Ab-coated plates for up to 5 days. Cell proliferation and cytokine production were measured. Induction of cTreg was monitored via the increase in IL-10 and granzyme B expression by T cells in the cultures activated with anti-CD46 Ab. Increases in IL-10 and granzyme B were not observed in cultures activated with Ab to CD3 (alone) or CD3/CD28 (Fig. 1A). To monitor for the cellular source of the IL-10 observed in the culture supernatants, we also performed IL-10 Secretion Assays at days 2 and 4 post-activation. At day 2, about 10% of CD3/CD46-activated T cells cultured alone produced IL-10, whereas only a negligible number of B cells activated and cultured alone produced IL-10. In co-culture, generally a quarter to a third of T cells secreted IL-10 at day 2 of activation while 5–7% of B cells now stained positive for IL-10. At day 4, most cells ceased to secrete IL-10 (Supporting Information Fig. 2). Thus, although a number of B cells produce IL-10 upon interaction with T cells, the main source of this cytokine during co-culture appears to be indeed cTreg.
To assess for cTreg-mediated killing or suppression of B-cell activation, we examined the absolute numbers of viable CD4+ T cell and B cells as well as absolute numbers of propidium iodide-positive (dead) cells at different days of culture (Figs. 1B and C) and performed CFSE dilution assays to monitor B-cell division (Fig. 1D). As expected, T-cell numbers in the co-cultures increased over time, with CD3/CD46-activated T cells proliferating slightly faster than CD3 or CD3/CD28-activated T cells (Fig. 1B). This increased proliferative capacity of cTreg in the presence of IL-2 has previously been reported 5, 7, 25. B cells underwent an initial proliferation burst peaking at day 3 of culture, then declined at day 5 with no difference in viable (Fig. 1C) or dead (data not shown) cell numbers in the culture conditions analyzed. Accordingly, the CFSE dilution profiles of B cells from all co-cultures with activated T cells were indistinguishable at the analyzed time points. Experiments performed with increased (equal ratios of B and T cells) numbers of Treg versus Teff yielded similar results (data not shown).
These data suggest that cTreg do not affect the viability or proliferative behavior of activated B cells in an autologous in vitro co-culture system.
cTreg promote B cell Ab production
Having established that cTreg do not kill B cells in our co-culture system, we next analyzed if these cells impact B cell Ig production. We isolated naïve CD27− B cells from PBMC and cultured them with autologous CD4+ T cells in Ab-coated plates as described for the experiments shown in Figure 1. Ig production was analyzed at day 10 of culture. For these extended culture periods, T cells were irradiated prior to co-culture with B cells to prevent an overgrowth of the faster proliferating T cells that would lead to a rapid nutrient depletion in the cultures. In addition, this method ensured equal T-cell numbers throughout the culture period for each condition tested. We observed a wide range of Ig levels produced by B cells from different donors upon stimulation (Table 1). However, Ab levels were in almost all cases significantly higher in co-cultures that included CD46 stimulation, as compared with CD3 and CD3/CD28 stimulation (Fig. 2A). CD46 co-stimulation induced about ninefold higher production of IgM, fivefold higher production of IgA and a sevenfold increase of IgG1 production in naïve B cell cultures when compared with CD3/CD28-activated cultures (Fig. 2A and Table 1). Although we did not observe statistically significant differences in IgA or IgG1 production among CD3, CD3/CD28 and CD3/CD46-activated cultures in eleven experiments (Fig. 2A), IgA and IgG1 levels were consistently highest in CD3/CD46-activated cultures (with the exception of one culture in which no IgG1 production was detected) (Fig. 2A and Table 1). A similar trend was seen for IgM and IgA production in memory B-cell cultures with autologous CD4+ T cells (data not shown), although differences in Ig production were less pronounced, and the donor to donor variations considerably higher. Ig isotypes, IgG2, IgG3, IgG4 and IgE, were absent in most cultures tested, or produced only at low levels, displaying no significant differences between the different culture conditions (data not shown). As the high proliferative potential of cTreg represents most likely an important characteristic of their in vivo phenotype, we also performed the above-described experiments utilizing non-irradiated T cells with a number of different donors. The usage of proliferating T cells also induced strong B cell Ab production and were comparable to those obtained with irradiated T cells (data not shown).
