T cell mediated immune responses to Toxoplasma gondii in pregnant women with primary toxoplasmosis

The aim of this study was to investigate T cell immunity to Toxoplasma gondii (Tg) in pregnant women with primary toxoplasmosis. This issue has never been addressed before in humans and available information derives from murine models. Peripheral blood mononuclear cells (PBMC) from pregnant women with primary Tg infection were stimulated with Tg tachyzoites, excretory-secretory antigens (ESA) or recombinant surface antigen-1 (rSAG-1), and tested for proliferation, immunophenotype, cytokine production and antigen specific cytotoxic activity. Pregnant women with primary toxoplasmosis displayed a significant decrease of the CD4/CD8 T cell ratio and a significant increase of circulating T cell receptor (TCR) gammadelta+ cells as compared to their uninfected counterparts. T cells from Tg infected pregnant women proliferated to Tg tachyzoites, ESA or rSAG-1. Most tachyzoite and ESA specific T cell blasts were CD4+, whereas SAG-1 specific blasts were CD4+ and CD8+. ESA and tachyzoite specific T cell blasts displayed a Th1 or Th0 cytokine profile with overexpression of IFN-gamma. This pattern was unchanged upon in vitro exposure of T cells to progesterone, tested at a concentration close to that reached in vivo at the maternal-fetal interface. Finally, tachyzoite or ESA specific T cell blasts lysed, through a granule exocytosis dependent mechanism, autologous lymphoblastoid cell lines presenting Tg antigens. In conclusion, pregnant women with primary toxoplasmosis mounted in vitro Tg-specific Th1/Th0 responses whose impact on neonatal infection warrants further investigation.     

T cell clones for antigen selection and lymphokine production in murine Schistosomiasis mansoni.

The role of T cells in immunity to murine schistosomiasis was examined through the use of T cell clones that recognize the live schistosomulum stage of Schistosoma mansoni. T cell clones of three different phenotypes were isolated and expanded into long term culture from lymph nodes of C57B1/6J mice vaccinated with irradiated S. mansoni larvae. They were characterized by surface markers, lymphokine production, and functional assays. The m.w. range of the Ag recognized by one clone was identified through nitrocellulose blotting and confirmed with a preparation of the putative protein made by immunoaffinity purification. All but one of the clones were CD4+, CD5+, Th cells. One clone, 35, produced Il-2 and IFN-gamma and was designated a TH1 clone. The others were designated TH2 clones because of Il-4 production. One clone was CD8+ and failed to show help. Clone 35 recognized live schistosomula and produced Il-2 when presented a 27-kDa protein from nitrocellulose. It proliferated in response to purified Ag. Clone 35 participated along with macrophages to induce up to 98% killing of live schistosomula in vitro. IFN-gamma and TNF-alpha were essential to the killing mechanism whereas Il-1, Il-2, and Il-4 were not required. This study has approached Ag identification for vaccine development from the point of view of T cells and showed that TH1 cells are essential to in vitro macrophage killing of schistosomula in murine schistosomiasis.     

Isolation of protective T cells from BALB/cJ mice chronically infected with Leishmania donovani

BALB/cJ mice, which are homozygous for Lshs on chromosone 1, are genetically susceptible to Leishmania donovani (S3), but spontaneously reduce their parasite burdens late in the course of infection. Spleens from chronically infected mice (5 to 8 mo) were found to have T cells that responded to leishmanial Ag by proliferating and secreting immune IFN. An IFN-secreting T cell line derived from the spleen of a chronically infected mouse, after boosting with leishmanial Ag and treatment with Pentostam to kill residual parasites, was able to activate macrophages to kill amastigotes in vitro. Furthermore, after adoptive transfer of this cell line, naive BALB/cJ mice challenged with amastigotes demonstrated a 42-fold reduction in parasite burden compared to controls. Four of five clones derived from the protective T cell line secreted IFN and proliferated between days 7 and 14 after Ag stimulation. All clones were Ly1+2-, L3T4+. Another T cell line, which had Ly1+2-, L3T4+ phenotype, was derived from the lymph nodes of s.c. immunized, uninfected mice. It multipled but never produced IFN in response to leishmanial Ag and failed to protect macrophages against L. donovani infection in vitro or in vivo. This nonprotective T cell line and the fifth clone from the protective line, which also did not secrete IFN, proliferated between days 2 and 7 after Ag stimulation. In summary, leishmania-responsive helper/inducer T cells, which produced IFN but were slow to proliferate in response to Ag in vitro and which were able to activate macrophages to reduce amastigotes in vitro and in vivo, are present in chronically infected BALB/cJ mice and may mediate the decrease in parasite burden during chronic infection.     

Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.     

A systematic molecular analysis of the T cell-stimulating antigens from Mycobacterium leprae with T cell clones of leprosy patients. Identification of a novel M. leprae HSP 70 fragment by M. leprae-specific T cells

Both protective immunity and immunopathology induced by mycobacteria are dependent on Ag-specific, CD4+ MHC class II-restricted T lymphocytes. The identification of Ag recognized by T cells is fundamental to the understanding of protective and pathologic immunity as well as to the design of effective immunoprophylaxis and immunotherapy strategies. Although some T cell clones are known to respond to recombinant mycobacterial heat shock proteins (hsp) like hsp3 65, the specificity of most T cells has remained unknown. We therefore have undertaken a specificity analysis of 48 well defined Mycobacterium leprae- and/or Mycobacterium tuberculosis-reactive (Th-1-like) T cell clones. Most clones (n = 44) were derived from different leprosy patients, and the remainder from one healthy control. Their HLA restriction molecules were DR2, DR3, DR4, DR5, DR7, DQ, or DP. T cell clones were stimulated with large numbers (n = 20 to 40) of mycobacterial SDS-PAGE-separated fractions bound to nitrocellulose. Each clone recognized a single fraction or peak with a particular Mr range. Some of the clones (n = 7) recognized the fraction that contained the hsp 65 as confirmed with the recombinant Ag. Most clones (n = 41), however, responded to Ag other than the hsp 65. Nine clones responded to a 67- to 80-kDa fraction. Five of them responded also to an ATP-purified, 70-kDa M. leprae protein, but only one of these five (that was HLA-DR2 restricted and cross-reactive with M. tuberculosis) recognized the recombinant C-terminal half (amino acids 278-621) of the M. leprae hsp 70 molecule and also recognized the recombinant M. tuberculosis hsp 70. We therefore have used the 5' part of the M. leprae hsp 70 gene that we have cloned recently. This fragment (that encodes amino acids 6-279) was indeed recognized by the other four M. leprae-specific T cells that were all HLA-DR3 restricted and did not cross-react with the highly homologous (95%) M. tuberculosis hsp 70. These results suggest that this novel fragment is a relevant T cell-stimulating Ag for leprosy patients. A panel of other recombinant Ag, including hsp 18 was tested. The majority of T cell clones appeared to recognize antigenic fractions distinct from hsp. In conclusion, T cells of leprosy patients see a large variety of different Ag including non-hsp, and one newly recognized moiety is the N-terminal M. leprae hsp 70 fragment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thymocyte antigens do not induce tolerance in the CD4+ T cell compartment.

