Alan E. Beer Medical Center for Reproductive Immunology

It might be well to liken the immune system to a rifle where a target such as a bacterium is first sighted and then destroyed. Were the immune system more like a shotgun or even more dramatically like a stick of dynamite, it still would be possible to eliminate the target but at the cost of significant collateral damage. Thus the immune system must be able first to recognize a pathogen before it can effect its elimination. Immunologists have come to recognize two broad types of immune response. Both can identify an appropriate target, however, they utilize different strategies for identifying their target.

The first type of immune response is called the innate immune response, which is the oldest system evolutionarily and is found in both insects and mammals. This system possesses a limited set of components that can specifically recognize pathogens. It earned the name "innate" because the recognition elements are hardcoded into the system, making immune cells inherently ready to function when encountering pathogens. Over millions of years of gradual evolution, the ability to distinguish between foreign pathogens and self has developed. The limited repertoire consists of recognition elements that bind to specific markers found only on pathogens, not on the body's cells. An essential component of the innate immune system is the natural killer cell, which we will discuss further below.

The second is the adaptive immune response. Unlike the innate immune response, the recognition components are not hard-wired into the system but are generated by a genetic mechanism that randomly reshuffles immune-related genes permitting the generation of a vast repertoire of recognition components. The repertoire so constituted is of sufficient size that at least one component can bind to any potential pathogen invading from the outside world. However, the enormous size of the repertoire necessitates that cells representing each component must be few so that the total number of cells constituting the entire repertoire can be accommodated within the immune system. As a consequence, there are too few pathogen-specific cells present at the moment of pathogen encounter. A period of immunologic latency follows, during which these pathogen-specific cells increase in number. The system is said to be adaptive because of this latency period. This system includes the B cells and T cells.

Lymphocytes are crucial cells involved in both the innate and adaptive immune systems. Under a microscope, all lymphocytes look the same. However, we now understand that there are various types of lymphocytes, which we can differentiate by analyzing specific protein markers on their surfaces using a flow cytometer, a laboratory instrument.

Adaptive immunity involves two types of lymphocytes. One type is called B cells, and they are responsible for producing antibodies. There is a wide range of B cells, each producing a unique antibody. These B cells remain inactive until a pathogen arrives. Once a pathogen is detected, the immune system selects a specific B cell that can interact with the pathogen and trigger its replication, leading to the production of enough antibodies to eliminate the pathogen.

The other type of lymphocyte involved in adaptive immunity is the T cell. Like B cells, T cells have a diverse repertoire. However, they recognize pathogens through receptors fixed on their surfaces. When faced with a pathogen, T cells are stimulated to multiply, increasing their numbers to effectively eliminate the invading pathogen.

Another type of lymphocyte is the Natural Killer (NK) Cell. As mentioned above, it is a member of the innate immune system. It was named for a very interesting observation made at the time of its discovery. Unlike cells of the adaptive immune system, natural killer cells or NK cells do not require a latency period needed for expansion in their numbers like cells of the adaptive immune system to kill the pathogen. NK cells are naturally competent to effect immediate killing. In the years following their discovery, much has been learned about their function. We now know that they can also respond to a pathogen-infected cell by releasing cytokines (locally acting hormone-like molecules produced by immune cells) that can direct other cells to perform the killing. Researchers have found that these two functions reside in distinct and separable NK subtypes when found in the blood.

So far, we have noticed that T cells can be differentiated based on the molecules they carry on their surfaces to target specific threats. However, T cells can also be categorized by their functions. Researchers studying a specific type called T helper cells have discovered that they can be further classified based on the cytokines they produce. Presently, we recognize approximately six or seven types of these cells, each designated as Th1, Th2, Th3, TR1, Th9, Th17, and T regulatory cells (Treg cells).

They each produce different patterns of cytokines. However, when T cells were first characterized by their cytokine production patterns only Th1 and Th2 types of cells were thought to exist. In a broad generalization, Th1 cells were thought to drive other cells into inflammatory patterns of activity while Th2 cells were thought to oppose Th1 activity. The mystery of how T helper cells functioned became clear. When activated, they release these hormone-like molecules into the local environment and drive other cells into activities that are orchestrated according to the nature of the cytokine pattern of the T helper cell type.

It became possible to assess the balance between these initially defined T helper cell subtypes present in the blood of an individual by laboratory testing. Lymphocytes are purified from blood and then stimulated in a test tube, causing them to produce cytokines. Using the flow cytometer, a laboratory instrument that is capable of analyzing cells one at a time, it became possible to quantify the relative numbers of cells producing cytokines representative of the T helper subtypes, Th1 and Th2, and then to calculate a ratio of the two subtypes. The resultant ratio became known as the Th1/Th2 ratio.

