MHC antigens and haemopoiesis

Transplant Immunology

1994; 2:171-175

MHC antigens and haemopoiesis Ralf Huss and H Joachim Deeg Transplantation Biology Program, Fred Hutchinson Cancer Research Center,

Seattle, Washington Introduction The major histocompatibility complex (MHC) plays a central role in thymic selection, antigen recognition and cell-cell interactions. MHC class II molecules have also been identified as differentiation markers in haemopoiesis; however, it is still highly speculative to what extent MHC genes and antigens are involved in regulation of haemopoiesis and in haempoietic reconstitution such as after bone marrow transplantation. The search for the haemopoietic stem cell with the highest proliferative potential and long-term repopulating capability involves the question whether this cell is MHC class II positive, since MHC class II positive haemopoietic or accessory cells have been recognized as pivotal to posttransplant haemopoietic reconsititution.

M H C class 11 expression on haemopoietic cells Despite numerous recent studies, the question whether the earliest identifiable haemopoietic stem cell expresses MHC class II antigens (DR or its homologue) is still controversial. 1~6The fact that different species express MHC class II antigens differently contributes to the apparent inconsistencies reported in the literature and the inability to arrive at a generally agreed-upon concept. For example, dogs 7 and guinea pigs8 express class II antigens broadly, including resting T cells, whereas mice express hardly any class II antigens even after activation, and on human cells MHC class II expression, especially on T lymphocytes, depends on the stage of activation. 9 The work of Moore et al. ~ and Keating et al. z indicates that class II antigens are not expressed on early haemopoietic progenitors in humans, but appear in subsequent stages of differentiation and are maintained in some (e.g. monocytes) but not other lineages (e.g. granulocytes) during final differentiation. On the other hand, Fitchen et al. presented data suggesting that both human and murine pluripotent progenitor cells express class II antigens. 6 These investigators observed that cytolytic treatment of murine marrow cells with anti-I-E (homologous to HLA-DR), but not anti-I-A (homologous to HLA-DQ), prevented the formation of colonyAddress for correspondence: Ralf Huss, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, M318, Seattle, WA 98104, USA. © Edward Arnold 1994

forming units spleen (CFU-S) in vivo. These findings do not necessarily argue against the notion that haemopoietic stem cells are MHC class II negative since it has been recognized that the cells giving rise to CFU-S are not truly stem cells, 1° and may lack long-term repopulating ability. The terminology for stem cells, progenitor cells, or precursors has not always been used consistently. Generally, 'stem cell' describes a cell capable of generating an entire tissue or system, for example all cell components of the lymphohaemopoietic system. While the stem cell is the earliest identifiable progenitor cell, the term 'progenitor cell' is usually applied to somewhat more differentiated cells giving rise to one or several cell lineages. In part, the definition is affected by the in vivo or in vitro models used for read-out, which may allow for the detection of various stages of progenitors. The term 'precursor' applies to any cell at a stage of differentiation or maturation preceding that of the cell under discussion. Interesting in the context of a discussion of stem cells, precursor ceils and DR expression are recent experiments by Huang and Terstappen. H These investigators cloned human fetal marrow cells, and showed that single CD34÷DR CD38- cells can differentiate into haemopoietic precursors a n d stromal elements which in turn support the differentiation of haemopoietic precursors; in this D R - subset primitive mesenchymal elements were mixed with blasts. A subset of CD34*DR+CD38- cells, consisting of homogeneous primitive blasts, gave rise to all haemopoietic lineages but not to stromal cells. Whether this finding is restricted to fetal cells is not clear yet. Srour et al. showed recently that while CD34÷DR + marrow cells gave rise to more differentiated precursors, high proliferative potential colony-forming cells (HPP-CFC) were contained predominantly among CD34÷DR - progenitors; the HPP-CFC could be further enriched by selecting for c-kit positive cells.12 Other experiments emphasize that haemopoietic stem cells do not express MHC class 11.13 However, a report by Traycoff et al. 14 illustrates very clearly that among cord blood cells it is the CD34+DR ÷ Rhodamine 123 °ull fraction which contains the majority of long-term culture initiating cells and presumably the cells capable of long-term haemopoietic reconstitution in vivo. In addition, Huang and Terstappen have shown recently that even in adult marrow the earliest identifiable haemopoietic precursors express H L A - D R , although at a level (approx. 200 molecules per cell) which may not be detectable by routine analysis, a5 It will be interesting also to examine fetal liver cells, representing yet another level of devel-

