Direct cross-priming by th lymphocytes generates memory cytotoxic T cell responses

Under optimal Ag stimulation, CTL become functional effector and memory T cells. Professional APCs (pAPC) are considered essential for the activation of CTL, due to their unique capacity to provide costimulation and present exogenous Ags through MHC class I molecules. In this study, we report a novel means by which Th lymphocytes acquire and present MHC class I determinants to naive CTL. Although previous studies have looked at T cell Ag presentation to activated T cells, this study presents the first example of Ag presentation by Th cells to naive CTL. We report that activated Th cells can function as effective pAPC for CTL. Our results show that: 1) In addition to acquisition of cell surface molecules, including MHC class I/peptide complexes, from pAPC, Th cells can acquire and present MHC class I-binding peptides through TCR-MHC class II interactions with pAPC; 2) the acquired Ag can be functionally presented to CTL; and 3) Ag presentation by Th cells induces naive CTL to proliferate and preferentially differentiate into cells that phenotypically and functionally resemble central memory T cells. These findings suggest a novel role of Th cells as pAPC for the development of memory immune responses.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.     

Studies on the mechanism of suppression of primary cytotoxic responses by cloned cytotoxic T lymphocytes

We have shown in the accompanying companion paper that cloned cytotoxic T lymphocytes (CTL) can serve as veto cells in vitro, suppressing primary cytotoxic activity directed against antigens expressed by those cloned CTL but not against third party antigens. We now explore the mechanism of this antigen-specific suppression by cloned CTL, using as a model system the ability of G4, a BALB.B anti-H-2Dd CTL clone, to specifically suppress a primary in vitro anti-H-2b CTL response. G4 cells do not constitutively secrete a suppressor factor, because suppression cannot be mediated by supernatants removed from G4 cells at a time when they are routinely used as veto cells. Furthermore, medium removed from cultures suppressed by G4 will not suppress, indicating that the veto cell function of G4 is not mediated by soluble factors. Full suppression of primary anti-H-2b CTL responses requires that G4 be present throughout the 5-day mixed lymphocyte culture (MLC). Removal of G4 during the first 3 days of MLC results in a drastic reduction in the level of antigen-specific suppression, with a slight but reproducible loss of suppression after veto cell removal on day 4. The addition of G4 during the course of an ongoing MLC reveals that maximal suppression requires the presence of veto cells during the first 24 to 48 hr of culture. Thus, G4 cells must be present both early and late in an MLC to exert maximal veto cell suppression. Several experiments suggest that G4-induced veto cell activity is unlikely to be due to cytolysis of CTL precursors which are capable of recognizing G4. G4 cannot specifically recognize these CTL precursors, and G4 cells are inefficient at lectin-mediated lysis of non-tumor cell targets. Furthermore, we show that G4 cells cannot lyse CTL which recognize them. Finally, dilutions of anti-clonotypic antibodies which completely block both lectin-mediated and specific cytolysis by G4 do not block (and in fact enhance) G4-mediated veto cell activity.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

Differential inhibition of human antigen-specific T cell clone proliferative responses by distinct monoclonal anti-HLA-DR antibodies

The inhibition of diphtheria toxoid and varidase-specific T cell clones from a single DR 6/7 donor by eight distinct monoclonal anti-HLA-DR antibodies was tested in proliferative assays. These moAb were selected because they had been previously defined for their ability: 1) to react with Ia molecules; 2) to recognize similar or different epitopes; 3) to share or not share idiotypic specificities. Our results show a distinct inhibition pattern for each clone tested. Furthermore, the various moAb could be classified into three groups according to their inhibitory effect on T cell proliferation. These data suggest: a) an epitopic restriction by class II antigens of antigen-specific human T cell clone proliferation; and b) the recognition of functional epitopes on the human Ia-like antigens by some but not all moAb studied.     

A stochastic model of cytotoxic T cell responses

We have constructed a stochastic stage-structured model of the cytotoxic T lymphocyte (CTL) response to antigen and the maintenance of immunological memory. The model follows the dynamics of a viral infection and the stimulation, proliferation, and differentiation of na?ve CD8(+) T cells into effector CTL, which can eliminate virally infected cells. The model is capable of following the dynamics of multiple T cell clones, each with a T cell receptor represented by a digit string. MHC-viral peptide complexes are also represented by strings and a string match rule is used to compute the affinity of a T cell receptor for a viral epitope. The avidities of interactions are also computed by taking into consideration the density of MHC-viral peptides on the surface of an infected cell. Lastly, the model allows the probability of T cell stimulation to depend on avidity but also incorporates the notion of an antigen-independent programmed proliferative response. We compare the model to experimental data on the cytotoxic T cell response to lymphocytic choriomeningitis virus infections.     

