Bone marrow cultures for developing suppressor and stimulator cells for research and therapeutic applications

A method of treatment using immune system suppressor cells and immune system stimulator cells comprises providing stem cells; combining the stem cells with lymphoid-derived cells to produce a co-culture; adding lipopolysaccharide and a factor selected from the group consisting of granulocyte macrophage-colony stimulating factor, macrophage colony stimulating factor, granulocyte-colony stimulating factor and mixtures thereof to the co-culture; obtaining immune system suppressor cells and immune system stimulator cells from the co-culture; and introducing and the cells into a host.

TECHNICAL FIELD 
Previous experimental models have demonstrated the beneficial effects of 
donor specific blood, bone marrow or spleen cell transfusions and 
attributed the allograft survival advantage to a variety of cellular 
candidates, including veto-like cells. See, e.g., Transplantation 43:4332 
(1987). These models require intrathymic injections in immature 
recipients, bone marrow ablation, or the use of antilymphocyte serum 
("AIS"), in most instances administered prior to transplantation. These 
restrictions along with the modest effects of bone-marrow transfusion in 
prospective human trials have limited the clinical application of these 
methods. In the present invention, the use of donor specific transfusions 
("DST") using these cells can achieve better results to other models. 
While bone marrow cultured alone is known to generate natural 
immunosuppressant cells, the infusion of co-culture cells is substantially 
more effective in prolonging allograft survival. This is especially so 
when GM-CSF (granulocyte macrophage-colony stimulating factor) and LPS 
(lipopolysaccharide) are part of the culture conditions. It appears that 
the suppressor cell activity derived from this co-culture is not donor 
strain-specific (as third party MLRs (mixed lymphocyte reactions) are 
equally suppressed). Furthermore, its generation is not dependent on donor 
specific splenocytes, given the equal to greater graft survival seen with 
allogeneic (ACI marrow plus Lewis spleen) co-culture infusion. 
Factors, such as GM-CSF and LPS are found to induce maturation of bone 
marrow cultures and generate natural suppressor cells. GM-CSF can enhance 
the effect of splenic cells on allograft survival. Indeed, GM-CSF has 
numerous other functions including the expansion, stimulation and 
development of monocyte populations. The present invention combines these 
two cell sources and produces a co-culture using both LPS and GM-CSF to 
optimize suppressor cell generation and function. These cells can be 
subsequently administered as a form of pre-transplant or post-transplant 
DST. The present invention generates highly potent suppressor cells which, 
when given as a DST on the day prior to transplants, may result in a 
marked prolongation of allograft survival. The present invention also 
generates highly potent immune system stimulatory cells. 
BACKGROUND OF THE INVENTION 
Improved allograft survival, occasionally leading to a state of stable 
transplant tolerance in immunologically mature mammalian systems is 
achievable by a variety of methods, usually including the use of 
antilymphocyte antibody preparations and some form of donor cell infusion. 
Models vary with respect to type of allograft and the timing/route of drug 
and donor cell administration but many combinations have proved 
successful. Despite these advances, the clinical application of many of 
these methods is hampered by the lack of consistent and reliable results 
in higher primates and man, along with the realities of technical, 
logistical and chronological limitations inherent in human 
transplantation. There is, therefore, further need to examine options in 
the transfusion associated induction of improved allograft survival in 
solid organ transplantation. 
Certain factors have not been fully addressed. First, the avoidance of 
intensive immunosuppression to limit the incidence of opportunistic 
infection and tumor formation makes a protocol that avoids use of potent 
antibody preparations desirable. Second, the enrichment of an antigen/cell 
source allowing sufficient supply for the multiple recipients of organs 
from a single cadaveric donor is important. Lastly, the application of an 
in vitro culture method to enrich immunomodulatory cells could allow for 
the use of important cytokines and other agents not easily tolerated 
in-vivo. 
The use of either donor blood, bone marrow cells or splenocytes has proved 
most successful in the induction of prolonged graft survival. In some of 
these models, the generation of a suppressor-type cell appears to be 
operative. In other work, the generation of potent suppressor cell 
populations has been achieved by in-vitro culture of bone marrow with the 
use of stimulators, especially lipopolysaccharide (LPS). The 
administration of granulocyte macrophage-colony stimulating factor 
(GM-CSF) or granulocyte colony stimulating factor ("G-CSF") to donors of 
bone marrow DST enhances allograft survival. Furthermore, GM-CSF is known 
to activate marrow-derived natural suppressor cell function. The 
fractionation of both cell cultures and transplant cellular sources can 
isolate and enrich subpopulations of cells that retain the properties of 
suppressor cell function and equally prolong allograft survival. 
Transplantation 
A major goal in solid organ transplantation is the permanent engraftment of 
the donor organ without a graft rejection immune response generated by the 
recipient, while preserving the immunocompetence of the recipient against 
other foreign antigens. Typically, in order to prevent host rejection 
responses, nonspecific immunosuppressive agents such as cyclosporine, 
methotrexate, steroids and FK506 are used. These agents must be 
administered on a daily basis and if stopped, graft rejection usually 
results. However, a major problem in using nonspecific immunosuppressive 
agents is that they function by suppressing all aspects of the immune 
response, thereby greatly increasing a recipient's susceptibility to 
infections and other diseases, including cancer. 
Furthermore, despite the use of immunosuppressive agents, graft rejection 
still remains a major source of morbidity and mortality in human organ 
transplantation. Most human transplants fail within 10 years without 
permanent graft acceptance. Only 50% of heart transplants survive five 
years and 20% of cadaveric kidney transplants survive 10 years. (See Opelz 
et al., 1981, Lancet 1:1223; Gjertson, 1992, UCLA Tissue Typing 
Laboratory, p. 225; Powles, 1980, Lancet, p. 327; Ramsay, 1982, New Engl. 
J. Med., p. 392). It would therefore be a major advance if tolerance to 
the donor cells can be induced in the recipient. 
The only known clinical condition in which complete systemic donor-specific 
transplantation tolerance occurs is when chimerism is created through bone 
marrow transplantation. (See Qin et al., 1989, J Exp Med. 169:779; Sykes 
et al., 1988, Immunol. Today 9:23; Sharabi et al., 1989, J. Exp. Med. 
169:493). This has been achieved in neonatal and adult animal models as 
well as in humans by total lymphoid irradiation of a recipient followed by 
bone marrow transplantation with donor cells. The success rate of 
allogeneic bone marrow transplantation is, in large part, dependent on the 
ability to closely match the major histocompatibility complex ("MHC") of 
the donor cells with that of the recipient cells to minimize the antigenic 
differences between the donor and the recipient, thereby reducing the 
frequency of host-versus-graft responses and graft-versus-host disease 
("GVHD"). In fact, MHC matching is essential, only a one or two antigen 
mismatch is acceptable because GVHD is very severe in cases of greater 
disparities. In addition, it also requires the appropriate conditioning of 
the recipient by potential lethal doses of total body irradiation (TBI) or 
cytotoxic drugs. 
The MHC is a gene complex that encodes a large array of individually unique 
glycoproteins expressed on the surface of both donor and host cells that 
are the major targets of transplantation rejection immune responses. In 
the human, the glycoproteins are referred to as HLA (human leukocyte 
antigen) antigens. When HLA identity is achieved by matching a patient 
with a family member such as a sibling, the probability of a successful 
outcome is relatively high, although GVHD is still not completely 
eliminated. However, when allogeneic bone marrow transplantation is 
performed between two MHC-mismatched individuals of the same species, 
common complications involve failure of engraftment, poor immunocompetence 
and a high incidence of GVHD. Unfortunately, only about 20% of all 
potential candidates for bone marrow transplantation have a suitable 
family member match. 
The field of bone marrow transplantation was developed originally to treat 
bone marrow-derived cancers. It is believed by those skilled in the art 
even today that lethal conditioning of a human recipient is required to 
achieve successful engraftment of donor bone marrow cells in the 
recipient. In fact, prior to the present invention, current conventional 
bone marrow transplantation has exclusively relied upon lethal 
conditioning approaches to achieve donor bone marrow engraftment. The 
requirement for lethal irradiation of the host which renders it totally 
immunocompetent poses a significant limitation to the potential clinical 
application of bone marrow transplantation to a variety of disease 
conditions, including solid organ or cellular transplantation, sickle cell 
anemia, thalassemia and aplastic anemia. 
Immunosuppressive agents are also extensively used following organ 
transplantation for the prevention of rejection episodes. In particular, 
cyclosporine, a potent immunosuppressive agent, prolongs the survival of 
allogeneic transplants involving skin, heart, kidney, pancreas, bone 
marrow, small intestine, and lung in animals. Cyclosporine has been 
demonstrated to suppress some humoral immunity and to a greater extent, 
cell mediated reactions such as allograft rejection, delayed 
hypersensitivity, experimental allergic encephalomyelitis, Freund's 
adjuvant arthritis, and graft versus host disease in many animal species 
for a variety of organs. 
U.S. Pat. No. 5,514,364, Ildstad, issued May 7, 1996, discloses non-lethal 
methods of conditioning a recipient for bone marrow transplantation. In 
particular, it relates to the use of sub-lethal doses of total body 
irradiation, cell type-specific antibodies, especially antibodies directed 
to bone marrow stromal cell markers, cytotoxic drugs, or a combination 
thereof. The methods of the invention have a wide range of applications, 
including, but not limited to, the conditioning of an individual for 
hematopoietic reconstitution by bone marrow transplantation for the 
treatment of hematological malignancies, hematological disorders, auto 
immunity, infectious diseases such as acquired immunodeficiency syndrome, 
and the engraftment of bone marrow cells to induce tolerance for solid 
organ, tissue and cellular transplantation. 
U.S. Pat. No. 5,486,359, Caplan et al., issued Jan. 23, 1996, discloses 
isolated human mesenchymal stem cells which can differentiate into more 
than one tissue type (e.g., bone, cartilage, muscle or marrow stroma), a 
method for isolating, purifying, and culturally expanding human 
mesenchymal stem cells (i.e., "mesenchymal stem cells" or "hMSCs"), and 
characterization of and uses, particularly research reagent, diagnostic 
and therapeutic uses for such cells. The stem cells can be culture 
expanded without differentiating. 
None of these references individually or collectively teach or suggest the 
present invention. 
SUMMARY OF THE INVENTION 
The present invention relates to methods of producing distinct populations 
of cells capable of immune suppressor and stimulatory action. 
