Kinetics of the anti-recipient cytotoxic cell response of mice with minor histocompatibility antigen graft-vs-host disease

The kinetics of the anti-recipient cytotoxic cell response of spleen cells from mice undergoing graft-vs-host disease (GVHD) induced to minor histocompatibility antigens were studied. Two population of cytotoxic cells were identified. Cytotoxic T lymphocytes (CTL) were present in recipient spleens 2 and 3 wk after transplantation but disappeared from the spleens before the onset of clinical disease. Cytotoxic T lymphocyte precursors (CTLp) were first detected in recipient spleens 2 wk after transplantation and were present during clinical disease. CTL may function as effectors in GVHD induced to minor histocompatibility antigens.     

Priming for a cytotoxic response to minor histocompatibility antigens: antigen specificity and failure to demonstrate a carrier effect.

Spleen cells from normal mice do not give a detectable in vitro cytotoxic T cell (CTL) response to minor H antigens. Spleen cells from animals primed in vivo with minor H antigens give a strong CTL response when boosted in culture with the appropriate stimulating cells. Here I have studied the requirements for priming a CTL response to minor H antigens. It is shown that priming is just as antigen specific as is cytolytic effector function. That is, priming cells have to carry the same minor antigens as the challenge cells. Inducing a graft-vs-host reaction in vivo does not nonspecifically allow spleen cells to respond to minor H antigens in vitro. Using minor H congenic mice (congenic for H-Y and/or H-7) I have also tried, and failed, to demonstrate a carrier effect in priming. Female mice primed to H-Y were challenged in culture with cells bearing H-Y and H-7 antigens in the hope that a helper response to H-Y would augment a CTL response to H-7. This did not happen, however. Such primed and boosted cells gave a strong secondary CTL response to H-Y but none to H-7. It is concluded that in order to prime for a detectable in vitro response to minor antigens it is necessary to expose the CTL precursors to antigen in vivo. This either expands the size of the pool of precursors by cell division or changes them in some qualitative way.     

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis

CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.     

Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.

Graft-versus-host disease (GvHD) is a chief complication of allogeneic bone marrow transplantation. In HLA-identical bone marrow transplantation, GvHD may be induced by disparities in minor histocompatibility antigens (mHags) between the donor and the recipient, with the antigen being present in the recipient and not in the donor. Cytotoxic T lymphocytes (CTLs) specific for mHags of the recipients can be isolated from the blood of recipients with severe GvHD (ref. 3). A retrospective study demonstrated an association between mismatch for mHags HA-1, -2, -4 and -5 and the occurrence of GvHD in adult recipients of bone marrow from HLA genotypically identical donors. Tetrameric HLA-peptide complexes have been used to visualize and quantitate antigen-specific CTLs in HIV-infected individuals and during Epstein-Barr virus and lymphocytic choriomeningitis virus infections. Here we show the direct ex vivo visualization of mHag-specific CTLs during GvHD using tetrameric HLA-class and I-mHag HA-1 and HY peptide complexes. In the peripheral blood of 17 HA-1 or HY mismatched marrow recipients, HA-1- and HY-specific CTLs were detected as early as 14 days after bone marrow transplantation. The tetrameric complexes demonstrated a significant increase in HA-1- and HY-specific CTLs during acute and chronic GvHD, which decreased after successful GvHD treatment. HLA class I-mHag peptide tetramers may serve as clinical tools for the diagnosis and monitoring of GvHD patients.     

The target minor H antigen for F1 cytotoxic T lymphocytes induced by Igh-congenic parental spleen cells is coded for by gene linked to H-2

