Design and chemical evaluation of reduced machine-yield cigarettes.

Experimental cigarettes (ECs) were made by combining technological applications that individually reduce the machine measured yields of specific toxicants or groups of toxicants in mainstream smoke (MS). Two tobacco blends, featuring a tobacco substitute sheet or a tobacco blend treatment, were combined with filters containing an amine functionalised resin (CR20L) and/or a polymer-derived, high activity carbon adsorbent to generate three ECs with the potential for generating lower smoke toxicant yields than conventional cigarettes. MS yields of smoke constituents were determined under 4 different smoking machine conditions. Health Canada Intense (HCI) machine smoking conditions gave the highest MS yields for nicotine-free dry particulate matter and for most smoke constituents measured. Toxicant yields from the ECs were compared with those from two commercial comparator cigarettes, three scientific control cigarettes measured contemporaneously and with published data on 120 commercial cigarettes. The ECs were found to generate some of the lowest machine yields of toxicants from cigarettes for which published HCI smoke chemistry data are available; these comparisons therefore confirm that ECs with reduced MS machine toxicant yields compared to commercial cigarettes can be produced. The results encourage further work examining human exposure to toxicants from these cigarettes, including human biomarker studies.

Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay.

Cytotoxic effector T cells of F1 (BALB/c X BALB.B) (H-2d/b) mice immunized against the minor histocompatibility differences of C57BL/10 (H-2b) can lyse targets from C57BL/10, but cannot lyse B10.D2 (H-2d) targets. Despite this lack of cross-reaction in the cytotoxic assay, C57BL/10 cells do prime F1 (BALB/c X BALB.B) mice for a secondary cytotoxic response to B10.D2. C57BL/10-primed, B10.D2-boosted cytotoxic cells lyse B10.D2 targets but not C57BL/10 targets. DBA/2 (H-2d) spleen cells or thymocytes prime F1 mice for a secondary response to DBA/2, B10.D2, and C57BL/10 cells, but DBA/2 mastocytes, P815, do not prime for a response to C57BL/10. Whether H-2 congenic lymphoid cells express minor histocompatibility determinants which cross-react at the cytotoxic T-cell level or the helper T-cell level is discussed.

The major histocompatibility complex determines susceptibility to cytotoxic T cells directed against minor histocompatibility antigens.

Cytotoxic cells were generated by immunizing one strain of mouse with cells from an allogeneic strain which carries the same H-2 region. The effector cells assayed in a 4 h 51Cr release assay were shown to be T cells and indistinguishable, except in specificity, from cytotoxic T cells directed at H-2 alloantigens. Although the genetic differences between responder and stimulator cells responsible for the immunization did not code in H-2, the H-2 complex did restrict susceptibility of target cells. For example, BALB.B cytotoxic cells (H-2b) immunized against and capable of lysing C57BL/6 cells (H-2b) would not lyse B6.C/H-2d target cells. C57BL/6 and B6.C/H-2d are congenic and differ in the H-2 region. Two hypotheses are considered to explain the H-2 restriction of susceptibility to cytotoxic T cells generated by an H-2 identical alloimmunization. (a) The dual (self) recognition hypothesis states that the cytotoxic cell has two recognition units, one for H-2-coded structures and another clonally restricted receptor for the minor alloantigen. (b) The interaction antigen hypothesis states that all the surface alloantigenic determinants recognized by cytotoxic T cells are the result of interaction between H-2- and non-H-2-coded gene products. Two lines of evidence, one with F1 effector cells and the other a cold target competition experiment, are presented which argue strongly in favor of the interaction antigen hypothesis. The regions of H-2 required to be histocompatible were mapped to the D region and to the left of IC, probably the K region. These results, and recent work on the response to virus-infected and TNP-modified syngeneic cells, suggest that cytotoxic cells are restricted in specificity to preferentially recognizing alterations in structures that are coded in the major histocompatibility complex.

