SPECIFIC ADHERENCE OF IN VITRO DIFFERENTIATED LYMPHOCYTES TO TARGET CELLS

Blast cells which were derived from rat lymphocytes by stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM) transformed within 2–3 days into a new type of lymphocytes when plated without mitogen on embryo fibroblast monolayers. These lymphocytes were termed secondary lyrophocytes. Upon addition of PWM to PWM-secondary lymphocytes a marked adherence to fibroblast monolayers was observed. The degree of adherence was estimated (a) by direct count of the lymphocytes in the medium and in the trypsinized fibroblast fraction, and (b) by using ⁵¹Cr-labeled lymphocytes. The adherence process required incubation at 37°C. The process started immediately after the addition of PWM and reached a plateau at 6 hr. At this time more than 80% of the lymphocytes adhered. In the absence of PWM only 12% of the lymphocytes were found in the fibroblast fraction. Unlike PWM-lymphocytes. Con A-lymphocytes, PHA-lymphocytes, and ordinary lymphocytes taken directly from the rat lymph nodes adhered only slightly more in the presence of PWM (10–20% adherence of ordinary lymphocytes) than in its absence (8% adherence). The adherence of the secondary lymphocytes and the ordinary lymphocytes was also studied in the presence of Con A and PHA. These mitogens induced high rate of adherence and they did not demonstrate specificity in their action. The adherence was accompanied by transformation of the lymphocytes to blast cells endowed with target-cell lytic ability. This transformation occurred mostly in the adhering fraction of the lymphocyte population. The results support the notion that target-cell recognition and destruction in cellular immunity involve contact between the cells.     

CELLS MEDIATING SPECIFIC IN VITRO CYTOTOXICITY : I. DETECTION OF RECEPTOR-BEARING LYMPHOCYTES

Spleen cells from mice immunized with allogeneic tumor cells are incubated on different fibroblast monolayers. The nonadsorbed cells are tested for cytotoxicity against ⁵¹Cr-labeled target cells. The cytotoxicity of nonadsorbed cells is much lower after incubation on fibroblasts syngeneic to the immunizing tumor cells than after incubation on fibroblasts syngeneic to the immune cells. This specific decrease of cytotoxic activity depends on the duration and temperature of incubation on monolayers. After incubation the monolayers are trypsinized and pure populations of adsorbed lymphocytes isolated by density gradient fractionation. The cytotoxicity of such trypsin-eluted, gradient-purified lymphocytes is much higher when these lymphocytes are isolated from fibroblasts syngeneic to the immunizing tumor cells than when they are isolated from fibroblasts syngeneic to the immune cells. These experiments demonstrate specific adsorption of immune cells onto fibroblasts carrying the immunizing antigens, and thus prove the existence of specific receptors at the surface of these immune cells. Spleen cells from mice immunized with two types of allogeneic tumor cells bearing different H-2 antigen alleles are incubated on different fibroblast monolayers. The results of such experiments show a differential specific adsorption pattern, suggesting independent adsorption of two populations of immune cells bearing receptors directed against either one or the other immunizing H-2 antigen. The existence of at least a majority of cells, each of which is homogeneous as to the specificity of its receptors, makes it likely that specific receptors are synthetized by the cells that bear them. The role of specific receptor-bearing cells in the killing process is discussed.     

Absence of CD4+ T Lymphocytes, CD8+ T Lymphocytes, or B Lymphocytes Has Different Effects on the Efficacy of Posaconazole and Benznidazole in Treatment of Experimental Acute Trypanosoma cruzi Infection

