The Human Bone Marrow as an Immunoregulatory Organ1

It should be stated at the outset that this report does not purport to claim that a single process is responsible for the establishment of a state of immunological unresponsiveness. However, it does attempt to introduce a unifying hypothesis that may be applicable to the practical usage of protocols to establish such a state in transplantation and possibly in autoimmunity. THE EARLY IDEOLOGY It was 45 years ago that in a virtual revolution in thinking in immunology there developed the acceptance and the subsequent expansion of two new dogmas: (1) that to eliminate toxins and pathogens as the major mode of defense, individual immune cells were, in their ontogeny of differentiation, internally programmed to react singly and then clonally against the virtually limitless individual stimuli of the outside world (1-3); (2) that before this programming was manifested the immune system would fail to recognize any antigenic stimulus as foreign, thereby not differentiating non-self from self-recognition. This allowed for non-self-antigens, if introduced in this early stage, to be immunologically tolerated on subsequent testing. In the chronology of the evolution of these two dogmas, there were the earlier descriptions of specific immunological paralysis and unresponsiveness to certain defined polysaccharides and haptens demonstrated in adult mice and guinea pigs respectively by Felton (4) and Chase (5). It remained for Medawar and his colleagues (6) in allotransplantation experiments to clearly define "acquired specific immunological tolerance" as an ontogenic concept involving the lack of maturation or differentiation were tolerance to be evoked. As early as 1953, Billingham, Brent, and Medawar demonstrated that allogeneic donor bone marrow-derived cells could confer such a specifically acquired tolerant state to the immune system of the murine recipient before self versus non-self recognition occurred in immune ontogeny, the test being subsequent acceptance of skin allografts from the same donors. The developmental processes of positive versus negative selection only a decade later began to be demonstrated to predominantly involve the thymus gland in maturational events of cells of the recipient immune system in studies first performed in neonatal rodents (7). By experimental manipulation of the immune system predominantly involving immunoablation by whole body x-irradiation, these findings of specific immunological tolerance as a result of the infusion of bone marrow-derived cells could be extended to adult animals as the system regenerated from stem cells in the bone marrow of either that of the donor (8), the recipient (9), or both (10). Nonetheless, except in the setting of clinical bone marrow transplantation using major (lethal) immunoablation followed by the salvage and replacement of the recipient immunohematopoietic system with donor cell lineages and only in the context of little or no donor-recipient MHC disparity, with few exceptions (11-15), to date, specific immunological tolerance in organ transplantation in humans has not been possible. DOES THE PRESENCE OF MERELY A VESTIGIAL THYMUS IN ADULT HUMANS MEAN THAT PERMANENT SPECIFIC ALLOGENEIC UNRESPONSIVENESS IS NOT POSSIBLE? The prominence of the thymus in certain mammalian species in weight per body surface area until maturity and beyond, is unlike that which is seen in humans after the second decade. With the life expectancy in humans being more than four times this period of thymic prominence, it is inferred that the thymus becomes vestigial comparatively earlier in the life of the human. In fact, in adult (mature) mice and rats it became possible to induce a specific alloimmune unresponsive state by direct injection into the thymus both of allogeneic bone marrow-derived cells or even of allopeptides; the latter presumably operative by indirect alloantigen presentation only (vide infra) (16). It therefore became necessary in humans to attempt to elucidate postthymic or extrathymic processes if the generation of alloimmune unresponsiveness in organ transplantation or even life-long tolerance to self-antigens in the absence of continuous immunosuppression were to be possible. In this context, what has been seen in humans are other types of (nontransplant) unresponsiveness putatively acquired (years) after the participation of the human thymus in positive and negative selection (i.e., postthymic or extrathymic unresponsiveness), such as in the acquired inappropriate TH1 immune inhibition to certain infectious agents and in the loss of immune surveillance in cancer (17, 18). In parallel, in organ transplantation, seminal observations by Starzl et al. yet to be fully explained, have described the presence of functioning organ transplants in a few adult humans 20-30 years postoperatively and after decades of the total absence of continuing immunosuppressive therapy, with putatively normal immune responsiveness to all other non-alloimmune (non-self) stimuli for the universe of external pathogens and toxins. Microchimerism of donor bone marrow-derived cells appeared to accompany these observations by Starzl in the long-term organ transplant recipients (14, 19, 20), and also in newer studies by several groups, with some controversy involved concerning its importance (21-24); but, the participation of any of the microchimeric cell lineages as active inhibitors of specific allograft immunity has not been demonstrated until the present (25). Nonetheless, it appears difficult to explain in the immunologically mature adult human how the thymus gland would have played an active role. HYPOTHESIS: THE BONE MARROW AS AN IMMUNOREGULATORY ENVIRONMENT-OLDER AND MORE RECENT BACKGROUND WORK. Why do bone marrow-derived cells confer this state of specifically acquired tolerance?. A persistent observation appeared in all of these experiments to be the requirement for the continuing presence of the "tolerizing antigen" similar to that which occurs with self-antigens, in niches or compartments in the network of cells and organs of the immune system, presumably internalized into certain cell types that