Selective activation of Fas/Fas ligand-mediated cytotoxicity by a self peptide.

To study how MHC-associated self antigens may regulate the function of T cells in the periphery, we generated CD8+ T cell lines specific for a single residue variant of a self peptide. The self peptide (GAYEFTTL) was isolated from H-2-Kb class I MHC molecules immunopurified from tumor cells. CD8+ CTL lines from H-2b mice were generated against a variant peptide, pE4R, (arginine for glutamic acid at the TCR contact position 4). In short-term 51Cr-release assays, these CTL lysed H-2Kb targets that were pulsed with picomolar levels of pE4R but did not lyse target cells coated with the self peptide at micromolar levels. However, in overnight assays the CTL lysed Fas-positive target cells in the presence of nanomolar levels of the self peptide. This killing was shown to be entirely Fas/Fas ligand mediated by blocking with anti-Fas antibody and Fas-Fc chimeric molecules. While the self peptide was unable to induce serine esterase release from the CTL, it did induce secretion of IFN-gamma. By these criteria then, the unmodified self ligand served as a partial agonist for the CTL raised against a single-residue variant. CD8+ T cell lines raised by in vitro stimulation with the self peptide were likewise unable to kill self peptide-coated targets via the perforin pathway but did lyse targets via Fas. These and similar data from other groups show that self antigens (i.e., MHC/peptide complexes) may be recognized by mature peripheral T cells. The T cell population is tolerant of the self antigen in the sense that they do not respond to physiological levels of the MHC/peptide complex. However, when the level of self antigen is increased (by using synthetic peptide loading) CD8+ T cells may respond by proliferation, IFN-gamma secretion, Fas ligand upregulation, and Fas-mediated cytolysis but are still unable to respond by perforin-mediated cytolysis or granzyme release. The physiological significance of such partial activation in regulation of the immune system remains to be demonstrated.

Specificity of cytotoxic T cells from athymic mice.

In normal mice, self-H-2 antigens in the thymus have a profound influence on T cell specificity. We have therefore investigated the properties of cytotoxic T lymphocyte (CTL) precursors from athymic nude mice (5) with the notion that they may provide a model system for the study of T cells whose receptro specificity is closer to the germ-line-encoded repertoire. It was found that the precursors of nude CTL are, themselves, THy-1+ cells. The possibility that these nude t cells were derived from the phenotypically normal mother by placental transfer was ruled out. In the presence of T cell growth factor, nude CTL can be induced by polyclonal activation with concanavalin A or by stimulation with allogeneic or trinitrophenyl (TNP)-modified syngeneic stimulator cells, but not by stimulation with minor H antigens in the context of self-H-2. Alloreactive, nude CTL--like those from normal mice--recognize H-2K- and H-2D-region-encoded antigens in killer-target cell interactions, but, unlike normal CTL, did not cross-react on third-party target cells. Whereas the anti-TNP response of nude mice is H-2 restricted, it does not seem to be influenced by self-H-2 antigens in the same manner as in normal mice. This is suggested by the finding that the immunodominance of H-2k over H-2d in the anti-TNP-self response of normal (H-2d X H-2b)F1 mice is absent in (H-2d X H-2k)F1 nude mice. These observations are discussed in relation to the role of the thymus in the generation of the normal mature T cell receptor repertoire.

Self H-2 antigens influence the specificity of alloreactive cells.

We have tested Jerne's hypothesis (9) that the phenomenon alloreactivity is explained by the existence of T cells that express germline-encoded receptors specific for major histocompatibility complex antigens and that these cells undergo no change in specificity during thymic differentiation. T cells from [F1 leads to Parent] bone marrow radiation chimeras reactive to conventional antigens are known to have a self preference, i.e., [A X B leads to A] chimeras respond better to H-2A-plus-antigen than to H-2B-plus-antigen. We show here that alloreactive cells from such chimeras also have a self preference. Thus, H-2k-specific alloreactive T cells from [H-2b X H-2d leads to H-2b] and [H-2b X H-2d leads to H-2d] chimeras cross-react more on TNP-modified H-2b or H-2d targets, respectively. In contrast to Jerne's prediction, the results suggest that the receptor repertoire of alloreactive F1 cells is influenced by H-2 antigens on radiation-resistant cells present during T cell ontogeny. By this criterion of having a self preference in H-2 restriction, alloreactive T cells appear to be similar to T cells that respond to conventional antigens.

