Colonization with Helicobacter is concomitant with modified gut microbiota and drastic failure of the immune control of Mycobacterium tuberculosis.

Epidemiological and experimental observations suggest that chronic microbial colonization can impact the immune control of other unrelated pathogens contracted in a concomitant or sequential manner. Possible interactions between Mycobacterium tuberculosis infection and persistence of other bacteria have scarcely been investigated. Here we demonstrated that natural colonization of the digestive tract with Helicobacter hepaticus in mice is concomitant with modification of the gut microbiota, subclinical inflammation, and drastic impairment of immune control of the growth of subsequently administered M. tuberculosis, which results in severe lung tissue injury. Our results provided insights upon the fact that this prior H. hepaticus colonization leads to failures in the mechanisms that could prevent the otherwise balanced cross-talk between M. tuberculosis and the immune system. Such disequilibrium ultimately leads to the inhibition of control of mycobacterial growth, outbreak of inflammation, and lung pathology. Among the dysregulated immune signatures, we noticed a correlation between the detrimental lung injury and the accumulation of activated T-lymphocytes. Our findings suggest that the impact of prior Helicobacter spp. colonization and subsequent M. tuberculosis parasitism might be greater than previously thought, which is a key point given that both species are among the most frequent invasive bacteria in human populations.

Induction of protective immunity against Mycobacterium tuberculosis by delivery of ESX antigens into airway dendritic cells.

As the Bacillus Calmette-Guérin (BCG) vaccine does not confer long-lasting protection against lung Mycobacterium tuberculosis infection, the development of more efficient vaccines is greatly needed. Here, we used mycobacterial low-molecular weight proteins of the 6-kDa Early Secreted Antigenic Target (ESAT-6) protein family (ESX) antigens for the evaluation of a novel vaccine delivery strategy that enables versatile in vivo targeting of antigens into specialized dendritic cell (DC) subsets. ESX antigens were genetically fused to the tetramerizing core of streptavidin (SA) to form high-affinity complexes with biotin (biot)-conjugated antibodies recognizing DC surface receptors. When directed through the CD11b or CD11c ß2-integrins or diverse C-type lectins, the ESX-SA:biot-antibody complexes were efficiently captured and presented on major histocompatibility complex molecules of DCs to specific T-cell receptors. Robust ESX-specific T-cell responses were induced by immunization with as little as several picomoles of ESX-SA targeted to DC subsets. Moreover, directing of TB10.4-SA to airway CD205(+) cells enabled the induction of mucosal T-cell responses and provided significant protection against virulent M. tuberculosis.

Role of CD40 in a T cell-mediated negative regulation of Ig production.

To investigate the possible role of CD40 in a negative regulation of Ig production, we used the mouse Ig allotype suppression model. T splenocytes from IGH(a/a) mice are able in vivo to totally and chronically inhibit the production of IgG(2a)(b) (IgG2a from the IGH(b) haplotype). Accordingly, postnatal transfer of IGH(a/a) T splenocytes into histocompatible IGH(a/b) F(1) or congenic IGH(b/b) mice leads to a characteristic IgG(2a)(b) suppression. The helper action of anti-IgG(2a)(b) CD4(+) T cells is required for the recruitment of anti-IgG(2a)(b) CD8(+) T suppression effectors. The latter use perforin (pore-forming protein, Pfp)- and/or Fas-dependent cytotoxic pathways to continuously eliminate B cells recently committed to IgG(2a)(b) production. In the present study we first showed that in vivo agonistic anti-CD40 mAb treatment of IGH(a/a) mice, deprived of their CD4(+) T cell compartment, could bypass the help of Ig allotype-specific CD4(+) T cells and generate CD8(+) T effector cells able to strongly inhibit IgG(2a)(b) production. This result demonstrates the usefulness of CD40 triggering in setting up an immune regulatory mechanism. Furthermore, with regard to the suppression-effector mechanism, we demonstrated that B cell CD40 expression was required for full suppression establishment via the Fas-dependent pathway. Indeed, IGH(a/a) PFP(degrees/degrees) T cells (using exclusively the Fas pathway) induced full IgG(2a)(b) suppression against IGH(b/b) CD40(+/+) B cells, but only partial inhibition of IgG(2a)(b) production against IGH(b/b) CD40(degrees/degrees) B cells. This finding provides the first demonstration of direct involvement of B cell CD40 expression in in vivo negative control of an Ig production.