would be long-lived and would serve to maintain this state of specific unresponsiveness. These cells would somehow present this antigen as a tolerogen, a nonimmunogenic moiety, allowing the introduction of additional antigen without the development of the usually expected programmed acquired immunity. Experimental observations relevant to this gave rise to the concepts of clonal deletion versus clonal anergy (26, 27). Thus far, observations in transplantation have ascribed at least one of these lineages to be of bone marrow origin, as possibly an altered (or variant) professional (dendritic) antigen presenting cell (APC*) subset expressing donor allo-peptide (28, 29). Direct vs. indirect antigen presentation. The additional concept that antigens presented to naïve T cells required co-recognition of major histocompatibility complex (MHC) cell surface molecules on professional antigen presenting cells in a restricted joint recognition antigen self by T cell defined a of acquired immune (or of to in the universe of antigens versus that occurred in to antigens introduced by the of In transplantation, alloimmune recognition was demonstrated to by naïve T that could be to an absence of professional of the on molecules would be presented on the cell of the donor putatively with the on recipient T cells It to be as to such a process was as or more a of tolerance as its indirect recognition in the of allogeneic and their presentation to recipient T cells by molecules in professional of the recipient bone marrow do not present antigen in in observations be demonstrated in a subsequent and in an of these professional donor from the as by of of before transplantation allowed a of acceptance to the to in which would this In this experimental the of specifically acquired tolerance was seen to after the of the period The test of specifically acquired tolerance in the mature human immune system therefore involve of a in which could by or in or compartments of the immune system and in which this unresponsiveness to the specific antigens in would to more and in the and OF THE OF IN THE OF AND THE the in the bone marrow are several in the of differentiation of individual cell lineages and even in the development of in which molecules from cells in an earlier state than differentiation, to maintain a state of molecules which In these involve the inhibition of the of or of other molecules The of this is the to an by of cells of the of the into the to be programmed to into an in which all of these were in a state of or other of an maturational in such a from the cell have to for this differentiation to be by a process could not a of molecules operative in the the inhibition of With of a cell differentiation the of the may also as a result of a process usually involving an or the that has been the of the defined in this is the a experimental allowing the of and to of developmental events to cell this several cell have been in the importance of both positive and negative in the of cell of the in the inhibition of development by the It is that several similar to the molecules in are negative of cell differentiation in cell mammalian the development of cells of the immune system in humans these molecules of of differentiation are a of cells being in the of the then the bone marrow with its stem cells serve as a similar for such inhibition the of acquired immunity. This is the hypothesis of this OF and observations in rodents in the chronology of of other in the that at least as a of the inhibition of an expected acquired immune there was an active It was demonstrated that specifically acquired neonatal tolerance could be by recipient bone marrow-derived cells or of It was also that of allogeneic could with the of or This latter observation was then extended into similar with organ allogeneic transplants there was the of the of with the total of the allogeneic donor from the cells of the tolerant to a second recipient in this active tolerance These experiments in the observations that a of cell lineages of the recipient immune system could confer at least certain of specific unresponsiveness to other naïve recipients T cells were the predominantly subset even these T cells could not be defined as cells and the of specific (or by these cells could not be defined The in or as the network became a concept of with both and with the possible that at there be maturational unresponsiveness or However, from that the network involve the of that in the of acquired immune is the with which such could be demonstrated with immune cells (or in the was in the bone a of cells than of cells of therefore not only appeared to be more early in the life of the whole also in the early of differentiation of cell lineages This even the observation that to be present as early in the ontogeny of cell immunity In other observations in cell ontogeny cell appears to either or cell differentiation by a of molecules and A of this on in cells in the bone marrow that could be as the of these cells at a more of differentiation to be a of than more the of cells from appears to be a of this that if in the ontogeny of would be with differentiation, immunological are with the it would be expected that of the immune would be as if not in than would the of differentiation that result in the to eliminate external As all acquired immune would from earlier (vide of immune network be later than and even that cell T be more and specific than their AND THE OF IN THE BONE MARROW it became a clinical as a of human allogeneic bone marrow transplantation, was the of acquired immunity by mature immune cells of the with to establish a state of allogeneic tolerance in these donor This is the that the tolerant state have occurred in the in to the as immune ontogeny was in the after The of this have been demonstrated to be either MHC or are the context of MHC histocompatibility and cells have been to be of this the of MHC on the cells has been to be a for the generation of by mature donor T It that the would be in in which are present a of cells that these MHC molecules and The is that this is not the in if the human bone marrow is in recipients of bone marrow