Allogenic benefit in stem cell therapy: cardiac repair and regeneration.

Stem cell (SC)-based therapies are a developing mean to repair, restore, maintain, or enhance organ functioning through life span. They are in particular a fast track to restore function in failing heart. Various types of SCs have been used in experimental and clinical studies showing the potential of these cells to revolutionize the treatment of heart diseases. Autologous cells have been privileged to overpass immunological barriers. The field has progressed tremendously and the hurdles, which have been largely overlooked in the excitement over the expected benefit the immunogenicity, have been revealed. Also, manufacturing of patient-specific clinical grade SC product, whether adult stem or reprogrammed induced pluripotent SCs, and the availability of these cells in sufficient amounts and status when needed is questionable. In contrast, adult SCs derived from healthy donors, thus allogeneic, have the advantage to be immediately available as an 'off-the-shelf' therapeutic product. The challenge is to overcome the immunological barriers to their transplantation. Recent research provided new insights into the mode of action and immune behavior of SCs in autologous as well as allogeneic settings. Lessons are learned and immune paradigms are changing: allogenicity, if balanced could be part of the dynamic and durable mechanisms that are critical to sustain cardiac regeneration and repair. We discuss the hurdles, lessons, and advances accomplished in the field through the progressive journey of cardiac-derived stem/progenitor cells toward allogeneic cardiac regenerative/reparative therapy.

Dose effect of cis- and trans-encoded HLA-DQ alpha beta heterodimers in IDDM susceptibility.

Insulin-dependent diabetes mellitus (IDDM) in whites is strongly associated with particular HLA-DQ alpha beta heterodimers composed of a DQ alpha chain with an arginine at residue 52 (Arg52+) combined to a DQ beta chain lacking an aspartic acid at residue 57 (Asp57-). With the aim of confirming this association, clarifying which heterodimers account for the highest risk of IDDM and explaining the excess risk of DR3-DQw2/DR4-DQw8, 115 unrelated white IDDM patients and 108 unrelated healthy nondiabetic control subjects were studied. With polymerase chain reaction and sequence-specific oligonucleotide probes, both patients and control subjects were typed for their HLA-DQA1 and DQB1 alleles and their DQA1-DQB1 haplotype and genotype frequencies were compared. Four major findings emerged from our analysis. 1) Arg52+ DQ alpha/Asp57- DQ beta heterodimers, formed in cis and/or in trans, are strongly associated with susceptibility to IDDM; 97% of patients and 46% of control subjects had at least one such susceptibility heterodimer (relative risk [RR] 32, confidence interval [Cl] 14.25-71.86, P less than 10(-7). 2) The degree of disease susceptibility depends on the number of such DQ heterodimers that a subject can express according to his or her DQA1-DQB1 genotype. The highest RR was observed in patients with four susceptibility DQ heterodimers (RR 41, Cl 17.05-95.9). 3) Only part of the susceptibility DQ heterodimers were significantly increased in patients, conferring IDDM susceptibility of different strength. The strongest association was with the DQA1*0501-DQB1*0302 combination formed in trans position (RR 35.2, CI 12.88-96.78, P less than 10(-7).(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-DP genotyping in HLA-A,B, and DR identical intrafamilial bone marrow transplantation.

In a study carried out for patients receiving intrafamilial HLA-A,B,DR identical, MLC negative bone marrow transplants, RFLP profiles of HLA-class II for 27 donor recipient pairs were analyzed. Twenty-four pairs were found HLA-class II identical while three pairs were HLA-DP incompatible. The patients of these three pairs did not reveal any acute GVHD greater than or equal to grade II. The seven cases of acute GVHD greater than or equal to grade II found in our panel were HLA-DR, DQ, and DP compatible. Thus, in practical terms pretransplantation HLA-DP typing does not seem necessary for intrafamilial HLA-identical, MLC negative BMT. On the other hand, this work confirmed that it is possible to type for HLA-DP using molecular biological techniques, and this in itself may have some important implications for unrelated BMT.

Expression of functional major histocompatibility complex class II molecules on HMC-1 human mast cells.

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC-1 human mast cell line, umbilical cord blood-derived mast cells express HLA-DR, -DP and -DQ molecules on their surface. MHC class II molecules expressed on HMC-1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural changes, suggesting their degranulation. Recognition of SEA-bound MHC class II molecules on HMC-1 mast cells by the T cell receptor of K25 cells, an SEA-specific murine T cell hybridoma, triggers significant IL-2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity.

HLA DQB1*0301 allele is involved in the susceptibility to erythema multiforme.

