The immune system self-regulates itself through many mechanisms, for example, thymic deletion of autoreactive cells, the need for co-stimulation for activation of T cells, and also through T regulatory (Treg) cells that recognize proteins that belong to the body and stop other T cells from attacking these proteins.  Another way in which activated T cells control themselves is by increasing expression of a molecule called CTLA-4.

This molecule works through several means.  One is outcompeting CD28 for access to CD80 and CD86 on the antigen presenting cell, the other is by sending a suppressive signal to the antigen presenting cell.

To activate a T cell, generally speaking, one requires 2 main signals, the T cell receptor (TCR) has to recognize a major histocompatibility complex (MHC) molecule that has a peptide inside of it.  If the TCR appropriately “fits” into the MHC-peptide complex, then the TCR sends a signal into the T cell.  However this first signal is not enough to activate the T cell.  The T cell requires another signal from the antigen presenting cell.  This second signal, also called costimulatory signal, is usually in the form of CD80 and/or CD86 from the antigen presenting cell, which both bind to CD28 on the T cell.  The activation of CD28 and the TCR on the T cell results in T cell activation.  When only the TCR is activated without CD28, then the T cell either goes into a state of prolonged unresponsiveness (called anergy), or in some situations actually can start expressing immune suppressive properties.  CTLA4 is turned on by the T cell after the T cell is activated. CTLA4 binds with much higher affinity to CD80 and CD86 than does CD28.  Therefore, after T cell activation, the T cell can “turn itself off” by expressing CTLA4.

Studies have demonstrated that activated T cells expressing CTLA4 can bind to dendritic cells (most potent antigen presenting cells), and that the CTLA4 on the activated T cell sents a negative signal to the dendritic cell, inducing the dendritic cell to secrete immune suppressive proteins and upregulate expression of the enzyme indolamine 2,3 deoxygenase (IDO) which in turn suppresses other T cells.

Interestingly CTLA4 is expressed not only by activated T cells but also by Treg cells.

Given the ability of CTLA4 to suppress immune responses via at least 2 mechanisms (more are known), scientists have tried to use CTLA4 therapeutically.  To make a soluble form of CTLA4 that can be administered, a pharmaceutical form has been developed by fusing the protein CTLA4 with the immunoglobulin domain of IgG, to make a chimeric molecule called CTLA4-Ig.  Currently CTLA4-Ig is sold by Bristol Myers Squibb under the name Abatacept (Orencia) for the treatment of rheumatoid arthritis that is nonresponsive to TNF-alpha inhibitors such as Remicade.

Given the immune modulatory properties of CTLA4-Ig, one question is whether it may inhibit suppress autoimmune responses in patients with multiple sclerosis.  A recent study (Viglietta et al. CTLA4Ig treatment in patients with multiple sclerosis: an open-label, phase 1 clinical trial. Neurology 2008 Sep 16;71(12):917-24) assessed this in a small clinical trial.

The investigators performed a Phase I trial, meaning that the primary endpoint was toxicity and ability to identify therapeutic dose for subsequent Phase II investigations.  A total of 20 patients with relapse remitting multiple sclerosis were treated with intravenous infusions of CTLA4-Ig and monitored for 3 months. 

CTLA4-Ig administration correlated with suppression of interferon gamma production and proliferation in response to myelin basic protein ex vivo.  The treatment elicited no serious treatment associated adverse events, although some mild adverse events were reported.

This study demonstrated feasibility of CTLA4-Ig administration in relapse remitting multiple sclerosis, and provides support for further Phase II trials.

Unfortunately CTLA4-Ig is still a non-specific immune suppressant in that it globally inhibits activation of T cells and theoretically could predispose to infections.  One important question is whether the CTLA4-Ig administration may help to “reprogram” the immune system so as to promote eventual tolerance to the myelin antigens while leaving immunity to extracorporal antigens intact. 

A possible approach would be to use CTLA4-Ig in combination with antigen-specific approaches such as vaccination with autoreactive T cells.  Additionally, it will be important to assess the effects of CTLA4-Ig on Treg cells.  For example, we know that interferon beta, which induces therapeutic effects in multiple sclerosis stimulates Treg activity.  We also know from animal studies (Salomon et al.  B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. 2000 Apr;12(4):431-40) that administration of CTLA4-Ig can actually block Treg generation, so if interferon beta works through Treg generation, the combination may not work.

The question of adult stem cells differentiating into needed tissue is not based on opinion but on over 2 decades of solid peer reviewed research at international academic institutions.  To state there is controversy regarding the ability of adult stem cells to therapeutically augment natural healing processes, is like saying there is controversy whether red blood cells transport oxygen. 

 

There is no question as to the scientific rationale of therapeutic benefits.  The only question is the extent of the benefits and the selection of patients for undergoing stem cell therapy. 

 

In order to understand these issues, we must first begin by understand a little bit about stem cells.  We will begin with the bone marrow stem cell.

 

1.  What is the bone marrow stem cell?

 

Traditionally, the bone marrow was viewed as the source of all blood cells, being responsible for production of trillions of cells per hour.  Essentially one type of cell in the bone marrow, called the hematopoietic (“hematopoietic” means “blood making”) stem cells, possesses the unique ability to make copies of itself, but also, depending on the needs of the body, to make other blood cell types as well.  At a molecular level we know that the human bone marrow hematopoietic stem cells expresses the markers CD34 and does not express markers of lineage commitment such as HLA-DR, CD38, CD11, CD31, etc [1].  This essentially means that bone marrow hematopoietic stem cells can be isolated and studied as a discrete entity. 

 

The bone marrow hematopoietic stem cell adapts to the needs of the body and accordingly produces specific cell types when the body needs them.  For example, when a person goes up to live on the mountains, the person’s body needs more red blood cells than usual since there is less oxygen at high altitudes.  In order to compensate for this, the kidney starts to produce more erythropoietin (a hormone that travels throughout the body), which instructs the hematopoietic stem cell to produce more blood cells [2].  The same occurs in situations of infections in which the body needs more white blood cells (such as neutrophils) to protect itself against the external pathogens.  In response to various molecular signals (G-CSF, GM-CSF) generated by the immune system of the person, the bone marrow hematopoietic stem cell starts to generate more white blood cells [3].