Table 1. Ig levels are increased in B-/T-cell cultures activated in the presence of anti-CD46 Ab. Co-cultures of naïve B cells and irradiated CD4+T cells were tested for the presence of IgM (top), IgA (middle) and IgG1 (bottom) at day 10 of culturea)
Fold increase over BCR/CD3/CD28
a) Ig concentrations are expressed in nanogram per milliliter. Fold increases of Ig levels in BCR/CD3/CD46-activated co-cultures over that of BCR/CD3/CD28-activated co-cultures are displayed in the right column. For donor 56, the fold increase in IgM was measured as BCR/CD3/CD46 over BCR/CD3 because of the unusual low levels of Ig produced in the BCR/CD3/CD28 condition (marked by *).
In conclusion, in contrast to their suppressive effects on Teff populations, cTreg did not inhibit B-cell functions in our autologous T-cell/B-cell co-cultures. Furthermore, cTreg appeared more potent in enhancing Ig production than conventional Teff.
CD46 engagement on B cells does not enhance Ig production
The above results demonstrate that Ab-mediated activation of CD46 during B-/T-cell co-cultures increases Ig responses. However, since B cells also express CD46, CD46 is not only engaged on T cells, but also on B cells in our experimental setup. To discriminate whether the heightened B-cell response is mediated via CD46-induced signals directly on the B cells or indirectly via CD46-activation of T cells, we cultured purified B cells (without T cells) in the presence of crosslinking Ab to the BCR, with or without simultaneous CD46 activation. No B-cell proliferation or Ig production was observed in either condition, indicating that concurrent BCR and CD46 crosslinking is not sufficient to induce B-cell blasting or Ig production (data not shown). We next modified this system and introduced additional stimulatory signals by activating the B cells via anti-CD40 Ab and pansorbin, conditions known to induce polyclonal B-cell activation. Additional crosslinking of CD46 in these cultures did not alter IgM or IgG production, while IgA production was marginally (and non-significantly) increased (Fig. 2B).
cTreg from a CD46-deficient patient do not provide efficient B-cell help
The previous experiments suggest that direct crosslinking of CD46 on B cells is likely not responsible for the increased Ig production observed in our cultures but rather is due to signals provided by CD46-activated T cells. We tried to substantiate this notion by using CD46-deficient B cells in this assay. Although we have not yet succeeded in silencing CD46 expression in primary human B cells, we were able to obtain a blood sample from a CD46-deficient patient 20.
To date, there are eight known cases of homozygous CD46 deficiency 19–21, 26, 27. These patients lack cell surface expression of CD46, which renders them more susceptible to complement attack under inflammatory conditions. All patients with complete CD46 deficiency develop hemolytic uremic syndrome, a triad of hemolytic anemia, thrombocytopenia and renal failure caused by thrombotic microangiopathy of the kidney 21. Surprisingly, in three of the eight known cases of complete CD46 deficiency, the patients also developed CVID (20 and unpublished data, V. F.-B.). Although the five remaining patients do not meet all criteria for a CVID diagnosis, two of them present with subnormal IgG1 levels (19, 20 and unpublished data, V. F.-B.). Based on our observation that cTreg provide B-cell help in our co-culture system, we hypothesized that T cells from CD46-deficient individuals fail to differentiate into cTreg and that these patients are missing a potential B-cell activation pathway.
We obtained cells from a CD46-deficient patient with hemolytic uremic syndrome but without CVID 20 to assess the role of CD46 activation on B and/or T cells in Ig induction. This patient has a mutation in an invariant dinucleotide of the splice site (IVS2+2T>G) between exons 2 and 3, resulting in the deletion of 144 bp and 48 amino acids in phase with the WT sequence. Expression of the mutated CD46 protein on granulocytes from this patient was less than 10% compared with healthy donors. The patient displayed normal CD4+/CD8+ T- and B-cell numbers, naïve versus memory B-cell populations as well as normal proportions of IgM+, IgA+ and IgG+ B cells in peripheral blood (data not shown). To co-culture CD46-deficient patient B/T cells with CD46-sufficient normal donor T/B cells, we first established that an allogeneic B/T-cell co-culture system leads to similar results as those obtained with the autologous system. Figure 2C shows that in both autologous and allogeneic B/T cell co-cultures Ig levels are significantly increased in the presence of cTreg. Similar to our previous experiments, cTreg induction under allogeneic culture conditions was monitored by high levels of IL-10 and granzyme B production (data not shown). Figure 2D shows CD46 expression on B and T cells from a healthy donor and the minimal residual expression on those from the CD46-deficient patient.