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opmental haemopoiesis, to further complement the emerging picture of MHC class II expression on haemopoietic cells. In addition to their role of a marker of differentiation, MHC class II antigen expression is also affected by maturation. Griffin et al. found that 'late' CFU-GM proceeding to monocyte morphology maintained high levels of D R expression, whereas maturation of granulocytie precursors was associated with a loss of class II antigens, a6 Results by TorokStorb et aL indicate that a monoclonal antibody (mAb) specific for a supertypic determinant on H L A - D R 4, 5, 7 and 9 reacted only with B lymphocytes, but not with monocytes or other lineages. 17 Furthermore, comparisons of syngeneic B lymphoblastoid and haemopoietic cell lines reveal expression of H L A - D R , DP and D Q on lymphoid cells, TMwhereas DQ is missing on nonlymphoid haemopoietic cells. In this context, Symington et al. pointed out that a haemopoietic cell line, HEL, even though expressing H L A - D R and DP, failed to stimulate allogeneic lymphocytes in cultures. ~9 This may be related to the absence of DQ or other accessory molecules, such as B7, a ligand for CD28 on responding T cells. DQ seems to have functions different from other class 1I molecules, since its regulation in regards to promoter responsiveness, D N A binding and inducibility is different from DR and DP. Furthermore, DQB shows allelic polymorphism in the promoter sequence which is not present in the corresponding sequence for DR. 2° Thus, the expression of MHC class II genes in early haemopoietic cells is developmentally regulated and lineage dependent. Expression includes H L A - D R and DP, but not DQ.

MHC class I and nonclassical MHC genes Haemopoietic cells express MHC class I molecules virtually in all stages of maturation and differentiation beginning during embryonic development. 2~ The only exception are haemopoietic precursors derived from the murine spleen (CFU-S), which begin to express H-2 antigens at a later stage than other tissues. This might be attributed to a change of the primary site of haemopoiesis from fetal liver and spleen to bone marrow. 22 Little is known about the expression of nonclassical or minor histocompatibility antigens on haemopoietic cells.23 What role class I and minor antigens play in haemopoiesis is, therefore, still unclear.

Marrow engraftment and graft failure

engraftment in mice, 26 results in humans are consistent with an effect of GVHD on the microenvironment which may interfere with engraftment. 27 It is also conceivable that MHC restriction is involved in GVHD2S; evidence for monocytedependent MHC restriction has been shown in marrow of allosensitized patients. 29 In a canine model, marrow transplants across MHC barriers are successful in only about 10% of animals and result in graft failure in 90%. 3o In conceptual agreement with these data is the observation that engraftment can be facilitated if dogs are pretreated with anti-MHC class II m A b pre-TBI (total body irradiation) and transplant, which results in sustained engraftment in 40-50% of dogs. 31 On the other hand, if monkeys or dogs are transplanted with marrow purged with anti-MHC class II antibodies to remove DR + cells, graft failure occurs.32 In mice with donorrecipient combinations in which resistance is not a problem, bone marrow transplantation across MHC barriers does not interfere with haemopoietic reconstitution, although G V H D may alter the pattern of extramedullary haemopoiesis. 26 MHC class II 'knockout' mice also show normal haemopoiesis, 33 suggesting that murine haemopoiesis does not require the presence of MHC class II genes or gene products. These data show some clear species differences, but illustrate that in some animal models MHC class II positive donor cells are required for haemopoietic reconstitution, whereas MHC class II positive recipient cells interfere with donor cell engraftment.

Autologous With autologous transplantation, MHC class II antigens should, a priori, not impact on haemopoietic recovery; a role for MHC class 11 is suggested, however, by some studies. Dogs given a lethal dose of TBI followed by autologous marrow infusion and anti-MHC class II mAb at appropriate doses immediately post-transplant experience delayed graft failure following an initial rise of peripheral blood granulocytes. 34 The effect of anti-MHC class II mAb is overcome by the concurrent administration of recombinant canine stem cell factor (SCF), a c-kit ligand. 35 This observation provides indirect evidence for an effect of anti-class II m A b on haemopoietic growth factor production or function, and suggests a link between MHC class II and growth factor expression (see below). The notion that MHC class 1I positive donor haemopoietic cells play a pivotal role is further supported by the observation that positively selected MHC class ll (DR) expressing marrow cells are sufficient for autologous haemopoietic reconstitution in dogs. 36

Alteration of growth factor expression

Allogeneic The role of MHC class II antigens in haemopoiesis has been studied in autologous and allogeneic bone marrow transplantation. In allogeneic bone marrow transplantation, MHC compatibility is of paramount importance for a high probability of transplant success. In clinical marrow transplantation, HLA-nonidentity between donor and recipient is associated with an increased probability of graft failure when compared to HLA-identical transplants; MHC class II (DR) molecules appear to represent a particularly severe barrier, z4 Graft failure may be due to the presence of anti-HLA antibodies (sensitization) or a resistance-like mechanism, z5 While graftversus-host disease (GVHD) does not seem to interfere with Transplant Immunology 1994; 2:171-175

Haemopoietic growth and differentiation are dependent on growth factors. The stage of differentiation and distance from the pluripotent stem cell determines the proliferative response to factors such as SCF, interleukins IL-3 and IL-6, or GM-CSF (granulocyte macrophage colony-stimulating factor). Marrow stromal cells are a main source of these growth factors. 37 In human and canine models, the administration of anti-MHC class II mAb exerts an indirect effect via class II positive accessory cells (possibly macrophages or endothelial cells) on the production and secretion of essential growth factors from stromat cells.34"3s Also, treatment of canine marrow stromal cells with anti-MHC class II m A b upregu-