Thymocytes fail to tolerize the developing T cell repertoire to self MHC class I (MHC I) Ags because transgenic (CD2Kb) mice expressing H-2Kb solely in lymphoid cell lineages reject skin grafts mismatched only for H-2Kb. In this study, we examined why thymocytes fail to tolerize the T cell repertoire to self MHC I Ags. The ability of CD2Kb mice to reject H-2Kb skin grafts was age dependent because CD2Kb mice older than 20 wk accepted skin grafts. T cells from younger CD2Kb mice proliferated, but did not develop cytotoxic functions in vitro in response to H-2Kb. Proliferative responses were dominated by H-2Kb-specific, CD4+ T cells rather than CD8+ T cells. Representative CD4+ T cell clones from CD2Kb mice were MHC II restricted and recognized processed H-2Kb. TCR transgenic mice were generated from one CD4+ T cell clone (361) to monitor development of H-2Kb-specific immature thymocytes when all thymic cells or lymphoid cell lineages only expressed H-2Kb. Thymocyte precursors were not eliminated and mice were not tolerant to H-2Kb when Tg361 TCR transgenic mice were intercrossed with CD2Kb mice. In contrast, all thymocyte precursors were eliminated efficiently in thymic microenvironments in which all cells expressed H-2Kb. We conclude that self MHC I Ags expressed exclusively in thymocytes do not induce T cell tolerance because presentation of processed self MHC I Ags on self MHC II molecules fails to induce negative selection of CD4+ T cell precursors. This suggests that some self Ags are effectively compartmentalized and cannot induce self-tolerance in the T cell repertoire.     

The induction and kinetics of antigen-specific CD8 T cells are defined by the stage specificity and compartmentalization of the antigen in murine toxoplasmosis

Toxoplasma gondii forms different life stages, fast-replicating tachyzoites and slow-growing bradyzoites, in mammalian hosts. CD8 T cells are of crucial importance in toxoplasmosis, but it is unknown which parasite stage is recognized by CD8 T cells. To analyze stage-specific CD8 T cell responses, we generated various recombinant Toxoplasma gondii expressing the heterologous Ag beta-galactosidase (beta-gal) and studied whether 1) secreted or cytoplasmic Ags and 2) tachyzoites or bradyzoites, which persist intracerebrally, induce CD8 T cells. We monitored the frequencies and kinetics of beta-gal-specific CD8 T cells in infected mice by MHC class I tetramer staining. Upon oral infection of B6C (H-2(bxd)) mice, only beta-gal-secreting tachyzoites induced beta-gal-specific CD8 T cells. However, upon secondary infection of mice that had received a primary infection with tachyzoites secreting beta-gal, beta-gal-secreting tachyzoites and bradyzoites transiently increased the frequency of intracerebral beta-gal-specific CD8 T cells. Frequencies of splenic and cerebral beta-gal-specific CD8 T cells peaked at day 23 after infection, thereafter persisting at high levels in the brain but declining in the spleen. Splenic and cerebral beta-gal-specific CD8 T cells produced IFN-gamma and were cytolytic upon specific restimulation. Thus, compartmentalization and stage specificity of an Ag determine the induction of CD8 T cells in toxoplasmosis.     

Theileria parva: CD4+ helper and cytotoxic T-cell clones react with a schizont-derived antigen associated with the surface of Theileria parva-infected lymphocytes.

Theileria parva is a protozoan parasite which infects and transforms bovine lymphocytes, resulting in a fatal lymphoproliferative disease. There is evidence that immunity to the intralymphocytic schizont stage is mediated by T cells. We have previously reported derivation of CD4+ T-cell clones which recognize parasite-derived antigens presented on the surface of infected cells in conjunction with MHC molecules and partial characterization of the antigens. The present study further evaluated one of these antigens, demonstrating that it could be derived from cells infected with different parasite stocks as well as from purified theilerial schizonts and that it was recognized by primed, but not unprimed, bovine lymphocytes including cytolytic CD4+ T cells. Using a cloned CD4+ cytolytic cell line, lysis of schizont-infected cells was shown to be MHC-restricted but not parasite-strain restricted. In addition we demonstrated that T cells which respond to the HSS antigen preparation were generated in cattle immunized with parasites from any of the three subspecies of T. parva. The antigenic material was fractionated by sequential subjection to anion-exchange chromatography, hydroxylapatite chromatography, and gel filtration using HPLC, which resulted in recovery of approximately 20% of the antigenic material with more than 10(6)-fold purification in selected fractions. To assess the molecular size of the proteins in the highly purified antigenic fractions, the T. parva-infected lymphocytes were metabolically labeled before fractionation with 3H-amino acids and the material was analyzed by SDS-polyacrylamide gel electrophoresis and liquid scintillation counting of gel slices. The major protein in these fractions had a molecular mass of 9-10 kDa.     

