Stimulation of alloreactive cytotoxic T cells by paternal major histocompatibility antigens during murine pregnancy

In an effort to elucidate T cell reactivity toward paternal major histocompatibility (MHC) antigens during pregnancy, the ability of pregnant mice to develop alloreactive cytotoxic T lymphocytes (CTL) was studied in individual multiparous females mated with MHC congeneic strains of B10 background. Spleen cells obtained from B10.BR females mated to allogeneic males manifested strikingly higher CTL than those from animals mated to syngeneic males or from virgins; syngeneically mated animals were equivalent to virgin controls in CTL responses. The augmented CTL response in allogeneic pregnancy was detected not only by stimulation with the paternal MHC antigens but also by an unrelated MHC haplotype. However, this augmentation was found only during pregnancy in that 2-5 days after the delivery the CTL activity in allopregnant animals returned to a level comparable to that of virgin controls. No suppressor cells were detected at this stage. These observations suggest that maternal T cells recognize MHC disparity with the fetus in some way during pregnancy. Anti-MHC antibodies, immunoglobulin (Ig) M, and IgGs of all subclasses were not detected in these animals throughout multiple pregnancies.     

Limiting dilution analysis of alloreactive cytotoxic precursor cells in aging mice

The quantitative minimal estimate of the frequency of alloantigen specific cytotoxic T lymphocyte precursor cells (CTL-p) was determined in young and old C57BL/6 mice by limiting dilution analysis. Supernatant from phorbol myristate acetate-induced EL-4 cells was used as a source of IL 2 in these assays, which therefore were independent of the presence of the Lyt-2-, IL 2-producing cells known to be deficient in aging mice. These studies showed that 24-mo-old mice had approximately 10-fold fewer CTL-p than their young counterparts. Comparison of the limiting dilution assay (LDA) with IL 2-supplemented primary MLC shows that estimates of the frequency of CTL-p do not consistently agree with cytotoxic activity detected in the higher cell density primary MLC, and that the LDA is most likely a better estimate of the effect of age on the development of CTL.     

Efficient in vivo priming of specific cytotoxic T cell responses by neonatal dendritic cells

In early life, a high susceptibility to infectious diseases as well as a poor capacity to respond to vaccines are generally observed as compared with observations in adults. The mechanisms underlying immune immaturity have not been fully elucidated and could be due to the immaturity of the T/B cell responses and/or to a defect in the nature and quality of Ag presentation by the APC. This prompted us to phenotypically and functionally characterize early life murine dendritic cells (DC) purified from spleens of 7-day-old mice. We showed that neonatal CD11c(+) DC express levels of costimulatory molecules and MHC molecules similar to those of adult DC and are able to fully maturate after LPS activation. Furthermore, we demonstrated that neonatal DC can efficiently take up, process, and present Ag to T cells in vitro and induce specific CTL responses in vivo. Although a reduced number of these cells was observed in the spleen of neonatal mice as compared with adults, this study clearly shows that neonatal DC have full functional capacity and may well prime Ag-specific naive T cells in vivo.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.     

Induction of alloreactive cytotoxic T cells by acute virus infection of mice

Alloreactive cytotoxic T lymphocytes (CTL) distinct from virus-specific CTL and activated natural killer (NK) cells were generated during acute lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6J mice. The alloreactive CTL shared similar antigenic markers (Thy-1.2+, Lyt-2.2+, and asialo GM1-) with the virus-specific CTL that appeared at the same time 7 days postinfection, but had different target specificities. These alloreactive CTL lysed allogeneic but not syngeneic or xenogeneic targets. These were distinct from activated NK cells, which lysed all target cell types, peaked 3 days postinfection, and had a phenotype of asialo GM1+, Thy-1 +/-, Lyt-2.2-. Cold target competition studies indicated that there were several subsets of alloreactive T cells with distinct specificities, and that these alloreactive T cells were not subsets of the virus-specific T cells. Similar types of alloreactive CTL were induced at much lower levels in C3H/St mice. This may indicate that the generation of this "aberrant" T cell activity is under genetic control. Hence, the LCMV infection of C57BL/6J mice induces several cytotoxic effector populations including alloreactive CTL, activated NK cells, and virus-specific CTL. Virus infections therefore have the ability not only to polyclonally stimulate B cells, as previously described, but also to stimulate CTL.     