transplants with studies have such as organs cells and (mature) cells of and skin of bone marrow compartments in a of cells both and MHC both antigen as as molecules in such in organs are to be necessary for to be then the of by the recipient bone marrow would be This may be of the of these in cells of the bone with a as have seen in in these cells on the in of such differentiation on these and other cells (vide in the bone marrow which is more by stem cells and their and the as in in studies of of its cell lineages (vide to the marrow as a continuing organ of By the human and the of cell lineages and in their immune are organs in which is more OF THE BONE MARROW In similar to the first in the to stem cells be in the and et al. have that two of cells then the from the and from the two stem cell with the of these stem cells from the the bone which then becomes the major of life A of the of a the of the bone marrow in and A of and OF THE BONE As the is a of the generation of cell lineages that to the bone marrow in the more mature studies using cells in from or bone marrow of the development of a to cells (vide infra) these one could that donor bone marrow cells have the to cells that either or outside of the bone marrow to recipient even more would have been the The been demonstrated to be an even more organ from which the bone marrow cells are would also be as a for such et al. have even a more using than bone in a tolerance in RECENT BONE first described as cells that T cells that recognize and that some T cells even serve as cells cells have been to be of T cells specific for or MHC antigens in these earlier has been to types of T cells T cells T cell bone marrow and cells these cells do not all have the same cell surface there appears to be a of immune cells that have the to specific T cells their MHC or cells to be in all in which early of T cell development or such as bone marrow and cells to be as a result of their cell surface putatively of MHC molecules on the cell in a of in that antigen is presented in such a that the T cell of the immune is on by the MHC antigens on the cell is by some with the absence of an second present on or the in of antigen presenting cells of has been that at least a of cells may be in the such studies were also performed by et al. in bone marrow in to allograft tolerance and In that the is by a more and by a of cell not yet differentiation, than cell to be the of the of the of the such as and are as by the described a few years ago by et al. other to be such cells have also been to be lack possibly cell in a state of in this the observations by et al. and et al. of of bone marrow-derived cells immune in rodents are of human organ donor on allogeneic immune as immune in studies of the in of these in immune against donor cell a for as a of clinical allograft acceptance was to be necessary for this to by bone marrow cells and the to be a of of the as to cells of the or have cells from the cell system to that these cells (or a direct in have and allogeneic not altered by immunosuppressive in cells by and as as bone marrow cells and cells by cell and were as in of similar of donor have been by (vide infra) in the and bone marrow of allograft recipients at after infusion The the of several of cells in that are either the more cells or their direct lineages or unresponsiveness in of the of donor bone marrow on allograft recipient cell by a and The of was introduced from this for studies for after clinical bone marrow The has been described in This has allowed for the and of cell of donor (or bone marrow-derived cells in transplant recipients postoperatively with donor body bone marrow The has the of of donor or recipient cells that may have an and transplant recipients also donor body bone marrow were with similar immunosuppressive not donor bone marrow the of these the observations were (1) a at least two donor bone marrow cell lineages and in at by at and In recipient marrow using the there were of these two donor lineages and in a of and to from the bone marrow and years at and years postoperatively in these same with has demonstrated that has in the marrow and has not in the bone marrow from transplant recipients bone marrow cell (2) of cells at or after early a negative with the of seen with and organ transplant postoperatively of donor total cells as as and in recipient marrow at this as with of were in recipients were MHC with their of was the absence of in this This observation to a of with alloimmune It also is with of in humans and recipients were for MHC antigens and with a of allograft tolerance in pigs and in rodents with MHC are (4) The of the donor T cells in both and bone marrow of recipients to years as with the donor to an have that this subset in the donor marrow In to an expected unresponsiveness as described by and in the bone there was a and state of the recipients with the an immunosuppressive This was from observations of (1) the of in on immunity were (2) the of to and the observations in on T and which appears to have allowed a in in these without as with the more (i.e., versus the specific donor and versus of a were demonstrated in the bone (i.e., vs. donor were also and after bone marrow infusion and transplantation using and were presented at the of the of and in which the donor transplant either or followed for to and the not the same immunosuppressive were also with donor The recipients of were one donor and were followed from using the same immunosuppressive (vide both and the was by and for at the with similar cell in were performed by were in the and in the However, only recipients and were and in the and and in the two of were in the the was with one was In both groups, was not with As was with at least a in of and donor bone marrow-derived cells and with a in later seen in the recipient bone marrow at a of not seen with of any In not have recipient bone marrow an in and postoperatively in donor total of cells as as and cells from and to and more have demonstrated in some recipients as donor at the as by has to as as in the recipient bone marrow not in has to in In the and were only one recipient which in this occurred