Specifically, the methods relate to a combination of splenocyte and bone 
marrow cells, co-cultured with LPS and GM-CSF, which induce two cell 
subpopulations. One cell subpopulation has an immune system suppressory 
function capable of prolonging allograft survival when infused as a donor 
specific blood transfusions ("DST") with a low dose, eight day course of 
cyclosporine ("CSA"). Another cell subpopulation has an immune system 
stimulatory function capable of stimulating the immune system when infused 
as a DST. 
Previous experimental models have demonstrated the beneficial effects of 
donor specific blood, bone marrow or spleen cell transfusions and 
attributed the allograft survival advantage to a variety of cellular 
candidates, including veto-like cells. See, e.g., Transplantation 43:4332 
(1987). In the present invention, the use of donor specific transfusions 
("DST") using these cells can achieve better results to other models. 
The present invention combines these two cell sources and produces a 
co-culture using both lipopolysaccharide ("LPS") and granulocyte 
macrophage-colony stimulating factor ("GM-CSF") to optimize suppressor 
cell generation and function. These cells can be subsequently administered 
as a form of pre-transplant DST. The present invention generates highly 
potent suppressor cells which, when given as a DST on the day prior to 
transplants, may result in a marked prolongation of allograft survival. 
The present invention also generates highly potent stimulatory cells. 
The method of their isolation comprises the steps of providing a tissue 
specimen containing stem cells, adding cells from the tissue specimen to a 
medium which contains factors that stimulate stem cell growth and allows, 
when cultured, for the selective differentiation of the stem cells into 
two distinct subpopulations of cells, one with an immune suppression 
function and one with an immune stimulation function. Any suitable cell 
sorting method may then separate the cells as known in the art. 
In another aspect, the present invention relates to a medium for the 
selective differentiation of the stem cells into two distinct 
subpopulations of cells, one with an immune suppression function and one 
with an immune stimulation function, wherein the medium comprises cells 
and/or factors that stimulate stem cell differentiation. 
In a further aspect, the present invention relates to a kit for 
differentiating stem cells from a tissue specimen. The kit comprises a 
medium containing factors and/or cells which stimulate the differentiation 
of the stem cells into two distinct subpopulations of cells, one with an 
immune suppression function and one with an immune stimulation function. 
In an additional aspect, the present invention is directed to various 
methods of utilizing the differentiated stem cells produced by the present 
invention for therapeutic and/or diagnostic purposes. For example, the 
differentiated stem cells may find use in: (1) regenerating tissues which 
have been damaged through acute injury, abnormal genetic expression or 
acquired disease; (2) treating a host with damaged tissue by removal of 
small aliquots of bone marrow, differentiating them in vitro, isolating 
the desired subpopulation of differentiated cells and reintroducing the 
differentiated cells back into the host (3) detecting and evaluating 
growth factors relevant to the immune system; (4) detecting and evaluating 
inhibitory factors which modulate the immune system. 
The present invention also relates to methods of inducing at least partial 
tolerance to an antigen comprising the steps of administering to the 
recipient animal a tolerogenic amount of a dendritic cell population, and 
administering to the recipient animal a tolerogenic amount of a suppressor 
cell population, substantially contemporaneously with the dendritic cell 
population. The antigen may be an alloantigen, autoantigen, or 
xenoantigen. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to methods of producing distinct populations 
of cells capable of immune suppressor and stimulatory action. 
Specifically, the methods relate to a combination of splenocyte and bone 
marrow cells, co-cultured with LPS and GM-CSF, which induce two cell 
subpopulations. One cell subpopulation has an immune system suppressory 
function capable of prolonging allograft survival when infused as a donor 
specific blood transfusions ("DST") with a low dose, eight day course of 
cyclosporine ("CSA"). Another cell subpopulation has an immune system 
stimulatory function capable of stimulating the immune system when infused 
as a DST. 
The present invention combines these two cell sources and produces a 
co-culture using both lipopolysaccharide ("LPS") and granulocyte 
macrophage-colony stimulating factor ("GM-CSF") or granulocyte colony 
stimulating factor ("G-CSF") to optimize suppressor cell generation and 
function. These cells can be subsequently administered as a form of 
pre-transplant DST. The present invention generates highly potent 
suppressor cells which, when given as a DST on the day prior to 
transplants, may result in a marked prolongation of allograft survival. 
The present invention also generates highly potent stimulatory cells. 
The method of their isolation comprises the steps of providing a tissue 
specimen containing stem cells, adding cells from the tissue specimen to a 
medium which contains factors that stimulate stem cell growth and allows, 
when cultured, for the selective differentiation of the stem cells into 
two distinct subpopulations of cells, one with an immune suppression 
function and one with an immune stimulation function. Any suitable cell 
sorting method may then separate the cells as known in the art. 
In another aspect, the present invention relates to a medium for the 
selective differentiation of the stem cells into two distinct 
subpopulations of cells, one with an immune suppression function and one 
with an immune stimulation function, wherein the medium comprises cells 
and/or factors that stimulate stem cell differentiation. 
In a further aspect, the present invention relates to a kit for 
differentiating stem cells from a tissue specimen. The kit comprises a 
medium containing factors and/or cells which stimulate the differentiation 
of the stem cells into two distinct subpopulations of cells, one with an 
immune suppression function and one with an immune stimulation function. 
In an additional aspect, the present invention is directed to various 
methods of utilizing the differentiated stem cells produced by the present 
invention for therapeutic and/or diagnostic purposes. For example, the 
differentiated stem cells may find use in: (1) regenerating tissues which 
have been damaged through acute injury, abnormal genetic expression or 
acquired disease; (2) treating a host with damaged tissue by removal of 
small aliquots of bone marrow, differentiating them in vitro, isolating 
the desired subpopulation of differentiated cells and reintroducing the 
differentiated cells back into the host (3) detecting and evaluating 
growth factors relevant to the immune system; (4) detecting and evaluating 
inhibitory factors which modulate the immune system; (5) treatment of 
infections, cancer, and burns in a patient in need of such treatment; (6) 
treatment of autoimmune diseases, e.g., rheumatoid arthritis; and (7) for 
pre- or post-transplantation therapy. 
Factors, such as GM-CSF and LPS are found to induce maturation of bone 
marrow cultures and generate natural suppressor cells. GM-CSF can enhance 
the effect of splenic cells on allograft survival. Indeed, GM-CSF has 
numerous other functions including the expansion, stimulation and 
development of monocyte populations. In one embodiment of the present 
invention, the method combines these two cell sources and produces a 
co-culture using both LPS and GM-CSF to optimize suppressor cell 
generation and function. These cells can be subsequently administered as a 
form of pre- or post-transplant DST utilizing highly potent suppressor 
cells generated which, when given as a DST on the day prior to 
transplants, may result in a marked prolongation of allograft survival. 
The present invention also generates highly potent stimulatory cells that 
can be subsequently administered as a method of stimulating the immune 
capability in an immune compromised patient. 
While not being bound by theory, it is believed that the action of the 
splenocytes on the bone marrow cells is likely derived from a secretory 
product; other embodiments may utilize a culture system where the 
splenocytes are replaced by other secretory cells. Still other embodiments 
may utilize a culture system where the splenocytes are replaced by 
cytokines, cytokine-producing cells, or lymphoid derived cells. 
While bone marrow cultured alone is known to generate natural suppressor 
cells, the infusion of co-culture cells is substantially more effective in 
prolonging allograft survival. This is especially so when GM-CSF and LPS 
are part of the culture conditions. It appears that the suppressor cell 
activity derived from this co-culture is not donor strain-specific (as 
third party MLRs are equally suppressed). Furthermore, its generation is 
not dependent on donor specific splenocytes. 
Bone marrow is the soft tissue occupying the medullary cavities of long 
bones, some haversian canals, and spaces between trabeculae of cancellous 
or spongy bone. Bone marrow is of two types: red, which is found in all 
bones in early life and in restricted locations in adulthood (i.e., in the 
spongy bone) and is concerned with the production of blood cells (i.e., 
hematopoiesis) and hemoglobin (thus, the red color); and yellow, which 
consists largely of fat cells (thus, the yellow color) and connective 
tissue. 
As a whole, bone marrow is a complex tissue comprising hematopoietic stem 
cells, red and white blood cells and their precursors, mesenchymal stem 
cells, stromal cells and their precursors, and a group of cells including 
fibroblasts, reticulocytes, adipocytes, and endothelial cells which form a 
connective tissue network called "stroma". Cells from the stroma 
morphologically regulate the differentiation of hematopoietic cells 
through direct interaction via cell surface proteins and the secretion of 
growth factors and are involved in the foundation and support of the bone 
structure. Studies using animal models have suggested that bone marrow 
contains "pre-stromal" cells that have the capacity to differentiate into 
cartilage, bone, and other connective tissue cells. (Beresford, J. N., 
Clin. Orthop., 240:270 (1989)). Recent evidence indicates that these 
cells, called pluripotent stromal stem cells or mesenchymal stem cells, 
have the ability to generate into several different types of cell lines 
(i.e., osteocytes, chondrocytes, adipocytes, etc.) upon activation. 
However, the mesenchymal stem cells are present in the tissue in very 
minute amounts with a wide variety of other cells (i.e., erythrocytes, 
platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, 
adipocytes, etc.), and, in an inverse relationship with age, they are 
capable of differentiating into an assortment of connective tissues 
depending upon the influence of a number of bioactive factors. 
The methods of the present invention may use bone marrow cells obtained 
from iliac crest, femoral, tibiae, spine, rib or other medullary spaces. 
Alternatively, the methods could use stem cells from other sources of 
human stem cells, e.g., from embryonic yolk sac, placenta, umbilical cord, 
fetal and adolescent skin, and blood. 
Mesenchymal stem cells are the formative pluripotential blast cells found 
inter alia in bone marrow, blood, dermis and periosteum that are capable 
of differentiating into any of the specific types of mesenchymal or 
connective tissues (i.e., the tissues of the body that support the 
specialized elements; particularly adipose, osseous, cartilaginous, 
elastic, and fibrous connective tissues) depending upon various influences 
from bioactive factors, such as cytokines. In a further embodiment, the 
bone marrow cells may consist entirely of mesenchymal stem cells. 
The hematopoietic microenvironment is primarily composed of hematopoietic 
cells and stromal cells. Hematopoietic cells include pluripotent 
uncommitted stem cells and unipotent committed stem cells, and 
differentiate into macrophages, monocytes, lymphocytes, erythrocytes, 
platelets, basophils, neutrophils and eosinophils. The stromal cells 
occupy much space of the bone marrow environment and they include 
endothelial cells that line the sinusoids, fibroblastic cells such as 
adventitial reticular cells, perisinusoidal adventitial cells, 
periarterial adventitial cells, intersinusoidal reticular cells and 
adipocytes, and macrophages (Dorshkind, Annu. Rev. Immunol. 8:111, (1990); 
Greenberger, Crit. Rev. Oncology/Hematology 11:65, (1991)). In another 
embodiment, the bone marrow cells may comprise hematopoietic cells or 
stromal cells. 
