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Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.

Splenic T cells, engaged in an in-vivo "response" to transferred syngeneic lymphoblasts, displayed an extensive proliferative activity if cultured in vitro. These cells (blocked by mitomycin C) were "stimulator" cells in a one-way syngeneic mixed lymphocyte culture (S-MLC). "Responder" cells were derived form spleens of syngeneic (sex and age matched) mice. Fractionation studies showed that "stimulator" and "responder" cells in the investigated S-MLC were nylon-wool non-adherent and sensitive to treatment with anti-Thy-1 antiserum and complement, i.e. were cells of the T lineage. Coculture experiments demonstrated that direct cell contact is required to trigger a proliferative "response" in the S-MLC. Fetal calf serum-supplemented tissue-culture medium supported "responder" cell proliferation, normal mouse serum-supplemented medium did not. The in-vivo generation of a T-cell population, which could "stimulate" a syngeneic T-cell "response" in the S-MLC in vitro, was investigated.

A number of different modified exogenous protein antigens can efficiently prime CTL responses. The biological significance of this for priming of normal CTL responses is unknown. Processing of exogenous antigen for MHC-I presentation seems an attractive possibility to bypass the necessity that all foreign antigens have to be translated within the cytosol of professional APC in order to prime CTL responses. Many microbes have a select host cell specificity that does not include APC. They kill their host cells or severely disturb the sophisticated cellular functions that are required in APC (84xNorkin, L.C. Clin. Microbiol. Rev. 1995; 8: 293–315PubMedSee all References, 45xHaywood, A.M. J. Virol. 1994; 68: 1–5PubMedSee all References). Another scenario for a virus-specific CTL response is thus that infected cells disintegrate (possibly as apoptotic bodies) and exogenous antigen from them is transferred to APC for further processing in different cellular compartments.There are many unresolved issues regarding processing of exogenous antigens for MHC-I presentation and the priming of CTL responses. First, which are the most important uptake mechanisms? Second, is there a special (MHC-II-like) compartment for “endosomal” processing, or do all APC “leak” antigen into the cytosol and thereby into the classical MHC-I processing pathway? Third, in a proposed endosomal-like compartment, which are the proteolytic enzymes involved in processing? Do they generate a spectrum of MHC-I-bound peptides distinct from those generated in the cytosolic pathway? Finally, how do MHC-I molecules gain access to the endosomal-like compartment, and how are trimeric peptide MHC-I–β2-microglobulin complexes transported to the cell surface and their membrane expression regulated?If exogenous antigens are instrumental in priming normal CTL responses, processing in noncytosolic, vesicular compartments, distinct from the endogenous classical class I processing pathway, might be important. If so, it may be relevant to reevaluate the endogenous/MHC-I and exogenous/MHC-II processing dogma and instead classify pathways in functional terms. In CTL activation, induction (by exogenous antigen) and tar- get cell recognition (by endogenous antigen) by class I–restricted peptide presentation would result from different processing pathways. This is an important issue in terms of vaccine development, as these should primarily be expressed in the most optimal processing compartment. If exogenous antigens are of major importance in generating CTL responses, vaccines based on live, replicating vectors (and nucleic acids) induce their protective effect in a nonviable form. In this transfer process, from the infected host cell to the professional APC cell, there might be a lesson to be learned in terms of formulations for direct uptake into APC cells. The potent clinical effect of the first human recombinant vaccine licensed for clinical use, which was based on self-assembly of the hepatitis B virus small surface antigen into 22 nm subviral particles, might be a “lesson of nature” in this context (Schirmbeck et al. 1996xSchirmbeck, R, Bohm, W, and Reimann, J. Intervirology, in press. 1996; See all ReferencesSchirmbeck et al. 1996).