MHC antigens and haemopoiesis

lates mRNA for GM-CSF, whereas message for TGF-13 (transforming growth factor-13) becomes undetectable. 39 Similar results were obtained in several other in vitro systems. If canine long-term marrow cultures were propagated in the presence of anti-class II mAb, the proportion of haemopoietic ceils and CFU-GM production declined; remaining cells consisted mostly of fibroblast-like stromal cells (DS Hong, personal communication). Similarly, in a human in vitro system, anti-MHC class I1 m A b interfered with high proliferative potential long-term culture initiating cells, predominantly affecting growth that was dependent on the presence of accessory cells and S C F . 38 These data suggest, therefore, that anti-MHC class II m A b either inhibits SCF production or interferes with its effect, and that this block can be abrogated by excess exogenous SCF. Thus, stimulation of MHC class II molecules appears to induce a positive signal for the up- or downregulation of growth factors (or their receptors) required for haemopoietic development. On the other hand, a growth factor such as GM-CSF induces the expression of MHC class I I , 9 which may contribute to the expression of DR on early haemopoietic progenitors which co-express receptors for GM-CSF.

DR +

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DR +

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CD34 + DR- ~ commitment' CD34 + DR + Figure 1 MHC class It (DR) possibly marks the separation of haemopoietic stem cells (DR ÷) and cells of the microenvironment (DR-), both derived from a CD34÷DR - pluripotent stem cell. ~ Other DR * accessory cells as well as growth factors, e.g. stem cell factor (SCF), might also induce, support or direct haemopoietic maturation and differentiation. Anti-MHC class II monoclonal antibodies (mAb) can interfere either with the production of growth f a c t o r s 39 o r with the direct interaction between haemopoietic stem cells (SC) or progenitors with cells of the microenvironment.

MHC and haemopoietic differentiation MHC class II antigens represent differentiation markers of haemopoiesis in all species studied to date. The expression of class II on haemopoietic cells appears to be locus dependent insofar as H L A - D R and DP (or their homologues in other species) are expressed in most progenitor cells, whereas D Q (in man) or IA (in the mouse) appear to be present only on lymphopoietic cells. The effect of anti-class 1I mAbs on posttransplant haemopoiesis in dogs suggests that no MHC class II molecules by themselves, but secondary signals mediated by MHC class 11 are involved. This signalling event is to some extent dependent on the epitopes recognized by the antibody, since not every anti-class II antibody, even if allele specific, can block or activate class II bearing cells.4° However, while human in vitro data are in agreement with the canine in vivo model, the murine data are discrepant. Both canine and human studies indicate that not only haemopoietic, but probably also marrow microenvironmental and other accessory cells are affected by anti-MHC class II treatment. This notion provides the basis for a role of haemopoietic growth factors in anti-MHC class 1I mediated inhibition of haemopoiesis. This is further supported by the observation that patients with "bare lymphocyte syndrome', i.e. a lack of MHC class I1 expression in some respects similar to that seen in MHC class 1I knockout mice, do not show any haematological abnormality.~z Figure 1, which takes into consideration recent findings by Huang and Terstappen, n illustrates how a pluripotent stem cell may give rise to both haemopoietic progenitors and stromal elements under the influence of growth factors (e.g. SCF) and accessory cells, which directly or indirectly participate in haemopoietic maturation and differentiation. It is also possible that haemopoietic stem cells or other progenitors interact directly with cells of the microenvironment via MHC class 1I molecules. This interaction appears to be disrupted by the presence of anti-MHC class 1I mAbs. Some apparent discrepancies regarding the expression of MHC class 11 antigens on stem ceils or progenitors might be Transplant Immunology 1994; 2:171-175

due not only to interspecies differences, 42 but also to the fact that cells at different stages of development were studied. For example, in adult frogs, MHC class II expression shows a pattern as seen in the dog, whereas tadpoles express class II in a pattern similar to that seen in mice. 43 As already pointed out above, the pattern in man is different again with prominent DR expression on umbilical cord blood cells and very low expression on adult marrow cells) 4"~5This is interesting since experiments with human malignant cells, for example, from patients with chronic myelocytic leukaemia have shown stronger DR expression than normal cells.44 Malignant cells are generally arrested at an early stage of differentiation and, thus, may express a phenotype seen on embryonic but not on adult cells.

Conclusion Similar to other tissues, nucleated haemopoietic cells generally express MHC class I antigens. MHC class II expression is locus dependent ( H L A - D Q or homologous molecules are not expressed on haemopoietic cells) and developmentally regulated. Recent data suggest that in man H L A - D R is expressed on the earliest identifiable haemopoietic precursors, more so during fetal than during adult life. Interspecies differences are considerable. Whether MHC class II expression during haemopoietic recovery after marrow transplantation reflects the pattern observed during ontogeny has not been examined systematically. Similar to MHC class II molecules on lymphoid cells, class II molecules on haemopoietic precursors are capable of transmembrane signal transduction and thereby maybe involved in growth factor expression and regulation of haemopoiesis.

Acknowledgements This work was supported in part by grants C A 18221, C A 31787 and HL 36444 from the National Institutes of Health, DHHS. Ralf Huss was also supported by a grant from the

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Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany. W e thank Bonnie Larson and Harriet Hefton for typing the manuscript.

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