 

In addition to containing the cells that make blood, the bone marrow contains cells called “stromal cells” that support the hematopoietic stem cells.  Essentially, stromal cells are comprised of various actual cell populations The stroma includes adipocytes, osteoblasts, and mesenchymal stem cells [4].  The primary function of mesenchymal stem cells in the bone marrow is to control proliferation of the hematopoietic stem cell, through provided growth factor support.  This is why certain scientists are using mesenchymal stem cells to accelerate blood formation after administration of hematopoietic stem cells in patients [5].

 

Bottom line:  There are two main types of stem cells found in the bone marrow: 1) hematopoietic stem cells that make blood and 2) mesenchymal stem cells that provide support for the hematopoietic stem cell.

 

2.  Bone Marrow Transplantation: The first application of stem cell therapy.

 

Stem cell therapy is not new.  Ever since the discovery of the hematopoietic stem cell by Drs Till and McCulloch in the 1960s, the use of these cells for transplantation into patients with defective bone marrow was envisioned.  The first hematopoietic stem cell transplant, or bone marrow transplant, was performed in 1956 by Dr. E. Donnall Thomas using bone marrow cells isolated from an identical twin donor for a recipient who had leukemia.  The idea was that if the patient was irradiated with high doses, then the radiation would kill all of the leukemia cells.  Unfortunately, the radiation would also destroy the healthy bone marrow stem cells.  So the idea was to utilize donor bone marrow to replenish the recipient with healthy hematopoietic stem cells.   Dr. Thomas, along with Joseph E. Murray, won the Nobel Prize in 1990 for this discovery.  The usefulness of transplanting the hematopoietic system was not limited to leukemias, in 1968, bone marrow transplantation was performed successfully on a patient with a genetic mutation called severe combined immunodeficiency.  In this disease the bone marrow stem cells are deficient in ability to generate T and B cells, as a result the patient is immune compromised and is forced to live in a sterile environment.  The administration of healthy bone marrow cells resulted in the child being able to function normally as a result of a non-mutated hematopoietic stem cell that is capable of making T and B cells.

 

Since the initial bone marrow transplant procedure was developed, several hundred thousands procedures have been performed, literally giving a new lease on life to many patients whose diseases were previously considered lethal.  Variations on the theme of bone marrow transplantation have also been performed in order to increase efficacy.  For example, patients with some leukemias are known to have a higher probability of relapse (leukemia coming back) after the transplant.  In order to fight off the relapse, clinicians have started infusing lymphocytes from the donor as a type of cellular therapy.  These Donor Lymphocyte Infusions are currently part of the accepted medical practice for treatment of post-transplant relapsed CML[6].  Additional modifications to the transplant procedure have included the use of G-CSF mobilized donor stem cells.  Instead of performing puncture of the iliac crest for bone marrow aspiration, a gentler procedure that was developed involved “telling the bone marrow stem cells” to leave the bone marrow and enter systemic circulation, by the administration of the drug G-CSF.  The stem cells are collected from the blood using a procedure called leukopheresis [7].  Another advancement in the area of hematopoietic stem cell transplantation has been the use of cord blood as a source of blood-forming stem cells.  Cord blood stem cells express higher regenerative potential on a per-cell basis compared to bone marrow stem cells.  Although cord blood stem cells are generally safer in the sense that they do not evoke graft versus host (a side effect of transplantation) with the same severity as bone marrow stem cells, cord blood stem cells are available in small numbers and therefore their use in adults is still limited to certain patient subgroups [8].

 

Conclusion:  Stem cell therapy in the sense of hematopoietic stem cell transplantation, has been occurring since 1956 for treatment of disturbances of the blood making components of patients.  These disturbances can be caused by genetic abnormalities (ie severe combined immunodeficiency, sickle cell anemia, etc) or induced as a side effect of treatment (ie dose radiation for clearing leukemia).  The hematopoietic-reconstituting stem cell therapy should not be confused with stem cell therapy for regenerative medicine.  In regenerative medicine the stem cells are administered in absence of the destruction of the recipient’s bone marrow hematopoietic compartment. 

 

3.  Bone Marrow and Regeneration: The Bone Marrow Cells Can Become Different Cells besides Just Blood Cells

 

As we described above, transfer of bone marrow stem cells has been performed for decades.  Scientists have wondered, if the bone marrow stem cell possesses the potential to differentiate into all the different types of blood cells, maybe it can also differentiate into other cells as well.  This process was originally termed “transdifferentiation”.  The first report of transdifferentiation to appear in the major medical literature was a paper by Orlic et al. [9], in which mouse bone marrow derived stem cells were injected into mice that were given an experimental heart attack.  The interesting thing about this experiment was that the bone marrow stem cells used were labeled to glow green.  The donor animals were genetically engineered to express the green fluorescent protein (GFP) gene throughout their bodies.  This essentially means that all cells derived from the GFP donor mice were green.  Additionally, the experimenters purified the mouse equivalent of the human CD34 bone marrow hematopoietic stem cell.  The molecular markers used where positivity for stem cell antigen (SCA-1) and negativity for the lineage markers (lin negative).  Following induction of a heart attack by ligation of one of the coronary arteries, the researchers implanted the cells in the area of infarct.  The mice which received implanted hematopoietic stem cells, but not control cells, had increased pumping ability of the heart and decreased levels of heart damage.  Most interestingly, when mice where sacrificed, green cells were observed throughout the area of damage.

 

This paper served as a strong indication in animals that bone marrow derived cells are capable of differentiating into heart tissue and helping to repair injury.  Eventually scientists started finding that bone marrow stem cells can differentiate into other tissues.  For example, human bone marrow derived CD34 cells have been demonstrated to differentiate into cells expressing liver markers, and can actually generate human liver proteins when injected into animals [10].

