Genetic control of parasite clearance leads to resistance to Plasmodium berghei ANKA infection and confers immunity.

Unprecedented cure after infection with the lethal Plasmodium berghei ANKA was observed in an F2 progeny generated by intercrossing the wild-derived WLA and the laboratory C57BL/6 mouse strains. Resistant mice were able to clear parasitaemia and establish immunity. The observed resistance was disclosed as a combinatorial effect of genetic factors derived from the two parental strains. Genetic mapping of survival time showed that the WLA allele at a locus on chromosome 1 (colocalizing with Berghei resistance 1 (Berr1), a locus associated with resistance to experimental cerebral malaria) increases the probability to resist early death. Also, the C57Bl/6 allele at a novel locus on chromosome 9 (Berr3) confers overall resistance to this lethal Plasmodium infection. This report underlines the value of using wild-derived mouse strains to identify novel genetic factors in the aetiology of disease phenotypes, and provides a unique model for studying parasite clearance and immunity associated with malaria.

Susceptibility to experimental cerebral malaria induced by Plasmodium berghei ANKA in inbred mouse strains recently derived from wild stock.

The neurological syndrome caused by Plasmodium berghei ANKA in rodents partially mimics the human disease. Several rodent models of cerebral malaria (CM) exist for the study of the mechanisms that cause the disease. However, since common laboratory mouse strains have limited gene pools, the role of their phenotypic variations causing CM is restricted. This constitutes an obstacle for efficient genetic analysis relating to the pathogenesis of malaria. Most common laboratory mouse strains are susceptible to CM, and the same major histocompatibility complex (MHC) haplotype may exhibit different levels of susceptibility. We analyzed the influence of the MHC haplotype on overcoming CM by using MHC congenic mice with C57BL/10 and C3H backgrounds. No correlation was found between MHC molecules and the development of CM. New wild-derived mouse strains with wide genetic polymorphisms were then used to find new models of resistance to CM. Six of the twelve strains tested were resistant to CM. For two of them, F(1) progeny and backcrosses performed with the reference strain C57BL/6 showed a high level of heterogeneity in the number and characteristics of the genetic factors associated with resistance to CM.

Quantitative relationship between MHC class II-superantigen complexes and the balance of T cell activation versus death.

The binding of bacterial superantigens (SAgs) is profoundly affected by the nature of the MHC class II-associated antigenic peptide. It was proposed that this limitation in the density of SAgs displayed at the surface of APCs is important for efficient TCR serial triggering as well as for preventing apoptosis of the responding T lymphocytes. Here, we have addressed quantitatively the size of this SAg-receptive pool of HLA-DR molecules that are available to bind and present staphylococcal enterotoxin A (SEA) at the surface of B lymphocytes. Our binding curves, depletion experiments, and quantitative immunoprecipitations show that about half the HLA-DR class II molecules on B cells are refractory to SEA binding. Yet, as compared with typical nominal Ags, an unusually high amount of class II-SAg complexes can be presented to T cells. This characteristic appears to be necessary for SAg-induced T cell apoptosis. When <0.3% of the total cell surface MHC class II molecules are occupied by SEA, T cells undergo a normal sequence of early activation events. However, presentation of a ligand density beyond this threshold results in T cell activation that is readily aborted by apoptosis but only after a few cell divisions. Thus, we confirm the existence of MHC class II subsets that are structurally unable to present SEA and provide a quantitative framework to account for the ability of bacterial SAgs to induce peripheral activation vs tolerance in the host.

Affiliation to mature B cell repertoire and positive selection can be separated in two distinct processes.