The present invention includes a method of achieving stable engraftment of 
donor organs or tissues by conditioning a recipient for organ or tissue 
transplantation with a donor cell preparation containing stem cells 
conditioned to differentiate into immune system suppressory cells. 
In a further embodiment, the stem cell is a cell selected from the group 
consisting of endothelial cells, adventitial reticular cells, 
perisinusoidal adventitial cells, periarterial adventitial cells, 
intersinusoidal reticular cells, adipocytes, macrophages and hematopoietic 
facilitator cells. In another embodiment, mesenchymal stem cells are used. 
The methods of the present invention provide an isolated, homogeneous 
population of stem cells that are differentiated into cells of distinct 
subpopulations. Preferably, the stem cells are obtained from bone marrow. 
Most preferably, the stem cells are obtained from bone marrow of a donor 
who was previously a recipient of the differentiated cells. Alternatively, 
the stem cells are obtained from periosteum. 
In another embodiment, the present invention provides a composition 
comprising the stem cells and a culture medium; wherein said culture 
medium differentiates the said stem cells into two subpopulations of 
cells. 
In another embodiment, the present invention provides a therapeutic 
composition comprising one of the subpopulations of differentiated stem 
cells and a pharmaceutically acceptable carrier; wherein said 
subpopulation of stem cells is present in an amount effective to produce 
immune system regulation. 
Discussion 
The suppressor effect may be concentrated by PERCOLL fractionation and 
further enriched by FACS (fluorescence antibody cell sorter) sorting of 
the "nonlymphocyte" region of this fraction. In stark contrast to fraction 
3, cells isolated in fraction 4 are highly stimulatory. On light 
microscopy the fraction 3 cells appeared as highly activated foamy 
macrophages. The FACS signature of this cell population is bland, with few 
positive markers noted in the active region of fraction 3. Specifically, 
the cell is not a lymphocyte, showing no staining for CD, CD4, CD8 or the 
rat pan-T cell clone OX-52. The cells are positive for a rat macrophage 
antigen marker (clone R2-1A6A) and we found significant staining with the 
antibody clone OX-33 (marking for CD45RA) in these cells as well. This 
cell bears little similarity to veto cells, dendritic cells, facilitator 
cells, progenitor cells, or some of the previously described natural 
suppressor cells, but appears to be a highly suppressive macrophage 
derived from the conditions of the co-culture. 
Specific data regarding tolerance induction using these cells is not yet 
available, but individual graft survival can be greatly prolonged. We have 
found the ACI to Lewis strain combination to be remarkably stringent, with 
loss of graft function from acute rejection occurring reliably at about 7 
days in controls, and at 16 days in animals given DST and CSA. In animals 
receiving tolerance induction protocols, rejection is still possible, even 
after 100 days of allograft survival without drug therapy. Furthermore, 
while the choice of CSA is important for its potentiating effect on the 
DST of blood protocol, the low dose CSA used in this protocol confers no 
independent advantage for graft survival. 
These findings are of interest given the relative potency of the suppressor 
cell generated, with PHA and MLR suppression in-vitro or graft survival 
prolongation in-vivo using a 10-100 fold decrease in cell numbers as 
compared to previous reports. Similar cells (although less potent) have 
been described and the general phenomenon of increased effectiveness of 
fractionated cells is in agreement with previous studies. Donor specific 
bone marrow transfusion with previous ALS treatment is noted to reduce the 
number or function of donor-reactive cytotoxic T lymphocytes for the 
duration of graft survival. A myeloid-derived natural suppressor cell has 
also been described in human bone marrow, but the effect of GM-CSF or LPS 
on these cells is not currently known. When those untreated cells are used 
in in-vitro experiments, they are found to be immunostimulatory at "low" 
doses (.ltoreq.4.times.10.sup.5 cells added to 1.times.10.sup.6 responder 
cells) and suppressive at higher doses. Cells with potent, nonspecific 
natural suppressor function are also noted in the circulation of long term 
renal transplant recipients. Activated macrophages from other sources, 
such as lung lavage, are known to possess suppressor function, however, 
macrophages represent a large group of varied circulating and tissue 
associated effector cells with a bewildering repertoire of secretory 
products and functions. Different macrophages behave quite differently and 
accurate comparisons of co-culture derived cells with in-vivo derived 
cells cannot be made. Furthermore, the specific function of the cell in 
question can be variable, as both veto activity and natural suppressor 
activity can be demonstrated by human lymphokine activated cells depending 
on their concentration in the MLR. 
The use of spleen cells in the co-culture is important given the much lower 
allograft survival noted with pure bone-marrow culture infusion. Since 
both spleen and marrow are contaminated with peripheral blood white cells, 
but marrow cells cultured alone failed to enhance allograft survival, it 
seems likely that a cell or factor(s) secreted by a cell within the spleen 
is responsible for this added effect. Previous studies using transfused 
spleen cells show prolongation of graft survival only with intermediate 
doses of cells and show these spleenderived suppressor cells to be 
FcR.sup.+ non-lymphocytes. Whether the macrophage-like cell noted in 
fraction 3 is of splenic or marrow origin, whether splenic lymphocytes or 
other cells provide "help" in the culture, and why recipient specific 
spleen works as well (or better) than donor specific spleen is currently 
under study. The effectiveness of syngeneic spleen cells in this model is 
intriguing. In the past, only donor specific spleen has routinely been 
used with success (but not in co-culture). Use of recipient specific 
spleen to condition an organ donor is not easily applied clinically. The 
fact that the splenic strain is interchangeable in invention is a novel 
finding. 
The improvement in allograft survival seen with co-culture cell infusion is 
achieved with a clinically ineffective dose of CSA, without ALS, and in a 
non-irradiated recipient with a mature (adult) and very strong recipient 
immune system. The possibility of further enhancement of allograft 
survival with the addition of ALS exists, but the timing of ALS therapy 
with a day 1 DST may be difficult. In bone marrow DST studies, technique 
and timing of administration become important as graft vs. host disease, 
sensitization with hyperacute rejection, and complete loss of the DST 
effect can all occur. For example, the use of bone-marrow infusion on day 
0 with concurrent or subsequent ALS has been shown to cause sensitization 
rather than improved allograft survival. A number of ALS preparations 
including monoclonal antibodies (anti-CD3) are known to induce secretion 
of colony stimulating factors, possibly making administration prior to 
bone-marrow cell infusion important. Furthermore, the use of ALS after 
bone marrow cell infusion may alter the cytokine milieu and reduce the 
effect. Therefore, correlates might be found between the in-vivo and 
in-vitro timing of GM-CSF induction, cytokine secretion and cell infusion. 
While removing the need for prolonged, pre-transplant drug therapy and 
shortening the period between DST administration and transplantation is a 
significant advance toward clinical applicability, the use of any day--1 
protocol is still not always easily applicable to the cadaveric donor 
situation, especially when co-culture time is considered. Although 
suppressor cells are shown to be capable of inhibiting proliferation of 
actively dividing, prestimulated lymphocytes, whether such suppressions of 
an MLR or post-transplant in vivo sensitization are possible remains to be 
studied. The culture method described could however, obviate the need for 
marrow cryopreservation in protocols using the transfusion later in the 
post-transplant course, while enhancing the effect of the administered 
cells. Furthermore, suppressor cells may be obtainable with shorter 
culture times, as marrow cells cultured only four days note inhibition of 
splenocyte proliferation. DST given only 12 hours pre-transplant and in 
many models multiple DSTs after transplants are also effective strategies 
allowing immediate clinical applicability of co-cultures maintained for 
shorter periods of time. 
The cytokine milieu developed within the co-culture is undoubtedly 
significant in the development and therefore function of the suppressor 
cells created. The use of LPS in bone marrow culture induces interferon 
("IFN") dependent production of nitric oxide, responsible for suppressor 
cell functions but other cytokines are likely involved in the development 
of the suppressor cell as well. We did note a high level of IFN present 
initially in our culture supernatant. As this mediator is shown to be 
important in suppressor cell function, activation, and the restoration of 
activation after impairment of suppressor function by glucocorticoids, it 
may be an important initial signal at the start of the culture conditions. 
The levels of TGF.beta. (transforming growth factor beta) are noted to 
rise over time, a consistent finding both with the development of natural 
suppressor cells in the culture and with their subsequent actions in 
prolonging the survival of allografts. In addition, IL-4 (interleukin-4) 
may be important for suppressor cell development or function (or as a 
consequence thereof) as concentrations are noted to progressively rise 
over the 7-day course of co-culture. Other cytokines, including IL-6 
(interleukin-6) may play an important role in the maintenance of 
suppressor function as well. In our study, IL-6 (interleukin-6) is low 
initially, peaked at day four, and remained relatively high on day seven. 
Low levels of TNF (tumor necrosis factor) are detected for all culture 
times. In other studies, TNF.alpha. (tumor necrosis factor alpha) has been 
shown to inhibit the function and division of human marrow progenitor 
cells in vitro. We have not measured cell-associated TNF in this model, 
however. As anticipated, significant elevations in PGE.sub.2 
(prostaglandin E.sub.2) levels are noted over time with peak levels at day 
seven. This finding is consistent since PGE.sub.2 can be immunosuppressive 
and is a secretory product of activated suppressor cells. Overall, the 
culture supernatants show an initial pro-inflammatory profile over the 
first four days, converting around that time to include a different class 
of regulatory cytokines associated with suppressor cell function. 
The effect of co-culture supernatant on the MLR response is of interest, 
especially since inhibition is not seen until day four and early 
supernatant (day 0) is stimulatory, suggesting that the cells placed in 
culture are initially primed to be pro-inflammatory. However, suppression 
of peripheral blood monocyte response to PHA by normal alveolar 
macrophages is thought to be dependent upon cell-cell contact. Those cells 
are obtained from healthy subjects, used fresh, and not cultured with 
stimulatory factors. Several other studies have demonstrated and initially 
characterized potent, soluble, suppressive factors secreted by marrow and 
spleen derived suppressor cells. Clinically important soluble factors have 
been described from other cell sources as well. The possibility that veto 
and suppressor mechanisms are not due to separate cells, but represent a 
spectrum of functions has some support in the literature. It seems likely 
that cytokines, like those noted in the co-culture supernatants 
(PGE.sub.2, TGF.beta. and others) are responsible for at least a part of 
the MLR suppression, and may represent the soluble factors noted when 
highly activated suppressor cells are studied. Whether a lower state of 
activation or a reduced effective concentration of these cells could 
result in veto-like functions remains to be studied. 
