Analyses of H-2 restriction specificity of helper T cells in fully allogeneic bone marrow chimera in mice

Using irradiation bone marrow chimeras to analyze restriction specificity of helper T cells, we found that recipient H-2 type dictated the H-2 type which the T cells recognize as self (adaptive differentiation). T cells from (H-2b----H-2k) chimeras cooperate with non-T cells bearing Iak to generate a vigorous PFC response to sheep erythrocytes (SRBC) in vitro, but not with genetically identical H-2b cells. However, when T cells from the chimeras and H-2b non-T cells were adoptively transferred into irradiated (donor X recipient) F1 mice with SRBC, marked responses were seen in recipient spleens where radio-resistant F1 macrophages might exist and act as antigen presenting cells (APC). From these in vitro and in vivo observations, we considered that in the primary antibody response to a T dependent antigen such as SRBC, only T cell-macrophage (APC) matching is required. In contrast, when T cells from H-2 incompatible chimeras which had been primed with SRBC in vivo were analyzed in vitro, these cells cooperated also with H-2b non-T cells. These findings indicate that there may be two separate stages of T cell differentiation during which the self restriction specificity is acquired: one appears to be responsive to intrathymic influences and is not associated with antigenic stimuli, and the other shows signs of being responsive to post-thymic stimuli and of involving antigenic presentation. Moreover, the latter appears to utilize the influence of donor type macrophages.     

Analyses of Ia restriction specificity of helper T cells in H-2 subregion compatible bone marrow chimera in mice

Using irradiation bone marrow chimeras which had partial compatibility in H-2 subregions between donor and recipient mice, we found that H-2I matching was sufficient for the chimeras to generate anti-sheep erythrocyte plaque-forming cell (PFC) responses. In such chimeras, T cells appeared to encounter appropriate partner cells bearing the same Ia antigens as those which they had learned to recognize as self in the recipient micro-environment. Furthermore, the PFC number seen in I-A compatible chimeras was only about half of that seen in I-A, I-E compatible chimeras, suggesting the existence of two independent subpopulations of helper T cells. When incompatibility of donor and recipient mice existed on the left side of the H-2I region, the responses were very weak. However, even in such chimeras, marked responses were observed for both IgM and IgG type PFC following a sufficient period after immunization. This observation appears to indicate the existence of a minor subpopulation of helper T cells which can expand and interact effectively with antigen presenting cells of donor type.     

H-2-controlled, dose-dependent suppression of the response that expels adult Trichinella spiralis from the small intestine of mice.

H-2 congenic strains of mice expressing the H-2k, H-2q or H-2f haplotype were tested for their ability to expel Trichinella spiralis from the gut following infection with either 100, 150, 200, 400, 500, or 600 L1 infective larvae. H-2q and H-2f mice expelled worms more quickly than H-2k mice when 100-200 L1 larvae were given, but this H-2-controlled effect was much reduced when mice received 400 L1 larvae, and completely eliminated when 500 or 600 L1 larvae were given. The observed dose-dependent delay in the expulsion response was paralleled by a concurrent suppression of lymphocyte responsiveness. Lymphocytes from H-2q mice infected with 100-200 L1 larvae incorporated more [3H]thymidine than did cells from H-2k mice. However, this H-2-controlled difference was not apparent in cells from mice receiving 400-600 L1 larvae. The strongest proliferation response in each case was associated with mice infected with the smallest number of worms. Since strains of mice expressing H-2q or H-2f alleles were suppressed at high doses to a much greater extent than were mice expressing H-2k, H-2 genes must influence this dose-dependent response. Many earlier studies, which failed to demonstrate marked H-2 effects on immunity to T. spiralis, employed infective doses which are shown here to be preferentially suppressive to otherwise resistant strains of mice.     

Experimental myasthenia gravis in congenic mice. Sequence mapping and H-2 restriction of T helper epitopes on the alpha subunits of Torpedo californica and murine acetylcholine receptors.

Immunization of mice with nicotinic acetylcholine receptor from Torpedo electric organ (TAChR) causes a disease similar to human myasthenia gravis (experimental autoimmune myasthenia gravis, EAMG). Susceptibility to EAMG correlates with the H-2 haplotype. In this study we used overlapping synthetic peptide corresponding to the complete sequences of the alpha subunits from TAChR and murine muscle AChR (MAChR) to map T helper epitopes in congenic murine strains of different H-2 haplotype. C57BL/6 and BALB/B mice (highly susceptible to EAMG) and BALB/c and CB17 mice (less susceptible to EAMG), immunized with TAChR, developed similar anti-TAChR antibody titers and L3T4+ (T helper) cell sensitization. Different sequence segments of the TAChR alpha subunit were recognized by L3T4+ cells from strains of H-2b and H-2d haplotype. The sequence segments recognized by the H-2d strains have the highest predicted propensity to form amphipatic alpha helices, while those recognized by the H-2b strains do not. We investigated whether in EAMG T helper cells cross-react with autologous AChR sequences, and a true break of the tolerance occurs. Overlapping synthetic peptides, corresponding to the complete sequence of MAChR alpha subunit, were used to test L3T4+ cell from mice immunized with TAChR. L3T4+ cell strains did not cross-react with any murine peptide sequence, while L3T4+ cells from H-2d mice were strongly stimulated by the peptide sequence Ma alpha 304-322, which is very similar to the homologous Torpedo peptide.     