ITM2A is induced during thymocyte selection and T cell activation and causes downregulation of CD8 when overexpressed in CD4(+)CD8(+) double positive thymocytes.

To identify novel genes that are involved in positive selection of thymocytes, we performed polymerase chain reaction (PCR)-based subtractive hybridization between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) null background are efficiently selected into the CD8 lineage in H-2(b) mice (RAG-2(-/-)OT-1, selecting thymi), but are not selected on a transporter associated with antigen processing (TAP) null background (RAG-2(-/-)TAP-1(-/-)OT-1, nonselecting thymi). We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus. The expression pattern of ITM2A in thymocyte subsets correlates with upregulation during positive selection. In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation. ITM2A expression was also induced in RAG-2(-/-) thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent M(r) of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surface. Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial downregulation of CD8 in CD4(+)CD8(+) double positive (DP) thymocytes, and a corresponding increase in the number of CD4(+)CD8(lo) thymocytes. Possible roles for this novel activation marker in thymocyte development are discussed.

A reexamination of the role of LYT-2-positive T cells in murine skin graft rejection.

We have investigated which T cell subclass defined by cytolysis with monoclonal anti-Lyt-1.2 and anti-Lyt-2.2 antibodies is required to adoptively transfer the ability to reject skin grafts. B6.Thy-1.1 spleen cells immune to graft antigens were fractionated with antibody plus C' and transferred to adult thymectomized, irradiated, bone marrow-reconstituted (ATXBM) B6.Thy-1.2 hosts that were simultaneously grafted with BALB.B skin. We found that when the ATXBM hosts were used 6 wk after irradiation and marrow reconstitution, both Lyt-1-depleted and Lyt-2-depleted immune spleen cells could transfer the ability to promptly reject skin grafts. However, such ATXBM recipients of Lyt-2-depleted cells that had rejected skin grafts were found to contain graft-specific CTL that were largely of host (B6.Thy-1.2) origin. When ATXBM hosts were used for the experiment 1 wk after irradiation and marrow reconstitution, no host-derived graft-specific CTL could be detected. However, graft rejection occurred in recipients of anti-Lyt-1- or anti-Lyt-2 plus C'-treated immune cells and specific CTL were generated from spleen cells of both groups. Thus, in the absence of a host-derived response, adoptively transferred immune Lyt-2+ cells, either resistant to, or that escaped from, antibody plus C' treatment, are able to expand in response to the antigenic stimulus provided by the graft. A more complete elimination of specific T cell subclasses is therefore needed to assess the relative contribution of a particular subset to the graft rejection process.

CD8+ T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo.

Class I major histocompatibility complex (MHC)-restricted CD8+ T cells have been demonstrated to be effective mediators of both acquired and adoptive immunity to the intracellular bacterium Listeria monocytogenes. We have recently determined that L. monocytogenes-infected H-2d mice recognize a nonamer peptide, residues 91-99, of the secreted protein listeriolysin O (LLO), in a H-2Kd-restricted fashion. In this report we have generated CD8+ T cell lines with specificity for LLO 91-99 in the context of H-2Kd by in vitro stimulation with P815 (H-2d) cells transfected with LLO. These CD8+ lines have been generated from immune donors after sublethal infection with L. monocytogenes, or after in vivo immunization with syngeneic spleen cells coated with synthetic LLO 91-99 peptide. LLO-specific CD8+ T cells derived from either protocol were capable of significant protection against L. monocytogenes infection. The in vivo protection by these CD8+ T cell lines has been shown to be solely due to recognition of LLO 91-99 in the context of H-2Kd. These studies demonstrate that CD8+ T cell immunity to a single, naturally produced peptide epitope has the potential for significant protection in a bacterial infection. Thus, the allele-specific motif approach to epitope prediction has identified a naturally produced bacterial epitope with biological relevance.