The current model shows a macrophage, derived from seven days of LPS and 
GM-CSF stimulated co-culture of bone marrow cells and splenocytes, with 
the ability to inhibit both in-vitro and in-vivo immune responsiveness, 
resulting in significant prolongation of rat cardiac allograft survival. 
This cell demonstrates natural suppressor functions at low doses in vitro 
(a single fraction three macrophage per 200 responder splenocytes) and 
remarkable potency when infused in a transplant model (&lt;1.times.10.sup.7 
whole culture cells/kg). These results are achieved with low dose 
(clinically ineffective) CSA as the only immunosuppressive agent. Further 
purification and characterization of these cells and determination of 
their mode of action should lead to a better understanding of the 
mechanisms of their suppressive effect and immune tolerance in general. 
Bone Marrow Preparation. Whole, unfractionated bone marrow is typically 
obtained from rib or long hones by flushing with Hank's Balanced Salt 
Solution ("HBSS"), or vertebral body bone marrow obtained by crushing with 
a bone rongeurs and elution with buffer solution, is filtered through 
nylon mesh and the viability assessed by trypan blue exclusion using a 
hemocytometer. The cells are collected by centrifugation at 1400 rpm for 
five minutes and then lysed with 25 mL of sterile, distilled water 
followed immediately by 25 mL of double strength HBSS. Cells are then 
recollected by centrifugation and pooled into one conical tube using a 
horizontal pipetting technique to exclude agglutinated material. Cells are 
resuspended in Roswell Park Memorial Institute Media ("RPMI") at a 
concentration of 1-2.times.10.sup.6 large mononuclear cells /mL and 
incubated at 37.degree. C. in 5% CO2 for one hour (to adhere cells). The 
nonadherent cells are collected and washed in HBSS and the viability 
rechecked. Cells are stored at 4.degree. C. until used. 
Splenocyte Preparation. Typically, splenic tissue is cut into small 
(approximately 0.5-1 gm) pieces and crushed using any convenient method 
(between glass slides and using a scraping technique with a sterile 
disposable blade), and collected in HBSS. The tissue is filtered through 
nylon mesh and collected by centrifugation. The cells are pooled and 
resuspended in 15 mL of HBSS, beneath which is layered 7.5 mL of 
FICOLL-HYPAQUE. This gradient is centrifuged at 1000 rpm for 45 minutes 
(no brake) and the interface cells collected and pooled. The pellet and 
solution are discarded. The pooled cells are washed three times in HBSS 
and resuspended in RPMI, and viability and cell counts checked. Cells are 
stored at 4.degree. C. until used. 
Culture Set-Up. Splenocytes and bone marrow cells are resuspended in RPMI 
or similar medium to a concentration of 2.times.10.sup.6 cells/mL and 
added together in equal amounts. The media is supplemented with the 
appropriate growth factor or stimulant and incubated at 37.degree. C. in 
5% CO.sub.2 (minimum but not to exceed 10%) at a concentration of 
3.3.times.10.sup.5 cells/square cm floor surface. Cultures are maintained 
using standard humidity, for at least four days (and up to 14 days) 
undisturbed and unshaken. 
Supplemental Factors: Specific supplementation with colony stimulating 
factors is necessary. The current technique utilizes species specific 
granulocyte macrophage-colony stimulating factor (GM-CSF). Where species 
specific factor is unavailable (i.e., the rat) murine GM-CSF is used and a 
concentration of 100 units/mL is added at the start of the culture. 
Additional agents for the stimulation and maturation of cells are added. 
The current model employs lipopolysaccharide (LPS or bacterial endotoxin) 
obtained from E coli and added at 1 .mu.g/mL at the start of the culture. 
Additional growth/factors that may be used in addition or in place of 
GM-CSF include macrophage colony stimulating factor ("M-CSF"), 
granulocyte-colony stimulating factor ("G-CSF") and the FLT3 ligand. 
Additional stimulating factors that may be added or used in place of LPS 
include interluekin-6, interleukin-4, tumor necrosis factor alpha 
("TNF.alpha."), and transforming growth factor beta ("TGF.beta. "). Again, 
all factors used are species specific unless unavailable, in which case 
either murine or human factors can be employed. 
Cell Harvest. Typically, cells are harvested and pooled by first obtaining 
nonadherent cells and rinsing the culture container and collecting the 
cells and washes in separate tubes. Following centrifugation, supernatants 
may be saved or frozen for analysis. The cells are resuspended and counted 
and their viability checked using trypan blue exclusion. Cells may be used 
unfractionated or following a number of fractionation techniques. The 
current method employs the use of unfractionated cells resuspended in 
sterile, pH-balanced medium. For fractionation, a discontinuous PERCOLL 
gradient is used with a 100%, 70%, 60%, 50%, 40% and 0% stepwise dilution. 
The cells are suspended in 100% PERCOLL and equal volumes of diluted 
PERCOLL are layered sequentially above the cells. The gradient is 
harvested after centrifugation at 1000 g (2300 rpm) for 60 minutes. Cells 
are obtained separately at each interface, counted, and the viability 
checked. The individual subfractions may be used alone or in combinations 
for research or therapeutic purposes. 
The methods of the present invention may be used to enhance allograft 
survival in a transplant patient and also to obtain specific populations 
of cells with stimulatory or suppressive capacities using fractionation by 
the PERCOLL method, FACS sorting or other suitable cell separation method 
as known in the art. These subpopulations may be used in in vitro testing 
using lymphocyte proliferation assays. This technique can provide cell 
cultures in animal and human systems for use in specific clinical/medical 
applications involving malignancy or inflammation. Whole cultures or 
derived subpopulations can be applied (by standard or intraportal venous 
transfusion techniques) for the treatment of immune system dysfunction, 
infection prevention and modulation of systemic inflammation in patients 
with thermal injury, infection, multiple trauma, and sepsis. The 
transfusion of donor-specific cell cultures can be used for the prevention 
or reduction in organ transplant rejection. Subpopulations of culture 
cells will be useful in the treatment of autoimmune disorders and 
malignancies. 
The use of different growth factors and stimulatory agents/cytokines can be 
tailored to the specific therapeutic advantage (see above), the use of 
these same or other growth factors may mobilize marrow cells into donor 
peripheral blood as a source of culture cells. The use of growth factors 
of stimulatory agents to enhance the effects of the cultured cells before, 
during or after administration is also a direct extension of this work. 
Co-culture Technique: Fresh spleens and lower extremity long bones are 
harvested aseptically and kept on ice in Hank's balanced salt solution 
(HBSS, Gibco Life Technologies, Gaithersburg Md.). Spleens are crushed 
between glass slides and sterile, filtered, splenic tissue is washed in 
HBSS, centrifuged on a FICOLL-HYPAQUE (HISTOPAQUE 1077, Sigma Diagnostics, 
St. Louis, Mo.) gradient at 1000 RPM.times.45 min. and the white cell 
layer harvested, washed twice, resuspended in Roswell Park Memorial 
Institute (RPMI) medium (Gibco) with 10% fetal calf serum (FCS) (Hyclone 
Labs, Logan, UT) and 100 units/mL penicillin, 100 .mu.g/mL streptomycin 
sulfate, and 0.25 .mu.g/mL amphotericin B (Gibco), to 2.times.10.sup.6 
cells/ml. Bone marrow harvests are performed as previously described by 
Ogle et al., Inflammation 18:175 (1994). The marrow cells are resuspended 
in RPMI+10% FCS, counted and diluted to 1.times.10.sup.6 large, 
mononuclear cells/ml. Twenty-five mL aliquots are incubated for 60 min. at 
37.degree. C. and 5% CO.sub.2 in 250 mL, 75 cm.sup.2 sterile, vented 
culture flasks (Costar, Cambridge Mass.). Nonadherent cells are then 
collected, washed and resuspended to 2.times.10.sup.6 cells/mL. 
Co-cultures consisted of equal volumes of spleen and marrow cells (total 
1.times.10.sup.6 of each cell stock/mL), most with added LPS (1 .mu.g/mL, 
E. coli 055:B5, Sigma Chemical, St. Louis, Mo.) and murine GM-CSF (100 
units/mL, R and D Systems, Minneapolis, Minn.) and are incubated for up to 
seven days at 37.degree. C. and 5% CO.sub.2 in 25 ml volumes in 250 mL 
sterile culture flasks. All cell counts are based on viable cells by 
trypan blue exclusion. Nonadherent co-culture cells are harvested at 
predetermined times, washed, resuspended and counted. 
Some initial cultures using nonadherent bone marrow only are performed with 
various combinations of LPS, murine GM-CSF, recombinant human TNF.alpha. 
(100 units/ml, Endogen, Boston Mass.) or unmodified media and harvested at 
day four or seven and used in mixed lymphocyte and phytohemagglutinin 
(PHA, Sigma, St. Louis, Mo.) experiments. 
Cell Fractionation: Cells are washed twice in HBSS, pelleted in a 15-ml 
conical tube at 1400 RPM.times.5 min. and resuspended to 2 ml in PERCOLL 
(Pharmacia, Uppsala, Sweden) solution (0.95 ml 10.times. HBSS in 9.05 ml 
sterile 100% PERCOLL, pH titrated to 7.35) over which are layered 2 ml 
each of 70%, 60%, 50%, 40% and 0% (plain HBSS) PERCOLL dilutions. After 
centrifugation at 2800 RPM.times.30 min., fractions at gradient interfaces 
are retrieved, washed and resuspended in RPMI with 10% FCS. Fractions (Fr) 
are labeled sequentially from the 100-70% interface (Fr 1) to the 40-0% 
interface (Fr 5). The fractionation of untreated bone marrow yielded four 
fractions (lacking cells at the 50-40% interface), while seven-day 
co-cultures yielded five fractions. Fraction five contained 97% dead 
cells, while Frs 1 and 2 contained very few but mostly viable cells (these 
are submitted for FACS analysis). Fraction 3 contained the majority of 
total cells (66%) at 95% viability, while Fr 4 contained somewhat fewer 
cells at similar viability (Frs 3 and 4 are used in further experiments). 
Subsequent separation of Fr 3 cells is accomplished by sterile, viable 
cell sorting on an Epics 753 cell sorter (Coulter, Miami Fla.) into 
subpopulations based on scatter pattern (debris, lymphocyte, and 
macrophage regions). An average of 60% of cells is lost during sorting. 
This is attributed to fragility acquired by some cells over time in 
culture. Macrophage region cells are used in further experiments. 