Alloantibodies prevent the induction of transplantation tolerance by enhancing alloreactive T cell priming

Circulating alloantibodies in transplant recipients are often associated with increased Ab-mediated as well as cellular rejection. We tested the hypothesis that alloantibodies facilitate cellular rejection by functioning as opsonins to enhance T cell activation using a BALB/c to C57BL/6 heart or skin transplant model. Long-term heart and skin survival induced with anti-CD154 alone or in combination with donor-specific transfusion (DST), respectively, was abrogated by the presence of anti-K(d) mAbs, and alloreactive T cell activation as well as acute rejection was observed. The prevention of graft acceptance in the skin model was dependent on anti-K(d) binding to and converting DST from tolerigenic to immunogenic. Adoptive transfer of CFSE-labeled TCR-transgenic T cells into B6 recipients treated with anti-CD154/DST revealed the ability of anti-K(d) to enhance the proliferation of anti-K(d)-specific T cells via the indirect pathway as well as of non-K(d)-reactive, recipient MHC-restricted CD4(+) and CD8(+) T cells. Thus, alloantibodies with restricted specificity are able to facilitate the indirect presentation as well as the cross-presentation of a larger repertoire of "linked" donor-derived Ags. These observations highlight the ability of alloantibodies to function not only in classical humoral rejection but also as opsonins that facilitate the CD40-CD154-independent activation of alloreactive T cells.     

Frequencies of alloreactive and self-restricted cytotoxic T cell precursors during lymphatic regeneration

The previous observation that during lymphatic regeneration precursors for MHC-restricted cytotoxic T cells appear before alloreactive precursors, was re-examined in macroculture and microculture experiments. Our macroculture experiments confirmed the observation that treatment with cyclophosphamide decreases the ratio of alloreactivity vs self-restricted reactivity. This shift in reactivity, however, did not result from a change in the T cell specificity repertoire, but was obviously mediated by regulatory effects in macrocultures. Spleen cells from cyclophosphamide-treated mice had normal ratios of CTL precursor frequencies for the three different specificities tested, but they contained a suppressive activity that inhibited the responses of normal spleen cells in macrocultures. Note Added: After submission of this paper we became aware of the paper of Sihvola and Hurme (J. Immunol. 130:1077, 1983) who also showed that although macrocultures from cyclophosphamide-treated mice were negative for the alloresponse, the same spleen cells showed only a approximately threefold decrease in CTL precursors when analyzed in limiting dilution cultures.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Co-recognition of endogenous antigens with HLA-DR1 by alloreactive human T cell clones

The fine specificity of anti-HLA-DR1 alloreactive, human T cells was investigated by using DR1-expressing human and murine stimulator cells. All three bulk cell lines and six out of seven T cell clones proliferated in response to DR1-expressing mouse L cells. In addition to these species non specific T cells, three clones were identified which proliferated only in response to DR1 expressed by human or by murine stimulator cells. The patterns of response of these clones may reflect specificity for species or lineage-specific peptides with DR1. The results of aldehyde fixation and cytotoxicity experiments suggested that some of the T cell clones which proliferated in response to human and murine DR1 stimulators also required to recognize species-specific antigens. The responses of four of the six clones were abolished by fixation of DR1-L cells but not of a DR-1 EBV transformed lymphoblastoid cell line before co-culture. In addition, these clones were also cytotoxic for DR1-expressing human targets. The same clones which failed to recognize fixed L cells also failed to lyse DR1-L cells in a short term chromium release assay. Taken together these results suggest that some alloreactive anti-DR1, T cells are specific for peptides of cellular proteins seen in the context of the allo-MHC molecule. It is envisaged that L cells when co-cultured with human T cells, process and present peptides derived from proteins that are shed or secreted by the human cells, for co-recognition with DR1 on the L cell surface. The presentation of multiple peptides derived from endogenous proteins by allogeneic cells may contribute to the high precursor frequency of allo-reactive T cells.     