Graft-vs-host reaction (GvHR) induced in (B10.BR X CWB)F1 (BWF1; H-2k/b, Ighb/b) by i.v. injection with CWB (H-2b, Ighb) spleen cells resulted in complete suppression of cytotoxic T lymphocyte (CTL) responsiveness of the F1 host spleen cells (GvHR-associated immunosuppression). In contrast, GvHR induced in BWF1 mice with CSW (H-2b, Ighj; Igh-congenic to CWB) spleen cells did not affect CTL responsiveness of the F1 host spleen cells at all. The BWF1 hosts undergoing the CSW-induced GvHR generated anti-CSW CTL in their spleens, and the subsequent culture of such BWF1 spleen cells with CSW stimulator cells, augmented the CTL activity. The BWF1 anti-CSW CTL lysed both Con A- and LPS-induced splenic blasts from mouse strains carrying the Ighj allele in the context of self H-2Kb. However, determination of the Igh haplotype in the serum IgG and of the susceptibility of the splenic lymphocytes to the BWF1 anti-CSW CTL on backcross mice, which carry either Ighb/j or Ighb/b in the context of H-2b/b or H-2b/k, showed clearly that Ighj and the gene coding for the target antigen for the BWF1 anti-CSW CTL segregated at ratios close to 1:1. The study in which linkage between the gene(s) coding for the target antigen for the BWF1 anti-CSW CTL and H-2 was examined on CWB X (C3H X CWB)F1 backcross mice and (B10.BR X CSW)F1 X B10 mice demonstrated that the gene, most likely a single gene, coding for the target antigen for the BWF1 anti-CSW CTL is located at 8.5 +/- 4.3 cross-over units to the right or left of the H-2 complex. We designated the minor H antigen to be recognized by the BWF1 anti-CSW CTL as H-X+, and we discuss the distinction between the H-X+ locus and the other minor H loci on chromosome 17.     

Molecular and functional dissection of the H-2Db-restricted subdominant cytotoxic T-cell response to lymphocytic choriomeningitis virus

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.     

Cytotoxic T lymphocyte response to minor H-42a alloantigen in H-42b mice: clonal inactivation of the precursor cytotoxic T lymphocytes by veto-like spleen cells that express the H-42a antigen

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by veto cells contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Generation of cytotoxic T cells specific for minor histocompatibility antigens by cross challenge in vitro with H-2 disparate adherent cells

Although it is well known that an H-2-restricted cytotoxic T cell response to minor histocompatibility antigens (MIHA) can be primed in vivo with H-2 disparate spleen cells, it has not been previously possible to induce cytotoxic T lymphocyte (CTL) precursors (CTLp) in vitro by this type of challenge. In this work, we demonstrate that the inability to cross challenge in vitro is due to the existence of inhibitory effects that can be obviated by cell fractionation, and to insufficient priming in vivo. BALB/c CTLp (H-2d) that have been repeatedly primed in vivo with B10.D2 can be challenged in vitro with C57BL10/J (H-2b) or B10.BR (H-2k)-adherent cells to generate CTL able to lyse B10.D2 (H-2d) target cells. The H-2 restriction properties of the cross-challenged CTL specific for MIHA were analyzed by using the technique of cold target competition. Within the limits of detection in bulk cultures, the entire response appeared to be H-2 unrestricted, whether the cross challenge was with intact C57BL10/J-adherent cells, or with membrane fragments of C57BL10/J presented by BALB/c adherent cells. The frequency of CTLp responsive to cross challenge was analyzed by limiting dilution, with cold target competition at each cell number to establish the restriction properties of the MIHA-specific CTL induced. We were able to detect two subsets of H-2-unrestricted CTLp responsive to intact C57BL10/J-adherent cells; one present at high frequency (1/250 T cells) and subject to suppressive effects at high cell number, and a second present at lower frequency (1/9800 T cells). There appeared to be a relatively infrequent subset of H-2-restricted CTLp as well (1/52,500 T cells). The frequency of CTLp responsive to cross challenge is of comparable magnitude to the frequency of H-2-restricted CTLp responsive to H-2-matched cells bearing MIHA. These observations are discussed in relationship to immunodominance and clonal dominance effects in the response to MIHA.     

Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2K(b) and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8(+) T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2K(b)-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.     

Immunodominance in the graft-vs-host disease T cell response to minor histocompatibility antigens

Immunodominance controls the generation of CTL in the C57BL/6By (B6) anti-BALB.B H-2b-matched strain combination. Despite the potential of responding to numerous individual minor histocompatibility (H) Ag on BALB.B APC, the focus of the CTL response is largely specific for only a limited number of target Ag. These minor H Ag could be distinguished by their differential expression on a panel of target cells from the CXB recombinant inbred strains, the E, G, I, J, and K (all H-2b), which express different composites of the original BALB minor H Ag. A hierarchy was observed in which first-order immunodominant Ag were present on both CXBK and CXBG cells, whereas second-order dominant Ag were found on CXBE, CXBJ, and CXBI cells. To test whether immunodominance also plays a role in the development of lethal graft-vs-host disease (GVHD) directed to multiple minor H Ag, B6 T cells were transplanted along with T cell depleted bone marrow, to irradiated (825 rad) recipients of either the BALB.B or CXB recombinant inbred strains. The results indicate that a hierarchy of immunodominance does exist in GVHD, but it differs from that predicted from the in vitro CTL studies. GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients. Presensitization of B6 donor mice to CXBG or CXBK splenocytes 3 wk before transplant did not significantly increase the overall GVHD potential in the corresponding CXBG or CXBK recipients. Evidence for second-order immunodominance was provided by the transfer of CXBE T cells and ATBM to irradiated CXBG and BALB.B recipients with resultant, potent GVHD.     