The intravenous injection of polyclonally activated lymphoblasts elicited a proliferative T cell reaction in the spleens of syngeneic recipient mice. In the non-fractionated cell populations obtained from these spleens 6 days after lymphoblast transfer, the LPS-induced proliferation and differentiation of B cells in vitro was suppressed. This suppressive effect was mediated by T cells, as i) treatment with anti-Thy-1 antiserum plus complement restored responsiveness of B cells to LPS in the spleen cell population that had responded in vivo to a syngeneic lymphoblast graft, and ii) the responsiveness of B cells to LPS was not impaired in non-fractionated spleen cell populations of nu/nu mice injected with syngeneic lymphoblasts. The relationship of this nonspecific T suppressor cell activity to the previously described nonspecific T helper cell activity for B cell activation is discussed.

Alloreactive T cells recognize polymorphic nonself MHC determinants that are expressed by different members of the same species. They are detected in specifically stimulated cytotoxic/proliferative responses in vitro (allogeneic mixed leukocyte reaction, allo MLR), and in the graft-versus-host (GvH) or host-versus-graft (HvG) reactions of various transplantation systems in vivo. These primary cell-mediated immune responses are easier to induce and are apparently of a different nature than MHC-restricted T-cell-mediated immune responses to conventional antigens or pathogens. This has always intrigued immunologists (and frustrated transplantation activists). Attempts to explain this preponderance of the T-cell reactivity to allogeneic MHC determinants focused either on a particular “antigenic nature” of MHC-encoded glycoproteins, or assumed a large pool of precursor T cells in normal individuals that specifically recognized allogeneic MHC antigens.

Polyclonally activated splenic lymphocytes (generated in mitogen-stimulated cultures) were transferred to syngeneic or autologous recipient mice. Injection of cells into the footpad of syngeneic recipients induced a regional response in the ipsilateral popliteal lymph node; intravenous cell transfer elicited a systemic splenomegaly reaction. These reactions displayed a linear log number of transferred cells/response relationship in syngeneic and autologous systems. The kinetic and magnitude of the regional response to syngeneic lymphoblasts and to allogeneic spleen cells were comparable. No difference was apparent in the phenomenology of the in-vivo responses to syngeneic lymphoblasts induced by various T- or B-cell mitogens. The in-vivo response was: 1. stimulated by large-size lymphoblasts; and 2. mediated by host T cells. Data excluded a direct involvement of mitogen or heterologous serum constituents in tissue culture medium in the described reaction. Experimental evidence argues against the involvement of virus components in the observed phenomenon.

The function of major histocompatibility complex (MHC) molecules remains elusive. Our detailed knowledge of their structural (genetic and biochemical) features contrasts sharply with our essentially speculative ideas concerning their function(s), which furthermore seem at present to be locked into sets of mutually exclusive hypotheses.

The intravenous transfer of polyclonally activated lymphoblasts elicited a proliferative response of host-derived Lyt1 T cells in the spleens of syngeneic recipient mice. These Lyt1 T cells (responding in vivo to a syngeneic lymphoblast graft) stimulated proliferation of resting, syngeneic splenic Lyt1 T cells, derived from uninjected mice, in a one-way syngeneic mixed lymphocyte culture in vitro. The data are discussed in the context of T cell activation induced by lymphoblastogenesis in a syngeneic system.

