HY immune tolerance is common in women without male offspring.

BACKGROUND: Sex difference is an established risk factor for hematopoietic stem cell transplantation (HSCT)-related complications like graft versus host disease (GVHD). CD8pos cytotoxic T cells specific for Y chromosome-encoded minor Histocompatibility antigens (HY) play an important role therein. Prior to HSC donation, female donors may encounter HY antigens through fetomaternal or transmaternal cell flow, potentially leading to the induction of HY-specific cytotoxic or regulatory immune responses. Whether HY priming occurs independent of parity, and whether HY priming is dependent on the presence of male microchimerism, is as yet unknown. METHODS: We investigated the presence of HY-specific regulatory T cells (Treg) and male microchimerism in 45 healthy women with a fully documented pregnancy and family history. HY peptide-induced linked suppression, a commonly reported functional feature of CD4pos and CD8pos Treg, was measured by trans vivo Delayed Type Hypersensitivity testing. As source of HY antigens, male microchimerism was analyzed by real-time PCR and defined by the presence of male DNA in at least one purified leukocyte cell type. RESULTS: HLA class I or class II restricted HY-specific Treg were detected in 26/42 (62%) women eligible for analysis. The prevalence of HY-specific Treg was significantly higher in women who had never given birth to sons than in women with male offspring (p = 0.004). Male microchimerism could be detected in 24 out of 45 (53%) women but did not correlate with the presence of HY specific Treg. CONCLUSIONS: HY-specific Treg in women with male offspring have been described previously. Here we show for the first time that, in fact, HY specific Treg are more common in nulliparous women and in parous women with female offspring. Their presence is independent of the presence of male microchimerism. Whether HY-specific Treg presence in female stem cell grafts might decrease the GVHD incidence in male HSCT recipients needs to be investigated.

Transmaternal cell flow leads to antigen-experienced cord blood.

Umbilical cord blood (UCB) is used for HSCT. It is known that UCB can comprise Ag-specific T cells. Here we question whether solely transmaternal cell flow may immunize UCB. Twenty-three female UCB samples were collected from healthy mothers and analyzed for minor histocompatibility Ag HY-specific responses. Forty-two of 104 tetramer(pos) T-cell clones, isolated from 16 of 17 UCB samples, showed male-specific lysis in vitro. Male microchimerism was present in 6 of 12 UCB samples analyzed. In conclusion, female UCB comprises HY-specific cytotoxic T cells. The immunization is presumably caused by transmaternal cell flow of male microchimerism present in the mother. The presence of immune cells in UCB that are not directed against maternal foreign Ags is remarkable and may explain the reported clinical observation of improved HSCT outcome with younger sibling donors.

Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members.

Bidirectional cell transfer during pregnancy frequently leads to postpartum persistence of allogeneic cells and alloimmune responses in both the mother and in her offspring. The life-long consequences of naturally acquired alloimmune reactivity are probably of importance for the outcome of allogeneic stem cell transplantation. We investigated the presence of CD8(pos) minor histocompatibility (H) antigen-specific cytotoxic T lymphocytes (T(CTL)) and CD8(pos) minor H antigen-specific T regulator cells (T(REG)) in peripheral blood cells obtained from 17 minor H antigen-disparate mother-offspring pairs. Absence of minor H antigen-specific T(REG), as marked by the feasibility to expand T(CTL) from isolated tetramer(pos) populations, was observed in 6 mothers and 1 son. The presence of minor H alloantigen-specific T(REG) was observed in 4 mothers and 5 sons. These T(REG) were detected within isolated tetramer(dim) staining fractions and functioned in a CTLA-4-dependent fashion. Our study indicates that both T(CTL) and T(REG) mediated alloimmunity against minor H antigens may be present in healthy female and male hematopoietic stem cell donors, potentially influencing graft-versus-host reactivity in different ways.

Hypomethylating drugs convert HA-1-negative solid tumors into targets for stem cell-based immunotherapy.

Clinical responses of solid tumors after allogeneic human leukocyte antigen-matched stem cell transplantation (SCT) often coincide with severe graft-versus-host disease (GVHD). Targeting minor histocompatibility antigens (mHags) with hematopoiesis- and cancer-restricted expression, for example, HA-1, may allow boosting the antitumor effect of allogeneic SCT without risking severe GVHD. The mHag HA-1 is aberrantly expressed in cancers of most entities. However, an estimated 30% to 40% of solid tumors do not express HA-1 (ie, are HA-1(neg)) and cannot be targeted by HA-1-specific immunotherapy. Here, we investigated the transcriptional regulation of HA-1 gene expression in cancer. We found that DNA hypermethylation in the HA-1 promoter region is closely associated with the absence of HA-1 gene expression in solid tumor cell lines. Moreover, we detected HA-1 promoter hypermethylation in primary cancers. The hypomethylating agent 5-aza-2'-deoxycytidine induced HA-1 expression only in HA-1(neg) tumor cells and sensitized them for recognition by HA-1-specific cytotoxic T lymphocytes. Contrarily, the histone deacetylation inhibitor trichostatin A induced HA-1 expression both in some HA-1(neg) tumor cell lines and in normal nonhematopoietic cells. Our data suggest that promoter hypermethylation contributes to the HA-1 gene regulation in tumors. Hypomethylating drugs might extend the safe applicability of HA-1 as an immunotherapeutic target on solid tumors after allogeneic SCT.