An endogenous antigenic peptide bypasses the class I antigen presentation defect in RMA-S.

The RMA-S cell line was derived from the Raucher virus-induced murine cell line RBL-5 by ethylmethane sulfonate mutagenesis and anti-H-2 antibody plus complement selection (Ljunggren, H.-G., and K. Karre. 1985. J. Exp. Med. 162:1745). RMA-S is defective in the ability to present endogenously synthesized antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) (Townsend, A., C. Ohlen, J. Bastin, H.-G. Ljunggren, L. Foster, and K. Karre. 1989. Nature [Lond.]. 340:443; Ohlen, C., J. Bastin, H.-G. Ljunggren, L. Foster, E. Wolpert, G. Klein, A. R. M. Townsend, and K. Karre. 1990. J. Immunol. 145:52). This defect has been attributed to the inability of RMA-S to deliver antigenic peptides derived from antigens in the cytosol into the endoplasmic reticulum (ER), where they can associate with class I MHC molecules (Townsend, A., C. Ohlen, J. Bastin, H.-G. Ljunggren, L. Foster, and K. Karre. 1989. Nature [Lond.]. 340:443). We show that RMA-S can present at least one endogenous antigen, vesicular stomatitis virus nucleoprotein (VSV-N), to class I MHC-restricted CTL. RMA-S presents VSV-N to CTL both when infected with VSV or transfected with the VSV nucleoprotein gene. The natural antigenic VSV nucleoprotein peptides purified from either RMA or RMA-S are indistinguishable when analyzed by high performance liquid chromatography. We also show that the genetic defect responsible for the RMA-S phenotype maps to the murine chromosome 17. This chromosome encodes the murine class I MHC genes as well as two genes, HAM-1 and -2, with homology to the adenosine triphosphate-dependent transporter superfamily (Monaco, J. J., S. Cho, and M. Attaya. 1990. Science [Wash. DC]. 250:1723). These results suggest that the system that delivers antigenic peptides from the cytosol to the ER in RMA-S may still be present and retain partial function.

Split tolerance induced by the intrathymic adoptive transfer of thymocyte stem cells.

The intrathymic transfer of semiallogeneic CD4/CD8 double-negative (DN) thymocyte stem cells into irradiated host mice resulted in a transient state of chimerism in adoptive host thymus, spleen, and lymph nodes. Host-derived T cells, isolated from the thymus and periphery of the chimeric mice, were found to be specifically nonresponsive to the MHC antigens of the semiallogeneic DN donor in cytotoxicity assays. This nonresponsiveness was not permanent, but persisted as long as appreciable numbers of Thy-1 alloantigen-positive progeny of the DN donor cells could be detected in the spleen and lymph nodes of adoptive host mice. FACS sorting of DN donor cells before intrathymic transfer indicated that nonresponsiveness could be induced by Thy-1+ cells and was therefore not attributable to contaminating thymic macrophages, dendritic cells, or B cells. When FACS-sorted Thy-1+ (bm5 x bm12)F1 DN cells were transferred intrathymically into C57BL/6 hosts, nonresponsiveness to DN donor MHC class I but not class II alloantigen (split tolerance) was observed. These experiments were repeated using FACS-sorted Thy-1+ DN donor cells that were semiallogeneic to the irradiated adoptive host at either MHC class I or class II locus with similar results. Limiting dilution analysis showed that host-derived CTL precursors were tolerant of DN donor MHC class I alloantigen and no evidence for the involvement of suppressor T cells was found. The data indicate that murine thymocytes themselves are capable of tolerizing to MHC class I but not class II alloantigen after intrathymic transfer. The implications for intrathymic T cell differentiation and maintenance of self tolerance are discussed.