Mechanisms of mouse T lymphocyte-induced suppression of the IgG2ab allotype and T lymphocyte tolerance to IgG2ab.

In mice of the Igha immunoglobulin allotypic haplotype we found, the presence of T lymphocytes with an inherent inhibitory activity against the expression of the IgG2a(b) allotype (IgG2a of the Ighb immunoglobulin allotypic haplotype). This constitutive anti-IgG2a(b) T lymphocyte activity can be enhanced in vivo by what we called "sensitization", which usually consists of one or two intravenous injections of B splenocytes from Ighb congenic mice. When injected at birth, the resulting anti-IgG2a(b) T splenocytes induce, with 100% success, total, specific and chronic (but experimentally reversible) suppression of IgG2a(b) in Igh(a/b) F1 hybrid mice prepared by mating Igh congenic mice. Even if restricted to IgG2a(b) expression, this experimental model, which deals with an unambiguous case of T cell-mediated down-regulation of immunoglobulin production, provides a clear and powerful tool to dissect finely the behavior of the partners (T and B lymphocytes) intervening in regulation within the immune system. For example, we observed that CD4 T lymphocytes were necessary to obtain full recruitment of anti-IgG2a(b) CD8 T lymphocytes during the sensitization, that suppression induction in anti-IgG2a(b) T splenocytes of newborn recipients required cooperation between CD4 and CD8 T lymphocytes, and that CD8 T lymphocytes were essential for suppression maintenance. We showed that this suppression was not characterized by an accumulation of B lymphocytes containing the allotype they could not secrete or Cgamma2a(b) mRNA they could not translate. The recipient's immune system was not involved in the suppession maintenance; this was done by donor T lymphocytes, which ensured the chronicity of IgG2a(b) suppression throughout the recipient's life. We demonstrated that the mechanism of this suppression implied an MHC-restricted presentation by target B lymphocytes of Cy2a(b) peptides to the T cell receptor (TCR) of anti-IgG2a(b) T lymphocytes. Notwithstanding the requirement of a CD4-CD8 T lymphocyte cooperation during the induction phase, we functionally determined that the suppression induction implicated an MHC class I-, but not class II-restricted interaction. We also demonstrated the existence in vivo of alternative or concomitant use of perforine- and Fas-mediated cytotoxicity pathways in this T cell-induced IgG2a(b) suppression. Thus this suppression did not imply silencing IgG2a(b) production, but B lymphocyte destruction by CD8 T lymphocytes. Always using our suppression model, we demonstrated that an agonistic anti-CD40 treatment helps in recruiting CD8 cytotoxic T lymphocytes, involved in immune regulatory functions and that CD40 expression on Ighb B lymphocytes confronted with CD8 T lymphocyte effectors only operating via the Fas pathway was involved in the total suppression of IgG2a(b) expression. The selection and maintenance of such normal T cell activity against the IgG2a(b) allotype in mice of different genetic backgrounds remain somewhat enigmatic. Indeed, we did not observe any similar activity against other immunoglobulin allotypes or isotypes. The intestinal flora had no influence on the emergence of this anti-IgG2a(b) T lymphocyte activity, as it was untouched in germ-free-Igha mice when compared with normal Igha mice. More recently, this model offered an opportunity to study problems pertaining to immune tolerance. For instance, we showed that the genetic elements involved in the building of anti-IgG2a(b) TCR were available in Igha and Ighb mice of different genetic backgrounds, but that somatic constraints, namely the perinatal presence of IgG2a(b), effectively prevented their acquisition, while its absence led to their spontaneous emergence. Consequently, we were able to induce anti-IgG2a(b) T lymphocytes into a tolerance state by injecting Igha mice with soluble IgG2a(b) during the perinatal period. However, the full T lymphocyte tolerance obtained in this manner was not definitively acquired, as it had reversed spontaneously when investigated 3 to 6 months after the end of tolerogen treatment, even when this treatment had been prolonged from the perinatal period to 9 months of age. The mechanisms (induction and reversion) of this tolerance involves the physical elimination or the irreversible inactivation of the natural anti-IgG2a(b) T lymphocyte clones and their resurgence, from bone-marrow precursors, as long as the thymus remains operational, but not the establishment of a reversible, functional unresponsiveness (anergy) or an active, cell-mediated inhibition of anti-IgG2a(b) T clones. We attempted to elucidate, in Ighb mice, whether the natural T lymphocyte unresponsiveness to IgG2a(b) involved a central tolerance mechanism and to identify the type of tolerogen implicated in this tolerogenesis. The experiments principally showed that this natural T lymphocyte tolerance to IgG2a(b) was mediated by a thymic mechanism; that the capacity to induce it was gradually acquired by Ighb thymuses and was most probably due to potentially IgG2a(b)-producing/presenting cells, progressively colonizing the developing thymus; and that a significantly decreased postnatal Cy2a(b) gene transcription correlated with the emergence of anti-IgG2a(b) T lymphocytes in Igh(a/b) F1 (postnatally deprived of their B lymphocyte compartment), which subjected them to autoimmune IgG2a(b)-allotype suppression.