A: Development of T Lymphocytes in the Thymus.- Introductory Remarks.- Development of T Lymphocytes Within the Thymus and Within Thymic Nurse Cells. With 3 Figures.- Thymus Development.- Major Histocompatibility-Restricted Cytolytic T-Lymphocyte Precursors from the Thymus of In Vivo Primed Mice: Increased Frequency and Resistance to Anti-Lyt-2 Antibody Inhibition. With 1 Figure.- Thymic Stem Cells: Their Interaction with the Thymic Stroma and Tolerance Induction.- B: Murine T-Cell Receptor Genes.- Organization, Rearrangement, and Diversification of Mouse T-Cell Receptor Genes. With 1 Figure.- Expression of T-Cell Receptor by a Mouse Monoclonal Antigen-Specific Suppressor T-Cell Line.- Somatic Variation of Antigen-Recognition Specificity in H-2b-TNP-Specific Cytotoxic T-Cell Clones.- The Change of Specificity, Karyotype, and Antigen-Receptor Gene Expression is Correlated in Cytotoxic T-Cell Lines.- C: Phenotype and Functional Potential of T-Cell Clones.- Introductory Remarks.- A Study of the Functional Potential of Mouse T-Cell Clones.- Generation, Propagation, and Variation in Cloned, Antigen-Specific, Ia-Restricted Cytolytic T-Cell Lines.- Significance of T4 or T8 Phenotype of Human Cytotoxic T-Lymphocyte Clones. With 1 Figure.- Natural and Unnatural Killing by Cytolytic T Lymphocytes. With 2 Figures.- Acquisition of Suppressive and Natural Killer-Like Activities Associated with Loss of Alloreactivity in Human "Helper" T-Lymphocyte Clones. With 3 Figures.- Expression and Function of Class II I-Ak Antigens on an Antigen-Specific T-Suppressor Cell Clone. With 2 Figures.- D: Signal Requirements for T-Cell Activation.- Introductory Remarks.- Heterogeneity of the Signal Requirements During the Primary Activation of Resting Lyt-2+ Cytotoxic T-Lymphocyte (CTL) Precursors into Clonally Developing CTL. With 2 Figures.- Regulation of Lytic Function by Recombinant IL2 and Antigen. With 4 Figures.- The Target Structure for T11: A Cell Interaction Molecule Involved in T-Cell Activation? With 3 Figures.- Antigen- and Lectin- Sensitized Murine Cytolytic T Lymphocyte- Precursors Require Both Interleukin 2 and Endogenously Produced Immune (?) Interferon for Their Growth and Differentiation. With 3 Figures.- Activation Requirements for Resting T Lymphocytes.- E: Self-Nonself Discrimination in the T-Cell Compartment.- Introductory Remarks.- Functional Clonal Deletion and Suppression as Complementary Mechanisms in T Lymphocyte Tolerance.- Human T Cell Clones, Tolerance, and the Analysis of Autoimmunity. With 1 Figure.- Antiself Suppressive (Veto) Activity of Responder Cells in Mixed Lymphocyte Cultures. With 6 Figures.- Analysis of T Suppressor Cell Mediated Tumor Escape Mechanisms. With 2 Figures.- The T-Cell Receptor Recognizes Nominal and Self Antigen Independently. A Theoretical Alternative to the Modified Self Concept. With 3 Figures.- F: T-Cell-Mediated Autoreactivity.- Introductory Remarks.- T-Cell Reactivity to Polymorphic MHC Determinants. I. MHC-Guided T-Cell Reactivity.- T-Cell Reactivity to Polymorphic MHC Determinants. II. Self-Reactive and Self-Restricted T Cells. With 6 Figures.- T-Cell Reactivity to Polymorphic MHC Determinants. III. Alloreactive and Allorestricted T Cells. With 9 Figures.- Appearance of New Specificities in Lectin-Induced T-Cell Clones Obtained from Limiting Dilution T-Cell Cultures. With 2 Figures.- Syngeneic Cytotoxicity and Veto Activity in Thymic Lymphoid Colonies and Their Expanded Progeny. With 4 Figures.- Functional Analysis of a Self-I-A Reactive T-Cell Clone Which Preferentially Stimulates Activated B Cells.- Indexed in Current Contents.

A recombinant retroviral vector with an inserted bacterial neomycin resistance (neo) gene was used to transfer in vitro neomycin resistance (neoR) into murine cytotoxic lymphocyte precursors (CLP). The infection protocol involved co-cultivation of mitogen-activated splenic T-blasts with irradiated cells that produced either the recombinant retrovirus plus a helper virus, or exclusively the recombinant retrovirus. Infected T-blasts were subsequently cultured under limiting dilution (LD) conditions that supported clonal in vitro development of a large fraction of murine CLP. In infected T-blast populations, frequency estimates were obtained for CLP that developed into functional cytotoxic T-lymphocyte (CTL) populations under G418-selected or non-selected conditions; from these frequency estimates, an efficiency of transduction of the neoR phenotype into murine CLP of 2-8% was calculated. Some conditions were defined that influenced transduction efficiency, i.e., the density of the infecting monolayer cells; the presence of interleukin 2-containing conditioned medium and mitogenic lectins during the co-culture period; a delayed onset of G418 selection after infection. It was demonstrated that the neoR phenotype of functional CTL populations derived from infected CLP resulted from expressed recombinant retrovirus.