Dissection and molecular analysis of alloreactive CD8+ T cell responses in allogeneic haematopoietic stem cell transplantation

We applied a cDNA expression screening procedure with cryopreserved non-clonal CD8+ T cell populations (Lennerz et al., Proc. Natl. Acad. Sci. USA 102:16013-8, 2005) to the identification of candidate antigens for graft-versus-host disease (GvHD) and graft-versus-leukaemia (GvL) effects in allogeneic haematopoietic stem cell transplantation (allo-HSCT). In a patient–donor model system with HLA class I disparities, we identified an HLA-B*44 mismatch allele, HLA-B*4405, as the dominant target of alloreactive T cells expanded in vitro from donor peripheral blood mononuclear cells (PBMC). HLA-B*4405-reactive T cells were detectable after multiple in vitro stimulations in the patient’s post-HSCT PBMC. In a patient–donor model with full HLA compatibility, the major target antigen of donor lymphocytes stimulated in vitro with the respective patient’s pre-HSCT PBMC was restricted by HLA-A*0201 and was encoded by TRIM22-442 C, a newly detected polymorphic allele of the tripartite motif family member TRIM22 (synonym: STAF50), preferentially expressed in cells of the haematopoietic system. An arginine(R)-to-cysteine(C) exchange at position 442 generated an immunogenic T cell epitope equivalent to a minor histocompatibility antigen (mHag). TRIM22-442C-specific T cells persisted long-term in the patient’s post-HSCT PBMC. Approximately, 1.3% of Caucasians carry TRIM22.442 C in association with HLA-A*0201. In particular, the knowledge of a large and diverse panel of such mHags may be crucial for further improvement of donor selection and adoptive T cell transfer strategies. The procedure applied herein will help to accelerate and facilitate their identification.     

Systemic administration of IL-15 augments the antigen-specific primary CD8+ T cell response following vaccination with peptide-pulsed dendritic cells

The systemic administration of IL-2 can act as a potent adjuvant for T cell-directed vaccine strategies. However, not only is the administration of IL-2 potentially toxic, but recent evidence suggests that it may also paradoxically limit the duration and magnitude of the cytotoxic T cell response. A recently identified cytokine, IL-15, shares many properties with IL-2 and may provide a preferential means of augmenting T cell-directed vaccine responses. Although well characterized in vitro, there are few data on the ability of IL-15 to augment T cell-mediated responses in vivo. We therefore evaluated the ability of systemic IL-15 to function as a T cell adjuvant in a murine vaccine model. To establish a population of easily identifiable Ag-responsive T cells, naive CD8(+) (OT-1) T cells were first adoptively transferred into mice. Vaccination with peptide-pulsed dendritic cells induced a modest expansion of OT-1 T cells. The addition of systemic IL-15 for 7 days following vaccination resulted in a significant increase in the expansion of responding T cells in the PBL, spleen, and lymph nodes. Importantly, the responding T cells were cytotoxic and maintained a Tc1-biased phenotype. We did not observe either enhanced resistance to activation-induced cell death or preferential generation of memory T cells as a result of treatment with IL-15 compared with IL-2. These studies show for the first time that IL-15 is capable of augmenting the primary CD8(+) T cell response to vaccination and contribute to the basis for future experiments exploring the clinical role of IL-15.     