The kidney-renal lymph node-system contributes to cross-tolerance against innocuous circulating antigen

Soluble Ags devoid of inflammatory stimuli, derived for example from self-serum or food proteins, induce T cell tolerance, predominantly in the spleen. In this study, we describe an additional role of the kidney-renal LN (rLN) system in tolerogenic presentation of circulating soluble Ags. Protein below albumin molecular mass constitutively passed the kidney glomerular filter and was concentrated in the tubular compartment. Enriched filterable Ag was endocytosed by kidney dendritic cells (kDCs). Simultaneously, it was transported cell independently within 2 min to DCs resident in rLNs. These DC phenotypically differed from kDCs carrying filterable Ag, and used a distinct mechanism, mannose receptor-mediated endocytosis, to internalize Ag. They activated specific CD8+ T cells, which subsequently proliferated without producing effector cytokines or developing cytotoxic activity, showed a curtailed lifespan and signs of apoptosis. Such T cell tolerization was independent of steady-state migratory kDC, because it occurred also when nephrectomy was performed soon after Ag injection. These findings demonstrate that the kidney dispatches concentrated blood-borne Ags to the rLNs, where they are captured by resident DCs, resulting in CD8+ T cell cross-tolerance. This mechanism may contribute to avoiding immunity against innocuous circulating protein Ags below albumin size.     

Functional maturation of CD4+CD25+CTLA4+CD45RA+ T regulatory cells in human neonatal T cell responses to environmental antigens/allergens

A number of laboratories have reported cord blood T cell responses to ubiquitous environmental Ags, including allergens, by proliferation and cytokine secretion. Moreover, the magnitude of these responses has been linked with risk for subsequent expression of allergy. These findings have been widely interpreted as evidence for transplacental priming and the development of fetal T memory cells against Ags present in the maternal environment. However, we present findings below that suggest that neonatal T cell responses to allergens (and other Ags) differ markedly from those occurring in later life. Notably, in contrast to allergen-responsive adult CD4(+) T cell cultures, responding neonatal T cell cultures display high levels of apoptosis. Comparable responses were observed against a range of microbial Ags and against a parasite Ag absent from the local environment, but not against autoantigen. A notable finding was the appearance in these cultures of CD4(+)CD25(+)CTLA4(+) T cells that de novo develop MLR-suppressive activity. These cells moreover expressed CD45RA and CD38, hallmarks of recent thymic emigrants. CFSE-labeling studies indicate that the CD4(+)CD25(+) cells observed at the end of the culture period were present in the day 0 starting populations, but they were not suppressive in MLR responses. Collectively, these findings suggest that a significant component of the reactivity of human neonatal CD4(+) T cells toward nominal Ag (allergen) represents a default response by recent thymic emigrants, providing an initial burst of short-lived cellular immunity in the absence of conventional T cell memory, which is limited in intensity and duration via the parallel activation of regulatory T cells.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

Alternate class I MHC antigen processing is inhibited by Toll-like receptor signaling pathogen-associated molecular patterns: Mycobacterium tuberculosis 19-kDa lipoprotein,CpG DNA,and lipopolysaccharide