Restriction in the function of single helper T cells.

T cells involved in specific and nonspecific co-operation belong to a long-lived population. However, under conditions of limiting dilution of T cells it is not a common event to detect both types of co-operative event in a single microculture well. Likewise, as far as specific co-operation is concerned, the simultaneous expression of both direct and indirect PFC responses in a single microculture well is also unusual. Estimates obtained of the number of PFC generated by one T cell in specific co-operation correspond well to those generated by a single B cell; and it would seem that under conditions of the microculture system there is a marked restriction in the number of B cells that a single T cell may interact with in the short term. This restriction seems to be of a one T-cell to one B-cell relationship.     

T helper cells,IL-2 and the generation of cytotoxic T-cell responses

CD8 T-cell immunity is thought to require helper activity derived from CD4 T cells. Nevertheless, under some circumstances, effective CD8-dependent T-cell responses occur in vivo without CD4 T-cell help. Several recent papers help to explain this paradox and lead to a refined view concerning the role of T helper cells and interleukin-2 receptor signaling in the production of cytotoxic T lymphocytes.     

In vitro studies on H-2 linked unresponsiveness. 1. Normal helper cells to (T,G)-A-L and GAT in low and non-responder mice.

Lymphoid cells from unprimed high responder (C57BL/10) and low responder mice (B10.Br, B10.A, CBA) to (T,G)-A-L and high responder (B10, B10.A) and non-responder (B10.G, DBA/I) mice to GAT can be induced to form antigen specific T-helper cells in vitro under identical culture conditions. The helper cells induced from high and low or non-responder mice appear to be identical in efficiency, antigen concentration requirement for induction and induction kinetics.     

Effect of carrier-specific tolerance on the generation of helper cell function

The effect of carrier-specific tolerance on the development of helper cell function was studied in irradiated rats reconstituted with normal or carrier-tolerant bone marrow. Bone marrow (BM) from normal rats or rats tolerant to sheep IgG (SGG) was transferred to lethally irradiated syngeneic recipients, which were challenged 3 to 5 weeks later when immunocompetence to T-dependent antigens was shown to have recovered. When recipients were challenged with the 2,4,6-trinitrophenyl (TNP) hapten coupled to SGG in adjuvant, groups which received SGG-tolerant BM were 70–80 % unresponsive in terms of anti-TNP plaque-forming cells compared to recipients of normal BM. This effect was carrier (SGG)-specific and was reversed when normal thymocytes were transferred with tolerant BM. Moreover, neither tolerant BM nor tolerant thymocytes were able to suppress the responsiveness of normal BM at a 1:1 ratio. These cell-mixing experiments imply that the reduction in helper cell function is due neither to suppressor cells nor antigen carryover in the tolerant BM. It is suggested that a BM precursor of the helper T cell may be rendered tolerant and therefore already possesses antigen specificity prior to thymic migration.     

Plasmodium falciparum-specific human T cell clones: evidence for helper and cytotoxic activities

This report describes the isolation, and the phenotypic and functional characterization of Plasmodium falciparum-specific human T lymphocyte clones (TLC) obtained from 2 acutely infected and 4 clinically immune donors. Approximately one third of the TLC obtained from the acutely infected patients had the phenotype CD8+/CD4-. No such clones were obtained from the clinically immune donors. P. falciparum-specific, major histocompatibility complex-restricted CD8+ clones can lyse unrelated tumor cell targets in the presence of anti-CD3 antibodies, suggesting that these clones have cytotoxic potential. Conversely no killing was obtained with two CD4+ P. falciparum-specific TLC, even in the presence of anti-CD3 antibody, In addition the helper function of a CD4+ clone was demonstrated in a T/B cell cooperation system.     