Characterization of dual-reactive H-2Kb-restricted anti-vesicular stomatitus virus and alloreactive cytotoxic T cells

Cross-reactive recognition of alloantigen by "self + X"-reactive cytotoxic T lymphocytes (CTL) has been documented in a variety of systems. It has been shown previously that the H-2Kb-restricted CTL response of C57BL/6 (B6) mice to vesicular stomatitis virus (VSV) infection is partially cross-reactive on uninfected target cells expressing the H-2Kbm8 mutation. In this report, we describe the isolation and detailed characterization of such dual-reactive CTL. By employing EL4 tumor lines transfected with genes encoding various VSV proteins, we demonstrated that the majority of dual-reactive CTL recognize the internal N protein of VSV and are also reactive against uninfected bm8 targets. Although the response of normal B6 mice to bm8 stimulators shows no measurable cross-reactivity on VSV-infected targets, the response of VSV-primed B6 mice to bm8 stimulation is almost entirely cross-reactive, lysing VSV-B6 targets and uninfected bm8 targets roughly equally. Furthermore, about 70% of CTL clones isolated from such mice by bm8 stimulation are dual-reactive with respect to effector function. Analysis at the population and clonal levels with cold target competition and antibody blocking suggests that the bulk of dual-reactive CTL have a higher avidity for VSV-B6 targets than for bm8 targets. The extreme case of this is illustrated by a fraction of CTL clones, isolated and maintained on bm8 stimulators, which lyse VSV-B6 targets but do not lyse bm8 targets. One such CTL clone is shown to be specific for the bm8 antigen in proliferation assays. These results demonstrate that: the specificity of an alloreactive CTL response may be dramatically altered by previous antigenic encounters; and dual-reactive CTL display a significant difference in affinity of the CTL receptor-determinant interaction, depending on the target which is recognized.     

Critical role for activation of antigen-presenting cells in priming of cytotoxic T cell responses after vaccination with virus-like particles

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo (51)Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.     

Lymphocyte-mediated cytolysis of allogeneic tumor cells in vitro. III. Enzyme sensitivity of target cell antigens.

Target tumor cells pretreated with high concentrations of papain or Pronase were resistant to lysis by cytotoxic T lymphocytes (CTL), whereas treatment with trypsin or neuraminidase had no protective effect. Parallel determinations of the H-2 content of target cells following enzyme treatment showed that approximately 80% of surface H-2 was removed by papain or Pronase, 40% by trypsin, and virtually none by neuraminidase treatment. Both susceptibility to lysis by CTL and content of surface H-2 after papain treatment were fully restored by 6 hr at 37 °C in nutrient medium. These findings suggest that lymphocyte-mediated cytolysis (LMC) determinants (target cell antigens bound by CTL) are sensitive to degradation by papain and Pronase but are resistant to the enzymatic action of trypsin and neuraminidase. That a similar pattern of enzyme sensitivity is shown by serologically defined H-2 antigens indicates that both functional classes, LMC and H-2, may have a structural association.     

Sensitization of lymphocytes against pooled allogeneic cells. II. Characterization of effector cells cytotoxic for autologous lymphoblastoid cell lines

Stimulation of human lymphocytes in mixed leukocyte culture (MLC) with x-irradiated pooled allogeneic normal cells (poolx) was previously shown to result in generation of effector cells cytotoxic for autologous Epstein-Barr virus- (EBV) transformed lymphoblastoid cell lines (LCL). This study was undertaken to determine whether lysis of the autologous EBV- transformed LCL cells by pool-stimulated cells is mediated by cytotoxic Tc lymphocytes (Tc) or natural killer- (NK) like cells, both of which are generated in MLC. In the first series of experiments, proliferating cells were eliminated by treatment of pool-stimulated cells with 5 X 10(-5) M 5-bromodeoxyuridine (BUdR) and light. The remaining cells failed to lyse allogeneic normal lymphocytes and autologous LCL cells, whereas cytotoxicity against NK-sensitive K562 leukemia cells was retained. In the second series of experiments, pool-stimulated effector cells were treated with monoclonal anti-human Tc cell antibodies, OKT3 or OKT8, and complement (C). The cells recovered after antibody and C treatment were diminished in their ability to lyse allogeneic normal lymphocytes as well as autologous LCL cells, whereas their cytotoxicity against K562 leukemia cells was unaffected. These combined results provide strong evidence that lysis of autologous LCL cells by lymphocytes stimulated with pooled allogeneic normal cells is mediated by Tc rather than NK-like cells.