We investigated the influence of CD4⁺ T lymphocytes, CD8⁺ T lymphocytes, and B lymphocytes on the efficacy of posaconazole (POS) and the reference drug benznidazole (BZ) during treatment of acute Trypanosoma cruzi infection in a murine model. Wild-type mice infected with T. cruzi and treated with POS or BZ presented no parasitemia, 100% survival, and 86 to 89% cure rates, defined as the percentages of animals with negative hemocultures at the end of the observation period. CD4⁺-T-lymphocyte-knockout (KO) mice infected with T. cruzi and treated with BZ or POS controlled parasitemia during treatment, although circulating parasites reappeared after drug pressure cessation, leading to only a 6% survival rate and no cure. CD8⁺-T-lymphocyte-KO mice infected with T. cruzi and treated with POS or BZ had intermediate results, displaying discrete parasitemia after the treatment was ended, 81 and 86% survival, and cure rates of 31 and 66%, respectively. B-lymphocyte-KO mice infected with T. cruzi and treated with BZ relapsed with parasitemia 1 week after the end of treatment and had a 67% survival rate and only a 22% cure rate. In contrast, the activity of POS was much less affected in these animals, with permanent suppression of parasitemia, 100% survival, and a 71% cure rate. Our results demonstrate that abrogation of different lymphocytes’ activities has distinct effects on the efficacy of POS and BZ in this experimental model, probably reflecting different parasite stages preferentially targeted by the two drugs and distinct cooperation patterns with the host immune system.     

SYNERGY DURING IN VITRO CYTOTOXIC ALLOGRAFT RESPONSES : I. EVIDENCE FOR CELL INTERACTION BETWEEN THYMOCYTES AND PERIPHERAL T CELLS

A mouse in vitro allograft system was used to evaluate the concept of T-T interaction in T cell-mediated cellular immunity. In analyzing the responsiveness of thymus-processed lymphocytes as obtained from different tissues, a heretogeneity within T cells was found in regard to their capacity to be immunized in vitro against transplantation antigens. Recirculating T cells were 10–20-fold superior to thymocytes, splenic T cells being intermediate. When few (1.5 x 10⁶) peripheral T cells, in numbers too small to yield good cytotoxic responses, were mixed with 14 x 10⁶ thymocytes and the cell mixture immunized in vitro against cell-bound alloantigens, cytotoxic activity was generated exceeding about 10–20-fold the values that could be explained by a pure additive effect. Synergy occurred also in a mixture of responder T cells derived from CBA (H-2^k) and AKR (H-2^k) mice. Thus AKR anti- C3H serum could be used for discriminating between thymus-derived and peripheral T cell-derived cytotoxic lymphocytes (CL). Cytotoxic activity produced during the synergistic interaction between thymocytes and peripheral T cells was about 70% T cell derived, the remainder being thymus derived. The synoptic interpretation of this finding and "limiting dilution" experiments of the responder cells suggested strongly that peripheral T cells provide the major source for precursor cells of CL, thymocytes acting mainly as helper (amplifier) cells.     

REGULATION OF AUTOSENSITIZATION : THE IMMUNE ACTIVATION AND SPECIFIC INHIBITION OF SELF-RECOGNIZING THYMUS-DERIVED LYMPHOCYTES

We studied the mechanisms underlying the natural tolerance of thymus-derived (T) lymphocytes for self-antigens. Lymphocytes from the thymus or lymph nodes of inbred rats were autosensitized in vitro against monolayers of autochthonous thymus reticulum cells or syngeneic fibroblasts. Receptors for self-antigens were detected by the specific adherence of normal lymphocytes to syngeneic cells. The achievement of active cell-mediated autosensitization was assayed by measuring the immunospecific lysis of syngeneic target cells in vitro, or graft-versus-host (GvH) reactions in vivo. The following observations were made using these systems. (a) A fraction of normal lymphocytes was found to have specific surface receptors that are able to recognize self-antigens which seem to be accessible in vivo. These potentially self-reactive lymphocytes were activated by incubation with syngeneic or autochthonous cells in vitro. Hence, the elimination of potentially self-reactive lymphocytes cannot be the only basis for natural self-tolerance. Therefore, the maintenance of self-tolerance in vivo appears to involve suppression of the immune reactivity of such self-tolerant lymphocytes. (b) We found that control of autosensitization depends upon the inhibition of the recognition of self-antigens. A GvH reaction in vivo could not be suppressed once recognition of self-antigens had occurred in vitro. Moreover, studies of the kinetics of antigen recognition indicated that several hours of incubation in vitro were needed for the inactivation of factors specifically inhibiting self-recognition. (c) We found that factors which inhibit self-recognition are present in fresh autologous serum. Treatment of the lymphocytes, but not syngeneic adsorbing cells, with autologous serum prevented recognition of syngeneic antigens. Allogeneic serum did not prevent self-recognition, and autologous serum did not inhibit the recognition of foreign antigens. These findings indicate that natural tolerance of T lymphocytes to self-antigens can be regulated by serum factors which act on the lymphocytes. The immunospecificity of the inhibitory effect suggests that these factors may be soluble self-antigens in a tolerogenic form.     