Using an 'oligoclonal' model, we have previously shown that mice transgenic for a mu chain (H3) and deficient for kappa chain expression display a mature B cell repertoire largely dominated by the H3/lambda1 pair, while the four H3/lambda available combinations can be observed in the immature B cell compartment. This led us to propose the existence of a positive selection process. To test this hypothesis, we have introduced the SJL lambda locus coding for a defective lambda1 chain (lambda1(s)) that creates a dysfunctional Ig receptor complex during B cell differentiation. Our results show that the lambda1(s) defect impairs the development of mature B cells when the H3-mu transgene insert is present in the hemizygous state. This suggests that the Gly --> Val substitution present in the C(lambda)1(s) chain at position 155 is sufficient to abrogate the selection of the H3/lambda1 pair. Unexpectedly, when the H3-mu transgene array is present in a homozygous state in lambda1(s) mice but not in 'wild-type' lambda1 mice (lambda1(+)), a significant number of mature B cells expressing all H3/lambda combinations can be developed. These results indicate that the overriding H3/lambda1 dominance observed in lambda1(+) mice is due to a positive selection process and not to a negative selection of other H3/lambda combinations. They also show that the export of B cells to the periphery can be controlled by the expression of the mu chain.

Liver CD4-CD8- NK1.1+ TCR alpha beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro.

Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4-CD8- NK1.1+ TCR alpha beta int cells and a down-regulation of CD4+ NK1.1+ TCR alpha beta int cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRV beta segment repertoire revealed that most of the expanded CD4-CD8- (double-negative) T cells presented a skewed TCRV beta repertoire and preferentially used V beta 2 and V beta 7 rather than V beta 8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-gamma was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4-CD8- NK1.1+ alpha beta T cells and down-regulation of CD4+ NK1.1+ TCR alpha beta int cells may contribute to the early immune response induced by the Plasmodium during the prime infection.

Tolerance to maternal immunoglobulins: resilience of the specific T cell repertoire in spite of long-lasting perturbations.

T cell tolerance is established and maintained through various mechanisms, the critical component being the persistence of the specific Ag. However, at the molecular level, the nature of the recovering TCR repertoire following breakdown of tolerance is unknown. We address this important question by following kappa light chain constant region (C kappa)-specific CD4+ T cells of kappa light chain knock-out (kappa-/-) mice born to kappa+/- mothers. These cells, which were in contact with maternal kappa+ Igs from early ontogeny until weaning, were strongly tolerized. Tolerance was reversible and waned with the disappearance of peptide C kappa 134-148 presentation in lymphoid organs, including the thymus. Whereas three specific V beta-J beta rearrangements emerged in the peptide C kappa 134-148-specific CD4+ T cell response of all regular kappa-/- mice, soon after breakdown of tolerance only one of these rearrangements was detected. The two others displayed a significant delay in reappearance and were still rare at 26 wk of age, while the control proliferative response had already recovered 3 mo earlier. At 52 wk of age, a complete recovery of the three canonical V beta-J beta rearrangements was observed. Thus, although profoundly perturbed for several months, the T cell repertoire returns to equilibrium, highlighting the resilient nature of this system.

T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR V(beta)8 during experimental cerebral malaria.

To characterize the T cells involved in the pathogenesis of cerebral malaria (CM) induced by infection with Plasmodium berghei ANKA clone 1.49L (PbA 1.49L), the occurrence of the disease was assessed in mice lacking T cells of either the alphabeta or gammadelta lineage (TCRalphabeta(-/-) or TCRgammadelta(-/-)). TCRgammadelta(-/-) mice were susceptible to CM, whereas all TCRalphabeta(-/-) mice were resistant, suggesting that T cells of the alphabeta lineage are important in the genesis of CM. The repertoire of TCR V(beta) segment gene expression was examined by flow cytometry in B10.D2 mice, a strain highly susceptible to CM induced by infection with PbA 1.49L. In these mice, CM was associated with an increase of T cells bearing the V(beta)8.1, 2 segments in the peripheral blood lymphocytes. Most V(beta)8.1, 2(+) T cells from peripheral blood lymphocytes of the mice that developed CM belonged to the CD8 subset, and exhibited the CD69(+), CD44(high) and CD62L(low) phenotype surface markers. The link between the increase in V(beta)8.1, 2(+) T cells and the neuropathological consequences of PbA infection was strengthened by the observation that the occurrence of CM was significantly reduced in mice treated with KJ16 antibodies against the V(beta)8.1 and V(beta)8.2 chains, and in mice rendered deficient in V(beta)8.1(+) T cells by a mouse mammary tumor virus superantigen.

