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.

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.

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.

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.

Emergence in C kappa knockout mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant kappa light chain region.

Allotype- or idiotype-specific CD4+ T cells have been reported to recognize immunoglobulin (Ig) peptides presented by class II molecules. In contrast, few data are available concerning the generation of Ig peptide-specific CD8+ T cells. We have therefore investigated whether T-depleted spleen cells from Ig kappa light chain-expressing 129/Sv mice (129 kappa +/+) could induce, in C kappa knockout mice (129 kappa -/-), the generation of Ig constant kappa light chain region (C kappa)-specific cytotoxic T lymphocytes (CTL). The determination of TCR beta chain expressed by nine CTL clones, together with the use of a library of overlapping peptides spanning the whole C kappa sequence, show that the B cells from kappa +/+ mice are able to elicit in C kappa knockout mice, the emergence of a diverse CTL repertoire that recognizes one single C kappa peptide presented by the H-2Kb class I molecule. In addition, these data support the notion that B cells are able to process and present on their class I molecules, peptides generated from their own kappa light chains.

Various V-J rearrangement efficiencies shape the mouse lambda B cell repertoire.

The diversity of the B cell repertoire of C kappa knockout mice is limited by the expression of four lambda light chain types. Among the spleen B cells, lambda 1 is expressed by the majority (58%) of cells, and lambda 3 by the minority (8%), while lambda 2 (V2) and lambda 2 (Vx) are expressed in intermediate quantities (18% and 16%, respectively). To assess the influence of mechanistic pressures on the lambda subtype distribution, the proportions of the different lambda rearrangements were determined in various B cell subpopulations divided on the basis of the lambda subtype expressed, and the V lambda J lambda junction sequences were studied at different steps of B cell differentiation (pre-B, immature and mature B cells). The data show that (1) the ratio of productive/non-productive VJ junctions is determined by the nature of the lambda segments that are rearranged as can be observed in the pre-B cells, (2) V1-J1 non-productive rearrangements are often found in the lambda 1-negative B cells in the periphery, and (3) V1J3 junctions are often non-productive regardless of the nature of the cells analyzed. Our results, therefore, suggest that a strong probability of initiating a V1-J1 rearrangement and a weak probability of giving a productive V1J3 junction are responsible for the lambda 1 dominance and the lambda 3 under-expression, respectively. The intermediate proportion of lambda 2(V2) subtype is most likely due to a probability of obtaining a productive joint that is better than that for V1J3 and a probability of initiating a rearrangement that is lower than that for V1J1. However, the lambda 2(Vx) cell proportion cannot be determined only by these parameters.

Expression and biological activity of interleukin-1 receptors in mouse gamma/delta thymocytes during ontogeny.

We have recently shown that the response of mouse thymocytes to interleukin (IL)-1 + IL-2 was maximal at birth and that the responding cells displayed a CD4-CD8- T cell receptor (TcR) gamma/delta + phenotype. Unexpectedly, despite their high proportion of gamma/delta + cells, fetal thymocyte populations responded only weakly to IL-1 + IL-2. In this report, we demonstrate that the discrepancy between the day 17.5 fetal and newborn sensitivities to the combined action of IL-1 and IL-2 is a consequence of the different patterns of high-affinity IL-1 receptor (IL-1R) expression displayed by these two cell subsets. Actually, high- and low-affinity IL-1R are found in TcR gamma/delta + newborn cells and, in contrast, only low-affinity IL-1R are detectable in day 17.5 fetal cells. Our binding and functional studies strongly support the hypothesis that high-affinity IL-1R on the one hand, and low-affinity ones on the other hand, are involved in the response to IL-1 + IL-2 of newborn and day 17.5 fetal thymocytes, respectively. In addition, the high-affinity IL-1R appear to be far more efficient than the low-affinity receptors in promoting IL-2 responsiveness of thymocytes.

Ontogeny of the response of mouse thymocytes to interleukin 1 and interleukin 2.

In the present report we demonstrate that the in vitro proliferative response of the newborn thymocytes to interleukin (IL) 1 and IL 2, which is remarkably stronger than the adult thymocyte response, is associated with a considerable increase of CD4-CD8- cells expressing a gamma/delta T cell receptor (TcR). By polymerase chain reaction analysis we show that the V gamma gene segment usage in the adult and newborn responding cells reflects the developmentally regulated expression of the V gamma gene segments, suggesting that the increase in TcR gamma/delta+ cells results from the polyclonal expansion of pre-existing clones. Surprisingly, although the fetal thymocyte populations contain higher numbers of TcR gamma/delta+ cells than the adult and newborn ones, the highest proliferative response to IL 1 and IL 2 is obtained with the newborn thymocytes. Non mutually exclusive hypotheses are discussed to explain these results.

Cellular basis of the resistance of newborn mice to the pathogenic effects of anti-CD3 treatment.

We have analysed the mechanisms underlying the differences in the susceptibilities of adult and newborn mice to the pathogenic effects of anti-CD3 mAbs. Our data show that the thymus cell number in adults is reduced by 93% 48 h after one single injection of 5 mg/kg of Ab whereas the same dose in newborns induces only a 30% decrease. In the adult, this effect is associated with a marked depletion of CD4+ CD8+ double positive (DP) cells and with the appearance of important areas of cell necrosis in the thymic cortex. In newborns, the DP cells are less affected and the thymic cortex does not present any cell necrosis even after an injection of 45 mg/kg of mAbs. Pre-treatment of adults with anti-CD4 and anti-CD8 Abs, while completely abolishing the toxic side-effects induced by anti-CD3 mAbs, does not protect the thymus from the depletion of DP cells. In vitro, anti-CD3 mAbs induce the proliferation of thymocytes and spleen cells from adults but not from newborns. Tumour necrosis factor-alpha (TNF alpha) is found in the serum of adults 90 min after injection of anti-CD3 but is never detected in the serum of anti-CD3 treated newborns. Taken together our data support the view that anti-CD3 mAbs act by two different mechanisms. The first one results from the binding of anti-CD3 on the CD3+ thymocytes which induces a direct toxicity for only the CD4+ CD8+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Junctional diversity of H and L chains allows the coexpression of two mutually exclusive idiotopes (IdI104 and IdI558).