Inhibition of Cyclooxygenase-1 and Cyclooxygenase-2 Impairs Trypanosoma cruzi Entry into Cardiac Cells and Promotes Differential Modulation of the Inflammatory Response

The intracellular protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasite''s life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host''s cyclooxygenase (COX) enzymes during T. cruzi invasion. Pharmacological antagonists for COX-1 (aspirin) and COX-2 (celecoxib) caused marked inhibition of T. cruzi infection when rat cardiac cells were pretreated with these nonsteroidal anti-inflammatory drugs (NSAIDs) for 60 min at 37°C before inoculation. This inhibition was associated with an increase in the production of NO and interleukin-1β and decreased production of transforming growth factor β (TGF-β) by cells. Taken together, these results indicate that COX-1 more than COX-2 is involved in the regulation of anti-T. cruzi activity in cardiac cells, and they provide a better understanding of the influence of TGF-β-interfering therapies on the innate inflammatory response to T. cruzi infection and may represent a very pertinent target for new therapeutic treatments of Chagas disease.     

Major Histocompatibility Complex Class I–restricted Cross-presentation Is Biased towards High Dose Antigens and Those Released during Cellular Destruction

Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I–restricted presentation to CD8⁺ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8⁺ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8⁺ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8⁺ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.     

Blocking and redistribution ("capping") of antigen receptors on T and B lymphocytes by anti-immunoglobulin antibody

Antigen-binding T and B lymphocytes were studied by combined autoradiography and immunofluorescence; mouse spleen lymphocytes binding the antigens, [¹²⁵I]MSH or [¹²⁵I]TIGAL, were incubated with rhodamine-labeled anti-Ig reagents or with a rhodamine-labeled IgG fraction of anti-θ serum. B cells were identified as Ig+ or θ-, T cells as Ig- or θ+. It was found that: (a) 20% (1–2 mo after priming) to 30% (3.5–4 mo after priming) of the antigen-binding cells were T cells. (b) The range of antigen molecules bound by B and T cells was similar. (c) Binding of antigen to B and T cells was inhibited by polyvalent anti-Ig, anti-µ, or anti-L reagents. Binding to T cells was more readily inhibited than to B cells. Normal rabbit serum, antimouse lymphocyte serum, or anti-θ did not inhibit antigen binding. (d) When Ig at the surface of B cells was induced, by noninhibiting concentrations of anti-Ig reagents, to redistribute into polar caps and the cells subsequently exposed to [¹²⁵I)antigen under noncapping conditions, the [¹²⁵I]antigen silver grains were distributed in caps superimposed on the Ig fluorescent cap. Of crucial importance, antigen was found in cap in the same proportion of T cells as B cells. Significant capping of antigen receptors was not induced in B or T cells with normal rabbit serum or by anti-Ig reagents absorbed with mouse Ig. The main conclusions of this series of experiments using direct visualization of antigen-binding B and T lymphocytes is that T cells have antigen-specific receptors, probably of IgM nature, and that the number of these receptors appears to range in the order of thousands.     