Central tolerance induction in natural immunoglobulin-allotype-specific T cells.

While self toleance is induced to IgG(b)(2a) in Igh(b / b) mice, an anti-IgG(b)(2a) T cell activity emerges in their Igh(a / a) congenic counterparts. This activity is revealed by postnatal transfer of Igh(a / a) T splenocytes into Igh(a / b) F(1), in which total suppression of IgG(2a)(b) expression is established. Here, we sought to determine whether the natural T cell unresponsiveness to IgG(2a)(b) in Igh(b / b) mice involved a central tolerance. Based on the kinetics of postnatal thymic C(gamma2a)(b) gene expression in Igh(b / b) mice, we transplanted thymi from Igh(b / b) donors of diverse ages into tolerogen-free Igh(a / a) nu / nu recipients. The state of T cell tolerance or responsiveness to IgG(2a)(b) in these reconstituted nu / nu hosts was determined by monitoring the capacity of their splenocytes to induce suppression in Igh(a / b) F(1). These experiments demonstrated that: (i) in the Igh(a / a) nu / nu recipients of adult Igh(b / b) thymi, 33 to 65 % T splenocytes were from nu / nu recipient origin, but these peripheral Igh(a / a) T cells were rendered tolerant to IgG(2a)(b) during their differentiation through the adult Igh(b / b) thymi, (ii) circulating IgG(2a)(b) was not a prerequisite for this tolerance induction, (iii) Igh(b / b) thymic epithelium was unable to induce tolerance to IgG(2a)(b) and (iv) IgG(2a)(b)-producing / presenting cells, colonizing the Igh(b / b) thymi, were certainly responsible of full tolerance induction to IgG(2a)(b).

Non-overlapping Fas- and BCL-2-regulated death pathways in IgG2a(b)-producing B cells.

Using perforin (Pfp)- and/or Fas-dependent cytotoxic pathways, T splenocytes from Igh(a/a) mice are able in vivo to totally and chronically eliminate congenic Igh(b/b) B cells committed to IgG2a(b) production. This phenomenon leads to a characteristic absence of serum IgG2a(b) expression (IgG2a(b) allotype suppression) in, for instance, histocompatible Igh(a/b) or Igh(b/b) mice, having neonatally received such T cells. Because the study of the protective role of BCL-2 oncoprotein against Fas-mediated cell death has generated contradictory findings, we examined the possible impact of constitutive overexpression of transgenic human BCL-2 protein in Igh(b/b) B cells when the latter were exposed in vivo exclusively with the Fas-dependent, anti-IgG2a(b) T cell activity of Igh(a/a) Pfp(0/0) mice. We observed that, despite high intracellular expression of functional transgenic BCL-2 and no up-regulation of the principal BCL-2 inhibitors in whole Igh(b/b) B cells, total, chronic and specific IgG2a(b) suppression was exerted by Igh(a/a) Pfp(0/0) cytotoxic T cells. These data show that, in this model of negative regulation of Ig production, Fas- and BCL-2-regulated mechanisms belong to non-overlapping death pathways at the level of IgG2a(b)-producing B cells, targets of Igh(a/a) T cell-mediated cytotoxicity. Thus, in these mature B cells, the Fas signaling-directly operating via caspase 8-does not involve a mitochondria-dependent pathway regulated by BCL-2.