Nylon wool-nonadherent spleen cells from three inbred mouse strains of H-2k (CBA), H-2d (BALB/c) and H-2b (C57BL/6) haplotype were co-cultured with 2,4,6- trinitro- phenyl (TNP)- modified or nonmodified allogeneic stimulator cells in a limiting dilution system. Using a recently described restimulation protocol, a surprisingly large number of splenic cytotoxic lymphocyte precursors (CLP) was clonally expanded in this primary in vitro response to allo - H-2 plus TNP determinants; measured CLP frequencies ranged from 1/30 to 1/300. The lytic specificity patterns of individual microcultures (selected for a high probability of clonality) were defined by split well analysis, and were furthermore followed up in time by sequentially reassaying microcultures at different time points of in vitro incubation. This analysis revealed the following: (a) a large fraction of cytotoxic T lymphocyte clones lysed TNP-modified but not nonmodified all ogeneic concanavalin A blast targets, i.e., were alloiestricted; (b) this was found in all 6 allogeneic strain combinations set up with b, k and d haplotype mice; (c) allorestricted lytic patterns predominated in microcultures with low numbers of responder cells per well, and at late time points of in vitro culture; (d) allorestricted lytic cultures were specific for the stimulating allogeneic H-2 plus T NP determinant(s); and (e) allorestricted lytic patterns were also found in microcultures stimulated by nonmodified allogeneic cells. To our knowledge, these are the highest CLP frequencies yet reported in limiting dilution systems that used a specific (re)stimulation protocol and measured the lytic responses obtained in a specificity- controlled readout.

We find rapid changes in the specificity of the cytolytic effector cells in a mixed lymphocyte culture. The lysis patterns produced by cytolytic effector cells generated near limiting dilution in murine mixed lymphocyte reactions of three types, F 1 anti-parent (F 1 (A × B) anti-A), allogeneic (C anti-F 1 (A × B)), and F 1 anti-modified parent (F 1 (A × B) anti-A-TNP), were investigated. Cultures were characterized by their ability or inability to lyse a panel of target cells (e.g., A, B, F 1 ). When individual cultures were tested at two different times, changes in lytic pattern were routinely seen, with some patterns reproducibly increasing in frequency and others reproducibly decreasing (e.g., patterns involving lysis of F 1 decreased in an F 1 anti-A response but increased in a C anti-F 1 response). X-linked isoenzyme analysis showed that changes can occur within a single clone of effector cells. These results imply that the T cell specificity repertoire continues to evolve during an ongoing immune response, a conclusion incompatible with clonal selection theory.

Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.

As revealed by flow cytometric analysis, about 30% of nylon wool nonadherent Lyt-2+ B6 spleen cells were F23+, i.e., were stained with the monoclonal antibody F23.1 directed against an allotypic T-cell receptor determinant. The specificity repertoire of splenic Lyt-2+/F23+ cytotoxic lymphocyte precursors (CLP) from B6 mice was investigated in a limiting dilution (LD) system designed to support clonal expansion in vitro of a representative fraction of this T-cell subset: in highly purified Lyt-2+ responder cells cocultured with mitomycin-treated F23 hybridoma cells in the presence of (recombinant) interleukin 2 under LD conditions, one out of three Lyt-2+/F23+ CLP gave rise to a functional cytotoxic T lymphocyte (CTL) clone. The split-well analysis of individual CTL populations demonstrated a clear-cut segregation of the lytic reactivities toward different allogeneic Con A blast targets. A large fraction of B6-derived CTL clones (3-10%) specifically lysed fully H-2 allogeneic (H-2k, H-2d), or H-2K mutant (bm1) targets. Self-reactive and allorestricted lytic patterns were not found.