Reactivity of chagasic antigal antibodies with noninfected cells treated with Trypanosoma cruzi secreted/excreted antigens

Here, we show that antigal antibodies from Chagas' disease patients react with noninfected host cells previously treated with antigens secreted by the trypomastigote forms of Trypanosoma cruzi. With the exception of human and Old World monkey cells, which are GAL-negative, cells of all mammals express the GAL epitope (Galα(1-3)Galβ(1-4)GIcNAc-R) on their surface. Thus only the former ones develop antigal antibodies. Antigal antibodies increase during infection with T. cruzi, which expresses GAL epitopes on the surface of the infective forms. Here, we show that incubation of noninfected, GAL-negative cells with antigens shed by T. cruzi renders these cells reactive to antigal antibodies purified from chagasic sera. Neither chagasic sera depleted of antigal antibodies nor antigal antibodies purified from normal sera display reactivity with treated cells. Cell reactivity of chagasic antigal was abolished in the presence of melibiose (Galα(1-6)Glc) or gal-gal (methyl 3-O-α-D-galactopyranosyl α-D-galactopyranoside). Since shedding of T. cruzi antigens can occur in vivo, these antigens may induce reactivity of chagasic antigal with noninfected human cells. The reactivity of noninfected, GAL-negative cells observed only with chagasic antigal antibodies can amplify the range of reactivity of these antibodies and consequently adds to their importance in the pathogenesis of human Chagas' disease. J. Clin. Lab. Anal. 12:108–114, 1998. © 1998 Wiley-Liss, Inc.     

Mechanisms of Hypergammaglobulinemia in Pulmonary Sarcoidosis: SITE OF INCREASED ANTIBODY PRODUCTION AND ROLE OF T LYMPHOCYTES

Pulmonary sarcoidosis is a disorder in which local granuloma formation is perpetuated by activated lung T lymphocytes. The present study suggests that lung T lymphocytes may also play a critical role in modulating local production of antibodies in this disorder. In untreated patients with pulmonary sarcoidosis, the numbers of IgG- and IgM-secreting cells per 10³ lung lymphocytes are markedly increased compared with those in normal individuals (P < 0.001 and P < 0.01, respectively); the numbers of IgA-secreting cells in lavage fluid of these patients are not increased (P > 0.2). In contrast to lungs, the numbers of IgG-, IgM-, and IgA-secreting cells in blood of patients with this disorder are similar to those in normal individuals (P > 0.2, each comparison). In patients with pulmonary sarcoidosis, there is a direct correlation between the percentage of bronchoalveolar cells that are T lymphocytes and the percentage of bronchoalveolar cells that secrete IgG (r = 0.79; P < 0.001); in normal individuals there is no such relationship (P > 0.2). When purified sarcoid lung T cells from patients with high proportions of T lymphocytes in their lavage fluid were co-cultured with blood mononuclear cells from normal individuals (without added antigens or mitogens), the B lymphocytes in these normal mononuclear cell suspensions were induced to differentiate into immunoglobulin-secreting cells (P < 0.01). In contrast, blood T lymphocytes from these same patients and lung T lymphocytes from sarcoidosis patients with low proportions of T lymphocytes in their lavage fluid did not stimulate normal B cells to produce immunoglobulin (P > 0.2, all comparisons). These findings suggest that in pulmonary sarcoidosis (a) the lung is an important site of immunoglobulin production; (b) activated lung T lymphocytes play an important role in modulating this local production of antibody, and thus are likely to modulate the polyclonal hyperglobulinemia observed in these individuals.     