Depletion of peritoneal CD5+ B cells has no effect on the course of Leishmania major infection in susceptible and resistant mice.

The mouse peritoneal cavity contains a unique self-renewing population of B cells (B-1) derived from fetal liver precursors and mainly producing polyreactive antibodies. Since B-1 cells are a potential source of IL-10, it has been suggested that these cells may contribute to the susceptibility of BALB/c mice to Leishmania major infection by skewing the T helper cell network towards a Th2 phenotype. Accordingly, L. major infection of B cell-defective BALB/c Xid mice (lacking B-1 cells) induces less severe disease compared with controls. However, in addition to the lack of B-1 cells, the Xid immune deficiency is characterized by high endogenous interferon-gamma (IFN-gamma) production. In the present study, the role of B-1 cells during L. major infection was investigated in mice experimentally depleted of peritoneal B-1 cells. Six weeks old C57Bl/6 and BALB/c mice were lethally irradiated and reconstituted with autologous bone marrow which allows systemic depletion of B-1 cells. Untreated BALB/c, C57Bl/6 as well as BALB/c Xid mice were used as controls. After reconstitution, mice were injected with L. major amastigotes and progression was followed using clinical, parasitological and immunological criteria. As previously reported, BALB/c Xid mice showed a significant reduction in disease progression. In contrast, despite the dramatic reduction of B-1 cells, B-1-depleted BALB/c mice showed similar or even worse disease progression compared with control BALB/c mice. No differences were found between B-1-depleted or control C57Bl/6 mice. Our data suggest that the B-1 cells do not contribute to the susceptibility of BALB/c mice to L. major infection.

Surface expression and functional competence of CD3-independent TCR zeta-chains in immature thymocytes.

In recombinase-deficient (RAG-2-/-) mice, double-negative thymocytes can be stimulated to proliferate and differentiate by anti-CD3 Abs. CD3 molecules are expressed on the surface of these cells in association with calnexin. In this study, we show that zeta-chains can be recovered as phosphorylated proteins in association with phosphorylated ZAP-70 from anti-CD3-stimulated RAG-2-/- thymocytes, even though they are not demonstrably associated with the CD3/calnexin complex. The lack of a physical association of zeta dimers with the CD3 complex in RAG-2-/- thymocytes and also in a pre-TCR-expressing cell line, as well as the efficient association of zeta dimers with ZAP-70 in the RAG-2-/- thymocytes, suggest that these zeta-chain dimers could contribute to pre-TCR signaling. This idea is supported by the finding that in RAG-2-/- zeta-deficient thymocytes, ZAP-70 and p120cbl were only weakly phosphorylated.

Cellular distribution of a mixed MHC class II heterodimer between DRalpha and a chimeric DObeta chain.

Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DObeta chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DObeta chain (DR/DObeta), containing 18 N-terminal residues of DRbeta, paired with the DRalpha chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DObeta chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DObeta chain along with DRalpha into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DObeta chimera and a DRa chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DObeta chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DOalpha chain and HLA-DM.

Conserved structural features between HLA-DO beta and -DR beta.

HLA-DO is a non-classical MHC class II molecule presumed to play a specialized role in the antigen processing pathway. We have modeled the HLA-DO beta-chain and found its overall structure compatible with the one of DR beta. Functional studies further highlighted the similarity between these beta-chains of the class II family of proteins. Indeed, a mixed heterodimer composed of the DR alpha and a chimeric DO beta-chains presented bacterial superantigens to T cells and was shown to interact with CD4. The implications of such structural conservation for the in vivo functions of HLA-DO are discussed.

Role of maternal Ig in the induction of C kappa-specific CD8+ T cell tolerance.