Mixed Lymphocyte Reactions: Spleens are harvested aseptically, crushed, 
filtered and washed in HBSS, then resuspended in 15 ml HBSS and separated 
on a FICOLL-HYPAQUE gradient at 1000 RPM.times.45 min. The white cells are 
harvested, washed, resuspended in RPMI with 10% FCS and cells incubated 
with 25 Fg Mitomycin C (Sigma, St. Louis, Mo.)/2.5.times.10.sup.7 cells/mL 
for 45 minutes at 37.degree. C. and 5% CO.sub.2 then washed 3.times. in 
RPMI are used as stimulator cells. Responder and stimulator cells are 
added to standard 96 well culture plates at 2.0.times.10.sup.6 cells/well 
to which are added titrations of whole co-culture, fr 3, or fr 4 cells or 
titrated amounts of co-culture supernatant. Cells are pulsed with 0.5 
FCi/well of .sup.3 H-thymidine (1 mCi/mL New England Nuclear, Boston, 
Mass.) and harvested on day 4. Activity is measured as counts per minute 
(cpm) with an LS600TA liquid scintillation counter (Beckman, Fullerton, 
Calif.). All reactions are performed in triplicate and results expressed 
as mean values. 
PHA (phytohemagglutinin) stimulation: Spleens are harvested, washed and 
separated as for MLR above. White cells resuspended in RPMI with 10% FCS 
are incubated in triplicate on standard 96 well culture plates with 1 
Fg/well PHA. Co-culture and Fr 3 cells are titrated in variable amounts 
and cpm are measured as for MLRs. All results are expressed as means of 
triplicate values in both MLR and PHA reactions. The percentage of 
suppression is calculated using the following formula: 
EQU % suppression=100.times.{cpm.sup.test cells .times.100/cpm.sup.control }. 
The % of activation is calculated simply as the multiple of increase over 
baseline.times.100. 
Antibodies and FACS Analysis: Biotin labeled IgG anti-pan-T (murine clone 
OX-52), IgG anti CD4 (OX-38), and IgG anti-CD8a (G28) with labeled isotype 
IgG.sub.2a ; biotin labeled IgG anti-CD45RA (OX-33) with labeled isotype 
IgG.sub.1 ; biotin labeled IgA anti-rat CD11b (wt.5) with labeled isotype 
IgA;phycoerytherin (PE) labeled IgG anti-rat RT1b (anti MHC-II, clone 
OX-6) and IgG anti-Thy 1.1 (CD90, clone OX-7) with labeled isotype, biotin 
labeled IgG anti-TCRa.beta. (R73) with biotin and PE labeled IgG.sub.1 
isotypes; and PE labeled IgG anti-CD3 (G4.18) with labeled isotype 
IgG.sub.3 are purchased from Pharimgen (San Diego, Calif.). Fluorescein 
isothiocyanate (FITC) labeled IgM anti-PMN and FITC labeled IgM 
anti-PMN/Mac (R2-1A6A) with labeled isotype are purchased from Caltag (San 
Francisco, Calif.). Biotin labeled wheat germ agglutinin (WGA) is 
purchased from Sigma Chemical Co. (St. Louis, Mo.) and streptavidin red 
670A is purchased from Gibco. Standard labeling technique using murine IgG 
blocker, 30 minute cold incubations and isotypic controls are performed 
prior to fixation of cells in paraformaldehyde. As a control, binding of 
biotin labeled WGA is inhibited by a pre-incubated in 2000 fold excess 
concentration of N-acetyl glucosamine (Sigma) to determine 
non-lectin-mediated fluorescence. FACS is performed on an EPICS XL scanner 
(Coulter), selecting separate lymphocyte and nonlymphocyte gated regions 
for analysis. 
Cytokine Analysis: Aliquots of co-culture supernatant are collected at 
predetermined times and frozen at -70.degree. C. for later analysis. 
Levels of IFN, IL-4, TGF.beta. (Genzyme, Cambridge, Mass.) and PGE.sub.2 
(Cayman Chemical, Ann Arbor, Mich.) are detected by enzyme-linked 
immunosorbent assays using standard, commercially available kits (murine 
IFN and IL4, human TGF.beta.). Bioassays for TNF and IL-6 (L 929 and 7TD 
cell lines, American Type Culture Collection, Rockville, Md.) are also 
performed using standard techniques. All studies are performed in 
triplicate and the results expressed as a mean value. 
Immunosuppression and Cardiac Transplantation: Heparinized whole blood is 
collected from ACI donors and administered fresh to anesthetized Lewis 
recipients via the penile vein on the day prior to transplantation in the 
DST control group (1 mL/recipient). Experimental groups received 
2.5.times.10.sup.6 viable cultured cells resuspended in 1 ml 
RPMI/recipient on the day prior to transplants. Heterotopic 
intra-abdominal ACI to Lewis cardiac transplants are performed using the 
method of Ono and Lindsey, J. Thorac. Cardiovasc. Surg 57:225 (1969). 
Briefly, the aortic root and pulmonary artery are anastomosed to the 
recipient infrarenal aorta and vena cava using standard microvascular 
techniques. Cyclosporine A, a gift of Sandoz Pharmaceuticals (East 
Hanover, N.J.), is dissolved in olive oil (Sigma) at a concentration of 5 
mg/mL and given at a dose of 10 mg/kg subcutaneously the day before 
transplantation with a daily dose of 2.5 mg/kg for seven days beginning on 
the day of engraftment. Allograft survival is assessed by daily palpation 
with rejection defined as a cessation of palpable contractions confirmed 
under general anesthesia by celiotomy. Graft survival statistics are 
expressed as a group means i the standard error. Animal groups included 
untreated, untransfused controls, standard DST controls and experimental 
groups. 
Statistics: Individual in vitro results are compared to baseline using the 
standard analysis of variance (ANOVA) followed by Tukey's test. Animal 
group survival data are compared by non-parametric Kruskal-Wallis test 
followed by pairwise comparisons using the Wilcoxon test. Significance is 
defined as a p value .ltoreq.0.05. 
Results 
The combinations including GM-CSF and LPS show near complete suppression 
remain at day seven (and are increased over day four effects). There is no 
significant increase in suppression when TNF.alpha. is added to GM-CSF and 
LPS. The combination of GM-CSF and LPS is used for the remainder of the in 
vitro experiments. White cell differentials determined by standard light 
microscopy with Wright-Giemsa stain show significant numbers of small 
mononuclear cells and occasional blasts. Clarification of monocyte 
phenotypes on day seven with double esterase staining show the majority of 
these cells are macrophages. 
Several control groups are done including one receiving no treatment (group 
1), a CSA group without DST (group 2), and a standard group given 
heparinized, whole ACI blood as a DST (group 3). Experimental groups 
performed include animals receiving infusions of 2.5.times.10.sup.6 (or 
roughly 7.5.times.10.sup.6 /kg) adherent or nonadherent bone marrow cells 
cultured with GM-CSF and LPS (groups 4 and 5). Subsequent co-cultures are 
performed with nonadherent bone marrow cells only; group 6 without LPS and 
GM-CSF, and group 7 with LPS and GM-CSF. A final group is given an 
allogeneic co-culture using ACI bone-marrow cells and Lewis splenocytes 
(group 8). In the absence of treatment, rejection occurs reliably around 
seven days post-transplantation. Infusion of bone marrow cells cultured 
without LPS and GM-CSF prove to be inferior to a DST of whole blood (9.3 
vs. 16.6 days), but when co-cultured with splenocytes, results are equal 
to standard DST. Allograft survival in groups 7 and 8 is significantly 
improved compared to all other groups. Both syngeneic (ACI marrow with ACI 
spleen) and allogeneic (ACI marrow with Lewis spleen) co-cultures 
prolonged mean allograft survival past five weeks, with some grafts 
lasting more than 100 days. 
The in-vitro effects of co-cultured cells demonstrate a marked suppression 
of the MLR and PHA responses in a dose dependent fashion with 
1.times.10.sup.4 cells showing significant suppression and near complete 
suppression noted with the addition of 5.times.10.sup.4 co-culture cells. 
To further isolate the suppressor cell population, seven-day co-cultures 
are then separated on a PERCOLL gradient and the fractions collected. 
Fractions 3 and 4 are titrated in MLR reactions and have different effects 
on MLR activity. Fraction 3 shows initiation of significant suppressive 
effects at the same starting cell concentrations as the whole coculture, 
while fraction 4 cause significant stimulation characteristic of an 
active, two-way MLR. The suppressive effects of ACI co-cultures are not 
strain specific as "third party" MLRs with Buffalo stain stimulators are 
also suppressed. Suppression of the PHA response by fraction 3 is somewhat 
more striking in that fewer added cells is required to suppress this 
response as compared to the MLRs. As noted above, fractions 3 and 4 
contain significant numbers of macrophage/monocyte populations. To 
identify serial changes in co-culture cell phenotypes, FACS analysis of 
monoclonal cell-surface markers is performed on fractions 3 and 4. Several 
changes in cell-surface markers are noted in the whole culture, yet 
fraction 3 cells are rather bland, losing most surface staining 
characteristics in the non-lymphocyte region. Scatter analysis of fraction 
3 shows a mixed population of cells, including a lymphocyte region as well 
as a distinct population of larger, more granular cells appearing in the 
macrophage region. This population is less evident in Fr 4. These cells 
are further sorted by scatter to test the hypothesis that the suppressive 
cells would be found in the nonlymphocyte (macrophage/monocyte) region. 
Sorting of unfixed Fr 3 cells produces 5.times.10.sup.5 macrophage region 
cells and 7.5.times.10.sup.4 lymphocyte region cells. The nonlymphocyte 
region cells are tested for their ability to suppress MLRs and demonstrate 
an increased potency over whole fraction 3 cells with complete suppression 
at 5.times.10.sup.3 added cells/well, indicating enrichment of the 
suppressor cells. Wright-Giemsa staining of cells from this region show 
them to be foamy, granulated macrophages, indicating a high degree of 
cellular activation. 
Early concentrations of IFN are high, but diminish by day 4. Conversely, 
IL-4 is found in low concentrations initially, but increase substantially 
over the 7-day culture period. Levels of TGF.beta. are also noted to rise 
over time. Bioassays for TNF show low levels throughout, while IL-6 peaks 
temporarily around day 4. MLRs performed with 50, 10 or 5FL of added 
supernatant show stimulation at day 0, and produced suppression at high 
concentration by day 4, and at all concentrations on day 7. 
Alternate Embodiment 
The success of a transplant depends on preventing the immune system of the 
host recipient from recognizing the transplant as foreign and, in some 
cases, preventing the graft from recognizing the host tissues as foreign. 