Reproductive immunologists have carried the study of these cell types to reproduction and, in particular, to the implantation of the early embryo into the lining of the uterus. A general theory emerged over the last ten years based on accumulated data proposing Th2 predominance as characteristic of successful pregnancy while Th1 predominance is characteristic of pregnancy failure (1) (2) (3) (4). The theory suggests that rebalancing the Th1/Th2 ratio toward an increase in the relative number of Th2 cells might result in improved reproductive success. To select patients with a history of repeated reproductive failure who might benefit from such rebalancing, the patient's peripheral blood needs to be assessed for using the Th1 Th2 Assay. Those patients with an abnormally high ratio would be considered candidates for immune-based therapy directed toward restoring a more optimal ratio.

More recently, information has emerged that has shown that the theory does not resolve certain questions regarding reproductive success and failure. For example, it is now well-recognized that the process of implantation of the early embryo into the uterine lining involves several Th1-type cytokines. Further, events that take place at the site of implantation do not involve either Th1 or Th2 lymphocytes but rather involve members of the innate immune system. The best characterized of these lymphocytes is the NK or natural killer cell. The NK cell is of particular interest today because we have come to recognize that NK cells resident in the uterine lining act in very different ways than those that circulate in the peripheral blood. The so-called decidual NK cell (decidua is the pregnancy-transformed lining of the uterus) is a cell that might be regarded as a hybrid of the two types of NK cells that circulate in the blood because it combines the two functions of blood NK cells: killing and cytokine release. In the uterine lining, the decidual NK cell, while fully armed for killing activity becomes active only to the extent that it releases cytokines that support the growth and transformation of the uterine lining for the support of the placenta. The inherent killing power of the decidual NK cell appears to be reserved until a danger is recognized, such as infection, whereupon it assumes the role of the killer cell, acting to eliminate infected cellular material.

Because of its dual function, the decidual NK might be regarded as distinct from the two types of NK cells circulating in the blood. Decidual NK cells serve a particularly important role in supporting the growth of the placenta producing a collection of different cytokines that direct blood vessel growth and transformation. Under normal conditions, the killing capacity that is inherent within decidual NK cells remains latent. Investigators have pointed to these distinctions, suggesting that laboratory study of blood NK cells is, theoretically, unlikely to shed light upon the activity of decidual NK cells. However, in medicine, theory alone is insufficient to make predictions. Publications cited below demonstrate high NK activity in blood NK cells correlates with reproductive failure.

Two therapies used by physicians practicing reproductive immunology are intravenously administered immunoglobulin (a biologic comprising the antibody-containing fraction obtained from the serum of multiple diverse donors) commonly known as IVIG and Humira. Humira is one of a new class of drugs that bind and render inactive one of the most potent cytokines released by Th1 cells known as TNFα. These drugs were selected because they were reasoned to be useful in patients with high levels of NK cell activity or Th1 predominance in the peripheral blood.

IVIG therapy has been used for a variety of immunologic conditions. The first demonstration that such preparations might be useful in treating conditions that involve an excessive immune response was made in 1981. Subsequently, the number of autoimmune and inflammatory diseases responding to IVIG therapy has been greatly expanded. A recent literature review appearing in the journal Trends in Immunology describes such uses in the general field of clinical immunology. The review cites literature indicating that IVIG therapy in part acts by diminishing TNFα.

The development of Humira represents one of the great achievements in translational medicine (the translation of basic science discoveries into clinical practice). It was recognized that several conditions characterized by over-activity of the immune system exhibit, amongst other features, excess quantities of the Th1 cytokines. Basic scientists reasoned that an agent that could bind and inactivate the most potent of these cytokines, TNFα, might mitigate the damage affected by excess Th1 activity. Humira was developed as an antibody that could be administered to patients reducing or eliminating excess levels of cytokine. Its success in clinical practice has been no less than phenomenal. It is now used in a variety of conditions, including rheumatoid arthritis, psoriasis, and inflammatory bowel diseases, to mention just a few.

A number of reproductive immunologists reasoned that these two drugs might be useful in patients suffering recurrent reproductive failure. A second literature review on IVIG, which appears in the journal Clinical Chemistry and Laboratory Medicine, describes its specific use in reproductive failure.