 

The concept of bone marrow cells differentiating into cells other than hematopoietic cells has subsequently been demonstrated in numerous laboratories for many types of tissues.  The table below summarizes some of the tissues.  We will include both hematopoietic and mesenchymal stem cells in the analysis since both are found in the bone marrow.  It is important to make a note that some researchers believe embryonic stem cells are the only cell types capable of differentiation into different tissues.  While it is true that embryonic stem cells indeed are “totipotent”, these cells are very far from clinical use.  Injection of embryonic stem cells into animals causes a type of aggressive tumor called “teratomas” [11], and furthermore, the differentiation of these cells is difficult to control.  In other words if you want to generate livers cells from embryonic stem cells, and you add liver-induction factors, such as hepatocyte growth factor, some cells become liver, but other cells still become neurons, kidney cells, and skin cells, thus until science advances, adult stem cells still seem to be superior for clinical applications. 

 

4.  Importance of Bone Marrow Resident Stem Cells for Natural Repair of Tissue Injury

 

The examples above, as well as the published literature, demonstrate in an unequivocal manner that bone marrow derived cells have a certain amount of plasticity, or ability or ability to differentiate into non-hematopoietic tissues. 

 

The main idea being that bone marrow can differentiate into a variety of tissues.  Now why would the bone marrow have this ability? One theory is that the bone marrow acts as a reserve of “regenerative cells” that are important in healing the body as the body ages.  The ability to regenerate is most potently seen in the salamander, which can regenerate whole limbs, based on potent stem cell activity.  Although humans do not have regenerative activity that potent, there are still evidences that bone marrow stem cells do in fact contribute to regeneration.  First we will provide an in vivo example of bone marrow “transdifferentiating” into other tissues in the human, and then we will provide examples of bone marrow helping in healing processes.

 

As we discussed above, bone marrow transplantation is performed for hematological disorders, one of which is leukemia.  Now if a female receives a male bone marrow transplant, one would imagine that the circulating blood cells of the female have the Y chromosome, which is correct.  What is more interesting is whether tissues in the female’s body actually start to express the Y chromosome.  In a very interesting study [12], researchers performed autopsy on female patients who had received male bone marrow transplants.  When they examined the heart tissue, the female heart tissue contained cardiac muscles that had the Y-chromosome !! This conclusively demonstrates that in normal situations bone marrow derived cells differentiate to become different parts of the body.  Similar findings were also reported in liver, pancreas, and kidney.

 

Now what about in situations of heart attacks?  One would imagine that during a heart attack, bone marrow derived stem cells would migrate into the heart.  If this is indeed the case, then taking blood samples of patients after heart attacks should demonstrate increases in stem cells.  Indeed numerous studies have shown this to be the case.  Below is an example from one study [13].

 

A similar argument can be made in stroke.  Interestingly, in stroke, there has been reported an association between higher degree of mobilization and improved recovery as assessed by the NIHSS score [14].

 

From the examples presented above, as well as numerous other publications, endogenous bone marrow stem cells have been demonstrated to play a regenerative role in humans.  The next question is whether the administration of bone marrow stem cell therapy can actually induce a clinical effect?

 

5.  Clinical Examples of Bone Marrow Stem Cell Therapy for Regeneration

 

Bone marrow stem cell therapy for regenerative (non-hematopoietic) purposes originally started with Japanese research when bone marrow cells were injected into the heart muscle of patients undergoing bypass surgery.  The idea was that the injected bone marrow cells will stimulate production of new blood vessels and thereby increase oxygenation to the heart [15].  The procedure, although highly invasive, was associated with no treatment related adverse effects and 3 out of the 5 patients had increased blood vessel production as assessed radiologically, as well as improved cardiac function.  This first demonstration in 2001, was repeated by numerous investigators.  In 2003, the study was repeated using CD133 purified bone marrow stem cells and published in the prestigious journal Lancet [16], reporting positive results.  Subsequently numerous studies have been conducted in the area of cardiology demonstrating that administration of a patient’s own bone marrow is associated with positive outcome.  For example, there are published pictures of myocardial activity before and after stem cell therapy from a clinical study [17].

 

Convincingly, statistically significant improvements in left ventricular ejection fraction have been observed in double blind trials.  For example there is published from a 200 patient trial [18].

 

In addition to the heart, bone marrow stem cells have been used for numerous other indications clinically.  One interesting indication is critical limb ischemia, which involves occlusion of blood flow to the lower limbs and is associated with need for amputation.  Below is a representative angiogram from a patient before and after bone marrow stem cell therapy as part of a double blind trial [19].  Additionally, in the same trial, clinical endpoints such as ankle brachial index (how much blood flows to the leg), pain-free walking, and transcutanous oxygen where all increased in a statistically significant manner.

 

Bone marrow stem cells have also been demonstrated to be effective in regeneration of other damaged/degenerated organs.  In a clinical trial with 9 patients suffering from liver failure [20], the administration of their own bone marrow cells intravenously elicited restoration in albumin production as seen in the figure below.  Each line in the figure represents one patient.  As well as overall decrease in clinical severity of disease (Child Pugh Score).

 

6.  Bone Marrow Derived Mesenchymal Stem Cells

 

The clinical examples above were aimed at fresh, non-cultured bone marrow derived stem cells.  Since it is difficult to enforce patents on medical procedures, numerous companies have developed “universal donor” off the shelf, stem cells that have been cultured in vitro and can be sold as “medicines”.  Of the companies activity in this area (Neuronyx, www.neuronyx.com in Phase I clinical trials, Pluristem, www.pluristem.com in late preclinical, and Osiris Therapeutics, www.osiris.com, 2 phase IIIs, several phase II trials), Osiris therapeutics has been the clear-cut leader, anticipated to have a product on the US market in the near future.  Below is a summary data of Osiris’s bone marrow mesenchymal stem cells used to treat Crohn’s Disease. Survival figures in patients treated with mesenchymal stem cells for a lethal inflammatory disease called are well published in the case of GVHD [21].