Differential calcium response in HeLa and HeLa-Fas cells by cytotoxic T lymphocytes

We constructed a CD95 overexpressing HeLa cell line which was extremely sensitive towards CD95 mediated apoptosis. In these CD95 overexpressing cells, CD95 blocks the nuclear calcium signal induced by perforin positive and CD95 ligand positive killer cells. This phenomenon is highly relevant in states of inflammatory syndromes such as systemic inflammatory response syndrome (SIRS) and sepsis which are associated with a high probability to reactivate latent viruses due to a functional deficiency of cytotoxic effectors.     

MHC-unrestricted cytotoxic and proliferative responses of two distinct human gamma/delta T cell subsets to Daudi cells.

Human V gamma 9/V delta 2 T cells, the major subset of gamma/delta T cells in peripheral blood of adults, mediate proliferative and cytotoxic responses to Daudi Burkitt's lymphoma cells without previous in vitro exposure to Daudi. Our experiments show that some gamma/delta T cells coexpressing V gamma 9 and V delta 1 genes also react to Daudi cells in cytotoxic and proliferative assays. Expression of V gamma 9 is not sufficient for the recognition of Daudi cells because most gamma/delta T cells expressing V delta 1 paired with V gamma 9 or other V gamma genes neither kill Daudi cells nor proliferate to Daudi. V gamma 9/V delta 2 T cells do not proliferate to other cell lines such as K562 or Molt4 that are sensitive to MHC-unrestricted cytolysis by NK cells and by most IL-2-activated gamma/delta T cell clones. Cold target inhibition assays demonstrate that Daudi cells are stronger inhibitors than K562 and Molt4 of MHC-unrestricted lysis by V gamma 9/V delta 2 clones. However, cold Daudi cells are relatively weaker inhibitors of MHC-unrestricted lysis by NK cell clones, most gamma/delta T cell clones expressing V delta 1 and alpha/beta T cell clones. Thus, recognition by V gamma 9/V delta 2 T cells and certain V gamma 9/V delta 1 T cells of Daudi appears to involve a specific triggering pathway that is distinct from recognition by these gamma/delta T cells of Molt4, K562, and other target cells. NK cell clones and most other gamma/delta and alpha/beta T cell clones derived from the same normal volunteer blood donors do not show this specific interaction with Daudi cells. These data show that distinct subsets of human gamma/delta T cells recognize Daudi cells and support the idea that the gamma/delta TCR may be directly involved.     

Differential survival of cytotoxic T cells and memory cell precursors

It is widely assumed that the development of memory CD8 T cells requires the escape of CTLs from programmed cell death. We show in this study that although serine protease inhibitor 6 (Spi6) is required to protect clonal bursts of CTLs from granzyme B-induced programmed cell death, it is not required for the development of memory cells. This conclusion is reached because memory cell precursors down-regulate both Spi6 and granzyme B, unlike CTLs, and they do not require Spi6 for survival. These findings suggest that memory CD8 T cells are derived from progenitors that are refractory to self-inflicted damage, rather than derived from fully differentiated CTLs.     

Differential expression of asialo GM1 on alloreactive cytotoxic T lymphocytes and lymphokine-activated killer cells

The present study was undertaken to examine the differential expression of asialo GM1 (AsGM1) on the responding cells and effectors of alloreactive cytotoxic T lymphocytes (CTL) and lymphokine-induced activated killers (LAK). It was found that AsGM1 was expressed on the 3-day-cultured LAK effectors. Its expression gradually disappeared to the extent that AsGM1 became undetectable after 5 to 6 days of culturing. In contrast, AsGM1 was detected on 3-day CTL generated in mixed-lymphocyte cultures (bulk cultures); however, the levels of AsGM1 expression remained the same for at least 7 days. When examining the expression of AsGM1 on the responding cells, the reciprocal results were obtained. AsGM1 was expressed the LAK responders, but we were unable to demonstrate AsGM1 on CTL responders. Depletion of AsGM1+ cells from the responding population reduced subsequent CTL responses; however, CTL responses could be restored by adding conditioned media containing both interleukin 2 (IL-2) and other helper-T-cell factors and could not be restored by purified IL-2 alone adding at comparable doses. Reconstituting the AsGM1-depleted responders with Lyt-2-depleted splenocytes also restored the CTL response. Furthermore, depletion of AsGM1 cells from the responding population did not reduce the precursor frequency of allo-CTL, whereas the precursor frequency of LAK cells was reduced 42-fold. These findings show that the reduction of CTL responses after depletion of AsGM1+ cells was not due to the removal of precursors; instead, the defect appeared to be in the helper population. We further found that the helper defect was not due to impaired IL-2 production, because the endogenous production of IL-2 AsGM1-depleted responders was not reduced. Therefore, AsGM1+ cells may play a role in the helper pathway other than IL-2 production.     

Rotavirus-specific cytotoxic T lymphocytes cross-react with target cells infected with different rotavirus serotypes.