TAP-inhibiting proteins US6, ICP47 and UL49.5 differentially affect minor and major histocompatibility antigen-specific recognition by cytotoxic T lymphocytes.

CTLs specific for hematopoietic system-restricted minor histocompatibility antigens (mHags) can serve as reagents for cellular adoptive immunotherapy after allogeneic stem cell transplantation (SCT). In the HLA-mismatched setting, CTLs specific for hematopoietic system-restricted mHags expressed solely by the non-self 'allo' HLA molecules could be used to treat relapse after HLA-mismatched SCT. The generation of mHag-specific allo-HLA-restricted CTLs requires antigen-presenting cells (APCs) expressing low numbers of endogenous peptides to avoid co-induction of undesired allo-HLA reactivities. In this study, we exploited viral evasion strategies to generate APCs expressing a controlled set of endogenous peptides. Herpesviruses persist lifelong following primary infection due to expression of viral gene products that hamper T-cell recognition of infected cells. The herpesvirus-derived proteins US6, ICP47 and UL49.5 down-regulate endogenous antigen presentation in human APCs via inhibition of the transporter associated with antigen processing. EBV-transformed B cell lines transduced with retroviral vectors encoding US6, ICP47 or UL49.5 exhibited a stable decrease in cell-surface HLA class I expression and were protected from lysis by mHag-specific CTLs. Exogenous addition of mHag peptide fully restored target cell recognition. UL49.5 showed the most pronounced inhibitory effect, reducing HLA class I expression and mHag-specific lysis up to 99%. UL49.5 also significantly diminished allo-HLA reactivities mediated by allo-HLA-specific CTLs. In conclusion, UL49.5 could be a powerful new tool to study and modulate endogenous antigen presentation.

Promiscuity of the AlloHLA-A2 restricted T cell repertoire hampers the generation of minor Histocompatibility antigen-specific cytotoxic T cells across HLA barriers.

Hematopoietic system-specific miHAs are ideal targets for adoptive immunotherapy after allogeneic HLA (alloHLA)-matched SCT. Adoptive immunotherapy with cytotoxic T cells targeting hematopoietic system-specific miHAs restricted by alloHLA molecules is an attractive strategy to treat relapsed hematologic malignancies after HLA-mismatched SCT. As a proof of principle, we exploited 2 new strategies to generate alloHLA-A2-restricted miHA-specific T cells from HLA-A2(neg) donors using a HLA/miHA multimer-guided approach. In one strategy, autologous DCs coated with HLA-A2/miHA complexes were used for in vitro generation of miHA-specific T cells from HLA-A2(neg) male donors. In the other strategy, miHA-specific T cells were directly isolated from the peripheral blood of HLA-A2(neg) parous females with HLA-A2(pos) offspring. Both methods introduced recombinant HLA-A2/miHA complexes as the sole allogeneic target antigen. However, neither method yielded high avidity miHA-specific T cells or prevented the emergence of peptide-dependent promiscuous T cells. The latter T cells resembled miHA-specific T cells so closely with regard to tetramer binding and cytokine production that only extensive testing at a clonal level revealed their nonspecific nature. Therefore, promiscuity of the alloHLA-A2 T cell repertoire of HLA-A2(neg) individuals hampers in vitro generation of genuine miHA-specific T cells and limits its use for adoptive immunotherapy after HLA-A2 mismatched SCT.

Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) specific for hematopoietic-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation. However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell-sized latex beads coated with HLA/mHag complexes HLA-A2/HA-1 or HLA-A2/HA-2 and recombinant CD80 and CD54 molecules can replace professional APCs. The artificial antigen-presenting constructs (aAPCs) effectively stimulated HA-1- and HA-2-specific CTL clones as shown by ligand-specific expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1-specific polyclonal CTL lines were enriched as efficiently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80, and CD54 may serve as tools for in vitro enrichment of immunotherapeutic mHag-specific CTL lines.

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.