Evidence of alternative or concomitant use of perforin- and Fas-dependent pathways in a T cell-mediated negative regulation of Ig production.

To study the possible involvement of perforin (Pfp)- and/or Fas-dependent cytotoxicity pathways in a T cell-mediated negative regulation of Ig production, we used the T cell-induced Ig-allotype suppression model. T splenocytes from Igha/a mice, when neonatally transferred into histocompatible Igha/b F1 or Ighb/b congenic hosts, are intrinsically able to totally, specifically, and chronically suppress the production of IgG2a of the Ighb haplotype (IgG2ab). It has not been established whether the suppression effectors, which are anti-IgG2ab MHC class I-restricted CD8+ T cells, cytolyse IgG2ab+ B targets or whether they only silence Ig production. In this study, using T cells from Igha/a Pfp+/+ or Pfpo/o mice, the latter obtained by crossbreeding, and B cells from Ighb/b Fas+/+ or Faslpr/lpr (lymphoproliferation) mice in appropriate adoptive transfer models, we demonstrated that: 1) under blockage of the Pfp-mediated pathway, Igha/a T cells were still able to induce suppression against wild-type IgG2ab+ B cells, 2) IgG2ab+ B cells with impaired Fas expression were also subjected to suppression by WT Igha/a T splenocytes, and 3) the suppression establishment was totally inhibited when both Pfp- and Fas-dependent mechanisms were simultaneously blocked, i.e., when Igha/a Pfpo/o T cells were used to induce suppression against Ighb/b Faslpr/lpr B cells. These results provide the first demonstration of the existence of alternative or simultaneous use of the major cytotoxic mechanisms in a T cell-mediated down-regulation of an Ig production.

Inhibition of IgG2ab production by Ig allotype-specific T cells can Be mediated without T-B cell contact.

The growth of IgG2ab-producing CB101 myeloma cells, subcutaneously or intraperitoneally inoculated into histocompatible BALB/c Igha mice sensitized against this Ig allotype, was delayed by 2-4 weeks compared to normal mice. While IgG2ab production was detected in the sera of 75-100% of normal mice, it was irreversibly inhibited in 100% of sensitized mice. IgG2ab suppression (IgG2ab sup) was also systematically obtained in sensitized but not normal recipients, implanted ip with a 0.1-micrometer-pore diffusion chamber (DC) containing CB101 cells. This time, the specific IgG2ab sup was reversible in vitro in the absence of anti-IgG2ab T cells. Adoptive transfer, of unfractionated or T but not B splenocytes from their sensitized counterparts into normal mice, 1 day before DC implantation, induced IgG2ab sup as well. These results indicate that, in these experimental circumstances, IgG2ab sup can also be mediated by diffusible suppressive factors produced by the effector T cells, without direct T-B-cell contact.

Breakdown of T cell tolerance to IgG2ab in Igha mice by de novo emerging anti-IgG2ab T cells and not anergy reversion.

The intrinsic T cell activity of Igha mice against IgG2ab (IgG2a from the Ighb haplotype) can be subjected to profound specific tolerance. In utero followed by post-natal exposure of Igha mice to soluble IgG2ab results in the loss of the capacity of their T splenocytes to induce specific and chronic IgG2ab allotype suppression in histocompatible Igha/b recipients. However, this full T cell tolerance has not been definitively acquired as it is spontaneously reversed when investigated 3-6 months after the end of the tolerogen treatment. Even when the IgG2ab tolerogen treatment was prolonged to 3, 6 or 9 months of age, T cell tolerance to IgG2ab vanished and the capacity of Igha T splenocytes to induce IgG2ab suppression in Igha/b recipients was systematically restored. The marked but partial thymus involution in 15-month-old Igha mice suggests the existence of some residual thymic output, capable of repopulating the anti-IgG2ab peripheral T pool subsequent to tolerogen clearance. In the present study, we showed that the mechanisms of this tolerance and its reversion involve, at the end of tolerogen treatment, the physical elimination or the irreversible inactivation of natural anti-IgG2ab T cell clones and their replacement, but neither the establishment of reversible anergy nor the recruitment of T cells which could actively maintain tolerance. The spontaneous breakdown of this T cell unresponsiveness was effectively prevented when de novo T cell maturation was inhibited by thymectomy at the end of tolerogen administration. Moreover, tolerance reversion did not occur in peripheral mature Igha T cells, parked in vivo, for up to 20 weeks in histocompatible tolerogen-free nu/nu mice.