Splenocytes from adult C57BL6 (H-2b) mice orally inoculated with nonmurine rotaviruses lysed syngeneic rotavirus-infected target cells. Cytotoxic T lymphocytes (CTLs) were responsible for this cytotoxic activity. Cytotoxic activity was (i) detected 7 days after primary oral inoculation; (ii) not detected in uninoculated animals; (iii) specific for rotavirus-infected target cells; (iv) eliminated by treatment with Thy 1.2-specific immunoglobulin M and complement; and (v) restricted at H-2Db. In addition, rotavirus-specific CTLs cross-reacted with target cells infected with different human or animal rotavirus serotypes. Heterotypic protection against rotavirus challenge may be mediated by cross-reactive CTLs.     

Phenotypic and functional characterization of allospecific and nonspecific (NK- and K-like) cytotoxic T lymphocytes generated in human mixed-lymphocyte cultures from noncytotoxic precursors

Highly efficient cytotoxic cells able to lyse unsensitized, antibody-coated, and relevant targets were generated de novo following sensitization in allogeneic human mixed lymphocyte cultures (MLC) of noncytotoxic IgG-Fc receptor (FcR)-negative T lymphocytes. The MLC-induced killers maintained the surface markers of T cells, as demonstrated by their reactivity with two monoclonal anti-T-cell antibodies. None of them reacted with a monoclonal antibody specific for myelomonocytic cells and up to 30% expressed DR antigens. The nonspecific (NK-and K-like) effector cells had lower affinity for sheep erythrocytes than the allospecific killers and appeared to have low-affinity FcR as compared to freshly isolated NK and K cells. Unlike fresh NK cells, the MLC-induced NK-like cells were not sensitive to trypsin treatment. However, both fresh NK and MLC-induced NK-like cells were sensitive to interferon treatment and were completely inactivated after preincubation with unlabeled K562 cells at 37 °C for 4 hr. The latter observation suggests the possible use of this system to deplete NK-like effects from the MLC-generated killers and to discriminate between nonspecific and allospecific cytotoxic T lymphocytes.     

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development. We have shown that an idiotype-bearing, hapten-specific suppressor factor suppresses priming for CTL in an H-2-unrestricted but allotype-restricted manner. The implication of these studies to regulatory networks is discussed.     

Differential effect of DHEA on mitogen-induced proliferation of T and B lymphocytes

Dehydroepiandrosterone (DHEA) is the predominant steroid hormone secreted by adrenal gland, and it has been proposed in recent years that DHEA has significant effects on immune function. We investigated the effect of DHEA (1 x 10(-5) - 1 x 10(-8)M) on proliferation of human T cells and B cells and on immunoglobulin production, a representative function of B cells. High doses of DHEA (1 x 10(-5)) significantly inhibited proliferation of peripheral blood mononuclear cells (PBMCs) and T cells induced by T cell mitogens hemagglutinin (PHA) and concanavalin A (Con A). Proliferation of PBMCs induced by B cell mitogens pokeweed mitogen (PWM) was increased by 1 x 10(-7) - 1 x 10(-6)M DHEA. Proliferation of PBMCs and B cells induced by Staphylococcus aureus Cowan strain I (SAC) was not significantly changed at any concentrations of DHEA. However, a concentration of 1 x 10(-7)M DHEA tended to potentiate their proliferation. This study suggested that DHEA acted on T and B lymphocytes differentially in immune system.     

Enhancement of cytotoxic and proliferative responses of lymphocytes from melanoma patients by incubation with monoclonal antibodies against ganglioside GD3

Summary Previous studies have shown that monoclonal antibodies (M.Ab) to the ganglioside GD₃ may induce partial remissions in tumour growth in patients with melanoma. In vitro studies demonstrated that M.Abs to GD₃ may also enhance lymphocyte responses to phytohemagglutinin and interleukin 2 (IL2). The present study extended these findings by showing that the IL2-dependent proliferative and cytotoxic response of T cell clones derived from a melanoma patient and a patient with the Vogt-Koyanagi-Harada syndrome were enhanced by pre-incubation of T cells with M.Ab to GD₃. The degree of enhancement increased with the duration of pre-incubation from 2 to 24 h and applied to both T4⁺ and T8⁺ clones. The potentiation of these responses was not specific for M.Abs to GD₃ but was also seen with M.Abs to GD₂ and the T10 structure on T cells but not with M.Abs to the transferrin receptor or an isotype control M.Ab. Incubation of lymphocytes from a melanoma patient with M.Ab to GD₃ during culture with autologous melanoma cells enhanced the proliferative response to the tumour. The expression of IL2 receptors (Tac epitope) on the T cells showed variable enhancement after incubation with M.Ab to GD₃ but the significance of these findings in relation to the mechanism of the enhanced responses is not known. These results suggest that certain M.Abs may stimulate cell-mediated immune responses against tumour cells and that this may provide an additional mode of action of M.Abs against tumours in vivo     

Differential sensitivity of cytotoxic T lymphocytes and lymphokine-activated killer cells to inhibition by L-ornithine

The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/2 mixed leukocyte cultures (MLC) by the amino acid L-ornithine (Orn) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from 10-1000 U/ml and measuring cytolytic activity against two tumor targets (P815 and YAC-1) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CD8- and ASGM1+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2.