The hematopoietic system-specific minor histocompatibility antigen HA-1 shows aberrant expression in epithelial cancer cells.

Allogeneic stem cell transplantation (SCT) can induce curative graft-versus-tumor reactions in patients with hematological malignancies and solid tumors. The graft-versus-tumor reaction after human histocompatibility leukocyte antigen (HLA)-identical SCT is mediated by alloimmune donor T cells specific for polymorphic minor histocompatibility antigens (mHags). Among these, the mHag HA-1 was found to be restricted to the hematopoietic system. Here, we report on the HA-1 ribonucleic acid expression by microdissected carcinoma tissues and by single disseminated tumor cells isolated from patients with various epithelial tumors. The HA-1 peptide is molecularly defined, as it forms an immunogenic peptide ligand with HLA-A2 on the cell membrane of carcinoma cell lines. HA-1-specific cytotoxic T cells lyse epithelial tumor cell lines in vitro, whereas normal epithelial cells are not recognized. Thus, HA-1-specific immunotherapy combined with HLA-identical allogeneic SCT may now be feasible for patients with HA-1(+) carcinomas.

Generation of minor histocompatibility antigen HA-1-specific cytotoxic T cells restricted by nonself HLA molecules: a potential strategy to treat relapsed leukemia after HLA-mismatched stem cell transplantation.

Successful stem cell transplantation (SCT) across HLA barriers can be performed with cord blood, megadoses of stem cells, or with nonmyeloablative conditioning strategies. Because the HLA-mismatched transplants are often T-cell depleted, leukemia relapse rates are high. Treatment of relapsed leukemia after HLA-mismatched SCT is difficult. A novel potential strategy to treat relapsed leukemia after HLA-mismatched SCT is the use of patients' mismatched HLA molecules as antigen-presenting molecules to generate hematopoietic system-specific cytotoxic T cells (CTLs) from the stem cell donor. Adoptive transfer of these hematopoietic system-specific CTLs that are restricted by nonself HLA molecules may eliminate leukemia without affecting the patient's nonhematopoietic cells or donor hematopoietic cells. We investigated the feasibility of this strategy using the hematopoietic system-specific minor histocompatibility antigen HA-1, which is known to induce HLA-A2-restricted CTLs. HLA-A2(-) peripheral blood mononuclear cells were stimulated with HLA-A2(+) T2 cells pulsed with synthetic HA-1 peptide or with dendritic cells transduced with the HA-1 cDNA. Tetrameric HLA-A2/HA-1 peptide complexes were used to monitor and enrich HA-1-specific CTLs. In the alloreactive cultures, HA-1-specific CTLs were enriched up to 7% by 3 rounds of antigen-specific stimulations and up to 87% by fluorescence-activated cell sorting of tetramer-positive T cells. The HA-1-specific CTLs showed specific lysis of the relevant target cells, including leukemic cells. Because the polyclonal CTL cultures also contained natural killer cells and allo-HLA-A2-specific CTLs, CTL clones were generated that showed the expected HA-1 specificity only. Thus, HA-1-specific CTLs restricted by nonself HLA-A2 molecules can be generated in an HLA-A2-mismatched setting.

General T-cell receptor antagonists to immunomodulate HLA-A2-restricted minor histocompatibility antigen HA-1-specific T-cell responses.

T-cell receptors (TCRs) of a series of minor histocompatibility antigen (mHag) HA-1-specific cytotoxic T-cell (CTL) clones isolated from 3 unrelated patients have been shown to use the same BV6S4A2 segment with conserved amino acids in the CDR3Vbeta region. This suggests that different HA-1-specific TCRs interact similarly to the HA-1 antigen presented by the HLA-A2 molecule. The mHag HA-1 forms an immunogenic complex with HLA-A2 and induces strong alloimmune responses after stem cell transplantation (SCT). It was questioned, therefore, whether clonal and polyclonal HA-1-specific CTL responses can be antagonized by a single TCR antagonistic peptide. Functional analysis and molecular modeling of single and double amino acid substitutions of TCR contact residues, adjacent residues, and HLA-A2 binding residues resulted in 4 peptides with high affinity for HLA-A2 and with the capacity to inhibit the lysis of endogenously HA-1-expressing EBV-BLCL by 3 different HA-1-specific CTL clones. These peptides also efficiently antagonized HA-1-specific polyclonal CTL lines derived from 3 patients and significantly reduced the number of interferon-gamma-producing HA-1-specific CTL of a patient with graft-versus-host disease after HA-1-mismatched SCT. These data show that general TCR antagonists can be developed that inhibit HLA-A2-restricted HA-1-specific CTL responses on the clonal and the polyclonal level and that TCR antagonists may modulate the immunodominant mHag HA-1 responses.