Erythema multiforme (EM) is an acute, episodic inflammatory disorder of the skin and mucous membranes of various etiology that could be related to immunologic hypersensitivity response. EM has been previously reported to be associated with serologically defined HLA-DRw53 and DQw3 antigens. In this report, we reevaluate the role of HLA class II alleles in EM manifestations. With use of the polymerase chain reaction, followed by sequence-specific oligonucleotide hybridization, 35 unrelated Caucasian EM patients and 80 randomly selected healthy subjects were studied, and the DRB3, DRB4, DQA1, and DQB1 alleles were analyzed. The comparison of frequencies of these alleles indicates that (i) susceptibility to EM disease is more associated with the HLA-DQ than the HLA-DR subregions and (ii) that the DQB1*0301 is the most frequent allele among EM patients. Sixty-six percent of the patients had the DQB1*0301 allele compared to 31% of the controls (RR = 4.1; p less than 0.001). An even stronger DQB1*0301 association was found in the patient group with herpes-associated EM (76%; RR = 6.5; p less than 0.001). Our data demonstrate a clear association between an HLA-DQB1 allele and susceptibility to EM.

HLA-DR, DQ, and/or DP genotypic mismatches between recipient-donor pairs in unrelated bone marrow transplantation and transplant clinical outcome.

Sixteen recipient-donor pairs who underwent unrelated BMT were analyzed for their HLA-class II identity by DNA-RFLP, in order to evaluate the importance of the genotypic HLA-DR, DQ, DP identity in the clinical outcome of unrelated bone marrow transplantation. From our study, a clear correlation between the HLA-DR, DQ, and DP genetic identity and acute GVHD (aGVHD) is not obvious since the number of studied cases is still limited. Nevertheless, it seems that the genetic identity influence the clinical outcome and patient survival. Six patients out of the ten who experienced severe aGVHD (greater than grade II) differed from their respective donors by HLA-DP mismatch in the GVH direction. Two patients rejected their grafts, and both presented HLA-DP incompatibilities in both GVH and HVG directions. Hence, HLA-DP may function as a transplantation antigen like the other HLA-class II molecules (DR, DQ) in unrelated BMT. Accordingly, we propose considering it in the pretransplantation histocompatibility testing. Nevertheless, further studies with larger numbers of cases should be done in order to confirm the role of HLA-DP. No correlation was observed between the mixed lymphocyte reaction (MLR) reactivity and the incidence of aGVHD. Accordingly, MLR response seems to be an incomplete indicator of GVHD, and a functional test is still to be found.

Ligation of MHC class II molecules differentially upregulates TNF beta gene expression in B cell lines of different MHC class II haplotypes.

Although the production of selected cytokines by B cells is important for their regulation, little is known about MHC class II-induced cytokine expression in these cells. We designed the present studies to investigate MHC class II-mediated TNF-beta gene expression in 19 EBV-transformed homozygote B cell lines at similar stage of differentiation but presenting different MHC class II haplotypes. Our results demonstrate that in contrast to PMA, engagement of MHC class II with staphylococcal enterotoxin A (SEA), a natural ligand, or with anti-HLA-DR mAb L243, stimulates TNF-beta gene expression in some but not all B cell lines. The differential stimulation of TNF-beta gene expression via MHC class II was not due to the cells MHC class II expression level, nor to their capacity to bind the ligands as evidenced by SEA binding affinity studies. Together these results demonstrate that ligation of MHC class II molecules can stimulate TNF-beta gene expression in a B cell line-dependent manner. The differential cytokine gene expression might be due to an influence of MHC class II haplotype either by a linkage disequilibrium with TNF-beta gene or by a differential association with effector or cell surface molecules.

Gene polymorphism of HLA-DPB1 and DPA1 loci in caucasoid population: frequencies and DPB1-DPA1 associations.