The molecular basis for the unexpected coexpression of the individual Id (IdI)558 and IdI104 Id by anti-alpha(1-3) DEX antibody (Ab) (126.33 and 414.2) derived from the MPW wild mouse strain has been investigated by the comparison of the structures of their VH and V lambda 1 chain regions with those of two other MPW-derived Ab (262.9 and 16.3) expressing either IdI558 or IdI104 Id. Our data show that 262.9 and 16.3 Ab display identical V lambda 1 and very similar VH regions when compared with BALB/c anti-alpha (1-3) dextran Ab expressing IdI104 or IdI558, respectively. The two Ab (414.2 and 126.33) that express both IdI104 and IdI558 Id display two main features. First, their VH CDR3 are different from those found in IdI104 or IdI558 expressing anti-alpha(1-3) dextran Ab. Second, their V lambda 1 are identical to those from BALB/c origin except for the presence of an additional residue, a phenylalanine at position 95A of CDR3. This additional residue is encoded by the V lambda 1 gene segment and results from a hitherto undescribed V lambda 1-J lambda 1 junction. The alteration of the length of the V lambda 1 CDR3 loop, in conjunction with particular residues within VH CDR3, allows the coexpression of two Id that were found to be mutually exclusive in laboratory mice.

Mouse V lambda x gene sequence generates no junctional diversity and is conserved in mammalian species.

The lambda x, a new mouse Ig lambda L chain, is produced by rearrangement of the V lambda x, J lambda 2, and C lambda 2 gene segments. The V lambda x amino acid sequence is as divergent to other V lambda as to Vk gene sequences. Additionally, its third hypervariable region (CDR3) is four amino acids longer than those of all other variable gene segments of murine L chain. We have cloned and sequenced the germ-line V lambda x gene and found that the unexpected CDR3 length is encoded by the V lambda x gene. Junctional diversity is prevented by a TAA termination codon localized at the V lambda x 3' extremity. Moreover, we show a striking conservation of the V lambda x sequence in various mammalian species. Portions of the V lambda x sequence display more than 70% of nucleotide sequence identity with rabbit and human variable regions. These results suggest that V lambda x predated the divergence of mammalian species.

Effects of neonatal injections of anti-CD3 epsilon monoclonal antibodies on the T cell functions of adult mice.

The effect of neonatal injections of anti-CD3 mAb on the subsequent immune responsiveness of adult mice has been investigated. The data indicate that neonatal treatment with three injections of 50 microliters of anti-CD3 epsilon ascitic fluid induce a profound depletion of T cells in the peripheral lymphoid organs but do not modify the absolute number of thymocytes. This treatment completely abolishes the T cell functions of mice at least 3 wk after the last injection of mAb. In the thymus, this suppression is associated with a decrease in the number of TcR/CD3 molecules in the dull CD3+ cells and with a drastic reduction the bright CD3+ population. However, the amount and the size of the TcR alpha,beta and CD3 epsilon mRNA transcripts are not modified, suggesting that the down-regulation of the TcR/CD3 complex induced by anti-CD3 mAb does not exert a feedback inhibition on the transcription of TcR genes. The suppression induced by neonatal injections of anti-CD3 epsilon mAb is reversible and the induction of immune responses requires the reappearance of a minimal number of bright CD3+ cells. However, this suppression can be maintained without side effects for several months provided that anti-CD3 mAb were administered at 7-day intervals from birth. This injection schedule should allow the study of the effect of anti-CD3 antibodies during the T cell ontogeny on the establishment of the B and T cell repertoires.

Mapping of Igk-V genes using backcrossed laboratory and wild mice.

In the mouse, the genes coding for the Ly-2 antigen, the beta chain of the T-cell receptor, and the immunoglobulin kappa light chain have been located on chromosome 6. Although a tentative order has been proposed for these genes, very few data have been reported concerning their genetic distance. To address this question, we have produced backcross mice between SJL and MAI (a wild-derived strain belonging to the Mus musculus), since these mice segregate for the Ly-2 and Igk-C proteins and for the Igk-V24, Igk-V21, Igk-V10, Igk-V8, and Igk-V4 genes. Twelve recombinants were obtained from 163 backcross mice studied. Two mice showed a recombination between the (Igk-V24, Igk-V10, Igk-V8, Igk-V4) and the (Ly-2, Igk-C, Igk-V21) groups, and ten mice displayed a recombination between the (Igk-V24, Igk-V10, Igk-V8, Igk-V4) group and the Tcrb-C loci. These data imply the following gene order: Tcrb-C .... (Igk-V24, Igk-V10, Igk-V8, Igk-V4) .... (Igk-V21, Igk-C, Ly-2). They indicate a distance of 6.1 cM between Tcrb-C and (Igk-V24, Igk-V10, Igk-V8, Igk-V4) and 1.2 cM between Igk-V24, Igk-V10, Igk-V8, Igk-V4 and the (Igk-V21, Igk-C, Ly-2) groups.