 

7.  Conclusion: Bone Marrow Stem Cell Therapy…Enhancing the Body’s Regenerative Potential

 

Dr. Thomas would be proud. From the initial bone marrow “cell therapy” transplant in 1956, the use of bone marrow stem cells has expanded to cover almost any indication one could imagine.  One only has to look at the www.clinicaltrials.gov database to see that clinical applications of bone marrow cells are being tested in conditions ranging from heart disease, to autoimmunity, to neurological conditions.

 

The wide applicability of bone marrow stem cells to so many diseases comes from the fact that stem cell therapy is only an augmentation of the natural regenerative processes.  It is established that injured or damaged tissue releases distinct factors that “call in” bone marrow stem cells.  By administering the cells in high concentrations intravenously, or locally, the bone marrow stem cell therapist is only helping the body to do what it is trying to do…to heal itself.

 

References

 

1.         Srour, E.F., et al., Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis. Blood Cells, 1991. 17(2): p. 287-95.

2.         Berglund, B., High-altitude training. Aspects of haematological adaptation. Sports Med, 1992. 14(5): p. 289-303.

3.         Lee, K.Y., et al., Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes. Exp Mol Med, 2000. 32(4): p. 210-5.

4.         Krebsbach, P.H., et al., Bone marrow stromal cells: characterization and clinical application. Crit Rev Oral Biol Med, 1999. 10(2): p. 165-81.

5.         Koc, O.N., et al., Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000. 18(2): p. 307-16.

6.         Dazzi, F. and C. Fozza, Disease relapse after haematopoietic stem cell transplantation: risk factors and treatment. Best Pract Res Clin Haematol, 2007. 20(2): p. 311-27.

7.         Levesque, J.P., et al., Mobilization of bone marrow-derived progenitors. Handb Exp Pharmacol, 2007(180): p. 3-36.

8.         Riordan, N.H., et al., Cord blood in regenerative medicine: do we need immune suppression? J Transl Med, 2007. 5: p. 8.

9.         Orlic, D., et al., Bone marrow cells regenerate infarcted myocardium. Nature, 2001. 410(6829): p. 701-5.

10.       Wang, X., et al., Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood, 2003. 101(10): p. 4201-8.

11.       Nussbaum, J., et al., Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J, 2007. 21(7): p. 1345-57.

12.       Deb, A., et al., Bone marrow-derived cardiomyocytes are present in adult human heart: A study of gender-mismatched bone marrow transplantation patients. Circulation, 2003. 107(9): p. 1247-9.

13.       Muller-Ehmsen, J., et al., The mobilization of CD34 positive mononuclear cells after myocardial infarction is abolished by revascularization of the culprit vessel. Int J Cardiol, 2005. 103(1): p. 7-11.

14.       Dunac, A., et al., Neurological and functional recovery in human stroke are associated with peripheral blood CD34+ cell mobilization. J Neurol, 2007. 254(3): p. 327-32.

15.       Hamano, K., et al., Local implantation of autologous bone marrow cells for therapeutic angiogenesis in patients with ischemic heart disease: clinical trial and preliminary results. Jpn Circ J, 2001. 65(9): p. 845-7.

16.       Stamm, C., et al., Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet, 2003. 361(9351): p. 45-6.

17.       Stamm, C., et al., Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: safety and efficacy studies. J Thorac Cardiovasc Surg, 2007. 133(3): p. 717-25.

18.       Schachinger, V., et al., Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med, 2006. 355(12): p. 1210-21.

19.       Tateishi-Yuyama, E., et al., Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet, 2002. 360(9331): p. 427-35.

20.       Terai, S., et al., Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells, 2006. 24(10): p. 2292-8.

21.       Ringden, O., et al., Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation, 2006. 81(10): p. 1390-7.

 

 

There is some evidence to suggest that pregnant women with multiple sclerosis experience a diminished frequency and severity of relapse in the last trimester of pregnancy.  This has prompted investigators to assess whether hormones such as progesterone are capable of inhibiting multiple sclerosis in animal models.  Indeed this seems to be the case. 

For example, Garay et al (Steroid protection in the experimental autoimmune encephalomyelitis model of multiple sclerosis. Neuroimmunomodulation 2008;15(1):76-83) used the B6 mouse model of multiple sclerosis (immunized with peptide from myelin oligodendrocyte protein 40-54) to demonstrate that administration of progesterone before induction of pathology led to suppressed disease severity index, inhibition of demyelination and increased expression of the sodium-potassium-ATPase gene in motor neurons.  Another study, (Correale et al. Steroid hormone regulation of cytokine secretion by proteolipid protein-specific CD4+ T cell clones isolated from multiple sclerosis patients and normal control subjects. 1998 Oct 1;161(7):3365-74) demonstrated that culture of T cells in progesterone upregulated ability to generate interleukin-4, a Th2 cytokine.  This is shown in the figure below.

Now we on the one hand we know that hormones affect immunological cells, but do hormones such as progesterone alter the ability of stem cells to modulate immune responses?  It appears that they do.  Ivanova-Todorova et al published (HLA-G expression is up-regulated by progesterone in mesenchymal stem cells. Am J Reprod Immunol. 2009 Jul;62(1):25-33) that treatment of mesenchymal stem cells with progesterone increased expression of the immune modulatory protein HLA-G.  This implies that ex vivo treatment of mesenchymal stem cells with progesterone may be useful in augmenting their ability to alter immune responses.  Additionally, it would be interesting to see if in vivo synergy may be obtained by treating patients with hormones and concurrently administering stem cells.

The ability to augment therapeutic activity of mesenchymal stem cells is very appealing since these cells are already in Phase III clinical trials by the company Osiris Therapeutics for treatment of Graft Versus Host Disease.  Once these cells are approved for marketing purposes (anticipated to be next year), then physicians will be able to use them on a more widespread basis and in many situations for off-label uses.  This will cause a great interest in methods of augmenting their efficacy, including methods as mentioned above.