The T/B cell interaction involved in induction of the mouse IgG2ab suppression is restricted by major histocompatibility complex class I, but not class II molecules.

To determine the major histocompatibility complex (MHC) restriction of the T/ B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2ab suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igha haplotype, when neonatally transferred into histocompatible Igha/b heterozygotes, are able to induce a specific and total suppression of the IgG2ab allotype. Nevertheless, only transfer of IgG2ab-primed Igha T splenocytes induces this suppression in Ighb/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8+ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4+CD8+ T cell cooperation and operated via the recognition by the involved TCR of C gamma 2ab-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Ighb mice genetically deficient for MHC class I (beta 2-microglobulin%, or beta 2m%) or class II (I-A beta%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2ab T cells transferred into Ighb H-2b I-A beta% mice carry out the suppression process normally, while in Ighb H-2b beta 2m% recipients, their suppression induction capacity is significantly inhibited. Moreover, the C gamma 2ab 103-118 peptide, identified as the sole C gamma 2ab-derived peptide able to amplify the anti-IgG2ab T cell reactivity in Igha H-2b mice, is also able to stabilize the H-2Db, but not the H-2Kb class I molecules at the surface of RMA-S (TAP2-, H-2b) cells. These results indicate that, despite the CD4+/CD8+ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2ab+ B cells for suppression establishment.

Spontaneous breakdown of T cell tolerance in the mouse IgG2ab-suppression model despite long-term tolerogenesis since the perinatal period.

T cell-induced IgG2ab allotype suppression provides a physiological model for the study of T cell responsiveness or tolerance to this Ig allotype. Normal, untreated mice of the Igha haplotype possess a basic and easily amplifiable T cell reactivity against the expression of IgG2ab, while their Ighb congenic mice produce substantial levels of this Ig and thereby are tolerant to this self-protein antigen. Therefore, the involved TCR repertoire in Igha and Ighb congenic mice is different. We have previously shown, in Ighb and Igha/b mice perinatally deprived of IgG2ab expression, that T lymphocytes bearing anti-IgG2ab TCR can emerge and induce an autoimmune suppression of IgG2ab. Correlatively, full and IgG2ab-specific T cell tolerance can be induced in Igha mice by their perinatal exposure to this Ig allotype. In this physiological model, which involves neither superantigens nor TCR-transgenic T cells, the responsive or tolerant state in Igha mice is assessed in vivo by the capacity to induce or not a T CD8(+)-dependent suppression of IgG2ab allotype production in Igha/b recipients of these cells. Taking advantage of this system, we were able to demonstrate here that, over the long term, this perinatally induced, IgG2ab-specific T cell tolerance was not definitively acquired, and that a spontaneous and total tolerance breakdown was observed by the age of 6 months. Furthermore, we showed that perinatal followed by prolonged tolerogen treatments up to 3, 6 and even 9 months of age were no longer sufficient to assure definitive T cell tolerance acquisition to IgG2ab, as the T cell suppression-induction capacity of Igha mice was partially and then entirely restored 3-6 months after the end of the tolerogen administration.

T cell-induced Ig allotypic suppression in mice. Basis for emergence or tolerization, during the perinatal period, of natural T cells specific to the IgG2ab allotype.

We have previously described an anti-IgG2ab T cell activity in normal Igha/a mice. Their congenic partner at the Igh locus (Ighb/b) and Igha/b hybrids bred from them, do not display this T cell activity but express IgG2ab. As these mice are supposed to possess the same genetic elements related to this potential T cell repertoire, only somatic selection mechanisms could be responsible for their different behavior. In this study, we investigated the basis for the emergence (in Igha/a mice) or tolerization (in Ighb/b-congenic mice and in Igha/b hybrids) of these natural anti-IgG2ab T cells. Stringent perinatal B cell deprivation in Ighb/b and Igha/b mice resulted in the emergence of anti-IgG2ab T cells, as these individuals could be subjected to autoimmune, T cell-mediated IgG2ab suppression. Furthermore, the acquisition of anti-IgG2ab T cell activity was drastically reduced in Igha/a mice, perinatally exposed to IgG2ab; thus, the presence of this allotype leads to tolerization of these specific T cells.