Lack of IL-4 receptor expression on T helper cells reduces T helper 2 cell polyfunctionality and confers resistance in allergic bronchopulmonary mycosis

T helper (Th)1 and Th2 cells play decisive roles in the regulation of resistance vs. susceptibility to pulmonary cryptococcosis. To study the function of interleukin (IL)-4 receptor (IL-4R) on Th cells in pulmonary cryptococcosis, we infected mice specifically lacking IL-4Rα on CD4(+) T cells (Lck(Cre)IL-4Rα(-/lox) mice) and IL-4Rα(-/lox) controls. Lck(Cre)IL-4Rα(-/lox) mice developed enhanced resistance accompanied by reduced pulmonary allergic inflammation and diminished production of the Th2 cytokines IL-4, IL-5, and IL-13 as compared with IL-4Rα(-/lox) mice. Polyfunctional antigen-specific Th2 cells producing simultaneously two or three Th2 cytokines were reduced in infected Lck(Cre)IL-4Rα(-/lox) mice, pointing to a critical role of polyfunctional Th2 cells for disease progression. Reduced Th2 polyfunctionality was associated with fewer pulmonary alternatively activated macrophages. This work is the first direct evidence for a critical contribution of the IL-4R on Th cells to Th2-dependent susceptibility during allergic bronchopulmonary mycosis. Moreover, the data demonstrate that the quality of the Th2 response has an impact on type 2 inflammation. The analysis of polyfunctional Th2 cells may be useful for monitoring the course of the disease.     

The role of macrophages in the generation of T helper cells. III. Influence of macrophage-derived factors in helper cell induction

Helper cell induction to soluble or particulate antigens in vitro requires the cooperation of T cells and macrophages. A direct contact between macrophages and T cells is not obligatory for this cooperation and factors released from macrophages are as effective in activating T cells as the cells themselves. Two different types of macrophage-derived factors where found. The supernatant obtained from purified macrophages incubated with antigen for several days generates helper cells in absence of macrophages or additional antigen, but only if obtained from macrophages which were identical at the I-A subregion of the H-2 complex as the T cells. This factor was called genetically related macrophage factor (GRF). The other factor(s), which is present in the supernatant obtained from macrophages incubated for several days without antigen, replaces macrophages only if the antigen is particulate. This factor(s), called nonspecific macrophage factor (NMF) is not restricted genetically and is also obtained from allogeneic macrophages. The importance of both these factors in helper cell induction is discussed.     

Prenatal exposure to bisphenol A up-regulates immune responses, including T helper 1 and T helper 2 responses, in mice

The effect of prenatal exposure to bisphenol A (BPA) on the immune system in mice was investigated. Virgin female mice were fed varying doses of BPA, on a daily basis, over a period of 18 days commencing on the day of pairing with stud males (day 0). On day 77, their male offspring of 8 weeks were immunized with hen egg lysozyme (HEL). Three weeks later, anti-HEL immunoglobulin G (IgG) in sera, and proliferative responses of spleen cells to the antigen, were measured. Anti-HEL IgG2a and interferon-gamma (IFN-gamma), secreted from splenic lymphocytes, were measured as indicators of T helper 1 (Th1) immune responses, while anti-HEL IgG1 and interleukin-4 (IL-4) were measured as indicators of Th2 responses. The results showed that fetal exposure to BPA was followed by significant increases in anti-HEL IgG as well as antigen-specific cell proliferation. Both Th1 responses (including anti-HEL IgG2a and IFN-gamma production) and Th2 responses (including anti-HEL IgG1 and IL-4 production) were augmented by prenatal exposure to BPA, although the augmentation of Th1 responses appeared to be greater than that of Th2 responses. Two-colour flow cytometric analysis showed that mice exposed prenatally to BPA had 29% and 100% more splenic CD3(+) CD4(+) and CD3(+) CD8(+) cells, respectively, than control animals. Similar results were obtained from females whose mothers had consumed BPA during pregnancy. These results suggest that prenatal exposure to BPA may result in the up-regulation of immune responses, especially Th1 responses, in adulthood.     

Absence of an antigen-specific helper T cell required for the expression of the T 15 idiotype in mice treated with anti-mu antibody

T-dependent anti-phosphorylcholine (PC) plaque-forming cell (PFC) responses were studied in BALB/c mice. Helper T cells derived from normal, carrier-primed donors induced anti-PC PFC responses dominated by the T15 idiotype. Helper T cells derived from carrier-primed mice that had been treated from birth with anti-μ antibody, so that they were lacking B cells and circulating antibody bearing the T15 idiotype, also provided helper cell function for the anti-PC antibody response. In contrast to the response induced by helper T cells from normal mice, T helper cells from μ-suppressed mice induced an anti-PC antibody response which was mainly non- T15 in character. The failure to induce antibody formation by T15⁺ B cells was not due to suppressor cells but rather to the lack of an Lyt-1⁺ helper T cell population which is necessary for predominant T15 production. This latter cell population was shown to be present in carrier-primed normal but missing or diminished in carrier- primed anti-μ-treated BALB/c mice. It required carrier priming for the expression of its helper function, but its function did not require the antigen (carrier) to be physically linked to the hapten. From this, we conclude that dominant production of the T15 idiotype involves the synergistic activity of two antigen-specific helper T cells. The helper cell population which is required for predominant T15 production is greatly diminished in mice treated with anti-μ antibody from birth. Hence, the production of circulating T15⁺ antibody induced by environmental antigens or the appearance of T 15-bearing B cells would appear to be required for the induction of helper T cells which enhance T15⁺ anti-PC antibody synthesis.     