Although the influence of maternal Ig on the B cell repertoire and subsequent Ab response has been extensively studied, much less attention has been devoted to their effects on T cell responses of the offspring. To address this question, we have studied the influence of maternal kappa-positive Ig (Ig kappa) on the C kappa-specific CD8+ T cell response of kappa knock-out (kappa-/-) pups resulting from various crosses and foster nursings. These systems allowed control of physiologic transmission of Ig kappa at defined periods of ontogeny. Our data show that conventional transfer of maternal Ig via the placenta plus colostrum/milk or adoptive transfer via only the colostrum/milk were the most efficient at tolerizing C kappa-specific CD8+ responses. Surprisingly, tolerance was not detected in kappa-/- pups born to kappa+/- females obtained by cesarean delivery and suckled by kappa-/- mothers (transplacental supply only). Tolerance, which was strong until 5 wk of age, was reversible and waned with the decrease of Ig kappa serum concentration. Depletion of CD4+ T cells at the time of C kappa peptide immunization abolished the tolerance of C kappa-specific CD8+ T cells. These data suggest that an oral supply of Ig is very efficient at inducing and maintaining tolerance of C kappa-specific CD8+ T cells, at least for several weeks after birth, and that suppression rather than deletion is responsible for this tolerance. In addition, they strengthen the view that tolerance of CD8+ T cells to a soluble Ag is never permanently acquired even if it is present in large quantities during ontogeny.

Differential chronology of TCRADV2 gene use by alpha and delta chains of the mouse TCR.

The genes coding for TCR alpha and delta chains share the same genetic locus (TCRA/D). The rules governing the utilization of a V gene with the alpha and delta chains have not been established. More specifically, it is not known whether the position of a gene within the locus influences its utilization in alpha and delta TCR. To elucidate these points, we mapped ADV2 genes in the TCRA/D locus of BALB/c mice and analyzed their utilization in TCR alpha and delta transcripts from thymi isolated from mice of different ages. Our results show that all ADV2 genes can be used by the two chains, but with strikingly different patterns. Moreover, ADV2 utilization by the alpha chain proceeds in successive concentric waves during development, suggesting a progressive regulation of gene accessibility and utilization. These results support independent control of TCRA and TCRD gene assembly.

T cell tolerance to kappa light chain (L kappa): identification of a naturally processed self-C kappa-peptidic region by specific CD4+ T cell hybridomas obtained in L kappa-deficient mice.

In contrast to H-2d kappa light chain-deficient mice (kappa-/-), BALB/c (kappa+/+) mice fail to respond to kappa light chains (L kappa). This suggests that C kappa-specific T cells are tolerant to this self-antigen in kappa+/+ mice. To get insights into the cellular and molecular basis of this tolerance, we first characterized the presented L kappa-derived C kappa-peptidic region(s). Among a library of overlapping peptides spanning the whole C kappa sequence, only three consecutive peptides are recognized by CD4+ T cell hybridomas obtained in L kappa-immunized kappa-/- mice. This C kappa-peptidic region, which is also the only one containing the I-Ed-binding consensus motif, is immunogenic since it is able to prime lymph node cells of kappa-/- mice to subsequent in vitro proliferative response to either L kappa or kappa+/+ APC. Conversely, no kappa+/+ T cell proliferation is observed under the same conditions. Activation of our hybridomas by cells from central and peripheral lymphoid tissues reveals that this C kappa region is naturally expressed on BALB/c kappa+/+ APC. In addition to B cells, macrophages and dendritic cells are able to present this region. Taken together our data suggest that the described self-C kappa region is implicated in the C kappa-specific CD4+ T cell tolerization in BALB/c mice.

Does positive selection determine the B cell repertoire?