One of the reasons why fat stem cells seem to have therapeutic activity in animal models and early clinical trials is likely associated with their ability to modulate the immune system.  Specifically, the mesenchymal stem cell component of the fat is very interesting since administration of both mouse and human mesenchymal stem cells into animal models of multiple sclerosis has resulted in beneficial effects in the disease process.

How to mesenchymal stem cells affect multiple sclerosis?  There is some evidence that mesenchymal stem cells produce the enzyme indolamine 2,3 deoxygenase, which depletes local tryptophan and causes death of nearby T cells.  The importance of this enzyme is seen in studies in which stem cell mediated inhibition of multiple sclerosis is reversed by addition of a chemical inhibitor of indolamine 2.3 deoxygenase. In addition to inhibition of activated T cells, indolamine 2,3 deoxygenase causes production of various small molecules that can directly induce T cell apoptosis.  This enzyme is one of the mechanisms by which tumors escape immune attack and is also involved in the ability of the fetus (which has different genes than the mother) to grow up inside the mother without immunological rejection.

Mesenchymal stem cells express molecules such as HLA-G which are known to send inhibitory signals to T cells and prevent their activation.  Additionally, HLA-G is known to bind to immunoglobulin-like transcripts (ILTs) on dendritic cells and induce immune suppressive activities. We previously discussed that subsets of T regulatory cells express HLA-G.

Of course, besides indolamine 2,3 deoxygenase and HLA-G, mesenchymal stem cells modulate the immune system by secretion of cytokines.  Notable cytokines that have been implicated include TGF-beta, IL-10 and leukemia inhibitory factor (LIF).  Interestingly, the cytokines that are immune modulatory actually start getting produced in higher quantities when the mesenchymal stem cell is under allogeneic immunological pressure, such as in a mixed lymphocyte reaction (Nasef et al. Leukemia inhibitory factor: Role in human mesenchymal stem cells mediated immunosuppression. Cell Immunol 2008 May-Jun;253(1-2):16-22).  This would make sense since why would mesenchymal stem cells constitutively secrete immune suppressants? They would theoretically secrete them only when they are needed by the host, which is what seems to be the case.

Today we wanted to mention a new type of mesenchymal stem cell mediated immune modulatory mechanism: cleavage of the interleukin-2 receptor protein CD25.  The clinically used antibody daclizumab binds to anti-CD25 and has had some promising effects in multiple sclerosis patients.  In a recent paper (Ding et al. Mesenchymal Stem Cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of Matrix Metalloproteinase-2 and -9. Diabetes 2009 Jun 9) it was demonstrated that mesenchymal stem cells can cut and thereby inactivate CD25 on T cells via expression of MMPs 2 and 9.

The investigators took the study one step further and shown that while the mesenchymal stem cells could provide prolongation of allogeneic tissue survival, this was associated with their ability to reduce expression of CD25.

This paper is very interesting not only for the finding that mesenchymal stem cells can modulate this interesting area of T cell biology, but also because it suggests modulation of MMP activity by other means may be a useful method of controlling the immune system.  For example, numerous MMP inhibitory compounds have been developed for treatment of cancer (cancer needs angiogenesis, angiogenesis needs MMPs) but not many, well to my knowledge none, have worked in Phase III.  This means that there is a possiblity that MMP modulators that are feasible from a clinical trial perspective may already be in existance. 

Another interesting question that this study begs is whether the MMPs involved in angiogenesis of cancer are also involved in cleaving CD25 off immune cells and therefore may be one of the mechanisms by which cancer reduces the immune response.

Plasma exchange (also called plasmapheresis) is the procedure in which blood is taken from the patient, the plasma is removed and replaced with donor plasma (or sometimes plasma replacements such as albumin), and reintroduced into the body with the original cellular component.

The aim of the procedure is to deplete the blood of various immunological factors, such as antibodies, that may be associated with pathology, or causing of the disease process.  Obviously this is a short-term solution since antibodies are made by B cells and the original B cells that are making putatively harmful antibodies will still be in the patient after the plasmapheresis is complete.  However it may be that the plasmapheresis gives the immune system time to “re-adjust itself”.  An extreme example of the immune system re-adjusting itself is in the clinical trials where they patient’s hematopoietic (blood making) stem cells are extracted, the patient’s immune system is destroyed on purpose, and then the hematopoietic stem cells are placed back into the patient to create a “re-adjusted” immune system that hopefully will not be attacking the myelin sheath anymore.  Obviously this process has many possible side effects and at least theoretically, it seems more attractive to use your own fat derived stem cells for multiple sclerosis. 

In any case, plasma exchange offers the possibility of removing some of the pathological components so that the immune system may try to “self-regulate” itself.  How could this be?  One possibility of this is through giving T regulatory cells a chance by reducing the inflammation-creating cytokines and antibodies found in the plasma of multiple sclerosis patients.  On the other hand, the actual effector cells in multiple sclerosis are the CD4 T cells, which activate macrophages and myeloid cells to infiltrate the central nervious system, so would removal of antibodies and soluble components really have a significant impact?

The area of plasma exchange for multiple sclerosis appears to be rather controversial.  On its website, the National MS Society states: “It is not clear whether plasmapheresis is of benefit in the short- or long-term treatment of MS, and its use in MS remains controversial.”

We write about plasma exchange for multiple sclerosis today because a recent paper was published (Trebst et al. Plasma Exchange Therapy in Steroid-Unresponsive Relapses in Patients with Multiple Sclerosis. Blood Purif. 2009 Jun 11;28(2):108-115) in which 20 patients who were unresponsive to steroids were reported upon. 

The investigators from the Department of Neurology, Medical School Hannover, Hannover, Germany, reported ”a marked-to-moderate clinical response with clear gain of function in 76% of patients with uni- or bilateral optic neuritis and in 87.5% of patients with relapses other than optic neuritis was observed.”