The genetic polymorphism of the HLA-DPB1 and DPA1 loci was studied in 60 unrelated caucasoid individuals by PCR-RFLP. The polymorphic second exon of DPB1, the third exon of DPA1, and the transmembrane DPA1 exon were specifically amplified in vitro by polymerase chain reaction (PCR). Amplified DNAs were digested with selected enzymes. Twenty patterns were obtained with DPB1 defining 20 DPB1 alleles. Thirty-nine homozygous cell lines were used as HLA-DP reference cells. The results obtained with these cell lines were compared to those obtained by PLT, RFLP, and SSO. Although three subdivisions of the allele DPA1*01 were reported, DPA1*0103 was the only represented one in the caucasoid population. In the studied population, it was the most frequent DPA1 allele (76.6%), whereas DPA1*0201 frequency is 23.3%. DPB1*0401 and DPB1*0402 are the most frequent among the DPB1 alleles (40.0% and 13.3%, respectively). This may lead to a lower HLA-DPB1 diversity among caucasoids. Certain HLA-DPB1 alleles associate exclusively with one DPA1 allele (DPB1*0401, 0402, and 0301 with DPA1*01 and DPB1*0101, 0501, and 1701 with DPA1*0201) whereas the others can associate with both DPA1 alleles. This by itself can create another kind of polymorphism, indicating the importance of HLA-DPA1 typing. Thus, PCR-RFLP seems to be one of the best DNA typing methods: it represents direct, accurate, fast, and nonradioactive typing for both HLA-DPA1 and HLA-DPB1 alleles.

Evaluation of HLA-class II identity between unrelated individuals by serological typing, DNA-RFLP method, and mixed lymphocyte reaction.

Seven groups, each consisting of two to nine unrelated HLA-A, -B, and -DR serologically identical individuals, were analyzed by DNA-restriction fragment length polymorphism (RFLP) and mixed lymphocyte reactions (MLR) in order to evaluate HLA-class II identity between unrelated individuals and to assess the importance of HLA-class II incompatibilities detected by DNA-RFLP in the allogeneic reactions. It is clear that DNA-RFLP represents a powerful typing method for HLA-DR, -DQ, and -DP since the combinations of the RFLP band patterns define all the serological specificities and most of the cellular specificities to give a highly accurate typing. This report shows that an HLA-DP incompatibility induces proliferation in primary mixed lymphocyte culture (MLC) between unrelated HLA-A, -B, -DR, -DQ, and -DW identical individuals, which may suggest the importance of this molecule as a transplantation antigen, especially for unrelated bone marrow transplantations. Still, an isolated HLA-DPw4/HLA-DP a disparity did not induce any proliferation in MLC. Moreover, our results show that DQw7 (w3)/DQw8 (w3) disparity associated with HLA-DR4 represents a nonfunctional incompatibility in MLR. The HLA-Dw subtypes of HLA-DR specificities can induce a high proliferative response in MLC. The HLA-Dw subtypes of HLA-DR specificities can induce a high proliferative response in MLC. Finally, DNA-RFLP typing represents a reliable method for the selection of histocompatible donor-recipient pairs and could potentially reduce many logistic problems and delays in live-donor transplantation, especially for unrelated bone marrow transplantation.

HLA-DP distribution in Shanghai Chinese--a study by polymerase chain reaction--restriction fragment length polymorphism.

One hundred and four normal unrelated Chinese were typed for HLA-DPA1 and DPB1 alleles by polymerase chain reaction-restriction fragment length polymorphism. Increased frequencies of HLA-DPA1*0201 and DPB1*0501 were found in this Chinese population as compared with those detected in Caucasoids and blacks.

Synergistic effect between CD40 and class II signals overcome the requirement for class II dimerization in superantigen-induced cytokine gene expression.

Although staphylococcal enterotoxin A (SEA), B (SEB), and toxic shock syndrome toxin 1 (TSST-1) bind to major histocompatibility complex (MHC) class II molecules, they differ in their mode of binding. Signaling induced by these toxins via MHC class II molecules seems to be largely mediated by their mode of interaction. In the present study, we have demonstrated that contrary to SEA, stimulation of the human monocytic cell line THP-1 with SEB or TSST-1 failed to induce interleukin-1 beta or tumor necrosis factor-alpha gene expression. Treatment of THP-1 cells with interferon-gamma increased the level of MHC class II expression but did not enhance the SEB and TSST-1 response. However, cross-linking of SEB or TSST-1 bound to MHC class II molecules with specific antibodies leads to cytokine gene expression, indicating that dimerization of class II molecules is a requirement for this superantigen-induced response. The presence of anti-CD40 antibodies in the course of SEB or TSST-1 stimulation overcomes this requirement, indicating that certain signal(s) induced via CD40 molecules can replace those induced by dimerization of class II molecules. Pretreatment with anti-lymphocyte functional antigen-1 (LFA-1) antibodies completely inhibited SEA-induced response as well as that induced by SEB or TSST-1 in the presence of CD40 antibodies, supporting the involvement of LFA-1 intercellular adhesion molecule system in these responses. The entirety of these results demonstrate clearly that dimerization of class II molecules is a prerequisite for superantigen-induced T cell-independent cytokine gene expression which can be replaced by signaling via CD40 in an LFA-1-dependent system.