For example, when a host receives a bone marrow transplant, the 
transplanted bone marrow may recognize the new host as foreign, resulting 
in graft versus host disease (GVHD). Consequently, the survival of the 
host depends on preventing both the rejection of the donor bone marrow as 
well as rejection of the host by the graft immune reaction. 
At present, deleterious immune reactions are prevented or treated by 
general immune suppression in that the suppression is not antigen 
specific. Nonspecific immune suppression agents, such as steroids and 
antibodies to lymphocytes, put the host at increased risk for infection 
and development of tumors. In recent years, unwanted immune reactions have 
been prevented or treated with more selective immune suppression, such as 
with Cyclosporine A (CsA). CsA was thought to inhibit the proliferation of 
cytotoxic T cells while having relatively little effect on the 
proliferation of suppressor T cells. In addition, immunosuppressive 
therapy with CsA leads to depletion of the thymic medullary dendritic 
cells, the principal antigen presenting cells of the adult thymus. 
Although CsA has significantly improved the overall success of transplants 
and has shown some success with autoimmune diseases, it must be 
administered for the life of the patient. As a result, patients receiving 
such long-term CsA therapy are constantly at considerable risk for 
infections and neoplasms, as well as toxicity from the CsA. 
Unwanted immune reactions which can result in autoimmune disease and 
transplant rejection can also be inhibited using steroids, azathioprine, 
anti-T cell antibodies, and more recently, monoclonal antibodies to T cell 
subpopulations. In addition to CsA, other selective immunosuppressive 
drugs that have been used include rapamycin, desoxyspergualine, and FK506 
(tacrolimus). Unfortunately, when such agents are withdrawn, the unwanted 
immune reactions often recur. Ideally, it is a primary goal of the methods 
of the present invention to reduce the amount of general immunosuppressive 
drugs given to the host. As a result, the tolerant host would remain 
capable of reacting appropriately to other antigens such as those 
associated with life-threatening infections or neoplasms. 
Therefore, in another embodiment of the present invention, it is possible 
to separately culture donor specific dendritic cells while, in parallel, 
culturing suppressor cells in separate media. In this embodiment, 
dendritic cells and suppressor cells are grown according to methods well 
known in the art. 
Typically, an immunosuppressant agent is administered substantially 
contemporaneously with the cells. A preferred element of the present 
invention is that the immunosuppressive agent is administered in only some 
relatively low dosages. 
The method of the invention is useful for preventing an immune reaction to 
alloantigens (the antigens of an allograft) of transplanted organs from 
other human donors (allografts). An allograft is a graft to a genetically 
different member of the same species. Allografts are rejected by virtue of 
the immunological response of T lymphocytes to histocompatibility 
antigens. Such tissues for transplant include, but are not limited to, 
heart, liver, kidney, lung, pancreas, pancreatic islets, brain tissue, 
cornea, bone, intestine, skin, and hematopoietic cells. The method of the 
invention is useful in preventing graft versus host disease in cases of 
mismatched bone marrow or lymphoid tissue transplanted for the treatment 
of acute leukemia, aplastic anemia, and enzyme or immune deficiencies, for 
example. 
The method of the invention is also useful for treatment of autoimmune 
diseases where the immune system attacks the host's own tissues. Such 
autoimmune diseases include, but are not limited to, type 1 
insulin-dependent diabetes mellitus, adult respiratory distress syndrome, 
inflammatory bowel disease, dermatitis, meningitis, thrombotic 
thrombocytopenic purpura, Sjogren's syndrome, encephalitis, uveitic, 
leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever, 
Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis, 
primary biliary cirrhosis, pemphigus, pemphigoid, necrotizing vasculitis, 
myasthenia gravis, multiple sclerosis, lupus erythematosus, polymyositis, 
sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNS 
inflammatory disorder, antigen-antibody complex mediated diseases, 
autoimmune haemolytic anemia, Hashimoto's thyroiditis, Graves disease, 
habitual spontaneous abortions, Reynard's syndrome, glomerulonephritis, 
dermatomyositis, chronic active hepatitis, celiac disease, autoimmune 
complications of AIDS, atrophic gastritis, ankylosing spondylitis and 
Addison's disease. 
In this alternate embodiment, the present invention provides methods of 
inducing at least partial tolerance to an antigen comprising the steps of 
administering to the recipient animal a tolerogenic amount of a dendritic 
cell population; and administering to the recipient animal a tolerogenic 
amount of a suppressor cell population, substantially contemporaneously 
with the dendritic cell population. The antigen may be an alloantigen, 
autoantigen, or xenoantigen. 
In yet another embodiment, the present invention includes a method of 
inducing at least partial tolerance to an antigen comprising the steps of 
administering to the recipient animal a tolerogenic amount of a dendritic 
cell population; administering to the recipient animal a tolerogenic 
amount of a suppressor cell population, substantially contemporaneously 
with the dendritic cell population; and administering an immunosuppressive 
agent for a time and under conditions sufficient to induce allograft 
tolerance wherein the immunosuppressive agent is administered 
substantially contemporaneously with the administration of dendritic cells 
and suppressor cells of the present methods. The antigen may be an 
alloantigen, autoantigen, or xenoantigen. 
Suppressor cells are isolated and cultured alone in order to generate 
natural immunosuppressant cells. The suppressor cells, which may be 
utilized in the present invention, include any of the cells that act to 
suppress the action of the host immune system. These include natural 
suppressor cells ("NSC"), suppressor T-cells ("CD8+" or "T8+"), any 
suppressor macrophage ("SM"), or any suppressor derived from bone marrow 
progenitor "immature" cells which are cultured into mature suppressor 
cells. The CD8+ cells may be isolated from peripheral blood. 
Dendritic cells ("DC") may be obtained from one of the sources known in the 
art by known methods. Typically, the cells are obtained from the bone 
marrow, blood, liver, fetal liver, or the spleen or may be obtained by 
differentiating bone marrow progenitor cells. 
The methods of the present invention may use bone marrow cells obtained 
from iliac crest, femora, tibiae, spine, rib or other medullary spaces. 
Alternatively, the methods could use stem cells from other sources of 
human stem cells, e.g., from embryonic yolk sac, placenta, umbilical cord, 
fetal and adolescent skin, and blood. 
These cells can be subsequently administered as a form of pre-transplant or 
post-transplant DST. When given as a DST on the day prior to 
transplantation, the treatment results in a marked prolongation of 
allograft survival. 
The fractionation of both cell cultures and transplant cellular sources can 
isolate and enrich subpopulations of cells that retain the properties of 
suppressor cell function and equally prolong allograft survival. The 
suppressor cells may be concentrated by PERCOLL fractionation and further 
enriched by FACS sorting. 
One or more of the hormones selected from the group consisting of GM-CSF, 
cytokines, interleukins (e.g., IL-2, IL-3, IL-4, IL-6), TNF, and LPS may 
be added as part of the culture conditions. 
A "cytokine" is any one of the groups of hormone-like mediators produced by 
lymphocytes. Representative cytokines include but are not limited to IL1, 
IL2, IL3, IL4, IL6 and gamma IFN. The term cytokine is generally accepted 
to extend to other trophic factors that share biological functions and/or 
receptor signaling properties with lymphocyte-derived mediators. 
As used herein, "an effective amount" or "tolerogenic amount" of the 
therapeutic cells is that the number of cells administered which includes 
enough cells to be capable of inducing at least partial tolerance to an 
alloantigen in a host animal. As used herein, "a safe and effective 
amount" of the therapeutic cells is pharmaceutically safe to a subject and 
that amount includes enough cells to cause an increase in allograft 
survival while causing no side effects or an acceptable level of side 
effects. 
Therapeutic cells of the present invention are grown in an appropriate 
growth medium. As used herein, the term "appropriate growth medium" means 
a medium containing nutrients required for the growth of cells. Nutrients 
required for cell growth may include a carbon source, a nitrogen source, 
essential amino acids, vitamins, minerals and growth factors. The pH of 
the medium is preferably maintained at a pH greater than about two and 
less than about eight, preferably at about pH 6.5. Methods for maintaining 
a stable pH include buffering and constant pH control, preferably through 
the addition of sodium hydroxide. Preferred buffering agents include 
succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Cultured 
mammalian cells are generally grown in commercially available 
serum-containing or serum-free media. Selection of a medium appropriate 
for the particular cell line used is within the level of ordinary skill in 
the art. 
The nutrient medium can contain, as elemental components, sodium, 
potassium, calcium, magnesium, phosphorus, chlorine, amino acid(s), 
vitamin(s), hormone(s), antibiotics, serum, or other chemical components 
depending on application purposes. The nutrient medium can contain a 
cytokine such as interleukin-1, interleukin-2, interleukin-3, 
interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, 
interleukin-9, interleukin-10, interleukin-11, interleukin-12, TNF (Tumor 
Necrosis Factor), gamma--interferon, a granulocyte macrophage 
colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating 
factor (G-CSF), or a macrophage colony-stimulating factor (M-CSF), 
depending on application purposes. 
The two separate cell cultures (dendritic cells and suppressor cells) may 
be propagated for about 1 day to about 21 days and are preferably 
propagated from about 2 days to about 10 days and most preferably for 
about 7 days. Typically, the cells will be propagated for about the same 
length of time. However, one type could be grown for a different length of 
time. At the end of the propagation phase, the dendritic and suppressor 
cells are recovered and delivered to a host. 
Introduction into Host 
It is contemplated that target cells will be located within an animal or 
human patient, in which case a safe and effective amount of the 
therapeutic cells, in pharmacologically acceptable form, would be 
administered to the patient. Generally speaking, it is contemplated that 
useful methods of the present invention will include the selected cells in 
a convenient amount, e.g., from about 1.times.10.sup.6 total cells/kg body 
weight to about 1.times.10.sup.9 total cells/kg body weight of the 
dendritic and suppressor cell mix, administered at a dosage of about 
1.times.10.sup.5 total cells/kg body weight to about 1.times.10.sup.8 
total cells/kg body weight of the dendritic and suppressor cell mix. 
Typically, the two populations of cells will be mixed prior to 
administration to a host but may be administered separately to the host. 
The cells are preferably administered in a ratio from about 2:1 to about 
1:20 dendritic cells:suppressor cells. The cells are more preferably 
administered in a ratio of from about 1:1 to about 1:10 dendritic 
cells:suppressor cells. 
Alternatively, the two populations may be administered separately over a 
course of about 1 day to about 14 days. 
Typically, the cells are diluted in a pharmacologically or physiologically 
acceptable carrier, such as, for example, phosphate buffered saline. The 
route of administration and ultimate amounts of material that is 
administered to the patient or animal under such circumstances will depend 
upon the intended application and will be apparent to those of skill in 
the art in light of the factors which follow. 