Identification of T cell peptides from C gamma 2ab involved in IgG2ab T cell-induced allotypic suppression. Relationship with the MHC haplotype of the suppressor T cell-donor Igha mice.

Using the algorithm of the peptidic self-model we selected, from the constant part of the IgG2ab polypeptidic heavy chain, four peptides susceptible to being recognized by the T cells of Igha mice that prevent IgG2ab expression. Among these four peptides, two were capable of enhancing in vivo this T cell activity of Igha mice almost as well as the whole IgG2ab allotype. One of these peptides, C gamma 2ab-248-263, located in the CH3 domain of IgG2ab, was effective in H-2d mice, while the other one, C gamma 2ab-103-118, located in the hinge region of IgG2ab, was efficient in H-2b mice. This demonstrates for the first time that this suppression involves the TCR of the effector cells and peptides derived from the allotype presented in the context of MHC molecules of the target cells, the presentation specificity being dependent on the MHC haplotype. The availability of these peptides will allow us to further understand the role of class I and/or class II MHC molecules in this suppression.

Role of CD4+ and CD8+ cell subsets during amplification of natural T cell activity against IgG2ab in Igha mice and during induction of IgG2ab allotype suppression in Igha/b mice.

Transfer into F1 Igha/b mice of splenocytes from Igha mice sensitized once against B cells from an Ighb congenic strain induces total, chronic, and IgG2ab (IgG2a of the Ighb haplotype)-specific allotype suppression in these recipients. We previously demonstrated that both the CD4+ and CD8+ T subsets were necessary for inducing suppression, but that CD8+, cells by themselves were sufficient for maintaining suppression. We have studied the suppression induction capacity of different mixtures of CD4+ and CD8+ subsets obtained by in vitro cytotoxic treatment of T splenocytes from normal or sensitized Igha mice, and we have established that suppression induction requires the cooperation between CD4+ and CD8+ populations, both of which have to be IgG2ab specific. In addition, Igha mice were sensitized in the absence of CD4+ or CD8+ cells by in vivo cytotoxic treatment performed before and after the sensitization in order to obtain an IgG2ab-specific CD4+ population that has arisen in the absence of CD8+ cells, and vice versa. We found that only IgG2ab-specific CD4+ cells from anti-CD8-treated mice (T'sens CD4+) had the ability to induce suppression in F1 Igha/b hosts. Nevertheless, the real effector cells in this suppression model display the CD8+ phenotype, as in vivo cytotoxic anti-CD8 treatment of Igha/b recipients of T'sens CD4+ abrogates the suppression induction capacity. Taken together, these results show that T'sens CD4+ have an important capacity to recruit CD8+ anti-IgG2ab effector cells from precursors that have been transferred with them into Igha/b hosts. These precursors are actually derived from the T'sens CD4+ cell preparation, because we have recently demonstrated that suppression is maintained by donor T cells throughout the recipient's life. CD4+ cells can have their anti-IgG2ab activity amplified only by means of target cells (i.e., B cells from Ighb congenic mice), whereas, in the absence of CD4+ cells, and despite the presence of target cells, CD8+ cells seem unable to acquire this amplified activity.

T cell-induced allotypic suppression. Origin of the CD8+ T cells maintaining IgG2ab suppression.

One of the problems raised by the T cell-induced allotypic suppression is the origin (donor or host) of the T cells responsible for the chronicity of the suppression. To address this point, we used T cells from Igha/a Thy-1.2 mice whose natural T cell activity against IgG2ab was enhanced in vivo. These T cells were injected into newborn Ighb/b Thy-1.1 mice where they induced complete suppression of IgG2ab expression in around 70% of these recipients. During a study that lasted more than 1 yr, we found that about 3% of the recipient splenocytes were T cells of the donor type. By means of suppression-transfer experiments, using either Thy-1.2+ or Thy-1.1+ cell-depleted splenocytes from mice suppressed in this manner we were able to unambiguously show that Thy-1.2+ cell-depleted splenocytes were incapable of transferring the suppression, whereas Thy-1.1+ cell-depleted splenocytes could. We thus demonstrated that suppression was maintained throughout the recipient's life by donor Thy-1.2+ T cells.