CD40- and HLA-DR-mediated cell death pathways share a lot of similarities but differ in their use of ADP-ribosyltransferase activities.

CD40 and HLA-DR molecules are two major components of the immune system, and their engagement on several cell types leads to various cellular events that modulate cell function. In this study, we demonstrate that signaling via these molecules leads to a rapid B cell death. CD40-mediated cell death was mainly observed in Epstein-Barr virus (EBV)-transformed B cell lines, whereas, HLA-DR-induced response can be triggered in normal activated B cells as well as in EBV-transformed B cell lines. Cell death induced via both molecules does not require de novo protein synthesis, but involves the integrity of the cytoskeleton. The sensitivity of CD40- and HLA-DR-mediated cell death to various inhibitors is very similar to that previously reported for tumor necrosis factor receptor (TNFR)- and Fas-triggered apoptosis; however, caspases leading to poly(ADP-ribose) polymerase cleavage are not implicated in this response. Both B cell death forms do not involve Fas-Fas ligand and TNF-TNFR systems, but require LFA-1-independent cell-cell interactions mediated by still undefined molecules. Although CD40- and HLA-DR-mediated cell death appears to follow a common pathway, inhibitors of poly- and mono-ADP-ribosyltransferase activity differentially affect these responses. Defining the molecules involved in CD40- and HLA-DR-mediated death will provide a possible interrelation between the different B cell death programs that can lead to a better comprehension of regulation of B cell functions.

CD20 is physically and functionally coupled to MHC class II and CD40 on human B cell lines.

Engagement of MHC class II and CD40 on B cell lines triggers intracellular signals that activates cell surface adhesion receptors, resulting in LFA-1-dependent and -independent cell-cell adhesion. In this study, a murine monoclonal antibody (mAb R21) has been produced against a LFA-1-negative human B cell line and proven to completely block MHC class II- and CD40-induced LFA-1-independent homotypic adhesion. However, this mAb failed to prevent MHC class II- or CD40-induced homotypic adhesion in LFA-1-positive Raji B cells, and alone, it triggered LFA-1-dependent cell-cell adhesion. Biochemical characterization indicated that the CD20 molecule, a tetraspan phosphoprotein expressed on B cells that functions as a Ca2+-conductive ion channel, is the target of mAb R21. Interestingly, further biochemical analysis demonstrated that CD20 is physically associated with MHC class II and CD40 molecules on the cell surface of LFA-1-negative and LFA-1-positive B cell lines. Although these three molecules are associated with each other, the complex formation between any two of them is not dependent on the simultaneous expression of the three molecules. Altogether, these results indicate that CD20 is physically and probably functionally coupled to the MHC class II and CD40 molecules; thereby it may have certain modulatory effects on their functions.

CD40 associates with the MHC class II molecules on human B cells.

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross-linking of MHC class II or CD40 molecules leads to the association as well as the co-association of both molecules to the NP-49-insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.

Molecular HLA-class II typing: advantages and clinical application.

The major histocompatibility complex (HLA system in Man) is characterized by its extensive degree of allelic polymorphism. After the serological and cellular methods, the molecular biological methods appeared to be a powerful tool in detecting HLA-class II polymorphism. Restriction fragment length polymorphism (RFLP), using restriction endonucleases, represented an efficient method of HLA-DR, DQ, and DP genotyping. Recently, the polymerase chain reaction (PCR) permitted the development of two major class II genotyping techniques: the PCR allele specific oligonucleotide typing and the PCR-RFLP typing. By these new methods, a refined and accurate determination of DRB, DQB, DQA, and DPB alleles is possible. The major application of a refined HLA class II typing is in transplantation especially for unrelated bone marrow grafts. Moreover, it permits the identification of the amino acids implicated in the cellular response which by itself is important for the fundamental immunological studies.

Signalling via MHC class II molecules selectively induces IL-1 beta over IL-1 receptor antagonist gene expression.

Activation of human monocytes or human monocytic cell lines by several types of stimuli coordinately induces IL-1 beta and its antagonist (IL-1Ra) gene expression; alterations in their balance seem to mediate the inflammatory response. Using the human monocytic cell line THP-1, we report that superantigens, such as staphylococcal enterotoxin A (SEA) and Mycoplasma arthritidis -derived superantigen (MAM) induce an increase in the level of IL-1 beta mRNA without any detectable effect on IL-Ra mRNA. Unlike MAM-induced IL-1 beta mRNA, SEA-induced IL-1 beta mRNA was adequately translated into protein. Superantigen-induced gene expression is mediated by signalling, via their receptors, the MHC class II molecules. Thus, it appears that this mode of signalling selectively induces the proinflammatory cytokine IL-1 beta gene expression which, by itself, can have major importance in disease pathology especially in autoimmune diseases.