Pathogen-associated molecular patterns (PAMPs) signal through Toll-like receptors (TLRs) to activate immune responses, but prolonged exposure to PAMPs from Mycobacterium tuberculosis (MTB) and other pathogens inhibits class II MHC (MHC-II) expression and Ag processing, which may allow MTB to evade CD4(+) T cell immunity. Alternate class I MHC (MHC-I) processing allows macrophages to present Ags from MTB and other bacteria to CD8(+) T cells, but the effect of PAMPs on this processing pathway is unknown. In our studies, MTB and TLR-signaling PAMPs, MTB 19-kDa lipoprotein, CpG DNA, and LPS, inhibited alternate MHC-I processing of latex-conjugated Ag by IFN-gamma-activated macrophages. Inhibition was dependent on TLR-2 for MTB 19-kDa lipoprotein (but not whole MTB or the other PAMPs); inhibition was dependent on myeloid differentiation factor 88 for MTB and all of the individual PAMPs. Inhibition of MHC-II and alternate MHC-I processing was delayed, appearing after 16 h of PAMP exposure, as would occur in chronically infected macrophages. Despite inhibition of alternate MHC-I Ag processing, there was no inhibition of MHC-I expression, MHC-I-restricted presentation of exogenous peptide or conventional MHC-I processing of cytosolic Ag. MTB 19-kDa lipoprotein and other PAMPs inhibited phagosome maturation and phagosome Ag degradation in a myeloid differentiation factor 88-dependent manner; this may limit availability of peptides to bind MHC-I. By inhibiting both MHC-II and alternate MHC-I Ag processing, pathogens that establish prolonged infection of macrophages (>16 h), e.g., MTB, may immunologically silence macrophages and evade surveillance by both CD4(+) and CD8(+) T cells, promoting chronic infection.     

Human autoreactive CD4+ T cells from naive CD45RA+ and memory CD45RO+ subsets differ with respect to epitope specificity and functional antigen avidity

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4+ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T cell clones from CD45RA+/RO- and CD45RO+/RA- CD4+ T cell subsets from the peripheral blood of multiple sclerosis patients and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA+ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA+ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA+ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA+ T cell subset as the source for immunodominant, potentially pathogenic effector CD4+ T cell responses in humans.     

Generation and functional characterization of T cell lines and clones specific for Schistosoma japonicum egg antigen in humans

T cell lines specific for Schistosoma japonicum egg Ag were established in vitro from patients with chronic schistosomiasis japonica, and investigated their possible immunopathologic roles by testing lymphokines production and in vitro granuloma formation assay. All lines tested had surface phenotypes of CD3+ CD4+ CD8-, and showed S. japonicum soluble egg Ag (SEA)-specific proliferation requiring HLA-DR-restricted Ag presentation. Of these fractions of SEA separated by gel filtration, Fraction II (m.w. 7,000 to 18,000) and III (m.w. 7,000) induced strong proliferation of T cell lines, whereas fraction I (m.w. 18,000+) failed to induce detectable proliferation to any T cell lines tested. One of the T cell lines was cloned by micromanipulation: two of eight clones responded only to fraction II, and six to both fractions II and III. We observed that four of eight clones tested produced IL-2 in response to SEA, and three of them were able to transfer S. japonicum egg-specific granulomatous hypersensitivity in vitro to an HLA haplo-identical individual without previous schistosome infection. These immunopathologic functions of T cell clones seemed to be activated by at least two distinct epitopes of SEA. Our present observations suggest that at least two distinct CD4+ human T cells, both of which recognize epitopes expressed on SEA molecules of less than 18 kDa, might have critical roles in granulomatous hypersensitivity to eggs of S. japonicum in humans.     

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.     

Altered CD4+ T cell phenotype and function determine the susceptibility to mucosal candidiasis in transgenic mice expressing HIV-1

The impairments of protective mucosal immunity which cause susceptibility to oropharyngeal candidiasis (OPC) in HIV infection remain undefined. This study used a model of OPC in CD4C/HIV MutA transgenic (Tg) mice expressing Rev, Env, and Nef of HIV-1 to investigate the role of transgene expressing dendritic cells (DCs) and CD4+ T cells in maintenance of chronic oral carriage of Candida albicans. DCs were depleted in the Tg mice and had an immature phenotype, with low expression of MHC class II and IL-12. CD4+ T cells were quantitatively reduced in the oral mucosa, cervical lymph nodes (CLNs) and peripheral blood of the Tg mice, and displayed a polarization toward a nonprotective Th2 response. Proliferation of CLN CD4+ T cells from infected Tg mice in response to C. albicans Ag in vitro was abrogated and the cells failed to acquire an effector phenotype. Coculture of C. albicans-pulsed DCs with CD4+ T cells in vitro showed that Tg expression in either or both of these cell populations sharply reduced the proliferation of CD4+ T cells and their production of IL-2. Finally, transfer of naive non-Tg CD4+ T cells into these Tg mice restored proliferation to C. albicans Ag and sharply reduced oral burdens of C. albicans. Overall, these results indicate that defective CD4+ T cells primarily determine the susceptibility to chronic carriage of C. albicans in these Tg mice.     