Helper T cells for cytotoxic T lymphocytes need not be I region restricted.

We investigated the antigenic requirements for restimulation of H-2- restricted cytolytic T lymphocytes (CTL) in vitro to determine whether H-2 I region-restricted helper T cells are required in these responses. In one set of experiments, we studied the in vitro response of (responder x nonresponder)F(1) female T cells to the male antigen H-Y. We chose to examine this response because it has been suggested that the defect in nonresponder strains is a failure of helper T cells to recognize H-Y in association with nonresponder I region determinants. However, we find that nonresponder male stimulator cells are as effective as F(1) male stimulator cells at inducing H-Y-specific CTL responses. This finding calls into question reports that secondary CTL responses to H-Y are dependent upon the activation of H-Y- specific helper T cells restricted to responder type I region determinants. In a second set of experiments, we examined the requirements for restimulation of H-2-restricted T cells specific for minor-histocompatibility antigens from long-term mixed lymphocyte cultures. These cultures were established by repeatedly restimulating cultures of specific T cells with H- 2-matched stimulator cells expressing foreign minor histocompatibility antigens. We found that H-2D-restricted T ceils, including CTL, could be restimulated with cells that were matched with the responding cells at only the D region genes. This response did not appear to result from positive allogeneic effects or from antigen processing and "representation" by responder type APC that might contaminate the cultures. Thus, we find no evidence for a requirement for I region-restricted helper T cells in these CTL responses. However, helper T cells are required because we find that CTL lines derived by limit-dilution cloning from these long-term MLC are absolutely dependent upon exogenous helper factors for growth. The most simple interpretation of these results is that the helper cells are restricted to H-2 antigens other than I region antigens or to antigens that code outside of the H-2 complex. Finally, we show that factor-dependent CTL lines must recognize their specific antigen to proliferate, even in the presence of exogenous factors. The requirement of activated CTL for antigen to proliferate provides an explanation for how specific CTL can be selectively enriched in MLC by specific antigen stimulation. Furthermore, it is at variance with reports that memory CTL or activated CTL require only interleukin 2 for restimulation.

H-2 antigens of the thymus determine lymphocyte specificity.

After immunization, normal H-2 heterozygous mice (for example H-2(b) x H-2(d)) generate two populations of cytotoxic effector T cells, one specific for target cells expressing H-2(b)-plus-antigen and the other specific for H- 2(d)-plus-antigen. With a multideterminant antigen, these two populations have about the same activity. We show here that the H-2 type of resident cells in the thymus determines the H-2 preference of cytotoxic T lymphocytes. F(1)(B 10 x B 10.D2) (H-2(b) x H-2 (d)) mice were thymectomized, lethally irradiated, and reconstituted with T-cell-depleted syngeneic hematopoietic cells. Groups of such ATXBM mice were grafted subcutaneously with neonatal thymus lobes from parental mice, either B10 (H-2 (b)) or B10.D2 (H-2(d)). 2-3 mo later, the mice were immunized against the minor histocompatibility antigens on F(1)(BALB/c x BALB.B) cells and assayed for cytotoxic T-cell activity. H-2(b) x H-2(d) ATXBM mice with H-2(b) thymus grafts responded to antigen-plus-H-2(b) much better than to antigen-plus-H-2(d), and vice versa for the mice with H-2(d) thymus grafts. As judged by antiserum treatment, the effector cells were of F(1) origin. To explore the possibility that the "thymus preference" may have been due to suppression of T-cell activity, nonimmune spleen and lymph node cells from normal H-2(b) x H-2(d) mice and cells from H-2(b) x H-2(d) mice bearing a homozygous thymus were mixed 1:1 and immunized in adoptive transfer. The mixture responded to antigen-plus-H-2(b) and antigen-plus-H-2(d) equally well, demonstrating that the cells that showed a "thymus preference" could not suppress a response to antigen in association with the nonthymic H-2 type. We conclude from these and other experiments that H-2 antigens present on resident cells of the thymus determine the spectrum of specificity of T cells which mature in that thymus and eventually make up the peripheral T- cell pool.