The concluded that “Plasma exchange is an effective and well tolerated therapeutic option for steroid-unresponsive MS relapses.”

It will be interesting to elucidate the immunological mechanisms by which plasma exchange may mediate its effects, and if it may be incorporated into other immune modulatory therapies.  Such a simple incorporation could be the combination of plasma exchange and antigen-specific immunization to achieve tolerance.  We previously discussed here that immunization in the presence of inflammation or “danger” is often associated with immune activation, whereas introduction of antigen in absence of inflammation can be associated with tolerance.  Therefore by “washing the body” of inflammatory agents, one may achieve even better effects with agents such as BioMS’s MBP8298 product or Bayhill Therapeutic’s myelin basic protein DNA vaccine called BHT-3009. 

It is possible that in the future be combined with agents that increase existing nerve condition such as fampridine and of course mesenchymal stem cells.

Why would the immune system “turn” on patients?  I figured I would write a couple of thoughts regarding the cells and mechanisms involved in how the immune system functions.  For many of you this may be obvious, but I thought I would review some of the concepts here at a very high level, with the hope of stimulating ideas/discussions.

The immune system shares with the nervous system the features of: memory, self-nonself recognition, specificity, and “knowing” when to respond or not respond based on quantity of stimulus.

For example, the immune system is does not “blindly” attack everything that is foreign.  As you know, sometimes the immune system does not even attack just what is foreign but alson turns against the body, such as in autoimmune conditions like multiple sclerosis, or type 1 diabetes, or rheumatoid arthritis, where the immune system attacks myelin basic protein, GAD, and collagen I, respectively.

Usually the immune system selects what it should attack or not attack based on recognition of what is “self” and what is “non-self”.  Classically, this process is thought to occur before birth.  The main cells of the “adaptive immune system”, the part of the immune system that learns based on exposure, are the T cells and the B cells.  The T cells can either be killer T cells (CD8), which recognize other cells that are virally infected or cancerous, and CD4 T cells, which provide chemical signals (cytokines) for recruitment of other immune cells (T helper cells).  Of CD4 cells broadly speaking, there are also T helper 1 cells, that contribute to immunity to cancer and viruses, Th2 cells that fight parasitic infections, and Th17 cells that cause inflammation.  There are also T suppressor/T regulatory cells, which inhibit ongoing T cell responses.  The B cells, on the other hand, are responsible for making antibodies, proteins that bind to bacteria or other cells and inhibit their activity or kill them. 

Lets first discuss how the T cells tell “self” from “non-self”.  The process is called thymic selection.  Each T cell has the capability of making approximately 10(10)-10(15) different types of receptors, which can each recognize different “antigens” or in other words, molecules found on other things.  This means that in the thymus T cells are made that can recognize pretty much anything in the universe !  So how is it then then the T cells that are made in the thymus do not attack the body? 

What happens is that in the thymus, many of the proteins or chemical compositions that are present in the body, or will be present in the body in the future as the person ages, are actually expressed in the thymus before birth.  So T cells that are generated before birth, when they encounter proteins in the thymus, they are instructed to die.  This causes the T cells that escape the thymus (because they didn’t recognize “self”) to be able to recognize everything else that is not the body.

So if T cells are deleted that recognize the body, then why do some people get autoimmunity?

There are a couple reasons.  Firstly the protein in the thymus that causes expression of every single body protein, called AIRE (stands for “autoimmune regulator”), is sometimes mutated.  Patients with a mutation in AIRE do not express all of the body proteins in the thymus and as a result autoreactive T cells enter the body and eventually cause autoimmunity.  Mind you, this is a pretty rare cause of autoimmunity.  AIRE mutations are found primarily in patients with the disease Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) which has autoimmunity against tissues that make hormones.

Another cause of autoimmunity is that the T cells that escape from the thymus, although at time of escape do not recognize “self”, upon encounter with a protein that is “non-self” they start to mutate so as to recognize “self”.  The ability of the T cell to mutate is critical since many pathogens mutate…so if the T cells could not recognize the mutated pathogens, then they would eventually kill the host.

So why is it then that not everyone gets autoimmunity? One reason is that recognition of proteins by T cells is not enough to stimulate the T cell.  The T cell also requires what are called “second signals”.  These are signals associated with “Danger”.  For example, the immune system intrinsically “knows” that certain things are potentially harmful for the host.  Bacteria have unique components that are not found in mammalian cells, such as lipopolysaccharide, which stimulate cells of the body such as dendritic cells, to provide a “second signal” to the T cell.  So if a bacteria has a “non-self” protein on it, but is not stimulating the “danger signal”, then the immune response will not be activated.  Only if there is: a) a unique nonself protein; and b) a “danger signal”, will be immune system be activated.

One way in which autoimmunity occurs is when there is inflammation at a site in the body that expresses proteins that may be recognized by the “self-reactive” T cells.

Let me provide an animal model example that proves this point.  Pamela Ohashi, now at the Ontario Cancer Institute, was the first author of a paper (Ohashi et al. Ablation of “tolerance” and induction of diabetes by virus infection in viral antigen transgenic mice. 1991 Apr 19;65(2):305-17) in which the cellular basis for self-nonself recognition was elucidated.

Ohashi developed two types of mice.  The first type expressed a foreign protein called LCMV glycoprotein (GP) selectively in the pancreas.  She did this by genetic engineering of the mouse so that the “foreign” protein is present in the mouse from fetal stages all the way to adulthood.  How as we discussed before, T cells capable of reacting against GP may theoretically be deleted by AIRE in the thymus.  Even if these cells are not deleted, they theoretically would not be able to cause autoimmunity (in this case very easily measurable by insulin levels because the autoantigen is in the pancreas) because there is no “danger signal”.   When she examined these mice, they grew old and as expected did not develop autoimmunity.