The allogeneic effect: M-locus differences substitute for differences in the H-2 major histocompatibility complex.

The primary immune response against sheep red blood cells in T cell-deficient spleen cell cultures from nude mice was tested in the absence and presence of allogeneic spleen cells. The allogeneic spleen cells differed either in regard to the major histocompatibility complex (H-2) or only with respect to the M-locus. Surprisingly the M-locus different spleen cells were almost as efficient in enhancing the anti-sheep red blood cell response in nude cultures as were the cells differing on the complete H-2 complex. Evidence is presented that AKR anti-theta serum sensitive T cells are responsible for the M-locus-dependent effect edscribed. This effect is shown to be mediated by a factor released from actived T cells stimulated in M-locus different mixed lymphocyte cultures. Since almost identical parameters have been observed in both the M-locus-dependent situation as in the "classical" allogeneic situation we concluded that an allogeneic effect can be induced by T cells responding to a complete set of the major histocompatibility complex (H-2) as well as to lymphocyte-activating determinants (M-locus) alone.     

H-41, a new minor histocompatibility locus. I. Histogenetic analysis

The B10.STA12 mouse congenic line inherited from the wild mouse parent not only the H-2w13 haplotype but also an allele at a minor H locus, which we designate H-41. This allele (H-41a) differentiates the B10.STA12 line from B10.STA10 and B10.LIB55, which carry identical H-2w13 haplotypes but a different H-41 allele (the H-41b, also present in the background strain C57BL/10Sn). The B10.STA12 and B10.STA10 lines reject each other's skin grafts and generate cytolytic T lymphocytes (CTL) after in vivo immunization and in vitro restimulation with cells of the partner strain. The B10.STA12 anti-B10.STA10 CTL react with B10.STA10, B10.LIB55, and B10.STA39 target cells and with cells of F1 hybrids between the responder strain B10.STA12 and strains C57BL/6, C57BL/10, C57L, BALB/c, A, AKR, WB, DBA/1, and DBA/2 but fail to react with (C3H x B10.STA12) F1 and (CBA x B10.STA12) F1 cells. The B10.STA10 anti-B10.STA12 CTL react with B10.STA12, B10.P, and C3H.NB cells but fail to react to (B6 x B10.STA10) F1 target cells. The CTL reactivity in both combinations is Dp restricted. The B10.STA10 anti-B10.STA12 CTL exhibit, in addition, a cross-reactivity with B10.SAA48 cells that may be directed at one of the alloantigens controlled by the H-2 haplotype of this strain.     

T helper cell lines that augment in vivo cytotoxic T-cell responses to minor alloantigens

We have investigated the ability of long-term cultured T helper (Th) cell lines to help an in vivo cytotoxic T lymphocyte (CTL) response to non-H-2 alloantigens (minor antigens). Th cell lines specific for various single or undefined minor antigens were selected by regular restimulation with antigen in vitro. They were antigen specific and H-2 restricted in proliferation assays and were found to be able to help primary CTL responses to multiple minor antigens and secondary CTL responses to single minor antigens. Although the Th were antigen specific they did not determine the specificity of the CTL. Th cells were both necessary and limiting for an effective CTL response indicating that "helper-independent" CTL are not in themselves sufficient to generate a strong in vivo response. Under conditions where a CTL response was clearly H-2 restricted, Th cells were not. Thus, the Th cells appeared to be activated by reprocessed antigen rather than antigen on the surface of the injected antigenic cells even though the CTL themselves reacted directly to the injected antigen.     

The T-cell antigen receptor: a logical response to an unknown ligand

The immune system can be roughly divided into innate and adaptive compartments. The adaptive compartment includes the B and T lymphocytes, whose antigen receptors are generated by recombination of gene segments. The consequence is that the creation of self-reactive lymphocytes is unavoidable. For the host to remain viable, the immune system has evolved a strategy for removing autoimmune lymphocytes during development. This review discusses how T lymphocytes are generated, how they recognize antigens, and how their antigen receptor directs the removal of self-reactive T cells.