CD46-deficient B cells produced similar amounts of IgM compared with CD46-sufficient B cells upon co-culture with CD46-sufficient T cells (Fig. 2E, upper panel). This observation supports our initial observation that CD46 crosslinking directly on B cells does not enhance Ig production (Fig. 2B) and substantiates our interpretation that the B-cell support in our co-culture systems is independent of CD46 signaling on B cells but delivered via signals from the CD46-activated T cells. Accordingly, CD46-deficient T cells from this patient did not develop into IL-10-producing cTreg upon CD3/CD46 engagement (data not shown) and were impaired in their ability to provide help to normal CD46-sufficient B cells under the tested culture conditions (Fig. 2E, lower panel). However, we noted that CD3/CD28-activated T cells from this patient also failed to provide B-cell support (Fig. 2E, lower panel). At present, it is unknown whether T cells from this CD46-deficient patient have other defects that contribute to the lack of B-cell help from both cTreg and Teff.
cTreg-mediated B-cell help requires IL-10 and cell–cell contact
Our results indicate that cTreg promote Ab responses upon co-culture with naïve B cells. cTreg B-cell help could be mediated by either soluble factor(s) released by cTreg, by a cell–cell contact-dependent mechanism, or by a combination of both. As IL-10 is a major factor required for optimal Ig production 13, we assessed if cTreg-derived IL-10 mediates B-cell activation in our co-culture system. B cells were cultured with cTreg or Teff with and without the addition of function-neutralizing Ab to IL-10 and reconstitution with rIL-10 (Fig. 3A). IgM levels were measured at day 10 of culture. Neutralization of IL-10 decreased cTreg-induced IgM production from B cells on average by about 60% (range: 42–84%). Reconstitution of cultures with rIL-10 not only restored IgM responses in CD3/CD46-activation settings but also increased IgM production in cultures with CD3 or CD3/CD28-activated T cells in most of the experiments performed (Fig. 3A). These results not only demonstrate the importance of cTreg-derived IL-10 in this system but also suggest that additional factors are involved because Ig production was never completely abolished by IL-10 neutralization.
To address whether direct T-cell/B-cell contact or crosstalk is necessary for the cTreg-mediated B-cell help, we set up co-cultures in a transwell system, which prevents direct cell contact but allows B/T-cell crosstalk via soluble factors. Separation of B and T cells abolished the enhancing effects of cTreg on Ig production, indicating that cell-contact-dependent mechanisms are required for optimal B-cell activation (Fig. 3B).
These experiments suggest that B-cell activation supported by cTreg is dependent on direct cell–cell contact but is further enhanced through IL-10 secretion.
Expression of co-stimulatory molecules by CD3/CD46-stimulated T cells
Potential cell–cell contact-dependent mechanisms during T-cell/B-cell interaction include the engagement of costimulatory molecules on B and T cells. To determine if cTreg provide superior B-cell help via the expression of a distinct co-stimulator profile, we compared the cell surface expression of a number of known T-cell co-stimulation and activation markers on cTreg and Teff after 1, 3 and 5 days of culture (Fig. 4A). CD46 co-stimulation did not induce significantly different expression levels of the costimulatory and activation markers CD40L, CD28, CTLA-4, CD200, OX40 and ICOS 28, 29 (Fig. 4A and data not shown). We did, however, note a consistently higher and prolonged expression of CD137, a member of the tumor necrosis receptor family known to be involved in T-cell activation/co-stimulation 30, and higher expression of CD25 on cTreg. We are currently investigating whether the changes in the expression of these two molecules account for the better B-cell help provided by cTreg compared with Teff.
The analysis of the co-stimulatory surface marker expression pattern of the T cells isolated from the CD46-deficient patient showed the expected down-regulation of CD3, up-regulation of CD28 and no significant differences in the up-regulation or expression of CTLA-4, CD200, OX40, LFA-1, ICOS or CD137, following stimulation with anti-CD3 Ab (Fig. 4B and not shown). However, we observed two differences: T cells from this patient did not express CD40L on the surface, a major hallmark for T-cell activation, and as expected, CD3/CD46 activation did not induce the high levels of CD25 and CD137 observed on CD4+ T cells from healthy donors – although CD46-deficient T cells responded normally to CD3 and CD3/CD28 activation with intermediate levels of CD25 and CD137 expression (Fig. 4B). The functional consequences of these observations are unclear and their further investigation is currently hampered by the limited access to blood samples from CD46-deficient patients.