The therapeutic cells can comprise, in addition to the cells, compounds 
and/or compositions that will also aid in relief of the symptoms of a 
target disease, such as immunosuppressant drugs, native hormones, adjuvant 
compounds or complementary drugs and hormones, in dosages useful for 
relief of the symptoms of the particular disease, as known to those 
skilled in the art. Dosages for the above-mentioned additional compounds 
are established and known to those skilled in the art. The ratio of 
therapeutic cells to additional agents is dependent upon the dose desired 
of each individual compound. Preferably, the additional agent will be 
administered as a pharmaceutically acceptable solution. 
The compound useful in the present inventive method may be administered by 
any suitable means. One skilled in the art will appreciate that many 
suitable methods of administering the compound to an animal in the context 
of the present invention, in particular a human, are available, and, 
although more than one route may be used to administer a particular 
compound, a particular route of administration may provide a more 
immediate and more effective reaction than another route. 
The cells should be administered such that a therapeutical number resides 
in the body. The number of cells administered to an animal, particularly a 
human, in the context of the present invention should be sufficient to 
effect a therapeutic response in the animal over a reasonable period of 
time. 
The cells may be administered in a pharmaceutically acceptable carrier. 
Pharmaceutically acceptable carriers are well known to those who are 
skilled in the art. The choice of carriers will be determined in part by 
the kind and number of cells delivered, as well as by the particular 
method used to administer the composition. Accordingly, there are a wide 
variety of suitable formulations of the pharmaceutical composition of the 
present invention. 
Formulations suitable for intravenous and intraperitoneal administration, 
for example, include aqueous and nonaqueous, isotonic sterile injection 
solutions, which can contain anti-oxidants, buffers, bacteriostats, and 
solutes that render the formulation isotonic with the blood of the 
intended recipient, and aqueous and nonaqueous sterile suspensions that 
can include suspending agents, solubilizers, thickening agents, 
stabilizers, and preservatives. 
The exact amount of such compounds required will vary from subject to 
subject, depending on the species, age, and general condition of the 
subject, the severity of the disease that is being treated, the particular 
compound used, its mode of administration, and the like. Since the cells 
may be administered intravenously, intra-arterially, or peritoneally, the 
choice will be determined by such factors as the location of the target 
site(s) within the body. The dosage will vary according to such factors as 
degree of compatibility of the donor and recipient, the health of the 
host, and the amount of immunosuppressant drugs given concurrently. Thus, 
it is not possible to specify an exact activity-promoting amount. However, 
an appropriate amount may be determined by one of ordinary skill in the 
art using only routine testing given the teachings herein. 
Typically, nonspecific immunosuppressive agents are administered in 
conjunction with the administration of the two cell populations. Such 
agents include cyclosporine ("CSA"); typically administered in about 6 
mg/kg body weight ("BW") to about 10 mg/kg BW initial dose), methotrexate, 
desoxyspergualine, rapamycin, steroids (e.g., prednisone), SOLUMEDROI 
(about 250 mg/kg BW to about 1000 mg/kg BW), ATGAM, OKT3, 
N-monomethylformamide (MMF), azathioprine (known in the industry as 
IMURAN) and FK506. 
The methods of the invention also comprise administering, to a host, an 
enriched dendritic cell population in combination with an enriched 
suppressor cell population suspended in a pharmaceutically-acceptable 
carrier. 
As used herein, the term "enriched" as applied to the cell populations of 
the invention refers to a more homogeneous population of dendritic or 
suppressor cells which have fewer other cells with which they are 
naturally associated. An enriched population of cells can be achieved by 
several methods known in the art. For example, an enriched population of 
dendritic cells can be obtained using immunoaffinity chromatography using 
monoclonal antibodies specific for determinants found only on dendritic 
cells. 
Enriched populations can also be obtained from mixed cell suspensions by 
positive selection (collecting only the desired cells) or negative 
selection (removing the undesirable cells). The technology for capturing 
specific cells on affinity materials is well known in the art (Wigzel, et 
al., J. Exp. Med., 128:23, 1969; Mage, et al., J. Imnmunol. Meth., 15:47, 
1977; Wysocki, et al., Proc. Natl. Acad. Sci. U.S.A., 75:2844, 1978; 
Schrempf-Decker, et al., J. Immunol Meth., 32:285, 1980; Muller-Sieburg, 
et al., Cell, 44:653, 1986). 
Monoclonal antibodies against antigens specific for mature, differentiated 
cells have been used in a variety of negative selection strategies to 
remove undesired cells, for example, to deplete T cells or malignant cells 
from allogeneic or autologous marrow grafts, respectively (Gee, et al., 
J.N.C.I. 80:154, 1988). Purification of human hematopoietic cells by 
negative selection with monoclonal antibodies and immunomagnetic 
microspheres can be accomplished using multiple monoclonal antibodies 
(Griffin, et al., Blood, 63:904, 1984). Enriched dendritic cell 
composition can be obtained from a mixture of lymphocytes, since dendritic 
cells lack surface Ig or T cell markers and do not respond to B or T cell 
mitogens in vitro. Dendritic cells also fail to react with MAC-1 
monoclonal antibodies, which reacts with all macrophages. Therefore, MAC-1 
antibodies provide a means of negative selection for dendritic cells. 
Procedures for separation of cells may include magnetic separation, using 
antibodycoated magnetic beads, affinity chromatography, cytotoxic agents 
joined to a monoclonal antibody or used in conjunction with a monoclonal 
antibody, for example, complement and cytotoxins, and "panning" with 
antibodies attached to a solid matrix, for example, plate, or other 
convenient technique. Techniques providing accurate separation include 
fluorescence activated cell sorters, which can have varying degrees of 
sophistication, for example, a plurality of color channels, low angle and 
obtuse light scattering detecting channels, impedance channels, etc. 
The immunosuppressive agent used according to the method of the invention 
is an agent that decreases the host's immune response to antigens. A 
preferred immunosuppressant of the invention is Cyclosporine A (CsA), 
however other agents, which cause immune suppression by depletion of 
thymic medulla dendritic cells, such as rapamycin, desoxyspergualine, and 
FK506 or functional equivalents of these compounds, may also be utilized. 
CsA is preferably administered by injection at a dose from about 0.3 to 
about 50 mg/kg/day, preferably from about 2.5 mg/kg/day to about 10 
mg/kg/day. The duration of CsA treatment may range from about two to about 
20 days, preferably about 14 days. 
The immunosuppressive agent is administered by any suitable means, 
including parenteral, subcutaneous, intrapulmonary, and intranasal 
administration, and if desired for local immunosuppressive treatment, 
intralesional administration (including perfusing or otherwise contacting 
the graft with the immunosuppressive agent prior to transplantation). 
Parenteral infusions include intramuscular, intravenous, intraarterial, or 
intraperitoneal administration. In addition, the immunosuppressive agent 
is suitably administered by pulse infusion, particularly with declining 
doses of the immunosuppressive agent. Preferably, the dosing is given by 
injections, most preferably intravenous or subcutaneous injections, 
depending in part on whether the administration is brief or chronic. 
As used herein, "substantially contemporaneously" refers to the time at 
which each of the therapeutic cells or immunosuppressant is administered 
to the recipient in relation to the time at which the others are 
administered. For example, a heart transplant recipient may receive 
enriched dendritic cells derived from donor spleen, during transplant 
surgery and receive CsA for a short time immediately following for about 
10-16 days, preferably about 14 days. In general, where transplant grafts 
are involved, the immunosuppressive agent can be administered from about 
one day to about 90 days before infusion of the tolerogenic cells until 
about seven days to about 90 days after the infusion of tolerogenic cells. 
Preferably, the immunosuppressive agent is administered from about seven 
days to about 28 days before infusion of tolerogenic cells until about 
seven days to about 28 days after infusion of tolerogenic cells. Where 
autoimmune disease is treated by infusion of foreign or altered 
tolerogenic cells, administration of immunosuppressive agent parallels the 
treatment times described for transplant grafts. 
According to the invention, an allogeneic bone marrow transplant recipient 
may have his own bone marrow harvested and processed to obtain a 
composition of enriched dendritic cells before transplantation of the 
donor bone marrow. The patient may receive immunosuppressive therapy 
followed by the infusion of transplanted bone marrow and dendritic cell 
composition previously harvested from the patient's own bone marrow. 
Enriched cells are administered in a physiologically acceptable solution. 
Preparations of enriched tolerogenic cells for parenteral administration 
include sterile aqueous or nonaqueous solutions, suspensions, and 
emulsions. Non-aqueous solvents include propylene glycol, and polyethylene 
glycol. Aqueous carriers include water, alcoholic/aqueous solutions, 
emulsions or suspensions, including saline and buffered media. Parenteral 
vehicles include sodium chloride solution, Ringer's dextrose, dextrose and 
sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles 
include fluid and nutrient replenishers, electrolyte replenishers, such as 
those based on Ringer's dextrose, and the like. Preservatives and other 
additives may also be present such as, for example, antimicrobials, 
anti-oxidants, chelating agents, and inert gases and the like. 
In an additional aspect, the present invention is directed to various 
methods of utilizing the cell mix produced by the present invention for 
therapeutic and/or diagnostic purposes. For example, the mix of dendritic 
cells and suppressor cells may find use in: (1) regenerating tissues which 
have been damaged through acute injury, abnormal genetic expression or 
acquired disease; (2) treating a host with damaged tissue by removal of 
small aliquots of bone marrow, differentiating them in vitro, isolating 
the desired subpopulation of differentiated cells and re-introducing the 
differentiated cells back into the host (3) detecting and evaluating 
growth factors relevant to the immune system; (4) detecting and evaluating 
inhibitory factors which modulate the immune system. 
Bone Marrow Preparation. Whole, unfractionated bone marrow is typically 
obtained from rib or long bones by flushing with Hank's Balanced Salt 
Solution ("HBSS"), or vertebral body bone marrow obtained by crushing with 
a bone rongeurs and elution with buffer solution, is filtered through 
nylon mesh and the viability assessed by trypan blue exclusion using a 
hemocytometer. The cells are collected by centrifugation at 1400 rpm for 
five minutes and then lysed with 25 mL of sterile, distilled water 
followed immediately by 25 mL of double strength HBSS. Cells are then 
recollected by centrifugation and pooled into one conical tube using a 
horizontal pipetting technique to exclude agglutinated material. Cells are 
resuspended in Roswell Park Memorial Institute Media ("RPMI") at a 
concentration of 1-2.times.10.sup.6 large mononuclear cells /mL and 
incubated at 37.degree. C. in 5% CO.sub.2 for one hour (to adhere cells). 