Heterogeneity of helper/inducer T lymphocytes. III. Responses of IL-2- and IL-4-producing (Th1 and Th2) clones to antigens presented by different accessory cells

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.     

Human breast carcinoma patients develop clonable oncofetal antigen-specific effector and regulatory T lymphocytes.

Oncofetal Ag (OFA) is a 44-kDa glycoprotein expressed during early to mid-gestation fetal development and re-expressed as a surface Ag by tumor cells soon after transformation. The Ag is detectable on all types of human and rodent tumors tested, but is undetectable on normal cells. In experimental animals it is autoimmunogenic and induces potentially protective T cell responses both after experimental immunization and during tumor development subsequent to carcinogenic insult. To determine whether this tumor-associated Ag is also immunogenic for human T lymphocytes, breast carcinoma patients' peripheral blood mononuclear leucocytes were stimulated in vitro with autologous tumor cells in the presence of IL-2, gamma-IFN, and IL-6 for 2 wk. The tumor-reactive cells were then restimulated and cloned by limiting dilution, and the clones were analyzed. We established 24, 19, 11, and 16 tumor-reactive clones from the four respective patients. Of those, 4, 6, 4, and 7, respectively, proliferated specifically to purified OFA. Both CD4 and CD8 OFA-specific clones were established, which responded equally well to purified OFA or 32- to 44-kDa immature laminin receptor protein. All were CD3+, TCR-alpha beta+. All CD4 clones secreted gamma-IFN, but neither secreted IL-4 nor IL-10. Both IFN-gamma-secreting cytotoxic CD8 clones and IL-10-secreting inhibitory CD8 clones were established. Thus, during human cancer development, the same types of OFA-specific effector and regulatory T cells are induced as during murine T lymphomagenesis.     

The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells

Induction of antitumor immunity involves the presence of both CD8(+) CTLs and CD4(+) Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34(+) hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8(+) CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4(+) Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4(+) T cells were cloned that released IFN-gamma upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8(+) and CD4(+) T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

Drug-specific T cells derived from patients with drug-induced allergic hepatitis.

Drug-induced allergic hepatitis is a tissue-specific inflammatory disease caused by hypersensitivity to a particular drug. Although the frequency of drug-induced allergic hepatitis appears to increase in proportion to the medicine, the mechanism by which tissue specificity is determined is still to be elucidated. In this study, we established CD4+ T cell clones specific for particular drugs from patients with drug-induced allergic hepatitis accompanied with mild blood eosinophilia and analyzed the possible role of liver protein as a directing factor of liver-specific inflammatory reactions. All CD4+ T cell clones obtained from two patients with this disease proliferated in response to a combination of the particular drug plus liver specific protein (LSP), which consists of over 30 proteins. Some T cell clones were responsive to an antigenic conformation consisting of the 200-kDa glycoprotein (partly purified LSP), a component of LSP, plus the causal drug. In contrast, all CD4+ T cell clones from a patient with simple drug-induced eosinophilia responded to the causal drug in the absence of LSP and partly purified LSP. These data suggested that LSP or partly purified LSP of the appropriate Ag is the target that leads to liver-specific inflammation in drug-induced allergic hepatitis. Furthermore, T cell lines derived from patients with drug-induced allergic hepatitis and simple drug-induced eosinophilia produced large amounts of IL-5 after the appropriate antigenic stimulation, whereas CD4+ T cell clones from donors with a normal amount of peripheral blood eosinophils secreted a much less IL-5. Taken together, these results indicate that overproduction of IL-5 by the allergen-sensitized T cells may result in blood eosinophilia.