MOUSE THYMUS-INDEPENDENT AND THYMUS-DERIVED LYMPHOID CELLS : I. IMMUNOFLUORESCENT AND FUNCTIONAL STUDIES

The simultaneous use on mouse lymphoid suspensions of heterologous antisera directed against thymus-derived (T) cell mouse-specific lymphocyte antigen and brain-associated theta antigen (MSLA and BAθ) or thymus-independent (B) cell mouse-specific bone marrow-derived lymphocyte antigen (MBLA) surface antigens allowed direct proof of the different specificity of these antisera by double immunofluorescence (IF) staining with selective visualization of fluorochromes. These antisera and antisera against mouse Ig and its different types of chains were then used with technique of either double IF staining or IF combined with radioautography, allowing the following conclusions: (a) Surface Ig (sIg) was found exclusively on B cells and never on T cells, but not all B cells had sIg. Cells containing detectable amounts of Ig were MBLA+, but had less sIg than other B cells or none at all. There was evidence for the existence of a significant number of MBLA+ lymphocytes, neither bearing nor containing detectable Ig. (b) µ-Chains were the most frequent but not the only heavy chains found on spleen cells; however, it could not be decided with the technique used, if a single cell can bear more than one type of heavy chain. No cell containing γ-chains was found to bear surface µ-chains, although a very few cells containing both µ- and γ-chains were observed. (c) The antigen-binding cells detected after immunization with bacteriophage T4, bovine serum albumin, Maia squinado hemocyanin, and sheep erythrocytes were analyzed for MSLA, MBLA or sIg using double IF, a combination of IF and radioautography, or inhibition of "rosette" formation. Practically all the antigen-binding cells detected were MSLA-, MBLA+, sIg+. (d) More B cells than T cells were found among short-lived lymphoid cells labeled by repeated in vivo injections of tritiated thymidine, but the results did not support a simplified concept equating T cells to long-lived and B cells to short-lived lymphocytes. (e) Cells dividing rapidly in the lymph nodes draining the sites of immunization with various antigens were predominantly T cells 2 days after immunization and in majority B cells a few days later. (f) Incubation of lymphoid cells at 37°C with rabbit anti-mouse Ig or anti-κ chains led to complete disappearance of sIg and to decrease of MBLA ("antigenic modulation"). In the same conditions, anti-MBLA gave partial modulation of MBLA and of sIg; MBLA, however, reappeared much faster than sIg. No modulation of T cell surface antigens by the appropriate antisera was observed. Cell treatment with Pronase could remove MBLA, sIg, MSLA, and BAθ, which reappeared within a few hours. Neuraminidase treatment was without detectable effect on these antigens.     

QUANTITATIVE STUDIES ON THE BEHAVIOR OF SENSITIZED LYMPHOCYTES IN VITRO : I. RELATIONSHIP OF THE DEGREE OF DESTRUCTION OF HOMOLOGOUS TARGET CELLS TO THE NUMBER OF LYMPHOCYTES AND TO THE TIME OF CONTACT IN CULTURE AND CONSIDERATION OF THE EFFECTS OF ISOIMMUNE SERUM

When lymphoid cells, derived from rats immunized with respect to homologous skin, were cultured with target cells originally of donor origin, cytocidal and cytostatic activities of the attacking lymphocytes became evident. By application of a sensitive and reproducible quantitative assay system, various aspects of the mechanism of this destructive interaction between target cells and lymphocytes were examined with the following results. 1. The degree of survival of target cells was inversely related to the number of sensitized lymphocytes. Graphic plots of the data indicated that this relationship was an exponential one similar to "single-hit" inactivation phenomena. One interpretation which could be placed on these results is that a single lymphocyte, if immunologically active, was sufficient to destroy or at least have a detectably adverse effect on one target cell. Furthermore, from such a model it could be computed that, of the lymphocytes derived from an immunized animal, approximately 1 to 2 per cent of the cells were immunologically active; i.e., capable of demonstrable destructive activities against homologous target cells in vitro. 2. Morphological studies on the effect of sensitized lymphoid cells on homologous target cells, aftervarious lengths of time in culture, showed that by 7 hours of incubation, the attacking lymphocytes firmly adhered to the target cells. The cytotoxic effect of these lymphocytes generally occurred after the 20th hour. Quantitative studies supported this conclusion; the latent period, i.e., the time required for detectable degrees of target cell destruction to occur, was approximately 20 hours. 3. A consequence of the incubation of target cells with normal lymphoid cells or even with small numbers of sensitized lymphoid cells was an increase in the rate of division of the target cells. As might be expected, this was reflected in a shorter doubling time of these cells. 4. Extracts prepared from sonically disrupted sensitized lymphocytes proved to be no more deleterious to target cells than similar preparations from normal lymphoid cells. Furthermore, no evidence could be obtained that sensitized lymphoid cells, separated from target cells by a Millipore membrane, were cytocidally effective. These data indicated that if a cell-bound substance is involved in the destruction of homologous cells, either it is not toxic by itself, or it cannot be detached from the sensitized cells. In any case, close apposition of the lymphocytes to the target cells is apparently required for the destruction of the latter in vitro. 5. Serum obtained from immunized animals, if heat-inactivated, did not adversely affect homologous target cells; if employed fresh, slight degrees of toxicity resulted. Specific isoimmune sera did not impart any detectable degrees of immunological reactivity upon otherwise normal lymphoid cells. Immune sera, even in high concentrations, did not augment the effect of sensitized lymphoid cells upon homologous target cells; rather a slight inhibitory effect of these sera was detected. 6. Attempts to detect the presence of complement activity, which might have been provided by the lymphoid cells in culture, were unsuccessful. On the basis of these results, it was suggested that the destruction of homologous target cells by sensitized lymphoid cells occurs as a two step process. First, the attacking lymphocytes attach to their targets via a non-toxic cell-bound substance having an immunologic specificity, and then, destruction of the target cells follows the result of some process dependent on the metabolic activity of the attacking lymphoid cells.     