In this study, we show that despite their suppressive effect on Teff activation and function, CD3/CD46-activated T cells supported B-cell responses during in vitro B-cell/T-cell co-culture experiments. B-cell help by cTreg required their IL-10 production but also cell–cell contact. T cells isolated from a CD46-deficient patient did not provide B-cell help upon CD3/CD46 stimulation.
IL-10-secreting adaptive regulatory CD4+ T cells represent an anti-inflammatory T-cell subpopulation with the capacity to down-modulate both Th1 and Th2 Teff responses 11, 31, 32. Recent work suggests that they play a central role in the maintenance of tolerance at specific locations involving host/environment interfaces, such as the gut, skin and the lung 33. Adaptive Treg are thus of considerable interest because of their therapeutic potential for the treatment of immune-mediated pathologies 34.
In humans, IL-10-secreting regulatory T cells can be induced ex vivo via polyclonal stimuli or antigen presented by APC 11, 35, in the presence of the glucocorticoid, dexamethasone and the active form of vitamin D, 1a25-dihydroxyvitamin D3 (1a25VitD3) (Tr1 cells) 35, 36, through stimulation via tolerogenic APC (Tr1 cells) 37 or through the concurrent activation of the TCR and the complement regulator CD46 in the presence of IL-2 (cTreg) 7. Although we and others have demonstrated the induction of cTreg by natural and pathogen-derived ligands for CD46 38–40, their in vivo existence and function have not yet been verified conclusively. This is mostly due to the lack of a suitable small animal model for the analysis of CD46 in T-cell function. Rodents such as mice, rats and guinea pigs do not express CD46 on their somatic cells 4, 41, suggesting that a CD46-induced cTreg pathway does not exist in these species. CD4+ T cells from transgenic mice expressing human CD46 in a human-like isoform pattern do not demonstrate increased IL-10 or granzyme B production upon concurrent TCR and hCD46 stimulation 42 (data not shown). We, therefore, are currently primarily expanding our understanding of CD46-induced Treg using human in vitro culture systems. A recent observation, however, strengthens the case for an in vivo role of cTreg in immune regulation: A study by Astier et al., established a connection between defects in the CD46-mediated induction of IL-10 in CD4+ T cells with MS in a proportion of patients indicating that CD46 may indeed play a role in the prevention of autoimmunity in man 38, 40. Excitingly, a connection between dysfunctional CD46-mediated IL-10 production in T cells has now also been linked with acute EAE, a mimic model fur human MS, in cynomolgus monkeys (which express CD46 naturally) 43.
An in vivo involvement of CD46 also in B-cell responses is suggested by the observation that one-third of the patients with complete CD46 deficiency develop CVID (20 and unpublished results, V. F.-B.). The phenotype of these patients raises an immediate question: Do these individuals show defects in Ig production because of the lack of CD46 signaling directly on B cells or because of “missing” CD46-activated T cells? To answer this, we performed experiments using a variety of B-cell stimulatory conditions. We were unable to show that signaling through B-cell-expressed CD46 directly affected B cell Ab responses under the conditions tested (Figs. 2B and E). Accordingly, B cells isolated from a CD46-deficient patient responded with normal Ig production upon proper T-cell help in our co-culture system. We cannot exclude that CD46 crosslinking and signaling directly on B cells modulate Ab responses under alternative activation conditions or in vivo. However, the data here suggest that the increased Ig production observed in our system is due to B-cell help provided by CD3/CD46-induced cTreg. This interpretation is supported by a recent study performed by Drouet and colleagues. In their study, Drouet and colleagues show that a C3-deficient patient (C3b is a principal ligand for CD46) presents with severe defects in DC function, reduced numbers of switched memory B cells and a defect in cTreg induction. The authors suggest that the poor cTreg function might contribute to the impaired B-cell responses found in this patient 44. The B lymphocyte sample from the CD46-deficient patient was helpful in determining that CD46 signaling within B cells is not vital to Ig production. The interpretation of the data from these assays regarding cTreg in vivo support of B-cell responses is much less straight forward; this patient did not develop CVID despite that fact that CD28- and CD46-activated T cells failed to provide B-cell help in our assays. The ability to mount quick and effective Ab responses upon infections is crucial. Thus, such important immunological functions are “covered” by several redundant pathways (one of which possibly being CD46 dependent) and it seems that one or several of these alternative T-cell help pathways (in addition to CD46) are affected in CD46-deficient patients with CVID. This, however, complicates a delineation of cTreg function using T-cell samples from these patients.