earlier than one postoperatively and not of the recipients that in was at than in followed at for However, the only of the total in the cells for vs. cells for these studies to (1) of an of histocompatibility on (2) a possible long-term of on allograft This appears to have also been by et al. in transplant recipients donor in the The of as a for in (4) a seen in the recipient marrow on the development of and after IN as of immunity. In a from an system was to if would similar on from positive normal and transplant was from either transplant recipients or their cells were with cell earlier from the same or were as in were performed using the cells and cell or In both the normal and transplant recipients there was a inhibition of generation by bone marrow These studies to the bone marrow in T cell against antigens in adult humans as an organ of such In a more indirect as seen a in in bone transplant recipients with recipients a similar immunosuppressive In the bone by the presence of bone marrow cells of this T cell was (vide of bone marrow cells on generation as immune as as in have observations that in in to allogeneic This may T which are not as by and as are postthymic T these studies in and were to the and of and individual vs. cells using allogeneic cells in the presence or absence of immunosuppression In the absence of immunosuppressive the in and in to to a than In these to as of both and cells from this to for of the of in the presence of the allogeneic cells as described in a report several experimental using to the as that cells of the were in as cells with allogeneic cells as such cells rise both to as as to cells of and singly or in no on the of and on of its or with the of cells as the described of on the of or cells were not by these immunosuppressive in the there appears to be a of cells that in antigen presentation by a negative lack these the that in which as cells in with allogeneic cells and which as a to to be cell of allogeneic be to these cells in the may be and to the immunosuppressive of of or bone marrow donor cells and were or of the cells with the system using with or were by cells were with allogeneic cells for and a in was are as T cells from cell using by not with to cells using by to T cells by with in which such by of both using the by immunosuppressive in of the of donor with cells of donor bone marrow cells for the of allograft acceptance in clinical to the of in and were this of T and (i.e., not were in with allogeneic a was seen in both and to cells as the with were of However, several experimental which MHC antigen on the appeared to their These (1) with allogeneic cells from the for (2) of and positive using and of which MHC on the and in more of cells from the by positive selection of cells from these allogeneic virtually allogeneic and by even using the that were the of et This was in to the of these same seen with cell This the requirement for indirect alloantigen presentation if a was to be to these allogeneic of the of these to by direct alloantigen direct antigen presentation by allogeneic bone marrow cells is the of cells of a in bone marrow is THE OF IN THE BONE MARROW As a in in recipients of bone marrow and it was that there was a that occurred the cells in the recipient bone marrow for after transplantation, as to the cells of the donor bone marrow infusion from the from were test the bone marrow cells expressing donor were from the recipient bone marrow one after transplantation using and The on of clearly that with donor or even to donor bone marrow cells also from the marrow of the after transplantation the same these cells of donor from the recipient bone marrow were more inhibitors of the recipient and to the donor (25). In it is that these donor cells from the recipient bone marrow were at least inhibitors of these than the donor marrow cells to similar protocols if to cell Thus it would that there is a direct in the marrow with alloimmune for of alloimmune of recipient to donor by donor cells from the recipient for clinical infusion have in and and transplant protocols in several from of a being by the the other major clinical the of donor bone marrow organ In transplant an early immunosuppressive is and the of the infusion is cell therapy, with and are the early T cells and early more than has been seen in the other This has the of on early and later immunosuppression has been in the In the into the the as as to after of two of recipient body weight per infusion on have been more than in studies from other (vide for In the only one infusion of donor bone marrow was possible into the It is also that as a result of more early an of naïve T the to some These recipient naïve cells and of the usually T the latter by or and are to a of bone marrow cells after into the of with and these the of that their maturation are also by and have been in these naïve T cells by their with the more allogeneic marrow of the events in this early as as in later years after transplantation, have to be what are the developmental of these recipient and the that these or does this to and T cell by a to the lack of programmed cell or even the of the bone marrow cells have the to negative involving it has also been on that the of immunosuppressive may the of tolerance in human transplantation, in of bone marrow cells if immunosuppressive protocols the hypothesis to be is the bone marrow as a these for this be as a to of the by the observations described in this (1) Why than with certain do bone marrow-derived cells to be necessary in the of allogeneic unresponsiveness (i.e., this be with whole organs except for a for (2) Why does in the bone marrow and not in the in clinical observations of after Why are cells in the bone marrow more in than bone marrow cells on the alloimmune of there not joint operative as a result of in cell of allogeneic of donor and recipient bone marrow cells in the recipient bone marrow allowing to does the of these cells their in this (4) Why do bone marrow cell from the human marrow to as as in the that be to the in the of would be as to the it is of the bone marrow to more and or into the marrow to in an organ in which a is one of