QUANTITATIVE STUDIES ON THE MIXED LYMPHOCYTE INTERACTION IN RATS : I. CONDITIONS AND PARAMETERS OF RESPONSE

Some of the conditions and parameters of the proliferative response in the mixed lymphocyte interaction have been studied with the use of culture inocula consisting of lymphoid cells from various immunogenetically diverse, isogenic strains of rats. Procedures are described by which consistent culture responses can be obtained which are measurable in terms of incorporation of radioactive thymidine into a trichloroacetic acid precipitable cell fraction. Results of experiments with this "mixed lymphocyte interaction" show that (a) Incorporation of thymidine into the mixed cultures is detectable only during and after the 4th day of incubation. Use of culture inocula derived from the thoracic duct or partially purified suspensions of lymphocytes from the peripheral blood provides further evidence that it is the small lymphocyte which synthesizes DNA in these cultures. The addition of peritoneal exudate cells, presumably containing some macrophages, to the mixed lymphocyte cultures does not alter the kinetics of the response. (b) Proliferative responses occur in mixed cultures of cells only when the donors differ by alleles at the important Ag-B histocompatibility locus. (c) Proliferative reactivity in mixed cultures of lymphocytes from parental and hybrid donors is unidirectional. With the use of chromosomally marked cells from donors of different sexes, it was established that only parental lymphocytes are dividing in mixed cultures of parental and hybrid cells. The results of these experiments strongly support the premise that the proliferative response in mixed cultures of lymphocytes represents a de novo immunologic reaction on the cellular level against histocompatibility antigens.     

PROLIFERATION OF B AND T CELLS IN MIXED LYMPHOCYTE CULTURES

Electrophoretically fractionated CBA/Ca spleen T cells alone respond to allogeneic cells in one-way MLC and to PHA. They do not respond to E. coli LPS. B cells alone do not respond to allogeneic cells nor to PHA, but do respond to LPS. When karyotypically distinguishable syngeneic mixtures of T and B lymphocytes are stimulated with allogeneic cells, at the most 5% of mitoses on 5–9th culture day are of B cell origin. This indicates that B cells are not substantially recruited to proliferate in the MLC.     

Induction of Resistance to Azole Drugs in Trypanosoma cruzi

Trypanosoma cruzi is the protozoan parasite that causes Chagas’ disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruzi infections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133–138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruzi infections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance in T. cruzi in vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruzi infection.     

QUANTITATIVE STUDIES ON THE MIXED LYMPHOCYTE INTERACTION IN RATS : VI. REACTIVITY OF LYMPHOCYTES FROM CONVENTIONAL AND GERMFREE RATS TO ALLOGENEIC AND XENOGENEIC CELL SURFACE ANTIGENS

The proliferative reactivity of lymphocytes from rat donors maintained under germfree or conventional conditions was examined in mixed lymphocyte cultures stimulated with allogeneic and xenogeneic cell surface antigens. The results show (a) that lymphocytes from conventionally maintained rats are less reactive to human, hamster, guinea pig, and mouse cell surface antigens than to the major H alloantigens, and (b) that lymphocytes from germfree rats display no demonstrable reactivity to xenogeneic cells, but are quantitatively normal in their response to allogenic cells. The conclusion drawn from these observations is that the circulating lymphocyte pool of an individual consists of a greater proportion of cells reactive to H alloantigens of other members of the same species than to the xenogeneic cellular antigens of members of other species and that this large number of cells is not generated by a mechanism involving immunization to cross-reactive environmental antigens.     