Also, the experiments performed here utilized B cells isolated from freshly drawn blood samples as this is so far the most reliable and accessible source for human naïve B cells. However, the phenotype of these B cells will likely differ from lymphoid tissue-resident B cells, representing the B-cell population directly interacting with activated T cells. Although we observed in experiments using B cells isolated from tonsils (which are part of the MALT), that blood-derived cTreg also support activation and Ab production by tonsillar naïve B cells in a comparable fashion (data not shown), cTreg may have distinct in vivo effects on other lymphoid B cells or on B cells that are activated in locations with a specialized environmental milieu.
We have initiated a screen of cTreg for specific activation markers or co-stimulatory molecules that could explain the increased B-cell stimulatory properties of these cells in comparison to Teff (Fig. 4A). One difference we noted between cTreg and Teff was a slightly higher and more prolonged expression of CD25 and CD137 upon CD46-stimulation. However, whether these molecules (in combination with the high IL-10 production by cTreg) are involved in the increased Ig production upon cTreg/B-cell contact needs to be addressed in future experiments.
Similar to other adaptive Treg, CD46-induced Treg suppress Teff via high IL-10 production 7. However, cTreg display features that set them apart from the “classic” adaptive Treg 3. CD3/CD46 activation induces strong granzyme B and perforin expression in CD4+ T cells 8. Granzyme A expression has been observed for natural CD25/Foxp3-positive Treg but not for adaptive Treg 8. Although granzyme B expressed by natural Treg is important in tolerance induction in transplantation 45, the functional role of CD46-mediated granzyme B expression is not clear. In addition, in contrast to other adaptive Treg, cTreg support DC responses 10 and, as shown in this study, also B-cell responses. Treg generally inhibit DC maturation 11 and, although the current knowledge of the effect of Treg on B cells is sparse, the few existing studies indicate a suppressive effect of Treg toward B lymphocytes 18, 46–48. Thus, one could argue that cTreg are distinct from other adaptive Treg in such that they specifically down-modulate Teff responses but support the other arms of the immune response.
The complement system is generally seen as a first line defense against invading pathogens. Its major function is to sense danger, destroy pathogens directly and immediately induce protective responses involving innate and adaptive immunity 49. Intuitively, complement proteins such as CD46 should transmit signals that support APC function, Ig production and Teff responses. Thus, the observation that cTreg not only support but also amplify APC and B-cell functions, which are essential in pathogen clearance, is therefore indeed in line with the idea that complement activation is a vital part in the initiation of an immune response. However, it is now clear that the proper and timely resolution (or suppression) of an immune response is also vital in the prevention of inflammation-mediated tissue damage and autoimmunity and cTreg may participate in this equally important down-modulatory phase in immunity/immune homeostasis. Thus, the interesting characteristics of CD46-induced cTreg may be an intriguing example as to how the complement system evolved to direct the initiation, effector and resolution phase of immune responses.
Materials and methods
Patient and donor samples
Patients and healthy donors participating in this study provided written consent in accordance with the Declaration of Helsinki. Blood was collected and processed with the approval and in accordance with the Washington University Medical Center Human Studies Committee guidelines and the Hôpital Européen Georges Pompidou Hospital Paris Review Board. The CD46-deficient patient has been described in a previous study 20.
Cell lines and Ab
T cells and B cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) with 10% FCS (Hyclone, Logan, UT, USA) and 200 mM L-glutamine in the presence of 25 U/mL recombinant human IL-2 (BioSource International, Camarilla, CA, USA) or additional components as indicated. CD40L-transfected J558L cells were a kind gift from Marco Colonna (Washington University). The anti-CD46 mAb TRA-2-10 50 was labeled with FITC (Sigma-Aldrich, St. Louis, MO, USA) using standard protocols. Unconjugated Ab to CD3 and CD28, and conjugated CD4, CD19, CD27, CD154, CD25, CD152, granzyme B, isotype control Ab and function-neutralizing mAb to human IL-10 were bought from BD Biosciences (San Diego, CA, USA). ICOS, CD137, CD134 and anti-CD40 clone 5C3 were obtained from eBioscience (San Diego, CA, USA). CD200 was from Coulter Immunotech and F(ab′)2 anti-human IgM/G/A from Jackson ImmunoResearch (West Grove, PA, USA). Recombinant human IL-10 was obtained from BD Biosciences.