Abundance of H-2 promiscuous T cells specific for mycobacterial determinants in H-2b/d F1 hybrid mice

A majority of immunodominant epitopes of mycobacterial antigens are known to be recognized by murine T cells in the context of several H-2 haplotypes. In this study, we established the frequency of T cells able to recognize these peptides promiscuously, i.e. in the context of allogeneic antigen-presenting cells, using hybridomas from peptide-immunized H-2 homologous and heterologous mice. The degree of promiscuity in homozygous mice varied between 4–27% between different specificities and genetic backgrounds. In particular, the results showed that promiscuity between A^b and A^d in respect to a peptide from the Mycobacterium tuberculosis 38-kDa protein (residues 350–369) was displayed by 22% of BALB/c and 4% of C57BL/10-derived hybrids, but by 42% of [BALB/c x C57BL/10] F1-derived clones. This represents a significant increase (p < 0.001) of T cell promiscuity compared to the parental haplotypes. It is noteworthy that considerably lower peptide concentrations were able to stimulate the promiscuous hybridomas compared to the H-2-restricted hybrids. This finding suggests a functional advantage of promiscuous T cells which enables them to expand preferentially in the initial stages of infections with M. tuberculosis and thus enables the host to mount a rapid protective immune response.     

Limiting dilution analysis of helper T-cell function. II. An approach to the study of the function of single helper T cells.

Procedures have been described which are suitable for experiments utilizing a microculture system whereby one can easily study the function of single helper T cells. These are: (a) the elimination of the background anti-hapten response by preliminary anti-theta + guinea-pig complement complement treatment of the 'hapten-primed' population; (b) the removal of any contaminating hapten-specific B cells from the carrier-primed population by passage through nylon wool; (c) irradiation of long-term primed 'nylon wool' enriched helper cells tp prevent cell division; (d) the partition of individual helper cells by limiting dilution.     

Effect of cyclosporin A on T cell immunity. II. Defective thymic education of CD4 T helper cell function in cyclosporin A-treated mice

Cyclosporin A (CsA) is an immunosuppressive agent that is widely used in transplantation. Recent animal studies indicate that CsA can affect the development of immunity so that autoreactive T lymphocytes are generated. In this study, mice were treated with CsA prior to irradiation and transplantation of syngeneic bone marrow to determine whether CsA pretreatment would affect the ability of the bone marrow recipients to develop normal T cell function. Our results indicate that (a) thymuses of CsA-treated mice do not contain single-positive thymocytes (i.e. L3T4+Ly-2- or L3T4-Ly-2+) during i.p. treatment with 15 mg/kg/day of CsA; (b) both populations of single-positive thymocytes reappear within 2 weeks of termination of CsA and (c) irradiation and bone marrow reconstitution of these CsA-treated mice results in reconstitution of normal numbers of L3T4+ and Ly-2+ cells, but the L3T4+ T cells to not provide T helper function, as determined by interleukin 2 production and cytotoxic T lymphocytes generation. These findings indicate that CsA can affect thymic microenvironment and may be important as a model for investigating intrathymic T cell maturation. Our results may also have clinical implications for T lymphocyte development in transplant patients receiving CsA.     

A fundamental role for interleukin-21 in the generation of T follicular helper cells

T cell help to B cells is a fundamental property of adaptive immunity, yet only recently have many of the cellular and molecular mechanisms of T cell help emerged. T follicular helper (Tfh) cells are the CD4(+) T helper cells that provide cognate help to B cells for high-affinity antibody production in germinal centers (GC). Tfh cells produce interleukin-21 (IL-21), and we show that IL-21 was necessary for GC formation. However, the central role of IL-21 in GC formation reflected its effects on Tfh cell generation rather than on B cells. Expression of the inducible costimulator (ICOS) was necessary for optimal production of IL-21, indicative of interplay between these two Tfh cell-expressed molecules. Finally, we demonstrate that IL-21's costimulatory capacity for T helper cell differentiation operated at the level of the T cell receptor signalosome through Vav1, a signaling molecule that controls T cell helper function. This study reveals a previously unappreciated role for Tfh cells in the formation of the GC and isotype switching through a CD4(+) T cell-intrinsic requirement for IL-21.