Staphylococcal superantigens as inducers of signal transduction in MHC class II-positive cells.

Staphylococcal superantigens (SEs and TSST-1) interact with and potentially activate two of the main subsets of the immune system: T lymphocytes and MHC class II-positive cells. Since the interaction of SEs and TSST-1 with MHC class II molecules is the first step in triggering immune cells activation, a detailed understanding of the nature of this interaction is essential for understanding its effect on the immune system and for designing therapeutic strategies for SEs and TSST-1-mediated injury. A series of events is induced in MHC class II-positive cells (B cells, activated T cells, monocytes, and synoviocytes) upon engagement with superantigens. Some of these events require monomeric forms of superantigens, whereas others are critically dependent on cross-linking of toxin-bound MHC class II molecules by a biochemical agent (biotin-avidin) or a natural physiological one such as the TCR. The ability of superantigens to induce polyclonal activation of MHC class II-positive cells may confer to the superantigen its capacity to trigger autoimmune diseases.

Superantigens initiate cognate CD4+ T cell/B cell interactions leading to early activation and proliferation of B cells.

Dimerization or even multimerization of various receptors is commonly required for signal transduction. We report here that clustering of major histocompatibility complex class II molecules in human B cells by biotinylated staphylococcal enterotoxin A (SEA) cross-linked with avidin induces an increase in the level of intracellular calcium. This response was abolished by prior treatment with protein tyrosine kinase (PTK) inhibitors, suggesting that SEA-triggered calcium mobilization in B cells is probably dependent on the activation of PTK. The implication of PTK in SEA-induced early B cell activation was then confirmed by demonstrating that cross-linked SEA induces a significant increase in the level of tyrosine phosphorylation in B cells. The requirement of biotinavidin cross-linking in SEA-induced calcium mobilization in B cells can be fulfilled by the addition CD4+ T cells, suggesting a role for CD4 molecules. Using the murine CD4- T cell hybridoma 3DT, or its derivative I1B3 transfected with human CD4 that both express SEA-specific TCR, we confirmed the CD4 requirement for B cell calcium mobilization and that both specific TCR and CD4 molecules are required in early events of B cell activation induced by SEA. The role of CD4 in SEA-induced B cell proliferation was then investigated. SEA-stimulated B cells proliferated in the presence of CD4+ T cells, whereas no response was observed in the presence of CD8+ T cells. The addition of clone I1B3 CD4+ T cells failed to fulfill the requirement of CD4+ T cells in SEA-induced B cell proliferation, indicating the possible involvement of other CD4+ T cell surface molecules in this response. This issue is currently under investigation.

Induction of chemokine gene expression by major histocompatibility complex class II ligands in human fibroblast-like synoviocytes. Differential regulation by interleukin-4 and dexamethasone.

Chemokines play a key role in recruiting leukocytes into inflamed synovial environment, and the cells of the synovial membrane, which express high levels of major histocompatibility complex (MHC) class II molecules, are a major source of these chemokines. Our data indicated that engagement of MHC class II molecules by staphylococcal enterotoxin A superantigen resulted in the induction of chemokine gene expression as well as protein synthesis. Pretreatment of the cells with cycloheximide potentiated the effect of superantigen on chemokine mRNA induction, suggesting that the expression of these genes may occur independently of prior protein synthesis. Ligation of MHC class II molecules in fibroblast-like synoviocytes by other ligands such as Mycoplasma arthritidis-derived superantigen and anti-class II antibody could also trigger an increase in the mRNA level of RANTES, MCP-1, and interleukin (IL)-8. The addition of dexamethasone to superantigen-treated fibroblast-like synoviocytes inhibited the mRNA expression of all three chemokines. IL-4 treatment decreased only the stimulating effect of superantigen on RANTES messanger suggesting that different mechanisms are involved in regulating these genes. The inhibitory effect of dexamethasone did not require a de novo protein synthesis, whereas that of IL-4 was protein-dependent. This report demonstrates that MHC class II ligands (superantigens and anti-MHC class II antibodies) may represent an important agent by which inflammatory chemokines can be induced and shows that this response can be modulated by the anti-inflammatory agents dexamethasone and IL-4.