Isolation of human T cells and B cells
CD4+ T cells and B cells were isolated from PBMC using CD4 and CD19 MicroBeads (Miltenyi Biotec, Auburn, CA, USA), respectively, according to the manufacturer's instructions. Where indicated, naïve B cells were isolated using the Naïve B-cell Isolation Kit (Miltenyi Biotec). Purity of isolated lymphocyte fractions was typically >95%.
In vitro stimulation of isolated CD4+ T cells was performed in 96-well culture plates coated with mAb to CD3, CD28 and/or CD46, each at 2.5 μg/mL. Purified CD4+ lymphocytes (1.5–2.0×105 cells/well) were added in 100 μL culture medium supplemented with 25 U/mL recombinant human IL-2. The plates were centrifuged at 50×g for 2 min, cultured and cell supernatants harvested at day 3 for cytokine analysis and for use in B-cell cultures.
To assess B-cell activation in the presence of T-cell supernatant, B cells were plated at 1×105 cells/well in culture medium containing 50 ng/mL rIL-10, 50 U/mL IL-2, 0.5 μg/mL anti-CD40 and pansorbin (heat-inactivated Staphylococcus aureus, used 1:2500, EMD Chemicals, Gibbstown, NJ, USA) in 50 μL volume. T-cell supernatant (100 μL) was then added and cell mixtures cultured at 37°C. Culture supernatants were collected at day 5 (wells then replenished with fresh media) and day 10 of cultures to measure Ig production. The effects of direct CD46 engagement on B cells were assessed by culturing B cells as described above but with or without the addition of pansorbin (Sigma-Aldrich) and in 96-well plates that had been coated overnight with 5 μg/mL anti-CD46 Ab.
B cells (50 000) were cultured with 100 000 irradiated (2000 rad) CD4+ T cells on 96-well plates coated with 5 μg/mL F(ab′)2 anti-human IgM/G/A and combinations of anti-CD3, CD3/CD28 or CD3/CD46. Culture media (containing 50 U/mL IL-2) was replaced on day 5 of co-culture and supernatants were analyzed on day 5 for cytokine content and on day 10 for Ig content. For neutralization of IL-10, 2.5 μg/mL soluble anti-IL-10 Ab was added at the start of the culture. Specificity of IL-10 neutralization was monitored via simultaneous treatment with anti-IL-10 Ab and excess amounts of rIL-10 (125 ng/mL). For assessing B- and T-cell proliferation and B-cell survival, B cells were labeled with CFSE (Sigma-Aldrich). Labeled B cells (100 000) were cultured with 50 000 autologous CD4+ T cells. CFSE dilution and numbers of live and dead B and T cells were assessed by FACS using anti-CD4 and PI staining.
Cytokine and Ig measurements
IL-10 production by T or B cells was measured using either the Th1/Th2 Cytometric Bead Array (BD Biosciences) or the Human IL-10 Secretion Assay Kit (Miltenyi Biotec) according to the manufacturer's instructions. IgM, IgA, IgG2, IgG3 and IgG4 were measured using the Human Ig FlexSet System (BD Biosciences). Samples were analyzed on a FACS Calibur and cytokine/Ig concentrations determined using the CBA software and the FCAP software (all BD Biosciences), respectively. IgG1, and in some experiments, also IgA and IgM, were analyzed by ELISA using Ab pairs from BD Biosciences and IgE levels were measured by ELISA (Bethyl Laboratories, Montgomery, TX, USA).
Statistical analyses were performed using the Student's two-tailed t-test (Excel software (Microsoft, Redmond, WA, USA)).
The authors thank Professor Jean-Pierre Grunfeld (Hopital Necker, Paris) and the CD46-deficient patient for generously providing us with a blood sample for our studies. They thank Alex Braun and Chris Evagora (Queen Mary University of London, ICMS Core Pathology), Graham Lord, Nicholas Powell and James Canavan (Biomedical Research Centre, King's College London) for expert help in immunohistology, and Daniel Hoft and Steven Truscott (Saint Louis University in St. Louis, MO, USA), and Tim Wilson (Washington University in St. Louis) for helpful discussion of the results and critical reading of the manuscript. This study was funded by the American Asthma Foundation (formerly SPAR) (A.F., J.P.A. and C.K.), the National Institute of Health grants 5 RO1 AI037618 (J.P.A.) and U19 AI070489 (J.P.A. and C.K.), the Kidney Patient Association UK (C.K.), and the Assistance Publique-Hôpitaux de Paris – Programme Hospitalier de Recherche Clinique [AOM 08198] 2008 (V. F.-B.).
Conflict of interest: The authors declare no financial or commercial conflict of interest.