Sensitization of lymphocytes against pooled allogeneic cells. I. Generation of cytotoxicity against autologous human lymphoblastoid cell lines

Lymphocytes sensitized in vitro to a pool of X-irradiated allogeneic normal lymphocytes from 20 individuals develop cytotoxic activity for autologous human lymphoblastoid cells (LCL). Whereas pool sensitized T lymphocytes lyse autologous LCL cells, they fail to lyse autologous B-enriched or T-enriched normal target cells nor autologous phytohemagglutinin (PHA) blasts. In contrast to pool sensitization, stimulation with normal cells of single allogeneic individuals rarely led to development of cytotoxicity against autologous LCL cells. We conclude that human Epstein-Barr virus transformed LCL cells express target antigens cross-reactive with allogeneic target antigens expressed on normal cells and that sensitization with a pool of allogeneic cells is an effective means of generating effector cells directed against autologous abnormal cells.     

FUNCTIONAL MATURATION OF THYMIC LYMPHOCYTE POPULATIONS IN VITRO

Mouse thymocytes were cultured for short periods of time either alone or with one of two supporting cell populations, splenic adherent cells or thymic epithelial cells. The thymus-derived (T) cell activity of thymocytes cultured on supporting cell populations increased dramatically during 2 days of culture, as assayed in the mixed lymphocyte interaction (MLI), response to phytomitogens, and helper cell activity in the in vitro antibody response. The level of activity attained was equal to that of spleen and lymph node lymphocytes and greater than that of steroid-resistant thymocytes. The cultured thymocytes had surface antigens characteristic of mature T lymphocytes with regard to θ and H-2. The appearance of functionally active lymphocytes in vitro depended upon cell division. Most of the active cultured cells arose from cells already undergoing maturation, i.e., from cells with reduced θ determinants and increased H-2 determinants. We therefore have generated a population of thymocytes indistinguishable from peripheral T lymphocytes using simple in vitro techniques. The extent to which the production of these active lymphocytes depends upon in vitro differentiation is discussed.     

The Route of Antigen Entry Determines the Requirement for L-selectin during Immune Responses

L-selectin, an adhesion molecule constitutively expressed on leukocytes, is important for primary adhesion and extravasation of lymphocytes at specialized high endothelial venules within lymph nodes and other leukocytes at sites of inflammation. We have generated L-selectin–deficient mice by targeted disruption, and have confirmed a previously reported phenotype which includes strikingly impaired contact hypersensitivity (CHS) responses to reactive haptens (Tedder, T.F., D.A. Steeber, and P. Pizcueta. 1995. J. Exp. Med. 181:2259–2264; Xu, J.C., I.S. Grewal, G.P. Geba, and R.A. Flavell. 1996. 183:589–598.). Since the mechanism of this impairment has not been clarified, we sought to define the stage(s) at which the CHS response is affected in L-selectin–deficient mice. We show that epidermal Langerhans cells in L-selectin– deficient mice are normal in number, migrate to peripheral lymph nodes appropriately, and are functional in presenting allogeneic and haptenic antigens. Moreover, T cells, as well as neutrophil and monocyte effector populations, are fully capable of entry into the inflamed skin sites in the absence of L-selectin. Thus, antigen presentation and effector mechanisms are intact in L-selectin deficient mice. In contrast, virtually no antigen-specific T cells can be found within draining peripheral nodes after a contact challenge, suggesting that the defect resides primarily in the inability of antigen-specific T cells to home to and be activated in these nodes. Indeed, L-selectin–deficient mice mount completely normal CHS responses when alternate routes of immunization are used. These studies pinpoint the lesion in CHS to a discrete stage of the afferent limb of the response, clarify the role of L-selectin on effector populations, and illustrate the critical importance of the route of antigen entry to the successful execution of an immune response.