She then developed a second strain of mice that have T cells that recognize the GP protein.  These mice, called T cell receptor (TCR) transgenic mice have a large quantity of their T cells capable of recognizing the GP protein.  When T cells from the TCR-trangenic mice are added to pancreatic cells that express GP, the pancreatic cells are destroyed in vitro.

So when she bred the mice, so that the offspring had: a) GP expressed in the pancreas; and b) Large numbers of T cell recognizing GP, the animals did not get diabetes !!

When she examined these mice, she found that because there was no “danger signal”, despite the large number of circulating autoreactive T cells, the mice did not get diabetes. 

When she infected the mice with the LCMV virus, which causes a potent “danger signal” then self-tolerance was lost and the animals rapidly developed diabetes.

Perhaps more representative of human autoimmunity are model systems in which administration of an autoantigen is given in the presence of a “danger signal”.  For example, if one wants to induce a disease that mimics multiple sclerosis, one takes myelin basic protein, or peptides of it, mixes it with adjuvant (serves as the “danger signal”) and administers it into mice or rats of certain genetic backgrounds.  The disease that follows resembles multiple sclerosis in terms of cellular pathology (infiltration of T cells and macrophages in the central nervous system), as well as physically in that it culminates in paralysis.  Some animal models of multiple sclerosis actually have a relapse-remitting component, thus further resembling human disease.

The interesting thing is that this sort of “induced” autoimmunity is not specific to multiple sclerosis but can be seen in other conditions.  For example, administration of type I collagen to mice on the SJL background together with adjuvant leads to induction of a disease similar to human rheumatoid arthritis, called “collagen induced arthritis”.  Even autoimmune cardiomyopathy can be induced (autoimmunity against heart muscle) by injection of myosin peptides together with adjuvant.

So the question is, how could multiple sclerosis actually start in people? and how could we even have a chance of curing it?

Of course the answer is not known.  There are some indications that various pathogens may be associated with initiation of disease.  Unfortunately, conclusive studies do not seem to support this.  One of the problems may be that a lot of pathogens are still not known because they can not be cultured.  Various cell wall deficient bacteria may be involved which are difficult to detect, or which cause a “hit and run” in the sense that they induce the autoimmune disease but are subsequently cleared by the immune system.

Some interesting work in this area was conducted at Vanderbilt University in the early 1990s.  There was speculation that Chlamydia pneumoniae was involved in multiple sclerosis because it was found in the cerebral spinal fluid of some patients, and other patients had high levels of antibody to it (Sriram et al. Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis. Ann Neurol 1999 Jul;46(1):6-14).  So the group of Dr. Sriram actually starting infecting mice with Chlamydia pneumoniae and inducing EAE by immunization with self-antigen.  As seen in the figure below, administration of the live bacteria result in acceleration of disease.

 

So at present it is still not known what causes multiple sclerosis in patients.  We do know that there are inflammatory components that appear to be associated with its onset, and also that there are certain genetic factors that contribute.

The next question is, even if we do not know how it starts, what type of treatments could work against it?

The obvious concept is that since MS is an immunologically mediated disease, it would make sense that suppression of the immune system would be therapeutically useful.  Indeed, this is how steroids work.  Numerous other agents that nonspecifically suppress the immune system are used in the treatment of MS, for example, cyclosporine, azathioprine and methotrexate.  These agents have various side effects in part because they knock-down the majority of T cell functions of the immune system.

Another class of agents used in the treatment of MS are biological response modifiers, the first generation being inhibitors of the protein Tumor Necrosis Factor-alpha (TNF-a).  Originally TNF was identified independently by Beutler and Cerami.  Beutler found that mice injected with the immune stimulant BCG made a protein that could cause necrosis of tumors, whereas Cerami found that mice bearing tumors had a protein that was responsible for weight-loss (cachexia).  Eventually it was found that both of them were working with the same protein.  Interestingly, while the protein ended up not have profound anti-tumor activity in humans, it was discovered that the protein played a significant role in the various inflammatory conditions that affect humans such as rheumatoid arthritis.  Interestingly antibodies to TNF-alpha instead of inhibiting multiple sclerosis actually appeared to accelerate the disease (van Oosten et al. Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2. Neurology 1996 Dec;47(6):1531-4).  This led to the search for other modulators of the immune system.

Currently used biologics or biological response modifiers are interferon beta and glatiramer acetate.  Although their mechanism of action is not entirely known even today, they appear to reduce severity of multiple sclerosis.  Other agents include antibodies such as natalizumab (Tysabri).  These agents do not suppress all aspects of the immune system, so have a better toxicity profile as compared to chemotherapeutics, however, are still relatively nonspecific and have adverse effects.  Newer treatments such as anti-CD25 antibodies are also in clinical trials, but still are not antigen-specific.

Antigen-specific approaches towards treatment of multiple sclerosis would specifically involve the neutralization of the T cells that are causing damage to myelin but not other T cells.  The level of immune specificity in multiple sclerosis is very high, as explained in this video, that demonstrates only certain T cell clones act as “driver clones” in that they maintain the autoreactive phenotype.  One way of killing these autoreactive cells is to use the immune system to kill them by vaccination with expanded autoreactive cells.

Another way is to “trick” the autoreactive cells into believing that they are no longer necessary.  For example, the immune system intrinsically knows that if there is too high concentrations of a protein that it is attacking, more often than not, it should not be attacking it.  So what happens when the body is overloaded with an autoreactive antigen, the T cells “decide” that they should stop their reactions against it.  This is the concept of “high dose” tolerance.  An interesting study from the NIH demonstrated that repeated administration of myelin basic protein into mice after induction of experimental allergic encephalomyelitis actually inhibited disease progression (McFarland et al. Amelioration of autoimmune reactions by antigen-induced apoptosis of T cells. Adv Exp Med Biol. 1995;383:157-66).

The concept of adding to the body something that could exacerbate an autoimmune response sounds risky, however, this was successfully used by the Canadian company BioMS in that they have performed clinical trials using peptides from myelin basic protein and administering them intravenously.  The company is currently performing pivotal Phase III trials in patients with secondary progressive multiple sclerosis.