The nonadherent cells are collected and washed in HBSS and the viability 
rechecked. Cells are stored at 4.degree. C. until used. 
Splenocyte Preparation. Typically, splenic tissue is cut into small 
(approximately 0.5-1 GM) pieces and crushed using any convenient method 
(between glass slides or using a scraping technique with a sterile 
disposable blade), and collected in HBSS. The tissue is filtered through 
nylon mesh and collected by centrifugation. The cells are pooled and 
resuspended in 15 mL of HBSS, beneath which is layered 7.5 mL of 
FICOLL-HYPAQUE. This gradient is centrifuged at 1000 rpm for 45 minutes 
(no brake) and the interface cells collected and pooled. The pellet and 
solution are discarded. The pooled cells are washed three times in HBSS 
and resuspended in RPMI, and viability and cell counts checked. Cells are 
stored at 4.degree. C. until used. 
Culture Set-Up. Splenocytes and bone marrow cells are separately suspended 
in RPMI or similar medium to a concentration of about 2.times.10.sup.6 
cells/mL. The media is supplemented with the appropriate growth factor or 
stimulant and incubated at 37.degree. C. in 5% CO.sub.2 (minimum but not 
to exceed 10%) at a concentration of 3.3.times.10.sup.5 cells/square cm 
floor surface. Cultures are maintained using standard humidity, for at 
least 4 days (and up to 14 days) undisturbed and unshaken. 
Supplemental Factors: Specific supplementation with colony stimulating 
factors is necessary. The current technique utilizes species specific 
granulocyte macrophage-colony stimulating factor (GM-CSF). Where species 
specific factor is unavailable (i.e. the rat) murine GM-CSF is used and a 
concentration of 100 units/mL is added at the start of the culture. 
Additional agents for the stimulation and maturation of cells are added. 
The current model employs lipopolysaccharide (LPS or bacterial endotoxin) 
obtained from E coli and added at 1 .mu.g/mL at the start of the culture. 
Additional growth/factors that may be used in addition or in place of 
GM-CSF include macrophage colony stimulating factor ("M-CSF"), 
granulocyte-colony stimulating factor ("G-CSF") and the FLT3 ligand. 
Additional stimulating factors that may be added or used in place of LPS 
include interleukin-6, interleukin-4, tumor necrosis factor alpha 
("TNF.alpha."), and transforming growth factor beta ("TGF.beta."). Again, 
all factors used are species specific unless unavailable, in which case 
either murine or human factors can be employed. 
Cell Harvest. Typically, cells are harvested and pooled by first obtaining 
nonadherent cells and rinsing the culture container and collecting the 
cells and washes in separate tubes. Following centrifugation, supernatants 
may be saved or frozen for analysis. The cells are resuspended and counted 
and their viability checked using trypan blue exclusion. Cells may be used 
unfractionated or following a number of fractionation techniques. The 
current method employs the use of unfractionated cells resuspended in 
sterile, pH-balanced medium. For fractionation, a discontinuous PERCOLL 
gradient is used with a 100%, 70%, 60%, 50%, 40% and 0% stepwise dilution. 
The cells are suspended in 100% PERCOLL and equal volumes of diluted 
PERCOLL are layered sequentially above the cells. The gradient is 
harvested after centrifugation at 1000 g (2300 rpm) for 60 minutes. Cells 
are obtained separately at each interface, counted, and the viability 
checked.

EXAMPLES 
Culture Technique: Fresh spleens and lower extremity long bones are 
harvested aseptically and kept on ice in Hank's balanced salt solution 
(HBSS, Gibco Life Technologies, Gaithersburg Md.). Spleens are crushed 
between glass slides and sterile, filtered, splenic tissue is washed in 
HBSS, centrifuged on a FICOLL-HYPAQUE (HISTOPAQUE 1077, Sigma Diagnostics, 
St. Louis, Mo.) gradient at 1000 RPM.times.45 min. and the white cell 
layer harvested, washed twice, resuspended in Roswell Park Memorial 
Institute (RPMI) medium (Gibco) with 10% fetal calf serum (FCS) (Hyclone 
Labs, Logan, UT) and 100 units/mL penicillin, 100 .mu.g/mL streptomycin 
sulfate, and 0.25 .mu.g/mL amphotericin B (Gibco), to 2.times.10.sup.6 
cells/ml. Bone marrow harvests are performed as previously described by 
Ogle et al., Inflammation 18:175 (1994). The marrow cells are resuspended 
in RPMI+10% FCS, counted and diluted to 1.times.10.sup.6 large, 
mononuclear cells/ml. Twenty-five mL aliquots are incubated for 60 min. at 
37.degree. C. and 5% CO.sub.2 in 250 mL, 75 cm.sup.2 sterile, vented 
culture flasks (Costar, Cambridge Mass.). Nonadherent cells are then 
collected, washed and resuspended to 2.times.10.sup.6 cells/mL. 
Co-cultures consisted of equal volumes of spleen and marrow cells (total 
1.times.10.sup.6 of each cell stock/mL), most with added LPS (1 .mu.g/mL, 
E. coli 055:B5, Sigma Chemical, St. Louis, Mo.) and murine GM-CSF (100 
units/mL, R and D Systems, Minneapolis, Minn.) and are incubated for up to 
7 days at 37.degree. C. and 5% CO.sub.2 in 25 ml volumes in 250 mL sterile 
culture flasks. All cell counts are based on viable cells by trypan blue 
exclusion. Nonadherent co-culture cells are harvested at predetermined 
times, washed, resuspended and counted. 
Some initial cultures using nonadherent bone marrow only are performed with 
various combinations of LPS, murine GM-CSF, recombinant human TNF.alpha. 
(100 units/ml, Endogen, Boston Mass.) or unmodified media and harvested at 
day 4 or 7 and used in mixed lymphocyte and phytohemagglutinin (PHA, 
Sigma, St. Louis, Mo.) experiments. 
Cell fractionation: Cells are washed twice in HBSS, pelleted in a 15-ml 
conical tube at 1400 RPM.times.5 min. and resuspended to 2 ml in PERCOLL 
(Pharmacia, Uppsala, Sweden) solution (0.95 ml 10.times. HBSS in 9.05 ml 
sterile 100% PERCOLL, pH titrated to 7.35) over which are layered 2 ml 
each of 70%, 60%, 50%, 40% and 0% (plain HBSS) PERCOLL dilutions. After 
centrifugation at 2800 RPM.times.30 min., fractions at gradient interfaces 
are retrieved, washed and resuspended in RPMI with 10% FCS. Fractions (Fr) 
are labeled sequentially from the 100-70% interface (Fr 1) to the 40-0% 
interface (Fr 5). The fractionation of untreated bone marrow yielded 4 
fractions (lacking cells at the 50-40% interface), while 7-day co-cultures 
yielded 5 fractions. Fraction 5 contained 97% dead cells, while Frs 1 and 
2 contained very few but mostly viable cells (these are submitted for FACS 
analysis). Fraction 3 contained the majority of total cells (66%) at 95% 
viability, while Fr 4 contained somewhat fewer cells at similar viability 
(Frs 3 and 4 are used in further experiments). Subsequent separation of Fr 
3 cells is accomplished by sterile, viable cell sorting on an Epics 753 
cell sorter (Coulter, Miami Fla.) into subpopulations based on scatter 
pattern (debris, lymphocyte, and macrophage regions). Averages of 60% of 
cells are lost during sorting. This is attributed to fragility acquired by 
some cells over time in culture. Macrophage region cells are used in 
further experiments. 
Mixed Lymphocyte Reactions: Spleens are harvested aseptically, crushed, 
filtered and washed in HBSS, then resuspended in 15 ml HBSS and separated 
on a FICOLL-HYPAQUE gradient at 1000 RPM.times.45 min. The white cells are 
harvested, washed, resuspended in RPMI with 10% FCS and cells incubated 
with 25Fg Mitomycin C. (Sigma, St. Louis, Mo.)/2.5.times.10.sup.7 cells/mL 
for 45 minutes at 37.degree. C. and 5% CO.sub.2 then washed 3.times. in 
RPMI are used as stimulator cells. Responder and stimulator cells are 
added to standard 96 well culture plates at 2.0.times.10.sup.6 cells/well 
to which are added titrations of whole co-culture, fr 3, or fr 4 cells or 
titrated amounts of co-culture supernatant. Cells are pulsed with 0.5 
FCi/well of .sup.3 H-thymidine (1 mCi/mL New England Nuclear, Boston, 
Mass.) and harvested on day 4. Activity is measured as counts per minute 
(cpm) with an LS600TA liquid scintillation counter (Beckman, Fullerton, 
Calif.). All reactions are performed in triplicate and results expressed 
as mean values. 
Immunosuppression and Cardiac Transplantation: Heparinized whole blood is 
collected from ACI donors and administered fresh to anesthetized Lewis 
recipients via the penile vein on the day prior to transplantation in the 
DST control group (1 mL/recipient). Experimental groups received 
2.5.times.10.sup.6 viable cultured cells resuspended in 1 ml 
RPMI/recipient on the day prior to transplants. Heterotopic 
intra-abdominal ACI to Lewis cardiac transplants are performed using the 
method of Ono and Lindsey, J. Thorac. Cardiovasc. Surg. 57:225 (1969). 
Briefly, the aortic root and pulmonary artery are anastomosed to the 
recipient infrarenal aorta and vena cava using standard microvascular 
techniques. Cyclosporine A, a gift of Sandoz Pharmaceuticals (East 
Hanover, N.J.), is dissolved in olive oil (Sigma) at a concentration of 5 
mg/mL and given at a dose of 10 mg/kg subcutaneously the day before 
transplantation with a daily dose of 2.5 mg/kg for seven days beginning on 
the day of engraftment. Allograft survival is assessed by daily palpation 
with rejection defined as a cessation of palpable contractions confirmed 
under general anesthesia by celiotomy. Graft survival statistics are 
expressed as a group means.+-.the standard error. Animal groups included 
untreated, untransfused controls, standard DST controls and experimental 
groups. 
In one embodiment of the invention a process for differentiating stem cells 
comprises providing a stem cell specimen, combining the stem cell specimen 
with a second cell specimen to produce a co-culture, and adding factors to 
the co-culture. The co-cultures are maintained for from about 4 days to 
about 14 days. 
Additional embodiments and modifications within the scope of the claimed 
invention will be apparent to one of ordinary skill in the art. 
Accordingly, the scope of the present invention will be considered in the 
terms of the following claims, and is understood not to be limited to the 
details of the methods described in the specification.