Alan E. Beer Medical Center for Reproductive Immunology

Summary

Lymphocyte Immunization Therapy or LIT is a procedure whereby white blood cells from the prospective father are injected into the skin of the prospective mother to prepare the maternal immune system for pregnancy. Because pregnancy tissues are the product of both the mother's and the father's genes, LIT assists the mother's immune system in the development of immunologic tolerance to the genetically foreign pregnancy tissues.

Below are some recent advances in the science of immunology. The paradox of immunization with paternally-marked cells to tame the mother's immune response to her paternally-marked fetus is explored. Immunologic science has come to embrace an entirely unexpected aspect of the immune system, what might be called the yang of the Chinese balance inherent in all things, the yin and yang.

The destructive forces inherent in the immunologic army are balanced by a second arm of the immune system protecting the self as well as the fetus from unfettered immune responses. Immunologists have long appreciated the importance of immune recognition of pathogens permitting their targeting for destruction. More recent science has begun to explain the capacity of the immune system to divide the recognition of antigens (substances that are recognized in an immune response) such as proteins into friends and foes. The decision is now known to take place at the time of their first encounter, the moment when proteins and the immune system are introduced. If the introduction is friendly, then the second arm of the immune system, the yang is switched into action and the immune system becomes tolerant in future encounters.

Lymphocyte immunotherapy (LIT) seen in the light of current science appears to enhance the friendly introduction of the immune system to paternal-gene programmed proteins, the same that the mother will encounter in placental tissues. The story that unfolds reveals new and important cells in the immune response. The numbers of these cells (T regulatory cells) in the blood have been shown to correlate with outcomes. Other cells (dendritic cells) are described that make the introduction. Appropriate interaction with these cells is paramount for success. Deceptively simple, proper LIT technique is very important for making the proper introduction and ultimate success.

How LIT Works

Our conventional understanding of the immune system might suggest that the mother's immune system would reject this foreign genetic material. However, during a healthy pregnancy, we see that the mother's immune system becomes tolerant to the fetus's paternally-determined proteins and this allows the pregnancy to continue to term. When Peter Medawar, an early Nobel Prize winner for the immunology of transplantation, considered the problem of maternal tolerance, he thought that the maternal immune system must somehow be oblivious to the fetus, either because of physical separation created by the placenta or by some other means that would avoid alerting the maternal immune system to the presence of the foreign fetal tissues. We no longer subscribe to this theory. We understand that the maternal immune system actively decides to tolerate rather than reject the fetus.

But how does the maternal immune system develop this tolerance? The immune system is confronted by a vast diversity of different substances most of which consist of genetically-encoded proteins. A decision must be made: are these proteins to be treated as friend or foe? The immune system is in constant contact with self-tissues. Were the immune system to recognize our thyroid gland or our pancreatic tissues as a foe, for example, an attack would ensue resulting in thyroiditis or diabetes. When such inappropriate recognition occurs, we refer to these diseases as auto-immune. The immune system has many different, and perhaps, redundant mechanisms to avoid these disastrous consequences. One common denominator of these mechanisms is a requirement that the immune system be introduced to self-substances in a manner that conveys the fact that these substances are friend, not foe. Throughout evolution, the immune system has developed a tool chest for the identification of foes as well as corresponding mechanisms for the identification of friends. The immune system is introduced to new substances, as it were, under the auspices of these mechanisms either as a friend or foe. The immune system follows the introduction responding to these substances in future encounters by attack and rejection or tolerance.

We now understand that many pregnancies are lost in their earliest stages because the immune system does not acquire this necessary tolerance to the foreign fetal tissues. Why tolerance does not occur remains a subject of considerable investigation and not inconsiderable debate. In the mid-1990s, an immunologist working in Japan named Shimon Sakaguchi reported the existence of a new type of immune cell. He named this new type of immune cell the T regulatory cell. When he combined these new cells with conventional immune cells, the activity of the conventional immune cells was markedly diminished. Over the last decade and a half, many immunologists have joined in the study of the behavior of T regulatory cells. A picture has now emerged that suggests that T regulatory cells recognize specific substances much in the same way as conventional immune cells. However, despite these similarities, their responses are very different. T regulatory cells turn off an immune response that conventional immune cells might start. It is thought that regulatory T cells comprise a major component of the immune system's ability to avoid self-attack or autoimmunity.

The importance of T regulatory cells has not escaped the attention of reproductive immunologists. Reproductive immunologists are specialized physicians and scientists who study the interactions of the immune system with the organs of reproduction. They have found a general increase in T regulatory cells in the blood during the period following ovulation as though anticipating the arrival of the embryo in the uterus with all its exposed paternal-gene encoded proteins. Studies of the endometrium (the uterine lining) further demonstrate an increase in these cells, but only in normal pregnancies. In those women who later go on to lose their pregnancies, the increase in these cells appears to be blunted. However, in healthy pregnancies, T regulatory cell numbers continue to expand upon the implantation of the embryonic tissues into the endometrium. Recent studies suggest the increase consists largely of specific T regulatory cells that recognize the embryonic tissues. These specific T regulatory cells are recruited from the blood, multiply after contact with embryonic tissues, and then are retained in placental tissues. It appears that these pregnancy-recognizing regulatory T cells might act as guardians of pregnancy tissue, protecting it against attack from conventional immune cells.