The effect of 2-mercaptoethanol on murine mixed lymphocyte cultures.

Mouse spleen cells which have been depleted of adherent cells do not respond to allogeneic lymphocytes in vitro. Their cytotoxic response can be restored by inclusion of mercaptoethanol in the medium. Mercaptoethanol is shown to have a stimulatory effect also on the response of normal (unseparated) spleen cells to alloantigens. The enhancement of the DNA-synthetic and cytotoxic response is similar, varying from 3.5-15-fold. Cytotoxic cells also appear in unmixed lymphocyte cultures in the presence of mercaptoethanol and fetal calf serum. The specificity of these background cytotoxic cells is not known.

Antigen recognition by cloned cytotoxic T lymphocytes follows rules predicted by the altered-self hypothesis.

Radiation chimeras prepared by injecting H-2 heterozygous F1 stem cells into lethally irradiated parental hosts show a marked, but not absolute, preference for host-type H-2 antigens in the H-2-restricted cytotoxic T lymphocyte (CTL) response to minor histocompatibility (minor H) antigens. We have selected for the anti-minor HCTL that are restricted to the parental H-2 type absent from the chimeric host and found that in two out of eight cases, such CTL lysed target cells of either parental H-2 type. From one of these CTL populations that lysed H-2d and H-2k target cells expressing BALB minor H antigens, clones were derived and further analyzed. The results showed that: (a) lysis of both H-2d and H-2k target cells was H-2 restricted; (b) H-2d restriction mapped to Dd, and H-2k restriction mapped to Kk; (c) testing against various H-2d and H-2k strains of different and partially overlapping minor H backgrounds as well as against the appropriate F1 crosses revealed that in Dd- and Kk-restricted killing, different minor H antigens were recognized. In a second system, a CTL population was selected from normal (H-2d x H-2k)F1 mice that was specific for H-2d plus minor H antigens and for H-2k plus trinitrophenylated bovine serum albumin. We interpret these findings in terms of the altered-self hypothesis: The association of one H-2 antigen with one conventional antigen X may be recognized by the same T cell receptor specific for the complex formed by a different H-2 antigen in association with a second conventional antigen Y. The implications of these observations for the influence of self H-2 on the generation of the T cell receptor repertoire are discussed.

High level expression of CD43 inhibits T cell receptor/CD3-mediated apoptosis.

In a screen designed to identify genes that regulate T cell receptor (TCR)/CD3-mediated apoptosis, we found that high level expression of CD43 protected T cell hybridomas from activation-induced cell death. The protection appears to result from its capacity to block Fas-mediated death signals rather than from inhibition of the upregulation of Fas and/or Fas ligand after T cell stimulation. We found that peripheral CD4(+) T cells can be divided into two subsets based on the level of CD43 surface expression. The CD4(+)CD43(low) subset exhibits a naive T cell phenotype, being CD62L(high)CD45RB(high)CD44(low), whereas CD4(+)CD43(high) cells exhibit a memory phenotype, being CD62L(low)CD45RB(low)CD44(high). Recent studies have demonstrated that engagement of TCR and Fas induces naive CD4(+) T cells to undergo apoptosis, and the same treatment enhances the proliferation of memory CD4(+) T cells. We confirm here that peripheral CD4(+)CD43(high) T cells are resistant to TCR/CD3-mediated cell death. These results suggest that the expression levels of CD43 on naive and memory CD4(+) T cells determine their susceptibility to Fas-dependent cell death and that high level expression of CD43 may be used as a marker to define CD4(+) memory T cells. Expression of CD43 provides a novel mechanism by which tumor cells expressing abnormally high levels of CD43 may escape Fas-mediated killing.

Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization.

CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.

Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo.

MHC class I-restricted T lymphocyte responses are usually directed to cellular antigenic components resulting from endogenous gene expression. Exogenous, non-replicating antigens, such as soluble proteins, usually fail to enter the class I pathway of antigen processing and presentation. Consistent with this notion, we have recently shown that soluble, exogenous proteins can be efficiently processed for class I presentation in vitro only if they are introduced directly into the target cell cytoplasm. In this report we extend this work to the in vivo situation by introducing soluble protein into the cytoplasm of mouse splenocytes via the osmotic lysis of pinosomes and then using these cells for in vivo immunization. Our results show that cytoplasmic loading of OVA and beta-GAL into H-2b and H-2d splenocytes respectively, resulted in effective in vivo immunogens for class I-restricted CTL. To our surprise, control spleen cell preparations simply incubated with the exogenous, native protein for 10 min at 37 degrees C in isotonic medium and then washed could also induce a comparable class I-restricted CTL response following intravenous injection. Experiments using (H-2b X H-2d)F1 mice showed that protein pulsed splenocytes from one parental strain could effectively "cross prime" T cells restricted to the MHC of the other parental strain. In all cases, target cell recognition by the effector CTL generated by immunization with spleen cell-associated antigen required the antigen to be present in the cell cytoplasm. Thus the CTL do not recognize target cells exposed to soluble, exogenous antigen. These results, reminiscent of analogous experiments with cross priming by minor histocompatibility antigens, argue that class I-restricted processing and presentation of exogenous antigen can occur in vivo following immunization with cell-associated antigen.

Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation.

In a depleted lymphoid compartment, naive T cells begin a slow proliferation that is independent of cognate antigen yet requires recognition of major histocompatibility complex-bound self-peptides. We have followed the phenotypic and functional changes that occur when naive CD8(+) T cells undergo this type of expansion in a lymphopenic environment. Naive T cells undergoing homeostasis-driven proliferation convert to a phenotypic and functional state similar to that of memory T cells, yet distinct from antigen-activated effector T cells. Naive T cells dividing in a lymphopenic host upregulate CD44, CD122 (interleukin 2 receptor beta) and Ly6C expression, acquire the ability to rapidly secrete interferon gamma, and become cytotoxic effectors when stimulated with cognate antigen. The conversion of naive T cells to cells masquerading as memory cells in response to a homeostatic signal does not represent an irreversible differentiation. Once the cellularity of the lymphoid compartment is restored and the T cells cease their division, they regain the functional and phenotypic characteristics of naive T cells. Thus, homeostasis-driven proliferation provides a thymus-independent mechanism for restoration of the naive compartment after a loss of T cells.

Requirements for bone marrow-derived antigen-presenting cells in priming cytotoxic T cell responses to intracellular pathogens.

Bone marrow (BM)-derived antigen-presenting cells (APCs) are potent stimulators of T cell immune responses. We investigated the requirements for antigen presentation by these cells in priming cytotoxic T lymphocyte (CTL) responses to intracellular bacterial and viral pathogens. [Parent-->F(1)] radiation BM chimeras were constructed using C57BL/6 donors and (C57BL/6 x BALB/c)F(1) recipients. Infection of chimeric mice with either Listeria monocytogenes or vaccinia virus expressing the nucleoprotein (NP) antigen from lymphocytic choriomeningitis virus (LCMV) primed H2-D(b)-restricted, but not H2-K(d)-restricted CTL responses, demonstrating the requirement for BM-derived APCs for successful priming of CTL responses to these pathogens. Surprisingly, this did not hold true for chimeric mice infected with LCMV itself. LCMV-infected animals developed strong CTL responses specific for both H2-D(b)- and H2-L(d)-restricted NP epitopes. These findings indicate that in vivo priming of CTL responses to LCMV is remarkably insensitive to deficiencies in antigen presentation by professional BM-derived APCs.