We published the first two papers demonstrating the success of Humira in patients suffering recurrent pregnancy failure who had elevated ratios of the Th1/Th2 ratio as demonstrated in lymphocytes taken from the blood:

Paper 1: Winger EE, Reed JL: Treatment with tumor necrosis factor inhibitors and intravenous immunoglobulin improves live birth rates in women with recurrent spontaneous abortion. Am J Reprod Immunol 2008; 60:8–16.

Paper 2: Winger and Reed et al., Treatment with Adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF, Am J Reprod Immunol. 2009 Feb;61(2):113-20. Epub 2008 Dec 3.

Several points of controversy exist amongst reproductive immunologists. While there are abundant studies correlating abnormalities in NK function as well as elevation in Th1/Th2 ratios, there is concern that therapies directed to their moderation might disturb the process of implantation of the nascent fetus in the uterine lining. The concern appears to be twofold. First, the implantation process itself appears to rely upon inflammatory cytokines. Second, the decidual NK cell is thought to require activation to alter its role in the transformative process permitting the uterine lining to support the growing placenta. It has been speculated that these processes might be blunted by the use of drugs such as Humira. However, we disclose an observation in our paper. For Humira to affect the process of implantation, it must be present at active concentrations during the time of implantation. We show that Humira can be administered well in advance of the time of embryo transfer during IVF therapy. By the time of embryo transfer, there is insufficient residual drug present to affect the cytokine balance at the implantation site, making moot these concerns. Decidual NK cells, likewise, are unaffected by drug administration. It has become clear that the immune system must balance the needs of activation of the immune system in response to challenges with the ever-present danger of attacking oneself, or, in the case of pregnancy, the embryo.

As noted above, cells may mature into Th1 and Th2 cells responding to immunologic challenges in the different ways that have been described. It is now clear that T cells may also mature into cells that perform a very different role. They suppress immune responses. Immune responses must be contained, particularly after a challenge has been dealt with, the immune response must be attenuated to limit collateral damage. Moreover, it is now recognized that such cells are essential to the prevention of immune attack on the fetus.

In addition to the immunotherapies described above, sub-anti-coagulant doses of heparin are also commonly used to prevent spontaneous abortion. It is believed that heparin acts as an anti-inflammatory agent in a manner analogous to Humira.

In summary, the field of reproductive immunology is a recognized academic and clinical field within the broader field of immunology. Translation of the concepts developed within the field has been practiced by a respected minority of clinicians practicing reproductive medicine. Their success has received considerable attention both within the field and by the public. The published data support the predictive value of immunologic testing.

References:

1. Paradisi R et al. T-helper 2-cytokine levels in women with threatened abortion. Eur J Obstet Gynecol Reprod Biol. 2003 Nov 10;111(1):43-9.
2. Daher S et al. Cytokines in recurrent pregnancy loss. J Reprod Immunol 2004 Jun;62(1-2):151-7.
3. Raghupathy R et al. Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent spontaneous abortion. Hum Reprod. 2000 Mar;15(3):713-8.
4. Kim, Joanne Young Hee Kwak; et al., Diagnosis and treatment of infertility, United States Patent Application 20040105858.
5. Tha-In, T et al., Modulation of the cellular immune system by intravenous immunoglobulin, Trends in Immunology (2008) 29(12):608-15.
6. Winger and Reed et al., Treatment with Adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF, Am J Reprod Immunol. 2009 Feb;61(2):113-20. Epub 2008 Dec 3.
7. Winger EE, Reed JL: Treatment with tumor necrosis factor inhibitors and intravenous immunoglobulin improves live birth rates in women with recurrent spontaneous abortion. Am J Reprod Immunol 2008; 60:8–16.
8. Omwandho CO, Gruessner SE, Roberts TK, and Tinneberg HR, Intravenous immunoglobulin (IVIG): modes of action in the clinical management of recurrent pregnancy loss (RPL) and selected autoimmune disorders, Clinical chemistry and laboratory medicine, CCLM / FESCC ( 2004)42(4):359-70.
9. Sundrud MS, Rao A, New twists of T cell fate: control of T cell activation and tolerance by TGF-beta and NFAT, Curr Opin Immunol. 2007 Jun;19(3):287-93.
10. Girardi G, Redecha P, Salmon JE: Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nat Med 2004; 10:1222–1226.
11. Borentsztajan K, Peppelenbosch MP, Spek A, Factor Xa: at the crossroads between coagulation and signaling in physiology and disease, Trends in Molecular Medicine (2008 September) 14(10):429-440.