Treatment of multiple sclerosis would ideally involve the combination of immune modulation and regeneration.  Both of these are important since current approaches do not address the fact that after damage to the central nervous system has occurred, the natural regenerative capacity, while present, is still relatively limited.

We have previously published results of 3 multiple sclerosis patients being administered their own fat derived stem cells, and subsequently having positive responses.  This procedure, which was described in a collaborative publication with the University of California San Diego, appears to be working via concurrent immune modulation and induction of regeneration.

Multiple sclerosis is associated with T cell attacks against components of the central nervous system, specifically the myelin sheath.  This is why immunizing mice with components of the myelin sheath induces an MS-like disease called experimental allergic encephalomyelitis (EAE), which interestingly enough can be induced to resolve by stem cell therapy.

An interesting idea would be to take the autoreactive T cells (T cells causing the damage) and “immunize” against them.  This would theoretically induce the immune system to fight the part of the immune system that is pathological.  Additionally, such an approach would have advantages over non-specific immune suppressants which non-specifically shut down all T cell responses.

A recent publication (Loftus et al. Autologous attenuated T-cell vaccine (Tovaxin) dose escalation in multiple sclerosis relapsing-remitting and secondary progressive patients nonresponsive to approved immunomodulatory therapies. Clin Immunol 2009 May;131(2):202-15. Epub 2009 Feb 18) took exactly this approach in a clinical trial.

Myelin-reactive T cells were generated by stimulation with peptides from myelin basic protein, proteolipid protein, and myelin oligodendrocyte protein and subsequently inactivated by irradiation so as to not proliferate.  This would prevent the autoreactive cells from causing damage when used for immunization.  The authors used the term “attenuated myelin reactive T-cells” (MRTC).

Vaccination was performed subcutaneously and patients were assessed for over 52 weeks. 

Reduction in autoreactive T cells in the patients as well as suppression of relapses was reported.  MRI studies did not reveal resolution of plaques, nor progression in patients that responded by clinical parameters such as EDSS. 

This study demonstrates the potential of inducing an immune response against pathology-causing T cells. It may be that future studies will seek to amplify the therapeutic response by combining this approach with mesenchymal stem cells.

Multiple sclerosis is caused in large part by T cells attacking the myelin sheath that serves as an insulator for neurons, however other cells, such as myeloid cells, are also postulated to play a role. 

Activated T cells express the protein CD25, which is the alpha chain of the IL-2 receptor.  Clinically, the use of antibodies against CD25 has been reported to induce immune suppression in conditions such as transplant rejection.  In fact, the anti-CD25 antibody, Daclizumab (drug name Zenapax) is approved by the FDA for prophylaxis of acute rejection in renal transplant patients for use as part of a cocktail with corticosteroids and cyclosporin. 

A recent clinical trial investigated the addition of daclizumab to interferon beta in patients with multiple sclerosis that did not respond satisfactorily to interferon beta (Bielekova et al. Effect of anti-CD25 antibody daclizumab in the inhibition of inflammation and stabilization of disease progression in multiple sclerosis. Arch Neurol 2009 Apr;66(4):483-9).

The study subjects where treated with interferon beta for three months and for another 5.5 months by the combination of interferon beta and daclizumab.  If after 5.5 months of daclizumab and interferon the patients had a >75% reduction in MRI contrast enhancing lesions, then they were given treatment with daclizumab alone for an additional 10 months.  If the patients did not experience a response, the dose of daclizumab was doubled.

Of 15 patients, 5 reported treatment associated adverse effects, with 2 patients having to discontinue daclizumab.  9 of the 13 patients that continued on daclizumab and interferon had disease stabilization, which was observed even after conversion to daclizumab therapy alone.   

According to the authors, this study suggests that “Daclizumab monotherapy is effective in most patients who experienced persistent MS disease activity with interferon beta therapy“.

This study is very interesting because CD25 expressing cells are not only activated T cells but also T regulatory cells, therefore some caution has to be used when administering agents that target CD25 since depletion of T regulatory cells would hypothetically accelerate progression of autoimmune diseases.  Nevertheless, this study supports the continued investigation of combinations between daclizumab and interferon.

Bristol, UK -

An interesting study was published by Gordon et al (Human mesenchymal stem cells abrogate experimental allergic encephalomyelitis after intraperitoneal injection, and with sparse CNS infiltration, Neurosci Lett 2008 Dec 19;448(1):71-3) describing the use of human bone marrow derived mesenchymal stem cells in the treatment of EAE, a mouse model of multiple sclerosis.

Previously people have demonstrated that administration of mouse mesenchymal stem cells into mice with EAE results in remission of disease.  In this current paper an interesting, and very relevant variation of the previous study was made….human stem cells were used.  This is important since human mesenchymal and mesenchymal-like stem cells are being developed by companies such as Osiris and Medistem as “universal donor” cells.  This means that theoretically these cells are not rejected by the immune system.  So the authors of this paper wondered whether the human cells would survive in the mouse, and whether they would actually mediate a therapeutic effect.

The investigators induced EAE through administration of the peptide MOG 35-55 together with an adjuvant in order to elicit immune responses against myelin.  They injected 1 million mesenchymal stem cells intraperitoneally and found a statistically significant reduction in disease score.  Disease score is measured on a scale of 0-5 (0 – Normal; 1 – Tail flaccidity or hind limb weakness; 2 – Partial hind limb paralysis; 3 – Complete hind limb paralysis, spastic paresis, impaired righting reflex; 4 – Complete hind and fore limb paralysis; 5 – Dead).

On day 50 the disease score seemed to have went in remission (about 0.5) in the mice receiving mesenchymal stem cells but was still active (2) in the control mice.  Interestingly tracking of the cells showed that few mesenchymal stem cells were found in the brain on day 50.

This paper was really nicely written and provides some good background references for people interested in this area.  It is available for free online at this link.