How do these investigations help us understand LIT? We have learned that the old theory that the immune system tolerates the foreignness of the embryo by simply avoiding contact with fetal tissues is probably not correct. Current science persuades us that immune recognition of fetal tissues occurs, but that it occurs under circumstances that identify embryonic tissues whether they be the product of maternal or paternal genetic programming as friends. Moreover, the most likely means for the generation of tolerance to embryonic tissues involves the generation of T regulatory cells. Failure to recognize embryonic tissues leads to insufficient development of tolerance mechanisms and, by extension, a decrease in the generation of tolerance-producing T regulatory cells. LIT may augment the development of tolerance possibly by increasing the numbers and appropriate distribution of T regulatory cells.

To understand how LIT works, it is also necessary to understand that T cells do not work alone but are coordinated in their function by other cells. Thus the immune response involves the interacting activity of different cell types. We have discussed two types of T cells. One is the effector T cell which recognizes a specific tissue or protein and attacks. The other is the regulatory T cell that recognizes the specific tissue recognized by the effector T cell but, as we have discussed, prevents the attack. A major question before immunologists is what process or cell type guides the decision as to which cell dominates the immune response. We have now come to learn that the decision may be made, in large part, by yet another cell type known as the dendritic cell.

The dendritic cell, like the T regulatory cell, has been the object of intense scrutiny over the last couple of decades. It has long been known to patrol the tissues of the body, particularly those tissues that make up the borders of the body where infectious agents may gain access to the body's interior, like the skin. There they act as scavengers of proteins from potential pathogens. Once they have consumed the invading and potentially dangerous organisms, they become mobile and move to regional immune centers known as lymph nodes where they can interact with T cells, selecting appropriate T cells and activating them thereby beginning the immune response.

More recent investigations have revealed, to our surprise and amazement that dendritic cells are far more versatile than we initially suspected. As we have discussed, early studies suggested that they functioned only to turn on conventional T cells to initiate an immune attack. Recent studies have revealed that they can also turn on T regulatory cells. In their role patrolling the body's tissues, they encounter an enormous quantity of self-gene-encoded proteins, those to which we must maintain tolerance. Studies now show us that dendritic cells participate in the maintenance of tolerance by turning on T regulatory cells that turn off immune responses to self-tissues, thereby avoiding dreaded autoimmunity. The recent and surprising discoveries about dendritic cells have shown us that they decide whether to turn on conventional immune cells or T regulatory cells by the way that they are introduced to the various substances they encounter. A friendly introduction is paramount to proper immune tolerance.

As we are now coming to better understand LIT, we are learning that LIT is all about making a friendly introduction to paternal proteins. White blood cells known as lymphocytes from the prospective father are collected and separated from the other blood constituents and injected into the prospective mother. The introduction of paternal proteins present on the paternal lymphocyte is thus consummated but the immunologic result remains to be determined by the nature of their introduction. As we have noted, dendritic cells take their cue from the nature of this introduction. Much of the controversy that has arisen about the efficacy of LIT appears to have been generated by a lack of understanding of this point.

An abundance of literature has been generated over the last two decades describing LIT procedures, mechanisms of action, and success as well as lack of success. Safety concerns have been raised in the past. However, recent studies by Kling and others have found little evidence to support these concerns.

The efficacy of LIT remains a concern of many reproductive immunologists. Based on a review performed by Ober and associates, efficacy was not demonstrated. A careful review of their study methods does not support their conclusions. While the LIT procedure itself seems quite simple, when it is not carefully followed, it can be expected to fail. Clark has shown that the lymphocytes that are collected from the father carry with them a marker that makes the introduction to the immune system a friendly one. It is technically known as CD200. It is found on the surface of fresh lymphocytes. Unfortunately, when lymphocytes are stored in the refrigerator, the CD200 marker is lost. Clark’s CD200 study.

If the lymphocytes used in the procedure lack the critical CD200 marker, a friendly introduction cannot be expected. In addition, where these cells are located is important as well. The cells should be injected into the skin, a site guarded by patrolling dendritic cells, not in the muscle or the vein. A review of the studies compiled by Ober does not appear to make any consistent distinction between the different procedures followed. Thus, it is not surprising that they did not find positive results.

While LIT is practiced in clinics and universities around the world, a ruling following the Ober study has been made by the FDA regarding the future practice of LIT in the United States. Currently, LIT is not practiced within the United States. Alan Beer practiced LIT until the publication of this letter in 2002. Upon cessation of LIT at his center, a significant increase in pregnancy loss was noted amongst patients being seen for recurrent pregnancy loss (16% to 36%). However, today doctors in the USA are unable to provide this therapy. Many patients, however, still wish to seek LIT in other countries where the therapy remains available.

We believe that the future for LIT remains bright. While there remain significant issues that need to be resolved, we feel that, when properly conducted, LIT offers significant assistance to patients suffering recurrent pregnancy loss and unexplained infertility. A variety of paths to introduce this therapy back into the United States may later be followed.

References

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