Fully H-2-incompatible chimeric mice were constructed by grafting lethally (950 rad) irradiated germ-free (GF) CBA (H2k) mice with anti-Thy 1 antibody plus complement-treated allogeneic C57Bl/6 (B6) (H2b) bone marrow cells. These chimeric mice were kept for more than 11 months, either under GF conditions or under barrier-sustained specific-pathogen-free (SPF) conditions. Controls included nonirradiated, nontransplanted, sex- and age-matched CBA and B6 mice raised under SPF conditions, and syngeneic chimeric mice of the CBA----CBA type kept under GF and SPF conditions. All chimeric mice were completely repopulated with donor-type lymphoid cells and showed no clinical or histological evidence of graft-versus-host disease. From the fully allogeneic chimeric mice, we enumerated the numbers of splenic cytotoxic T lymphocyte precursors (CTL-p) that could be clonally expanded under limiting dilution conditions in response to third-party alloantigens, or nonmodified and trinitrophenyl (TNP)-modified stimulator cells bearing host or donor H-2 antigens. The existence of high numbers of alloreactive and host- or donor-type H-2-restricted TNP-specific CTL-p in the spleens of fully allogeneic chimeras indicated almost normal immunocompetence. The surprising finding, however, was that large numbers of host (CBA)-reactive splenic CTL-p were inducible under limiting dilution conditions in healthy long-lived allogeneic chimeras, although these chimeric mice were devoid of any histological or clinical signs of graft-versus-host disease.

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.

The recently described cytokines IL-19, IL-20, and IL-24 share structural homology with IL-10 and are therefore classified as members of the IL-10 family of cytokines. Although it has long been speculated that signaling by their heterodimeric receptor complexes (IL-20R1/IL-20R2 and IL-22R/IL-20R2) influences immunological processes, the target cells for this group of cytokines are still unclear. By generating a knockout mouse strain deficient for the common IL-20R β-chain (IL-20R2), we show that IFN-γ and IL-2 secretion is significantly elevated after stimulation of IL-20R2−/−-deficient CD8 and CD4 T cells with Con A or anti-CD3/CD28 in vitro. IL-10 secretion by activated IL-20R2−/− CD4 cells was diminished. Consistent with our in vitro results, significantly more Ag-specific CD8 IFN-γ+ and CD4 IFN-γ+ T cells developed to locally applied DNA vaccines in IL-20R2-deficient mice. In a T cell-dependent model of contact hypersensitivity, IL-20R2 knockout mice were more sensitive to the contact allergen trinitro-chloro-benzene. Thus, IL-20R2 signaling directly regulates CD8 and CD4 T cell answers in vitro and in vivo. For the first time, we provide evidence that IL-19, IL-20, and IL-24 are part of a signaling network that normally down-modulates T cell responses in mice.

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8(+) T cell responses in IFN-beta (IFN-beta(-/-))- or type I IFN receptor (IFNAR(-/-))-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR(-/-) or IFN-beta(-/-) mice have normal numbers of CD4(+) and CD8(+) T cells, and CD25(+)FoxP3(+) T regulatory (T(R)) cells in liver and spleen. Twice as many CD8(+) T cells specific for different class I-restricted epitopes develop in IFNAR(-/-) or IFN-beta(-/-) mice than in normal animals after peptide- or DNA-based vaccination. IFN-gamma and TNF-alpha production and clonal expansion of specific CD8(+) T cells from normal and knockout mice are similar. CD25(+)FoxP3(+) T(R) cells down-modulate vaccine-primed CD8(+) T cell responses in normal, IFNAR(-/-), or IFN-beta(-/-) mice to a comparable extent. Low IFN-alpha or IFN-beta doses (500-10(3) U/mouse) down-modulate CD8(+) T cells priming in vivo. IFNAR- and IFN-beta-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4(+) T cells after polyclonal or specific stimulation in vitro or in vivo. CD8(+) T cell responses are thus subjected to negative control by both CD25(+)FoxP3(+) T(R) cells and CD4(+)IL-10(+) T(R1) cells, but only development of the latter T(R) cells depends on type I IFN.