The TH1 (T Helper 1) and TH2 (T Helper 2) Intracellular Cytokine Assay

The name of this test is nearly as complicated for patients as understanding what the test means. This short article will help you understand the importance of this assay for you as you prepare for a conception cycle.

In 1995, it became very clear to us that products of an activated immune system could damage the placenta and cause miscarriages as well as damage the embryo and cause implantation failure. The immune cells that could be counted and assayed at that time were of a family called natural killer cells with their CD56 marker (for more information, see Natural Killer (NK) Cell Assay for the Consumer). These cells produce a cytokine called tumor necrosis factor alpha (TNF alpha). When this molecule is released and becomes higher than normal in the body it enters the embryo, the placental cells and even cells of other active organs in the body and causes DNA damage in the cell, a process called apoptosis. The DNA of the rapidly dividing embryo cells is literally glued together and this stops cell division. The embryo or the baby "withers on the vine" and dies.

The first test to identify couples with this problem was the Reproductive Immunophenotype (for more information, see A Guide to Interpreting the Results of the Reproductive Immunophenotype). In this test we were able to identify women with too many NK cells and it was a great breakthrough for us. It soon became very clear that identifying these women and treating them was not successful in all. Simply counting the cell numbers was not specific enough.

We then developed the NK assay. This test cultures the NK cells of the patient with placental-like cells to determine how aggressively they kill the placental-like cells in 2 hours. We found that a killing power of above 15% at a ratio of 50:1 was too high and associated with infertility and pregnancy losses. We added one of the treatments given to patients, IVIG, to the NK assay. By doing this, we were able to determine if the IVIG would suppress the NK killing and also determine how much IVIG we needed to give the patient. This assay has been widely accepted and is now used worldwide.

It became clear to me over the years that there were some patients where treatment as I knew it simply did not work. Such patients entered a cycle of conception with a "green light, go ahead" from me, yet failed again and again. Certainly these patients were on an "Olympic team" of their own and required further treatment.

It was also becoming clear that the NK cells that I was evaluating are also the "bad guys" causing rheumatoid arthritis, and many of them prefer to leave the blood and live in the joints. I queried, "do women with implantation failures and recurrent losses, with optimal immune therapy as I knew it, have NK cells living as residents in the uterine lining, just as they do in the joint spaces with rheumatoid arthritis?" The answer to this question would turn out to be a definitive yes.

Natural Killer Cells in the Uterus

I began to do immune pathology on the pregnancy loss tissue and do endometrial biopsies on cycle day 26 of a normal cycle, or at the time of an implantation failure. Not surprising, 2% of my patients had NK cells surrounding the lost pregnancy and many had NK cells living in the uterus. At this time I began to explore the use of anti-rheumatoid arthritis (anti-TNF alpha) medications that direct their activity against the NK cells and TNF alpha molecules. I have studied three such medications that are FDA approved. Many women who had failed three IVF cycles before seeing me were successful during their next attempt following the anti-rheumatoid arthritis therapy. Many surprisingly became pregnant on their own. I have data on more than 200 babies born healthy in this program.

Since this time it has become clear that TNF alpha release by NK cells is really the fire alarm of the immune system. When elevated, it can indoctrinate other cells to perform aggressive roles of inflammation and even more TH1 cytokines to be released. These cells are not NK cells that are CD56+ but are CD3, and CD4 T cells that love to migrate to tissues including the uterus and the pregnancy where they can perform aggressive roles. We began to explore this possibility and thus five years later, the TH1/TH2 cytokine assay.

The immune system is balanced between a TH1 (autoimmune) and TH2 (pregnancy or suppressive response). Almost everyone is balanced in this system. Women with implantation failure or recurrent pregnancy losses are unbalanced; they are autoimmune with too much TH1. The TH1 (autoimmune "bad guys") produce TNF alpha, Interferon Gamma and Interleukin 2. Too much concentration of these cytokines kill cancer cells (a good thing) and a chronic state of too much of these can cause implantation failures, recurrent abortions and even thyroid, insulin and serotonin disturbances. (See Figure 1 below.)

Figure 1

The cells and the cytokines that balance this system and take over during pregnancy to protect it from being killed as cancer cells are the TH2 cytokine producing cells. These cytokines that protect the baby and down-regulate autoimmunity are Interleukin 4 (IL-4), IL-5, IL-6, IL-9, IL-10 and IL-13. During a normal pregnancy where the mother and baby experience no problems there are very high levels of TH2 cytokines and TH2 cytokine bearing cells in the blood and very low levels of TH1 cytokines and TH1 cytokine bearing cells in their body. Since high TH1 is also associated with the diseases Lupus and Rheumatoid Arthritis, these diseases often get much better during pregnancy and flare again following pregnancy. We have known this for years, but now understand the cellular basis for this phenomenon.

The TH1/TH2 intracellular cytokine assay simply counts the "good guys" and the "bad guys." We then give the results as two ratios:

1. the ratio of TNF alpha bearing cells to IL-10 bearing cells (TNFa/IL-10); and
2. the ratio of the Interferon Gamma bearing cells to IL-10 bearing cells (IFNg/IL-10).

The normal values are as follows:

We have found that women with the highest numbers are as follows:

1. Women with one live born child followed by secondary infertility or recurrent pregnancy losses. They are the coaches of the "Olympic team" with some of the highest values. Many of these women need the Rheumatoid arthritis drugs for 30 days before they begin a cycle of conception.

2. Second in line are women with three or more implantation failures following IVF.

3. Third in line are women with three or more pregnancy losses.

This new test is a godsend based on my experience. I feel strongly that we can use this test to find those infertile women and recurrent abortion women who will fail their next cycle without aggressive treatment. I look forward to the day when couples will stop the abuse of three or more implantation failures or recurrent losses before we get serious that there may be an immune problem that requires treatment. I strongly believe that we can find these couple before their first IVF cycle loss. Couples are waiting longer to start their families. Certainly pregnancy risk assessment is advocated in many areas of medicine. We must now add immune assessment to find these unfortunate couples that will spend a fortune in money, time and emotion and get nothing in return.

In women who find themselves on the list above, I recommend that minimal testing include:

1. Endometrial biopsy on cycle day 26.

2. Placental immune pathology of a D and C specimen done at the time of a loss.

3. Natural killer cell assay.

4. TH1/TH2 intracellular cytokine assay.

Couples can work with Dr. Stricker in person at our office in Los Gatos, or can consult with him long distance by phone consultations. Registered patients who need blood draw and shipping instructions for this test should contact us.

References
Kwak-Kim JY, Chung-Bang HS, Ng SC, Ntrivalas EI, Mangubat CP, Beaman KD, Beer AE, Gilman-Sachs A. Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF. Human Reproduction. 2003 Apr;18(4):767-73.

The information contained in this article is not intended to be a medical diagnosis, treatment or medical advice in any way, as it is general information and cannot be relied on without consultation with your physician. It is not intended nor is it implied to be a substitute for profession medical advice. As medical information can change rapidly, we strongly encourage you to discuss all health matters and concerns with your physician before embarking on new diagnostic or treatment strategies.

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Alan E. Beer Center for Reproductive Immunology & Genetics
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Date: 8-20-08, Time: 1:15 am.

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