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Memory CD8+ T cells require stronger TCR stimulation than naive cells to enter cell cycle due to reduced Zap70 activation and increased levels of protein tyrosine phosphatases., A hallmark of immunological memory is the ability of previously primed T cells to undergo rapid recall responses upon antigen reencounter. Classic work has suggested that memory T cells proliferate in response to lower doses of antigen than naive T cells and with reduced requirements for co-stimulation. In contrast to this premise, we observed that naive but not memory T cells proliferate in vivo in response to limited antigen presentation. To reconcile these observations, we tested the antigen threshold requirement for cell cycle entry in naive and central memory CD8+ T cells. Although both naive and memory T cells detect low dose antigen, only naive T cells activate cell cycle effectors. Direct comparison of TCR signaling on a single cell basis indicated that central memory T cells do not activate Zap70, induce cMyc expression, or degrade p27 in response to antigen levels that activate these functions in naive T cells. The reduced sensitivity of memory T cells may result from both decreased surface TCR expression and increased expression of protein tyrosine phosphatases as compared with naive T cells. Our data describe a novel aspect of memory T cell antigen threshold sensitivity that may critically regulate recall expansion.

Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4(+) T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4(+) T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed approximately 1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.

CD8+ T cells only require a brief stimulation with antigen in vitro to divide and differentiate into effector and memory cells upon transfer in vivo. The efficiency of clonal expansion and the functional characteristics of memory cells derived from briefly stimulated cells are poorly defined. We developed a system that allowed us to examine programming entirely in vivo. This was achieved by rapidly killing peptide-pulsed DCs carrying a diphtheria toxin receptor transgene with timed injections of diphtheria toxin without altering the course of an accompanying infection. The magnitude of clonal expansion, but not the functionality of the effector cells, correlated directly with the duration of antigen exposure. Furthermore, memory T cells were capable of mounting a secondary response, regardless of the length of antigen encounter during the primary response. These results indicate that the duration of initial antigen encounter influences the magnitude of the primary response, but does not program responsiveness during the secondary challenge.

In experiments to study the impact of deficiency in CD4+ T cell help on the magnitude of CD8+ cytotoxic T cell response to pathogens, it was noted that in CD4 gene knockout mice, the CD8 population made significant responses to several nominally major histocompatibility complex (MHC) class II–restricted epitopes in addition to the expected responses to MHC class I–restricted epitopes. A similar response by CD8+ T cells to class II–restricted epitopes was not observed in wild-type mice, or in mice that had been acutely depleted of CD4+ T cells just before the immunization. Coincident with this unexpected response to class II–restricted epitopes, it was also observed that the CD8+ response to the class I–restricted epitopes was consistently lower in CD4−/− mice than in wild-type mice. Further experiments suggested that these two observations are linked and that the CD8 population in CD4−/− mice may contain a majority of T cells that were actually selected by recognition of MHC class II molecules in the thymus. These results have implications for understanding CD4 versus CD8 lineage commitment in the thymus, and for the practical use of CD4−/− mice as models of helper deficiency.

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 β) and Ly6C expression, acquire the ability to rapidly secrete interferon γ, 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.

A previous report showed that the proliferative response of helper T cells to class II major histocompatibility complex (MHC)-restricted antigens can be inhibited by analogues of the antigen, which act as T cell receptor (TCR) antagonists. Here we define and analyze peptide variants that antagonize various functions of class I MHC-restricted cytotoxic T lymphocyte (CTL) clones. Of 64 variants at individual TCR contact sites of the Kb-restricted octamer peptide ovalbumin257-264 (OVAp), a very high proportion (40%) antagonized lysis by three OVAp-specific CTL clones. This effect was highly clone specific, since many antagonists for one T cell clone have differential effects on another. We show that this inhibition of CTL function is not a result of T cell-T cell interaction, precluding veto-like phenomena as a mechanism for antagonism. Moreover, we present evidence for direct interaction between the TCR and antagonist-MHC complexes. In further analysis of the T cell response, we found that serine esterase release and cytokine production are susceptible to TCR antagonism similarly to lysis. Ca2+ flux, an early event in signaling, is also inhibited by antagonists but may be more resistant to the antagonist effect than downstream responses.

We have used an in vitro system to study the effects of major histocompatibility complex class I binding peptides on thymic development. Fetal thymus lobes from mice deficient in the class I light chain (beta 2 microglobulin or beta 2 M-/-) were cultured for 10 d in vitro, during which time T cell precursors develop into mature T cells. In these organ cultures, as in the adult or neonatal beta 2 M-/- thymus, CD8+ mature T cells did not develop, demonstrating that the mature T cells seen during early murine thymic development are the result of the positive selection process. To these cultures we added various class I binding peptides with or without a source of exogenous beta 2M. CD8+ T cells developed to various degrees only in the presence of beta 2M and peptides. Using peptide mixtures of differing complexity, we showed that the efficiency of this process is dependent more on peptide complexity than on peptide concentration. These data argue for a specific role for peptides in the process of positive selection. Furthermore, this culture system should be useful in identifying peptides that can promote positive selection of cells expressing a specific T cell receptor (TCR) in TCR transgenic mice.

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.

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.

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.

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.

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.

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.

The leukocyte common antigen, T200, is expressed on all white blood cells but not on other differentiated cells. Within the hematopoietic lineage, specific cell types display characteristic structural forms of the molecule on their surface. We show that murine cytotoxic T lymphocyte clones and helper T cell clones contain different size mRNA for this molecule, and that the early precursors of T200 glycoprotein made in the helper and cytotoxic T cells differ in Mr. Thus, in addition to differences in posttranslational modifications, it is highly likely that a difference in protein structure contributes to the distinct forms of T200 glycoprotein found on these functional T cell subsets.

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.

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.

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.