To know whether each newly formed B cell has an equal chance of survival in the organism, we analyzed the composition of the B cell repertoire of extremely limited diversity by generating mu-transgenic kappa-knockout mice. Surprisingly, in both types of mice studied, the B cell repertoire is mainly composed of cells expressing the mu-transgene-encoded chain associated with only one out four available lambda types depending on the mu transgene. Moreover, B cell differentiation cultures in vitro show that newly formed B cells can express the various lambda types regardless of the presence or absence of the mu transgenes. These results show a drastic impact of the heavy chain on the lambda light chain repertoire expressed in the periphery. The overexpression of a unique heavy/light chain pairing therefore results from selective processes. The immature B cells may be positively selected to provide the immunocompetent B cells in the periphery.

Evidence for superantigenic activity during murine malaria infection.

TCR V beta usage was examined in C57BL/6 mice infected with Plasmodium yoelii. In addition to a polyclonal T cell activation, already described, a superantigenic-like activity was observed during the acute infection. This superantigenic activity induces a preferential deletion without prior expansion of CD4+ and CD8+ T cells bearing the TCR V beta 9 segment. The superantigen could be released by the parasite at different stages of its development since the deletion of V beta 9+ T cells was observed in blood and lymph nodes of mice infected either with sporozoites or with erythrocytic stages. Injection of sporozoite or parasitized erythrocytes to newborn mice led to a deletion and anergy of peripheral V beta 9+ T cells, without affecting thymic T cell populations. These observations suggest that the superantigen is released at very low concentrations during parasite development. The role of such parasite superantigenic activity in infectivity can be underlined by the observation that congenic BALB.D2 Mis1a mice lacking V beta 9 T cells are more susceptible to infection by P. yoelii.

V alpha domain modulates the multiple topologies of mouse T cell receptor V beta20/staphylococcal enterotoxins A and E complexes.

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major histocompatibility complex (MHC) class II molecules on two sites located on the alpha and beta chains. We have investigated the role of the T cell receptor (TCR) alpha chain in the modulation of the various topologies of TCR/SEA (or SEE)/class II complexes. For this purpose, we have used three mouse V beta20 T cell lines expressing different V alpha domains and two T cell hybridomas expressing mouse V beta1 or V beta11 segments. The response of these T cells to SEA and SEE was studied in the context of presentation by wild-type human MHC class II molecules; or by mutants on MHC, in each of the two superantigen binding sites (position alpha39K and beta81H) to which the superantigens can still bind but with an altered conformation. Although V beta20 T cell lines are efficiently stimulated using SEA and SEE presented by wild-type HLA-DR1 molecules, our results show that the nature of the TCR V alpha domain can affect differently the recognition of the toxins bound to mutant class II molecules. This suggests that various functional topologies exist for both SEA and SEE/class II complexes and that the T cell response to each of these complexes can be modulated by the V alpha domain of the TCR. Interestingly, the recognition of SEA and SEE is achieved in different fashions by a given V beta20 T cell line.

The lambda B cell repertoire of kappa-deficient mice.

Analysis of the B cell repertoire is complicated by the huge diversity inherent in the germ line determined combinatory. Making use of knockout technology, kappa-deficient mice have been obtained. They constitute a shrewd model to follow the expression of an Ig minilocus, such as the lambda one, in the normal condition compared with classical transgenic models. Indeed, in contrast to wild type mice, in which only 5% of lambda B cells are produced, these mutant mice exclusively produce lambda positive B cells. Although, the lambda locus is well characterized and has a relatively simple organization, the mechanistic and selective pressures that govern its utilization are still poorly understood. The analysis of the lambda B cell repertoire in kappa-deficient mice, should therefore bring more conclusive informations. Here we present the lambda subtype distribution in the various cellular compartments of the kappa-deficient mice, and discuss the rules that can be responsible for this distribution. Our recent data indicate that the lambda subtype proportions in the bone marrow and the spleen result, for the major part, from mechanistic processes (i.e., recombinase accessibility, production of V-J functional joint and H/L pairings) while the lambda proportions found in the peritoneal cavity ensue from selective processes. Finally, the capacity to respond to various antigens is discussed from such a generated lambda B cell repertoire.